You are on page 1of 75

STUDY OF

CYTOTOXICITY EFFECTS OF CISPLATIN AND GOLD DRUGS


ON HUMAN OVARIAN CANCER CELL LINES
WITH ATR-FTIR SPECTROSCOPY TECHNIQUES

A dissertation submitted to the University of Manchester for the degree of

Master of Science (Biotechnology) in the Faculty of Engineering and Physical


Sciences

2010

NIDHI KAPIL

School of Chemical Engineering and Analytical Science


CONTENTS

DESCRIPTION PAGE
NO.

TABLE OF CONTENTS 2

LIST OF FIGURES 3

LIST OF TABLES 5

NOMENCLATURE 6

ABSTRACT 7

DECLARATION 8

COPYRIGHT STATEMENT 9

ACKNOWLEDGEMENTS 10

CHAPTER 1: INTRODUCTION 11

CHAPTER 2: LITERATURE REVIEW 14

2.1 Cancer 14

2.2 Ovarian Cancer 15

2.3 Treatment of Ovarian Cancer 16

2.4 IR Spectroscopy 22

CHAPTER 3: EXPERIMENTAL TECHNIQUES AND 40


METHODOLOGY

3.1 A2780 cell line culture 40

3.2 Cell Fixation 40

3.3 ATR- FTIR Measurement 40

3.4 FTIR Measurement 43

CHAPTER 4: DATA ANALYSIS AND RESULTS 46

4.1 ATR-FTIR Data Results 46

4.2 FTIR Data Results 58

CHAPTER 5: CONCLUSIONS 64

REFERENCES 65

WORD COUNT 19,805

-2-
List of Figures

Figure Description Page


No. No.

1 Schematic diagram showing normal and cancerous cells 14


2 Schematic diagram showing cancer stages 15
3 Cisplatin 18
4 Taxol 20
5 Methotrexate 20
6 Fluorouracil 21
7 Olaparib 21
8 Chart of characteristics vibrations 22
9 Schematic diagram showing stretching & bending modes of CH2 23
Group
10 Schematic diagram showing different absorption bands 24
11 Schematic diagram showing Spectrometer Layout 25
12 Schematic diagram showing working of FTIR spectroscopy 26
13 Schematic diagram showing evescent wave 27
14 Schematic diagram showing working of ATR-FTIR spectroscopy 28
15 FTIR-ATR experimental set up 42
16a ATR Germanium crystal base 42
16b ATR Germanium crystal top 42
17 - 20 ATR spectra of CIS & KF(IC-30) and PCA scores Plots, 52
(950-3800 cm-1 )
21-22 ATR spectra of CIS & KF(IC-50), (950-3800 cm-1 ) 52
23-34 ATR spectra of CIS & KF(IC-50), PCA Score Plots & loading Plots 53-54
(950-3800 cm-1 )
35-38 ATR spectra of CIS & KF(IC-70), PCA Score Plots (950-3800 cm-1 ) 55

-3-
59-60

Figure60-61 Description Page


No.65 No.

39-44 ATR spectra of CIS (IC-30), (IC-50). & (IC-70) and PCA Score Plots 55-56
(950-3800 cm-1 )
45-52 ATR spectra of KF (IC-30), (IC-50). & (IC-70), PCA Score Plots and 56-57
loading Plots.
53-60 ATR spectra of CIS & KF(IC-50), PCA Score Plots & loading Plots 58-59
(Lipid Region )
61-68 ATR spectra of CIS & KF(IC-50), PCA Score Plots & loading Plots 59-60
(Protein Region )
69-76 ATR spectra of CIS & KF(IC-50), PCA Score Plots & loading Plots 60-61
(Carbohydrate & Nucleic acid Region )
77-80 FTIR spectra of CIS & KF(IC-30), PCA Score Plots and RMeiS 65
(Iteration 1 & 8)
81-84 FTIR spectra of CIS & KF(IC-50), PCA Score Plots and RMeiS 65-66
(Iteration 1 & 8)
85-88 FTIR spectra of CIS & KF(IC-70), PCA Score Plots and RMeiS 66
(Iteration 1 & 8)
89-92 FTIR spectra of CIS(IC-30, IC-50 & IC-70 ), PCA Score Plots and 67
RMeiS (Iteration 1 & 8)
93-96 FTIR spectra of KF(IC-30, IC-50 & IC-70 ), PCA Score Plots and 67-68
RMeiS (Iteration 1 & 8)
97-98 FTIR spectra of CIS(IC-30, IC-50 & IC-70 ), PCA Score Plots (950- 68
4000 cm-1 and RMeiS (Iteration 8)
99-100 FTIR spectra of KF(IC-30, IC-50 & IC-70 ), PCA Score Plots (950- 68
4000 cm-1 and RMeiS (Iteration 8)

-4-
List of Tables

Table Description Page No.


No.
1 Properties of infrared transmitting materials for ATR-IR 28
spectroscopy.
2 Details of samples and their spectrum 41

3 Details of Observed Peaks and their assignment in Cisplatin and 50


KF01-01 treated samples in Nucleic Acid & Carbohydrate and
Lipid Region.

4 Details of Observed Peaks and their assignment in Cisplatin and 51


KF01-01 treated samples in Protein Region.

-5-
NOMENCLATURE/ABBREVIATIONS

SYMBOL/ABBREVIATION DESCRIPTION

FTIR Fourier Transform Infrared

ATR Attenuated Total Reflectance

KF Gold complexes drug


(KF01-01)

CIS Cisplatin drug

IR Infrared

DNA DeoxyriboNucleic Acid

CO2 Carbon Dioxide

& And

PCA Principal Component


Analysis

PARP Poly ADP Ribose


Polymerase

UV Ultraviolet

MRI Magnetic Resonance


Imaging

PBS Phosphate Buffer Saline

RMieS Resonant Mie Scattering

EMSC Extended Multiplicative


Signal Correction

-6-
ABSTRACT

Attenuated Total Reflectance (ATR) and Fourier Transform Infrared (FTIR)


microspectroscopy has been used for studying the effect of cisplatin and KF01-01 drugs
on A2780 ovarian cancer cell lines as a function of concentrations . The A2780 cells were
exposed to KF01-01 and cisplatin drugs at different concentrations (IC-30, IC-50 and
IC-70) for 24 hrs. The spectra of these drug treated cells were acquired using ATR &
FTIR techniques. Further principle component analysis (PCA) was used for studying the
finer details of spectrums for evaluation of changes occurred in cell composition due to
these drugs at different concentrations..

Results from PCA analysis of ATR & FTIR data revealed that there are remarkable
differences in the spectra at all concentrations of KF01-01 and cisplatin. PCA analysis of
ATR and FTIR spectral data of samples treated with Cisplatin and KF01-01 drugs
revealed that drug samples at IC-50 concentration are clearly separable from the samples
of IC-30 and IC-70. Analysis of spectral data samples of both drugs corresponding to
lipid, protein and carbohydrate & nucleic acid regions revealed different clusters of score
points pertaining to these drugs . It is observed that clusters are more clear in
carbohydrate & nucleic acid region in comparison to lipid and protein region. These results
show that both drugs are inducing completely different chemical changes at different bands
indicating different working mechanism of both drugs. The analysis of spectra of KF01-01
& cisplatin treated cells at each concentration suggest that all the concentrations of KF01-
01 and cisplatin have an anticancer effect on A2780 cells . These results indicate similar
cytotoxic profile of KF01-01 and cisplatin at concentrations corresponding to IC-30 and
IC-70 and these produces important cell killing effect on A2780 cells at IC-50. Further
these results also indicate that cytotoxic effect of both drugs is non linear and IC-50
appears optimum dose.

It is observed that results of both ATR and FTIR techniques are in agreement . However
the spectrums measured with ATR revealed more details in comparison to spectral data
measured with FTIR system. FTIR spectral data had distortions and RMieS corrections
were applied for removal of distortions before PCA analysis. These correction techniques
sometimes can remove the important bands from the spectra. Accordingly ATR
spectroscopy appears better than FTIR spectroscopy.

-7-
DECLARATION

No portion of the work referred in the dissertation has been submitted in support of
an application for another degree or qualification of this or any other University or
other Institute of Learning.

-8-
COPYRIGHT STATEMENT

1. Copyright in text of this thesis rests with the author. Copies (by any process) either
in full, or of extracts, may be made only in accordance with instructions given by
the author and lodged in the John Ryland University Library of Manchester. Details
may be obtained from the Librarian. This page must for part of any copies made.
Further copies (by any process) of copies made in accordance with such
instructions may not be made without the permission (in writing) of the author.

2. The ownership of any intellectual property rights which may be described in this
thesis is vested in the University of Manchester, subject to any prior agreement to
the contrary, and may not be made available for use by third parties without the
written permission of the University, which will prescribe the terms and conditions
of any such agreement.

3. Further information on the conditions under which disclosures and exploitation


may take place is available from the Head of School of Chemical Engineering and
Analytical Science.

-9-
AKNOWLEDGEMENTS

A journey is easier when you travel together and Interdependence is certainly more
valuable than Independence. This project is the result of three months hard work whereby I
was accompanied & guided by many great people. Now I have the opportunity to express
my gratitude to all who helped me during this project.

First of all I would like to express my deepest thanks to Dr. Peter Gardner, Senior Lecturer,
CEAS for providing me this great opportunity of working in his lab in Manchester
Interdisciplinary Biocentre in the University Of Manchester.

I would also like to thank the PhD students Paul Bassan, Konrad Dorling, Intisar Khalifa
and Geraldine Monjardez who helped me throughout the project. They were always there
to listen and to give advice for doing the project well. I am also very thankful to them for
their immense help in planning and executing the work and as well as for the
encouragement, generosity and complete guidance to complete my project on time.

Last, but not the least I am thankful to my parents who have supported me throughout the
project and helped me in facing the challenges related to the project work. Honesty,
sincerity, and ambition with commitment in one's continued persistence towards the goals
are priceless values instilled in me by my parents. These insights and parables always
kept me enthusiastic and in inspired state through this project.

- 10 -
CHAPTER 1

1. INTRODUCTION

Despite of many advances in the medical sciences, cancer is continued to be a major health
problem. Cancer is becoming one of the common disease in today‟s scenario.Cancer is a
cell disease and it occurs due to alterations in the normal properties of the cell. These
alterations cause changes in nucleic acid, protein, lipid and carbohydrate quantities and / or
conformations (Mahadevan et al. 1997). These changes result in a faulty set of instructions
of cell function and cells do not undergo programmed cell death and they continue to
divide in an uncontrolled manner. These extra cells can form a mass of tissue called a
growth or tumor. The genes and proteins that control cell cycle and apoptosis are key in
understanding the mechanism of cancer and in development of anticancer drugs (Grey
2005). Most of anticancer drugs developed so far cause DNA damage through various
processes including distribution of cell cycle check points, growth factor and signal
transduction and ultimately cell death occurs by apoptosis . The design of anticancer drug
is a complicated issue and it should cover the inhibitory properties of drug alongwith its
delivery dosage and residence time in vivo. Finding the target of anticancer drug is
preliminary work of the discovery of its anticancer mechanism. Generally DNA is the
typical target for many anticancer drugs. Interaction between cancer cells and anticancer
drug is of prime importance in discovery of new drugs. The non invaging techniques
capable of providing molecular level information for investigation of functional groups,
bonding types and molecular conformations are very helpful in detection of cancer and for
development of anticancer drugs.

In recent years infrared spectroscopy has become a powerful tool for identifying
vibrational modes of various biomolecules of the cell which gives characteristic IR
spectrum and provides information about the structural and functional aspects ( Jackson
and Mantsch 1996). These IR vibrations can be correlated directly to the biochemical
composition . It has been found that IR spectrum alters in cancer disease and therefore IR
spectroscopy can detect and monitor characteristic changes in molecular composition and
structure that accompany transformation from normal to cancerous state. IR features of
various forms of cancer has been reported (Wood and et al. 2004). Furthermore IR
spectroscopy can be useful tool for monitoring the metastasis process and in cancer
grading ( Anderus et al. 1998). An interesting application of IR spectroscopy in biomedical
research is assessment of effects of drugs on cancer cells by monitoring biochemical
changes in cell before and after treatment with drugs. Sensitivity or resistance to drugs can
also be investigated by IR. Apoptosis or programmed cell death is of great
- 11 -
importance in understanding progression and treatment of cancer . As an active process,
apoptosis is accompanied by biochemical changes to three essential cellular components
i.e. DNA, protein and lipid. IR spectroscopy capable of analysing theses three cellular
components can provide unique insights into apoptosis processes, including analyses of the
effects of anticancer drugs ( Gaspari and Muzio ) 2003.

Spectroscopy has emerged as one of the major tools in biomedical applications and
significant development has taken place in this field related to cancer detection and
anticancer drug development. The development in IR instrumentation and in data
processing techniques in the past decade have demonstrated its potential in disease
diagnosis and treatment. Among the different spectroscopy methods that have been
evaluated for distinction between normal and neo-plastic tissues, Fourier Transform
Infrared (FTIR) spectroscopy and ATR-FTIR have shown huge potential in diagnosis of
normal and malignant cells (Sahu & Mordechai 2005). These vibration spectroscopic
techniques are relatively simple, reproducible and non destructive to the tissue. These
techniques provide molecular level information allowing investigation of functional
groups, bonding types and molecular conformations. The spectral bands in the spectrum
are molecule specific & provide direct information about the biological composition.

In FTIR spectroscopy time domain measurement of an interference pattern in the infrared


is utilized to form a frequency domain plot, showing the magnitude of each wavelength in
a given range. FTIR spectrums are generally distorted and do not appear as clear
absorbance spectrum due to Mie scattering. These distortions are corrected through various
processing techniques. Due to these problems special interest has arisen in ATR-FTIR
methods based on evanescent wave absorbance.

The minute changes encoded in the large spectral data of FTIR, ATR-FTIR are generally
difficult to visualize directly. Statistical methods are applied to extract important
information from the collected spectrum. Principle component analysis (PCA) is well-
known data compression method where large spectral data are reduced into small number
of independent variables. The principle components are linear combinations of variables
that describe the major source of variance between the spectra. The first principle
component (PC1) accounts for the most variance, and subsequent PCs account for
decreasing amounts of different source of variance. PCA has been utilized for pattern
recognition in the spectral data obtained through FTIR and ATR-FTIR measurements done
on various samples.

- 12 -
Cisplatin and its various analogues are widely used in treatment of ovarian cancer.
Cisplatin develops resistance to cancerous cells during course of treatment and it has
serious side effects. Extensive research is going on for discovering the new drugs for
cancer treatment with minimum side effects. Gold complexes compound based drugs have
shown great potential in cancer treatment and these have less side effects in comparison to
cisplatin.

It may be mentioned that cells treated with potential drugs will experience modifications
correlated with their cellular mode of action. Because infrared spectrum of cells yield a
precise image of all the chemical bonds present in the sample, different drug actions are
likely to yield a unique fingerprint characteristic of the mode of action of the drugs .
Accordingly the present work is undertaken to examine the application of FTIR and
Attenuated Total Reflectance (ATR) spectroscopy in studying the efficacy and working
mechanism of cisplatin and KF01-01 used in treatment of ovarian cancer . The A2780
ovarian cancer cells treated with cisplatin and KF01-01 drugs at different concentrations
i.e. IC30, IC50 and IC70 were utilized for FTIR & FTIR-ATR measurements. In all seven
samples of cisplatin and eight samples of KF01-01 treated with different concentrations
were analyzed by both techniques by taking ten spectra of each sample . The resulted
spectra reflected changes in biochemistry of A2780 ovarian cancer cells induced by
cisplatin and KF01-01 drugs. However these changes are difficult to detect in FTIR and
ATR-FTIR spectra. Therefore Principle component analysis (PCA) was applied for
extracting the hidden information from the collected spectra. ATR-FTIR spectral data
provided clean spectrum without any distortion and data was analyzed directly with PCA.
However FTIR spectral data reflected some distortions and therefore Resonant Mie
Scattering (RMieS) corrections were applied before principal component analysis. PCA
was performed for several combinations of spectral data of both drugs collected with
different concentrations.

The PCA analysis of these data indicates that chemical changes in A2780 cells induced by
cisplatin and KF01-01 drugs are different indicating that both drugs act differently. These
results suggest that all the concentrations of KF01-01 and cisplatin have anticancer effect
on A2780 cells . It is further inferred that both cisplatin and KF01-01 produces maximum
cancer cells killing effect at IC-50 in comparison to effect at IC-30 and IC-70
concentrations. Therefore the cytotoxic effect of both drugs is not linear at different
concentrations.

- 13 -
CHAPTER 2

2. LITERATURE REVIEW

2.1 Cancer

Cancer is a disease of the cell cycle. Some of the body‟s cells divide uncontrollably and
forms tumors. There are several factors that regulate the cell cycle and assure a cell divides
correctly. Chemical signals tell a cell when to start and stop dividing. Neighboring cells
communicate with dividing cells to regulate their growth. Normally, cells grow and divide
to form new cells as the body needs them. Before a cell divides, the DNA is checked to
make sure it has replicated correctly. If DNA does not copy itself correctly, a gene
mutation occurs and this DNA mutations disrupt the cell cycle. Due to this disruption in
cell cycle new cells grow when the body does not need them and old cells do not die when
they should. These extra cells can form a mass of tissue called a growth or tumor. Tumors
can be benign or malignant. Benign tumors are not cancer. Generally, benign tumors can
be removed and they do not grow back. Cells from benign tumors do not spread to other
parts of the body and they do not invade the tissues around them. On the other hand
Malignant tumors are cancer .

Figure 1. Schematic diagram showing normal and cancerous cells (Ref. 69 )

Malignant tumors also can be removed. But sometimes they grow back. Malignant tumors
cells invade other organs and form new tumors that damage these organs. The spread of
cancer is called metastasis. The occurrence of cancer depends on variety of factors such as-
due to abnormalities caused in the genetic material of the transformed cells, or by some
type of carcinogens like tobacco, smoke, radiations etc. The two classes of genes that are
affected by the genetic abnormality are- oncogenes and tumor suppressor genes. The
oncogenes are activated in the cancer cells which helps the cells to have programmed cell
death, growth and division. Whereas the tumor suppressor genes are inactivated in the

- 14 -
cancer cells which losses the normal functioning of those cells such as DNA replication
and control over the cell cycle.

Figure 2. Schematic diagram showing cancer stages (Ref. 70 )

The type of cancer is resembled by the type of the cell that resembles the tumor. The
different tumor includes:

 Carcinoma : These are the tumors derived from the epithelial cells and include
breast cancer, prostate, lung and colon cancer.
 Sarcoma : These are the tumors derived from the connective tissue.
 Lymphoma and Leukemia : These are the tumors derived from the blood forming
cells.
 Germ Cell Tumor : These are the tumors derived from the totipotent cells (the cells
that are able to divide and produce an organism). This category includes the testicle
cancer or the ovary cancer.
2.2 Ovarian Cancer

Ovarian cancer is a type of cancer that occurs in the females. This cancer usually occurs in
the ovaries of a women. Ovaries are the reproductive glands in which ova or eggs are
formed. When there is division and rapid growth of cells in one or both the ovaries of a
women then ovarian cancer occurs. The loss of the growth control of the cells leads to this
type of cancer as the uncontrolled growth leads to the formation of tumor.

The three types of cells which forms ovarian cancer are:-

 Epithelial cancer : This is the most common category of cancer which arises from

- 15 -
the cells that are covering the ovaries.

 Germ cell cancer : This cancer starts from the cells that are designed to form eggs
in the ovaries.

 Sex cord cancer : This type of cancer begins in the cells that are used to hold the
ovaries together and produce the female hormones.

Epithelial tumor is present on the surface of epithelial of the ovaries and accounts for about
90% of all the ovarian cancers. This tumor is subdivided into four different tumors which
include- serous, endometrioid, mucinous and clear cell tumors.

Most common ovarian cancers are ovarian epithelial carcinomas. It begins in the tissue
that covers ovaries. Ovarian cancer can invade, shed, or spread to other organs. When
cancer spreads from its original place to another part of the body, the new tumor has the
same kind of abnormal cells and the same name as the original tumor. If ovarian cancer
spreads to the liver, the cancer cells in the liver are actually ovarian cancer cells and it is
treated as ovarian cancer, not a liver cancer. Doctors call this new tumor “distant” or
metastatic disease.

Following are the stages of ovarian cancer:

 Stage I: Cancer cells are found in one or both ovaries. Cancer cells may be found on
the surface of the ovaries or in fluid collected from the abdomen.

 Stage II: Cancer cells have spread from one or both ovaries to other tissues in the
pelvis. Cancer cells are found on the fallopian tubes, the uterus, or other tissues in the
pelvis. Cancer cells may be found in fluid collected from the abdomen.

 Stage III: Cancer cells have spread to tissues outside the pelvis or to the regional lymph
nodes. Cancer cells may be found on the outside of the liver.

 Stage IV: Cancer cells have spread to tissues outside the abdomen and pelvis. Cancer
cells may be found inside the liver, in the lungs, or in other organs.

2.3 Treatment of Ovarian Cancer

Following are various ways of treating the ovarian cancer disease.

 Surgery : This is the most common treatment for cancer. In this a particular cancer
tumor is removed. Sometimes the nearby lymph nodes are also removed so as to
- 16 -
control the spread of cancer.
 Radiation Therapy :This is also known as radiotherapy treatment and can be applied
either before the surgery or sometimes after the surgery. The radiation rays are given to
the area where cancer is present. This helps in control of division and the growth of
the cells . This is done before the surgery. But if person has undergone the surgery then
the rays can be given for killing the remaining cells which can form cancer.
 Chemotherapy : In this anti cancer drugs are used for the treatment of the cancer. This
is used when the cancer is spread in large area. These drugs are either given orally, or
through injection in the vein or muscle. These drugs reach the cancer cells and kills
them. Most of anticancer drugs used in chemotherapy cause DNA damage through
various processes and ultimately cell death occurs by apoptosis. Most anti-cancer
drugs used include agents that target the cell cycle in order to inhibit the hyper
proliferation state of cancer cells and subsequently to induce apoptosis . Based on their
mode of action chemotherapeutic drugs can be classified into distinct groups ( i) drugs
that interferes with DNA synthesis (e.g. cisplatin) (ii) drugs that introduce DNA
damage (e.g. Fluorouracil, Methotrexate) and (iii) drugs that inhibit the function of
mitotic spindle (e.g. Taxol).

The most popular anti cancer drugs that are used for treating ovarian cancer are-

 Cisplatin and its various analogues like carboplatin


 Gold complexes
 Taxol
 Methotrexate
 Fluorouracil
 Olaparib

These drugs are different in nature and act differently in the body of the patient suffering
from ovarian cancer.

2.3.1 Cisplatin

Metals are essential for the normal functioning of living organisms and these occupy an
important role in current medical application (Bruijnincx & Sadler 2008). One of the major
medical breakthroughs for metal–based drugs was the accidental discovery of the
anticancer properties of cisplatin and its clinical introduction in the 1970. Cisplatin and
its analogues are widely used in treatment of ovarian cancer. The success of platinum
complexes in killing cancer cells mainly results from their ability to form various types of
adducts on DNA and this triggers apoptosis process that lead to cell death.
- 17 -
Later on it was observed that some tumors develop resistance during the course of therapy.
It is demonstrated that resistance might be mediated through two mechanisms : first, a
failure of a sufficient amount of platinum to reach the target DNA; and second, a failure to
achieve cell death after platinum–DNA adduct formation (Messori and Marcon 2004).
However the successes of the effectiveness of cisplatin against cancer further opened new
way of research for development of new drugs on other metal-based compounds
(Kartalon and Essigmann 2007).

Cisplatin is the most commonly used drug in chemotherapy for treatment of cancers of
the head, neck, bladder, ovary & testis. It is a platinum based drug and can be used for
treating different types of cancer. The compound cis-PtCl2(NH3)2 (Fig.3) was first
described by M. Peyrone in 1845 and was known for a long time as Peyrone's salt.
Cisplatin is colourless fluid in nature. Usually this drug is given through drip (infusion) to
the patient.

Chemotherapy is done in several sessions or cycles over a period of time. This drug is
given alongwith some other chemotherapy drugs for the treatment. Cisplatin acts as
alkylating agent inside the body and it replaces the hydrogen by the alkyl group in a
molecule necessary for tumor growth.

Fig. 3: Cisplatin (Ref. 71 )

This drug kills the cancer cells by binding to the DNA and interfering in the cell
mechanism thereby causing the cell death. The binding of cisplatin to DNA changes the
secondary structure of DNA & consequently the metabolism of cell. Unfortunately many
cancer cells are resistant to cisplatin treatment. The only way to overcome acquired
resistance to cisplatin in cancer cells is by increasing dosage, which results in higher
toxicity in normal body cells. Resistance to cisplatin treatment can be due to lack of the
p53gene, by the activation of cell survival genes such as nuclear factor KB and by the
reduced cellular concentration to cisplatin. Recently (Haitao et al. 2010) has reported

- 18 -
that Kaempferol enhances cisplatin‟s effect on ovarian cancer cells through promoting

apoptosis caused by down regulation of cMyc. Kaempferol is a dietry flavonoid that is


widely distributed in fruits & vegetables. Epidemiology studies have revealed a protective
effect of Kaempferol against ovarian cancer risk. It has been reported that Kaempteral is
effective in reducing vascular endothelial growth factor (VEGF) expression in ovarian
cancer cells.

2.3.2 Gold Complexes

Gold (I) and gold (III) complexes are potentially attractive as anticancer agents (Messori
and Marcon 2004). Both Gold(I) and Gold(III) complexes are isoelectric and isostructural
with platinum Pt(II) complex and are potentially attractive as anti cancer drug. The
category of the gold complexes which reacts well with the cancer is square planar gold(III)
complexes. These complexes have been allowed for in vitro pharmacological testing under
the physiological conditions. The gold (III) complexes constitutes five different gold
complexes-Au(en)2, Au(dien), Au(cyclam), Au(phen) and Au(terpy). A series of
organogold (III) complexes were also screened for the anti cancer activity with having
positive results in the case of human ovarian cancer cell line A2780.

Another category of gold complexes which showed cytotoxicity properties to the human
ovarian cancer cell line A2780 was dinucclear oxo gold (III) compounds. The design of an
effective anticancer drug is a complicated issue that must cover not only the drug's inherent
inhibitory properties but also its delivery, dosage, and residence time in vivo. Gold (I) and
gold (III) complexes overcome some of these challenges by forming strong covalent
attachments to targets. The gold(III) compounds have emerged as potential metal drugs
and this has resulted in synthesis of number of gold (III) compounds . These compounds
produce effect at molecular level such as direct DNA damage, modification of the cell
cycle, alterations of mitochondrial functions and induction of apoptosis. On the basis of
some new experimental evidence it is suggested that gold based compounds promote
apoptosis to a greater extent than cisplatin (Barnard et al. 2007).

2.3.3 Taxol

Taxol is used for curing different types of cancers (Fig. 4). This drug interferes with the
cancer cells and slow down their growth in the body. This drug can be used for curing
breast cancer, ovarian cancer and also lung cancer. It is present as a white powder and
when prepared for treatment it becomes a colourless and clear liquid. It is given
intravenously.

- 19 -
Fig 4: Taxol (Ref. 72)

2.3.4 Methotrexate

This drug is used for curing cancer disease and also some autoimmune diseases (Fig. 5). It
is also known as amethopterin. This drug is used for treatment of breast cancer, skin
cancer, lung cancer and ovarian cancer. It acts by inhibiting the metabolism of the folic
acid.

Fig 5: Methotrexate (Ref. 73 )

2.3.5 Fluorouracil

It is a pyrimidine drug used for curing cancer. This drug comes under the category of
antimetabolites. This is used in treatment of colon cancer, breast cancer, ovarian
cancer, stomach cancer and the pancreas cancer. This drug interferes with the growth

- 20 -
of cancer cells thereby leading to decrease the growth of the cancer cells in the body.

Fig 6: Fluorouracil (Ref. 74 )

2.3.6 Olaparib

Recently the first of a totally new type of drug for cancer treatment has been clinically
tested and results were published in the lancet. The trials were led by Dr. Andrew Tutt,
consultant clinical oncologist and director of the Breakthrough Breast Cancer Research
unit at King‟s College London.The drug investigated in the trials, olaparib, is a new class
of drug called PARP inhibitors and comes in pill form (Fig. 7) . It targets cancer cells
caused by faulty BRCA1 or BRCA2 genes.

Olaparib uses the synthetic lethality approach to kill cancer cells with faulty BRCA1 or
BRCA2 gene. It does this by blocking a protein called PARP and is therefore known as a
PARP inhibitor. Olaparib causes cancer cell with a BRCA fault to lose control of their
DNA stability. This causes the cancer cell to die and means that the tumour should either
stop growing or get smaller. Due to the drug working in a targeted way, it kills cancer
cells while leaving healthy cells relatively unaffected, which means fewer side effects for
patients.

Fig 7: Olaparib (Ref. 75 )

- 21 -
The results are for two separate Phase II international, multi-centre clinical trials – one for
breast cancer and one for ovarian cancer patients. The ovarian cancer trial looked at a
group of 57 women with advanced ovarian cancer who had already received
chemotherapy. Twenty four patients took 100 mg doses of olaparib while another 33 took
400 mg closes. Over 33 per cent of tumours in the higher dose group reduced significantly
in size and tumours were prevented from progressing for an average of six months. The
patients had only relatively minor side effects, such a fatigue and nausea.

2.4. IR SPECTROSCOPY

Infrared (IR) spectroscopy is one of the most common spectroscopic techniques used in
biology. Simply, it is the absorption measurement of different IR frequencies by a sample
positioned in the path of an IR beam. The main goal of IR spectroscopic analysis is to
determine the chemical functional groups in the sample. Different functional groups absorb
characteristic frequencies of IR radiation (Fig.8).

Fig. 8 Chart of characteristics vibrations (Ref. 76 )

- 22 -
IR radiations does not have enough energy to induce electronic transition as seen with UV.
Absorption of IR is restricted to compounds with small energy differences in the possible
vibrational states. For a molecule to absorb IR, the vibration within a molecule must cause
a net change in the dipole moment of the molecule. If the frequency of the radiation
matches with the vibrational frequency of the molecule, radiation will be absorbed causing
a change in the amplitude of molecular vibration.

Figure 9. Schematic diagram showing stretching &


bending modes of CH2 Group (Ref. 77 )

IR radiation promotes vibration of the covalent bonds of molecules within the samples that
absorbs it. The wave length of the IR radiation that is absorbed depends upon the nature of
the covalent bond & the strength of any intermolecular interactions. Various bimolecular
components give a characteristic IR spectrum that is akin to a bio chemical fingerprint of
that tissue. This helps in measurement of complex molecular vibration modes which
contain valuable information on changes occurring due to disease. The position of atoms
in molecules are not fixed and they are subject to a number of different vibrations falls into
two main categories of stretching and bending. Stretching occurs due to change in inter
atomic distance along bond axis. These can be two types symmetric and asymmetric.
Bending occurs due to change in angle between two bonds. These are four types : Rocking,
Scissoring, Wagging & Twisting (Fig.9) .

- 23 -
Therefore, the spectrum of each compound is unique similar to fingerprint. Intensities at
many wave numbers are simultaneously measured, but only a few of these wave numbers
of spectral reasons show diagnostic potential. In general the vibration frequency decreases
with increase in atomic weight and it increases with increase of bond energy. Specific
wave numbers for regions of absorbance in the IR spectra of cells / tissue are most affected
due to disease. Spectral bands in the spectra are molecule specific and provide direct
information about the bio-chemical composition (Fig.10). FTIR peaks are relevantly
narrow and in many cases can be associated with the vibration of a particular chemical
bond in a molecule. The physical effect of infrared is created by absorption and mainly
influences the dipole and Ionic bond.

Figure 10. Schematic diagram showing different absorption bands (Ref.78


)

2.4.1 Fourier Transform Infrared Spectroscopy (FTIR)

FTIR stands for Fourier Transform InfraRed, the preferred method of infrared
spectroscopy. In infrared spectroscopy, IR radiation is passed through a sample. Some of
the infrared radiation is absorbed by the sample and some of it is passed through . The
resulting spectrum represents the molecular absorption and transmission, creating a
molecular fingerprint of the sample. Like a fingerprint no two unique molecular structures
produce the same infrared spectrum. This makes infrared spectroscopy useful for several
types of analysis.

FTIR spectroscopy is the study of the interaction of IR radiation with matter. Earlier
instruments were dispersive and thus intensity at each wave number was measured
separately and this method was time consuming. On the other hand FTIR spectroscopy
system contains no monochromaters but an optical element composed of an
- 24 -
intrerferometer, allowing simultaneous measurements of complete region of wave numbers
in a short time (Fig. 11) . The Fourier transform computerized methodology transfers the
time domain spectrum frequency domain. This helps reduction in noise levels and make
data collection more rapid.

Figure 11. Schematic diagram showing Spectrometer Layout


(Ref. 79)

FTIR spectroscopy is typically used for viewing the IR radiations from 3µm to 15µm
wavelength. It is now used extensively in biological sciences and in many fields including
chemistry, physics, astronomy, semiconductor processing & even in forensics. It can be
applied to solids, liquids or gases. In last decades infrared spectroscopy has demonstrated
its potential as a novel technology for diagnosis and discovering new drugs for cancer.

The prominent areas where FTIR spectroscopy may be used in cancer diagnosis in the
future are ( Sahu and Mordechai 2005)

 Differentiation of normal and diseased tissues in organs – breast, colon, liver,


ovarian and cervix ; detection of early stages of malignancy.

 Monitoring abnormal cell growth and proliferation in tissue sections – colonic

- 25 -
crypts cervical epithelium.

 Distinguishing between normal and abnormal cell-cell scrapings (cervix), biopsies


from smaller organs like the prostate , thyroid and body fluids.

 Differentiation between cancer and other pathologic conditions with similar clinical
manifestation – IBD and colon cancer.

 Monitor the effect of anticancer therapy – chemotherapy in leukemia and tumor


grading - lymphoid tumors.

2.4.2 FTIR Instrumentation

Fourier transform spectrometers now almost replaced dispersive instruments for most
applications due to their superior speed and sensitivity. They have greatly extended the
capabilities of infrared spectroscopy and have been applied to many areas that are very
difficult or nearly impossible to analyze by dispersive instruments. Instead of viewing each
component frequency sequentially, as in a dispersive IR spectrometer, all frequencies are
examined simultaneously in Fourier transform infrared (FTIR) spectroscopy (Fig. 12).

Figure 12. Schematic diagram showing working of FTIR


spectroscopy (Ref. 80)

There are three basic spectrometer components in an FTIR system : radiation source,
interferometer, and detector. In FTIR an interferometer is used to differentiate and measure
the absorption component frequencies. The interferometer divides radiant beams and
generates an optical path difference between the beams. Further it recombines them for
producing repetitive interference signals measured as a function of optical path difference
by a detector. The interferometer produces interference signals, which contain infrared
spectral information generated after passing through a sample. When IR beam is directed
through the sample , the amplitude of set of waves are reduced by absorption if the

- 26 -
frequency of set of waves is same as one of the characteristics frequencies of the sample.

The interferogram is the record of the interference signals recorded in time domain. It
contains information over the entire IR region to which the detector is responsive. A
mathematical operation known as Fourier transformation converts the interferogram to the
frequency domain spectrum showing intensity versus frequency or wave number.

During FTIR measurement Mie scattering significantly distort the spectrum and these
spectrums are can not be used directly for any meaningful interpretation. Various
processing techniques are applied for correcting these distortions for extracting the
information corresponding to various absorption bands.

2.4.3 Attenuated total reflectance ( ATR) - FTIR

In view of the distortion due to Mie scattering in FTIR rapid developments have taken
place in ATR based spectroscopy in which measurements are free from these scattering
effects . In ATR, special processing techniques are not required and measured spectra can
be used directly for interpretation of various absorption bands.

Attenuated total reflectance (ATR) is especially useful for obtaining IR spectra of difficult
samples that cannot be readily examined by the normal transmission method. It is suitable
for studying thick or highly absorbing solid and liquid materials. ATR requires little or no
sample preparation for most samples and is one of the most versatile techniques.

Figure 13. Schematic diagram showing evescent wave (Ref. 81 )

ATR occurs when a beam of radiation enters from a more-dense (with a higher refractive
index) into a less-dense medium (with a lower refractive index). The fraction of the
- 27 -
incident beam reflected increases when the angle of incidence increases. All incident
radiation is completely reflected at the interface when the angle of incidence is greater than
the critical angle. The beam penetrates a very short distance beyond the interface and into
the less-dense medium before the complete reflection occurs. This penetration is called the
evanescent wave (Fig. 13) and typically is at a depth of a few micrometers (0.5µm to
5µm). Its intensity is attenuated by the sample in regions of the IR spectrum where the
sample absorbs.

Figure 14. Schematic diagram showing working of ATR-FT IR spectroscopy


(Ref. 82 )
The sample is normally placed in close contact with a more-dense, high-refractive-index
crystal such as zinc selenide, thallium bromide–thallium iodide (KRS-5), or germanium.
The IR beam is directed onto the beveled edge of the ATR crystal and internally reflected
through the crystal with a single or multiple reflections. Both the number of reflections and
the penetration depth decrease with increasing angle of incidence.

- 28 -
Table 1

Properties of infrared transmitting materials for ATR-IR spectroscopy

Material Comments Short Long Refractive pH Range


Wavelength Wavelength Index
(cm-1) (cm-1)
ZnSe Most 15000 461 2.40 5-9
common
Diamond Very costly 30000 <2 2.40 1-14
Ge Brittle 5500 432 4.0 1-14
Thallium Very toxic 17900 204 2.37 5-8
Bromide /
Thallium
Iodide

The attenuated energy of the evanescent wave from the sample is passed back to the IR
beam, which then exits the opposite end of the crystal and is passed to the detector in the
IR spectrometer for generation of infrared spectrum. The resulting ATR-IR spectrum
resembles the conventional IR spectrum, but with some differences: The absorption band
positions are identical in the two spectra, but the relative intensities of corresponding bands
are different.
2.4.4 Advantages of FTIR –ATR Spectroscopy
 These techniques are reagent free and can rapidly & non-invasively detect changes
in the biochemical composition of cells & tissues at the molecular level.
 In contrast to UV, X-Rays & Gamma rays, IR rays are non destructive to biological
samples and therefore the samples can be analyzed without any destruction and
measurement can be repeated if required.
 Compared to MRI & positron emission tomography (PET) these requires smaller
amount of samples.
 Faster than Raman spectroscopy for ex-vivo analysis.
 These are economic and simple to operate .
 Sample preparation does not require exclusive & special treatment which helps in
rapid diagnosis.
 Can be used in analysis of tissues, fluids and cells.
 Can help in identification of suitable common biomarkers for different types of
malignancies, irrespective of their nature & origin.

- 29 -
2.4.5 Application of FTIR and ATR-FTIR in cancer diagnosis and development of
anticancer drug

Cancer cell has defective cell cycle control and does not undergo programmed cell death.
Subsequently they continue to divide in an uncontrolled manner compared to normal cells.
These cells are characterized by increased nuclear material, an increased nuclear to
cytoplasmic ratio, increased mitotic activity, abnormal chromatin distribution and
decreased differentiation. These result in specific change in nucleic acid, protein, lipid and
carbohydrate quantities. The DNA protein interaction is also disturbed in malignant
transformations resulting in repeated duplication and amplification of DNA sequence
(Mahadevan 1997). Genes and proteins that control cell cycle and apoptosis are key in
understanding cancer and for development of potential anticancer drugs (Grey et al.
2005). The design of an effective anticancer drug is a complicated issue that must cover
not only the drug's inherent inhibitory properties but also its delivery, dosage, and
residence time in vivo.

DNA is the typical target for many anti-cancer drug. Different anti cancer drug interact
with cancer cells in different ways. Understanding the mechanism of anti cancer drugs
with cancer cells is becoming one of the focus in development and screening of new
anticancer drugs. Development of advance level IR instruments and computerized data
processing tools have accelerated the research for discovering new drugs for cancer
treatment.

In recent years, infrared spectroscopy has become a powerful tool for identifying
vibrational modes of various biomolecules of the cell which give a characteristic IR
spectrum (Jackson & Mantsch 1996). These infrared vibrations can then correlated
directly to the biochemical species and resultant IR spectrum can then be described as
biomarker. It has been found that the IR spectrum is altered in disease such as cancer.
Therefore, IR spectroscopy can detect and monitor characteristic changes in molecular
composition and structure in transformation of cell from normal to cancerous state.

FTIR and ATR-FTIR spectroscopy has been utilized successfully in the following
medical area

 Analysis of normal and cancerous tissues of esophagus cancer


 Fixation protocols for sub cellular imaging by FTIR
 FTIR used for prostate cancer diagnosis
 FTIR used for observing the biochemical changes in the Pancreatic cancer

- 30 -
 FTIR used in cancer detection
 Cancer histopathology by using FTIR
 FTIR studies on normal and oncogenes
 Vibrational spectroscopy used in cancer diagnosis
 Study of prostrate cell line using synchrotron based FTIR
 FTIR used in characterization of stem like cells
 FTIR used in discrimination of the factors influencing prostrate cell line
 FTIR and Raman spectroscopy used for Ovarian tissues
 Study of cisplatin resistance for the ovarian cancer cell line
 FTIR spectroscopy for biological tissues

The IR features of various forms of cancer have been widely reported (Salman et al. 2001,
Faolain et al. 2005). Infrared based techniques in biomedicine have become a reality with
a large amount of information accumulated from clinical studies. Among the different
spectroscopic methods that have been used , Fourier transform (FTIR) spectroscopy has
shown huge potential in biomedicine application (Wood et al. 2004) . FTIR spectroscopy
studies suggest that progression of normal cell to the metastasis state involves structural
modification in DNA. Moris et al. (1995) studied samples that contained cell collected
from cervical canals using FTIR and observed changes in the spectra at various stages of
cervical intraepithelial neoplasia that were of diagnostic potential . Another report dealt
with the distinction of normal and dysplastic cells in cervical samples using FTIR and
reported the similarity of the dyplastic cell spectra with the HeLa cells spectra (Wood et al.
1995). The important observation made utilizing principal component analysis was that
the glycogen absorbance was a prominent diagnostic feature. The development of
advanced computational methods to analyze the spectra further enhanced the use of FTIR
in distinction of normal and cancerous tissues (Cohenford et al. 1997, Romeo et al. 1998).
Sahu & Mordechai (2005) studied the application of FTIR in cancer detection . Wood et
al. ( 1998) reported FTIR spectroscopy as a biodiagnostic tool for cervical cancer. They
carried out an FTIR microscopic investigation of cell types and potential confounding
variables in screening for cervical malignancies. The aim of the study was to determine the
effectiveness of infrared spectroscopy in the diagnosis of cervical cancer and dysplasia. It
was found that leukocytes and in particular lymphocytes have spectral features in the
phosphodiester region ( 1300-900 cm-1).

Wang et al. (1997) focused on the microscopic FTIR studies of lung cancer cells in pleural
fluid. The results demonstrate significant spectral differences between normal, lung cancer

- 31 -
and tuberculous cells.

Binoy and others ( 2004) used the FTIR in studying the anticancer drug. Sensitivity or
resistance to drugs can be also investigated by the FTIR. Liu et al. (2007) examined the IR
spectral changes corresponding to protein, lipid and nucleic acid and demonstrated there is
a difference in spectra corresponding to drug resistant and drug sensitive leukemia cell
line. Understanding apoptosis or programmed cell death, has great importance in
progression of cancer and its treatment. As an active process, apoptosis is accompanied by
profound biochemical changes to three essential cellular components i.e. DNA, protein and
lipid. IR spectroscopy is capable of analysing these three essential cellular components
and simultaneously can provide unique insights into apoptosis processes, including
analyses of the effects of anticancer drugs (Gasparri and Muzio 2003).

Baker et al. (2009) used FTIR and histological pathology to compare the results of the
prostate tissue to know about the disease severity. Prostate cancer is the second most
common cancer of men. The biochemical changes involved with prostate cancer were also
evaluated using FTIR technique. The morphological features of the prostate cancer are
named according to the score with Gleason Grading System. This helps in dividing the
prostate cancer into five different grades. FTIR along with PC-DFA was done on the
formalin fixed prostate cancer tissue . These results were compared with those assigned to
the gleason grading. This grade system is divided into three categories depending upon the
stage of cancer-

 GD<7 least aggressive


 GS=7 intermediate potential
 GS>7 likely to progress

The tissue biopsies were differentiated into the above three categories. After this division
MATLAB along with PCA was done so as to differentiate between them. The analysis was
performed on the basis of three models-

 Model A- spectra vector normalized


 Model B- spectra vector normalized followed by first derivative
 Model C- spectra vector normalized followed by second derivative

Model B achieved highest overall sensitivity and Model A achieved highest specificity. It
was inferred that FTIR can not identify the specific protein, lipid or carbohydrate but can
help in estimating the concentrations of these molecules in the given sample.

- 32 -
Bhargava (2007) presented an excellent review on FTIR technology with reference to
prostate cancer. He has observed that it is critical to understand that data acquisition,
classification and its validation for translation of FTIR results to clinical pathology. He
also compared the manual clinical testing methods with FTIR and discussed advantage and
disadvantage of both techniques.

Gazi et al. (2009) used FTIR spectroscopy technique for the classification and
discrimination of the prostate cancer cell lines. In this study the FTIR spectra of the fixed
cancer cell line and also the primary epithelial cells were obtained from benign prostatic
hyperplasia(BPH). The analysis which gave the positive results in discrimination and
classifying the cell lines of the prostate cancer is called multivariate chemometric analysis.
This analysis gave the sensitivity and selectivity values of >94% and >98%. Alongwith
this examination two other results were also concluded. The effect of media on the cell line
and also the differences in nucleus to cytoplasm (N/C) was also examined. The major
contributing factor for the classification and discrimination of cell line was the biochemical
differences between the cell lines.

Gazi et al. (2008) used FTIR spectroscopy for the identification of clinically aggressive
prostate cancer. Alongwith the FTIR another process called principal component
discriminant analysis (PC-DFA) was applied for grading of the given tissues of prostate
cancer. This algorithm increased the sensitivity and selectivity of the gleason grades. The
main aim of this research was to determine the possible biochemical changes associated
with the progression of Prostate cancer. The gleason grading of the prostate cancer biopsies
is subject to both inter and intra variabililty, which reduces the value arising from the
grading system (Latouf and Saad, 2002).

Jacob et al. (2001) used FTIR for measuring the IR spectra of the normal genes and the H-
Ras gene of mouse fibroblasts. Both the genes have different results in the form of
spectrum. The absorption of the normal gene was higher than the malignant ones. The
spectrum was obtained in between 600- 3200 cm-1. Also the carbohydrates and phosphate
contents were higher in the normal cells as compared to the H-Ras genes. Moreover the
RNA/DNA content was high in the transfected cells which also concludes that
transcriptional activity increases in the case of cancerous cells.

The Ras oncogene are the first nonviral oncogenes to be recognized. The over expression
of Ras gene and Ras mutations is seen in lung carcinoma, head and neck carcinoma and
pancreatic carcinoma. This study was taken up by Lohr et al. (2000) and Schull et al.

- 33 -
(2000). The IR spectrum of the normal fibroblasts was obtained and two significant peaks
were seen in the spectrum. The first peak was at 1648 cm-1 which was amide I and the
other was at 1544 cm-1 corresponding amide II. The amide I band arises due to C=O
stretching and amide II band arises due to C-N stretching. The peaks at 1241 cm-1 and
1086 cm-1 were due to absorption of phosphate groups. The amount of absorption was
larger in the case of normal cells as compared to the cancerous cells. The band area of
phosphate was larger in the normal cells as compared to the Ras transfected cells. Also it
was seen that the DNA content of normal fibroblasts was higher than the Ras transfected
cells. After these studies it was concluded that FTIR absorption range from 600 cm-1 to
3200 cm-1 was low as compared to the tumorogenic Ras cells. Moreover the amount of
carbohydrate and phosphate content was high in normal cells as compared to the Ras cells.

Catherine et al. (2009) did work in the area of clinical diagnostics using both FTIR and
Raman spectroscopy. Raman Mapping and also IR imaging helped in identification and
classification of biochemical changes that are related to carcinogenesis. FTIR and Raman
were also used for the diagnosis of cancer and also for getting the information about the
changes that usually occur before cancer. FTIR is also used as a powerful tool for probing
the structure of lipids, nucleic acids, carbohydrates and also primary and secondary
structure of proteins (Jackson et al. 1991, Wang et al. 2008) . FTIR has been used in
variety of different fields which includes- probing the tissue, biological fluids etc and in
distinguishing between the different phases of the cell life cycle and also differenciating
between cancerous and normal cells (Deleris and Petibois 2003). The multivariate spectral
models showed better results of the different stages of cancer with high accuracy levels.
Raman and FTIR were used extensively for studying the different cancers like-
gastrointestinal, lung cancer, head and neck cancer etc.

Casson et al. (2009) used FTIR for characterization of stem like cell population in human
esophageal normal and adenocarcinoma cell lines. They worked in differentiating between
the normal and adenocarcinoma cell lines and also in evaluating spheroids. These are stem
like cell population which are derived from the parent cell line when grown in the serum
free media. The cell lines were discriminated using the IR spectrum. The stem like cells
were differenciated easily as compared to the adenocarcioma cells as they show a
absorbance spectrum at the range of 1000–1200 cm-1. FTIR and Raman spectroscopy has
been used in the identification of CSC(cancer stem cell) in human malignancy (Wang and
Dick 2005, Leedham 2008). The work by Martin et al. (2001) also indicated the possibility
of using infrared absorption band of the symmetric –PO2 stretching mode at 1080 cm-1.
This was assigned to DNA as a specific marker of the cancer stem cells.

- 34 -
The amide bands and the amount of protein played a important role in differenciating
among the cancerous samples and normal ones. The amide I and amide II were the two
significant bands in the case of breast carcinoma (Meurens et al.1996). The amide II region
was also used for differenciating between the colorectal cancer from the non cancerous
tissue (Fabian et al.2006). Major spectral changes were also seen in cervical cancer tissue
predominantly at the amide I region (Bamberry et al. 2006).

Chen et al. (2006) used FTIR for observing the changes in the biochemical imaging of the
pancreatic cancer. In this study the biochemical imaging changes of lipids, proteins and
nucleic acid in the pancreatic cancer tissue samples were inferred using imaging and line
scan technique. The intensities and frequencies of the absorption bands of the IR spectrum
was reduced at the amide bands of the proteins and also the stretching of the lipids. The
cancerous tissue contained large amount of protein and the distribution of lipids and DNA
was low. These substances are the high molecular mass glycoproteins which are produced
by the tumor cells. IR microspectroscopies techniques are also used for analysing and
detection of lipids, proteins and also detection of polytene chromosomes in cells and
tissues.

Wang et al. (2003) applied FTIR for analysis of normal and cancerous tissues of esophagus
and significant differences between normal and malignant tissues were observed. For the
bands of protein , amide bands I and II, amide band II at about 1550 cm-1 was weak and
broad in malignant tissues and sharp in normal tissue. It was shown that peak at 1080 cm-1
was stronger and higher in malignant tissues than the normal tissue indicating that DNA
content in malignant tissues is significantly higher than in normal tissues.

Jian et al. (2003) used FTIR for investigating and finding out the differences in the
cancerous and non cancerous tissues of the esophageal cancer. In this experiment 27
samples of the esophageal cancer were taken. Each tissue was divided into two parts and
both were stored separately. One was fixed in 10% formalin and the other was frozen with
liquid nitrogen. The spectra of the samples was recorded on Nicolet Magna 750 FTIR
spectrometer with mercury cadmium telluride (MCT) detector (Argov et al. 2002). Total
64 scans were recorded in the region from 900 to 4000 cm-1 . With the help of FTIR
spectrum different areas with different feature was diagnosed. The proteins bands amide I
and amide II were seen in the cancerous and non cancerous samples of the esophageus.

- 35 -
The amide II band was weak in the cancerous tissues and was strong and sharp in the
normal tissues. Also the lipid content was less in the cancerous tissues as compared to the
normal tissues. Moreover the amount of DNA was more in cancerous tissues due to
increased DNA replication as compared to the normal tissues.

Gazi et al. (2005) worked Synchroton based Fourier transform infrared for studying the
cellular events. In the process imaging mode was used to generate biospectroscopic
chemical maps of formalin fixed cells of Prostate cancer. The imaging mode of FTIR is
usually applied to single cells at spatial resolution. This helps in studying the amount of
lipid, protein and carbohydrate in the cell. The cells that are fixed on formalin at low
concentration of 4% give better signals of IR spectrum as compared to the unfixed cells.
The experiment took place in two different events – cytokinesis and cell locomotion.
Cytokinesis is the process in which the cell goes cell division. It means the cell divides into
daughter cells. The images obtained revealed that there is heterogeneous distribution of
lipids. The experiment resulted in the conclusion that the lipid intensity is higher due to
process of ruffling and lateral membrane flow which are the consequences of cell
migration.

Kohler et al. (2009) used synchrotron infrared microscopy for studying the single, isolated
cancer cell. The Mie scattering was used for getting a corrected spectrum of the given
cancer cell line. The FTIR was used for getting the spectrum having good signal to noise
ratio. The spectrum obtained of the nuclei from synchrotron based FTIR showed changes
in the lipids, DNA and proteins as compared to the spectrum obtained of the lung cancer
cells. The optical and chemical properties of the single cells and the nuclei was compared
using the Mie scattering technique.

Gazi et al. (2004) gave the different fixation protocols for subcellular imaging by using
FTIR. Synchroton based Fourier Transform Infrared spectroscopy was used for the
analysis of lipids, proteins, carbohydrates and some phosphorylated molecules of the cells
is called. This technique is used in the imaging mode and it gives the results in the form of
biospectroscopic maps which is different from the spectrum that is obtained from FTIR.
The experiment was done so as to differenciate between the results obtained from the cells
which were fixed by formalin to those cells which were not fixed. The vibrations that are
obtained during the process are due to the presence of lipids, proteins and carbohydrates.
The spectrum obtained is in the form of peaks which shows the chemical bonding. The
maps of single cell is generated by reducing the aperture size to the size of the cell. The
perfect size of the aperture to get good signals is 5×5 µm. The cells were fixed with
formalin so as to preserve the structural and biochemical constituents of the cell.
- 36 -
Formalin is also used to preserve lipids by reaction of the hydrated formalin with double
bonds of unsaturated hydrocarbon chains (Steward 1973). During the process, calcium
fluoride disks are used when FTIR is done in the transmission mode and if it is done in the
reflectance mode than we use MirrIR slides. MirrIR plates are thick and have large thermal
mass. The cells which were fixed on 4% formalin on to the MirrIR slides were washed
with deionized water for the removal of residual PBS from the surface of the cells. The
trypan blue stain was used to distinguish between the cells and also to obtain the intergrity
of the formalin fixed cells. The lipid localization in the formalin fixed cells was more in
the cytoplasm as compared to the nuclei. Formalin fixation is a simple and less time
consuming process for the sample preparation and it gives highly resolved
biospectroscopic images.

Fabian et al. ( 1995) made a comparative study on human breast tumors, human breast
tumor cell lines and xenografted human tumor cells and has been demonstrated that
significant difference exists in spectrums.

Ventao et al. (2006) used FTIR and Raman microspectroscopy to study normal, benign and
malignant formalin fixed ovarian tissues. FTIR and Raman spectroscopy was done so as to
investigate and understand the biochemical changes occurring in ovarian cancer. The
Raman and FTIR spectra of normal and benign showed lot of similarities as compared to
-1
malignant tissue. Raman spectra in the range of 700-1700 cm gave two groups
corresponding to one group of normal and benign and the second group of malignant.
These two groups were obtained after applying first derivative to the given data. Ovarian
cancers can also be discriminated on the basis of tissue autofluoroscence pattern (Malins et
al. 1998). Diagnosis by the proteomics method also been used for ovarian cancer (Krishna
et al. 2005). The tissues are formalin fixed so as to store for histopathology and these
tissues are studied using vibrational spectroscopies. The spectra obtained showed some
kind of spectral contamination due to formalin. This contamination was removed by
washing the tissue samples with saline (Haung et al. 2003). The normal and benign tissues
were easily studied and discriminated as compared to malignant sample. Normal tissue
spectra was characterized by higher protein content and on other hand more amount of
DNA and lipid was obtained in malignant tissues.

Godwin et al. (1993) worked for studying the cisplatin drug on ovarian cancer cell lines.

The study of Sukuta and Bruch (1995) was on factor analysis of cancer FTIR evanescent
wave fibroptical (FTIR-FEW) spectra. It has been demonstrated that FTIR-FEW technique
and chemical factor analysis has a potential of a clinical diagnostic tool.

- 37 -
Wong et al. ( 1995 ) carried out research on exfoliated cells and tissues from human
endocervix and ectocervix by FTIR and ATR/FTIR spectroscopy and it has been
demonstrated that ATR/FTIR is more desirable method than the transmission method to
obtain meaningful and good quality infrared spectra of tissues.

Clarke et al. (2009) did work in this removal of dispersion artefacts of the FTIR spectra
that was obtained from the single biological cells. The FTIR spectra of the single
biological cells in the transflection mode gives the outcome in the form of dispersion
artefacts (Lasch et al. 2002). The spectrum that is obtained can have a weighted sum of the
sample reflection and transmission and the dominance of the reflection spectrum in
optically dense regions can account for dispersion artefacts.

Attenuated total reflection is a promising tool used for the biochemical origin of the given
disease. In the case of the cells, the cytoplasm is relatively sparse whereas the nucleus is
dense in terms of biochemical content. The dispersion artefact can be corrected by back
transforming the spectra and extracting the real and the imaginary components of the
spectrum resulting in decreased dispersive components. The contribution of the reflection
spectrum can become dominant in regions of high optical density giving rise to “dispersion
artefacts”. In the transflection geometry the effect is real and there is no dispersion artefact
present.

2.5 Principal Component Analysis

Principal component analysis is a variable reduction procedure. It is useful when data is


taken on number of variables and some of the variables are correlated with one another.
Because of this correlation it is possible to reduce the observed variables into a smaller
number of principal components that will account for most of the variance in the observed
variables.

A principal component can be defined as a linear combination of optimally-weighted


observed variables. The first component extracted in a principal component analysis
accounts for a maximum amount of total variance in the observed variables and will be
correlated with at least some of the observed variables. Further the second component
extracted will have two important characteristics. First, this component will account for a
maximal amount of variance in the data set that was not accounted for by the first
component. The second component will be correlated with some of the observed variables
that did not display strong correlations with component 1.The second characteristic of the
second component is that it will be uncorrelated with the first component. This means that

- 38 -
the correlation between components 1 and 2 would be zero. The remaining components
that are extracted in the analysis display the same two characteristics: each component
accounts for a maximal amount of variance in the observed variables that was not
accounted for by the preceding components, and is uncorrelated with all of the preceding
components. A principal component analysis proceeds in this fashion, with each new
component accounting for progressively smaller and smaller amounts of variance.

PCA is used abundantly in biological sciences because it is a simple, non-parametric


method of extracting relevant information from confusing data sets. PCA is helpful in
identifying patterns in data for highlighting their similarities and differences . PCA
provides a roadmap to reduce a complex data set to a lower dimension and this helps in
identifying the hidden information in the data.

The first principle component (PC1) accounts for the most variance, and subsequent PCs
account for decreasing amounts of different source of variance. The data may be analyzed
and viewed in for clustering when viewed in a particular direction and other aspect of the
information that is derived from PCA is indication of the important of particular variable
for each PC. These properties can be displayed on loading plot and each PC loading plot
provide useful information about the possible important variables and the contribution of
these variables to each PC responsible for the patterns observed on a PC score plot.
Therefore loading plot can be used to identify the molecular factor that underlies the
grouping or discrimination of the original data (Heri et al. 2003, Walsh et al. 2008).

- 39 -
CHAPTER 3

3. EXPERIMENTAL TECHNIQUES AND METHODOLOGY

The aim of the experiment was to study the effect of cisplatin and KF01-01 drug on
A2780 human ovarian cancer cell lines using FTIR and ATR-FTIR spectroscopy. Total
15 samples consisting seven samples treated with cisplatin and eight samples treated with
KF01-01 were taken for the data analysis. These samples were treated with different
concentration of IC-30, IC-50 and IC-70 . The details of samples are given in Table no. 2 .

3.1 A2780 cell line culture

A2780 human ovarian cancer cell line was obtained from the section of ovarian tissue of
the patient. The cultured cells were washed with 4ml of phosphate buffer saline (PBS)
solution several times. After washing the cells were allowed to harvest after treating with
trypsin in 0.5ml solution. After this step the cells were allowed to grow on the MirrIR
slides till the volume of the cells became 70% onto the slide.

3.2 Cell Fixation :-

The cells were fixed onto the MirrIR slides. The MirrIR slides are used because they
reflect about 95% of mid IR radiation without any interfering absorption. The cells which
were grown on the MirrIR slides were exposed to different concentration ( IC-30, IC-50
and IC-70) of cisplatin and KF01-01 for about 24 hours . After the exposure these slides
were washed with PBS, so as to remove any extra culture media or dead cells from the
slide. For the fixation of the cells 4ml of formaldehyde was added on cells for about 40
minutes. After washing with PBS the fixed slides were washed with distilled water and
then allowed for drying.

3.3 ATR- FTIR Measurement

FTIR stands for Fourier Transform Infrared Spectroscopy and the results are directly
obtained in the frequency domain (Fig.15). ATR stands for Attenuated Total Reflectance
and in this system in addition to FTIR system, ATR crystal is also used for measuring the
spectrum. The measured spectrums are distortion free. In this technique the main
component is the crystal. There are different varieties of crystals that are used for the
experiment. The refractive index of the crystal should always be high as compared to the
refractive index of the sample that is to be measured . In the present measurement
Germanium crystal (Fig.16a) has been used for taking the spectra of all the
- 40 -
samples. The crystal was inserted into the microscope for taking the spectrum. ATR was
done in the reflection mode because the slides used were the MirriR slides. The measured
spectrum data was uploaded into Matlab software for PCA analysis for extraction of
hidden information from the spectra of A2780 ovarian cancer cell lines samples treated
with cisplatin and KF01-01 drugs. Details of the measured spectra of A2780 cell lines
treated with cisplatin and KF01-01 corresponding to their concentrations are given in
Table No. 2

Table No. 2 : Details of samples and their spectrum

S. Type Of Drug Sample No./ No.Of Spectrums


No. Concentration
ATR-FTIR FTIR

1 Cisplatin (CIS) Sample 1 - IC 30 10 10

2 Cisplatin (CIS) Sample 2 - IC 30 10 10

3 Cisplatin (CIS) Sample 3 - IC 30 10 10

4 Cisplatin (CIS) Sample 4 - IC 50 10 10

5 Cisplatin (CIS) Sample 5 - IC 50 10 10

6 Cisplatin (CIS) Sample 6 - IC 70 10 10

7 Cisplatin (CIS) Sample 7 - IC 70 10 10

8 Gold (KF0101) Sample 8 - IC 30 10 10

9 Gold (KF0101) Sample 9 - IC 30 10 10

10 Gold (KF0101) Sample 10 - IC 50 10 10

11 Gold (KF0101) Sample 11 - IC 50 10 10

12 Gold (KF0101) Sample 12 - IC 50 10 10

13 Gold (KF0101) Sample 13 - IC 70 10 10

14 Gold (KF0101) Sample 14 - IC 70 10 10

15 Gold (KF0101) Sample 15 - IC 70 10 10

- 41 -
Fig15: ATR-FTIR experimental set up (Ref. 83 )

The crystals that have high refractive index as compared to the refractive index of the
sample are used for measurement. The light is allowed to pass through crystal which has a
high refractive index of 4 as compared to the refractive index of the given sample. The
crystal is pressed against the sample to take the measurements.

Fig16a : ATR Germanium crystal base Fig16b : ATR Germanium crystal top

(Ref. 84 )

- 42 -
3.3.1 Measurement Procedure for taking ATR-FTIR Spectrum

The Germanium crystal is taken and adjusted just above the stage into the microscope.
Then a clear MirrIR slide without any sample is taken and adjusted onto the stage. The
background scans of 2056 were taken by touching the crystal on to the slide. After taking
the background measurement the crystal is removed and cleaned with the ethanol to

remove any particles from the crystal which can disturb the spectra. After cleaning the
crystal it is again placed into the microscope and sample slide is loaded on to the stage.
Aperture size adjustment is not required in the ATR experiment. Now with the help of joy
stick the particular area of cells is selected and that area is made clear with the help of
adjusting screw placed near the stage of microscope. This process is done without the
crystal. The moment a particular area is selected than the crystal is inserted in to the
microscope. Now the pre processing beam is checked on to the computer screen. Now
with the help of fine tune screw present on the joy stick we make the crystal to touch one
of the cells. The moment the crystal touches one of the cells a peak of amide I appears on
the screen of computer in the pre processing beam. Once that peak appears the spectrum of
the cell is measured. The total of 10 spectrum with 1056 scans for each sample were taken
at the resolution of 4cm-1. Everytime after measuring the spectrum the crystal was cleaned
with the help of ethanol and then inserted again in the microscope. The cleaning is done so
as to remove any dust or sample from the crystal. The presence of sample or dust might
cause disturbances in the spectrum of the sample. After the spectrum is obtained than the
file is converted from „bph‟ to the form of „csv‟ file. This file format is accepted by
Matlab software. The files are loaded in the Matlab software for further analysis. The data
is processed for extraction of hidden information by applying first or second derivative.
This is done so as to reduce the noise and for getting better results. After applying
derivative to the raw spectrum data , normalisation is done and then the data is passed for
the PCA analysis. PCA stands for principal component analysis and it reduces the data to
scores. These scores are present according to the components like PC1 and PC2. Both the
components have different characteristics according to which the particular data is
converted into scores. After PCA plot, the loading plot is plotted to get the exact location
of the peaks obtained in the spectrum.

3.4 FTIR Measurement

Spectra of all the samples was also taken with FTIR. The Infrared spectral data for the
A2780 human ovarian cancer cell lines was obtained using FTIR system. In this system the

- 43 -
microscope contains a detector known as liquid nitrogen cooled Mercury Cadmium

Telluride (MCT) detector. The spectrometer was purged using dry nitrogen for removing
any presence of water vapour and carbon dioxide (CO2). The spectrums were obtained in
reflection mode and then PCA (Principal Component Analysis) was done on the
spectrums. Before doing the PCA, spectrums were corrected with RMieS (Resonance Mie
Scattering) so as to remove the dispersion artefacts that usually occurs in the FTIR
experimental data. Dispersion Artefacts are observed as a sharp decrease in the intensity of
high wave number side of the absorption bands. This is seen in the Amide I band case at
1655cm-1, causing a downward shift of the real peak. The other algorithm that is used at
the place where the Mie scattering is weak and no strong distortion is seen in the Amide I
band region is called Extended Multiplicative Signal Correction (EMSC) algorithm. It has
been shown that the principal origin of dispersion artefacts is due to the process called as
Resonance Mie scattering and this is present in both FTIR transmission and transflection
mode. Dispersion artefacts affects both shape and the position of the Amide I band. After
applying RMieS on the measured data, PCA was done for getting the better separation of
the scores based on the two different components. Details of the measured spectra of
A2780 cell lines treated with cisplatin and KF01-01 corresponding to their concentration
are given in Table No. 2. The measured spectrum data was uploaded into Matlab software
for PCA analysis for extraction of hidden information from the spectra of A2780 ovarian
cancer cell lines samples treated with cisplatin and KF01-01 drugs.

3.4.1 Measurement Procedure for FTIR data

The MirrIR slides in which the sample was allowed to grow was taken and placed on the
stage of the microscope. Sample slide was kept near to the clean MirrIR slide without any
sample on it. In the measurement of FTIR spectrums a particular aperture size is adjusted.
For this experiment an aperture size of 100µm×100µm was adjusted in the microscope.
Then the background scans of 2056 were taken for the clean MirrIR slide. After taking the
background the spectrums of the given sample slide were taken. For each of the sample 10
spectrums of 1056 scans were taken at the resolution of 4cm-1 . The range of the spectrum
which was selected for taking the spectrums was from 700-4000cm-1 and the parameters
were controlled by the Varian Software. The area was selected with the help of joy stick
which was controlled by the computer and making that particular area having cells clear
with the help of the adjusting screw that was present near the microscope stage. The
experiment was conducted in the reflection mode of the microscope for the given MirrIR
slides. Once the spectrums are obtained then the spectrum file is converted from „bph‟ into

- 44 -
the file „csv‟ that is accepted by the Matlab software. This software uploads the data and

converts into simple data in the form of scores. The data is converted into scores in the
PCA software. PCA converts the data into scores that gives the result according to two
different components. Before applying PCA the data is run through RMieS (Resonance
Mie Scattering) for the reduction of noise and the scattering that is obtained during the
process of taking spectrum. Once the data is run through this command then it is allowed
to proceed for PCA analysis. In the PCA plot we get the scores that are present according
to the two components (PC1 and PC2). The PCA plot is then titled according to the data
and also the name of the samples are given in the legend box. After the PCA plot is
obtained than the loading plot is also plotted. The results are finally obtained for the
analysis and for concluding the data.

- 45 -
CHAPTER 4

4. DATA ANALYSIS AND RESULTS

The present work has been carried out for studying the effect of Cisplatin and KF01-01
drugs on A2780 ovarian cancer cell lines as a function of concentration utilizing the ATR-
FTIR and FTIR spectroscopy. The used drugs concentration of IC-30, IC-50 & IC-70
cover the cytotoxic range with 30%, 50% & 70% of cell growth inhibition respectively.
The spectrums obtained after ATR-FTIR and FTIR measurements only provide very
limited information regarding biochemical composition of the sample. Therefore all ATR-
FTIR and FTIR corrected spectra of the A2780 cells have been statistically analyzed.
Principal component analysis (PCA) was preformed through MATLAB software for
classification of cells based on their response to drugs. Initially these data have been pre-
treated to enhance the spectral features and to eliminate unnecessary artefacts generated
due to variations in sample thickness or due to some other impurities. Pre-treatment
process includes taking first derivative or second derivative, mean centring and vector
normalization respectively.

4.1 ATR-FTIR Data Results

In case of ATR-FTIR initially PCA was performed on spectral data from 950- 3800 cm-1
range. Cisplatin and KF01-01 combined sample data of (IC-30, IC-50 and IC70)
concentration was analyzed separately. PCA was done by taking raw data , first derivative
and second derivative for reducing the noise and for enhancement of signal. Cisplatin
combined data of concentrations (IC-30, IC-50 and IC70) was analyzed with PCA.
KF01-01 data of (IC-30, IC-50 and IC70) concentrations was also analyzed similarly with
PCA. Further cisplatin and KF01-01 data at ((IC-30, IC-50 and IC70) was analyzed
separately for different regions i.e. lipid region (2800- 3000 cm-1), protein region (1500-
1600 cm-1) and carbohydrate & nucleic acid region (850 -1300cm-1 ). In PCA analysis first
two components give maximum variation and therefore, only first two PCs were selected
for comparing observed patterns. Loading plot of scores for each PC were also plotted for
score plot. The loading plots allowed to identify the specific spectral features with
wavenumber location on each PC. Utilising the PCA score plots and loading plots of ATR
data of all Cisplatin and KF01-01 drug treated sample data all observed peaks at different
wave nos. were identified. The comical bonds or functions assigned to these waves nos.
are give in table no. 3 and 4. The assignment of bands of these waves nos. have been taken

- 46 -
from the database compiled by Monasaghi et al. (2008).

4.1.1 Cisplatin and KF01-01 drug treated samples at IC-30, IC-50 and IC-70

The spectra, PCA score plots and loading plots corresponding to all samples are given in
figure 17 to 76. The spectrum data of cisplatin and KF01-01 drug treated samples at
different concentrations (IC-30, IC-50 & IC-70) was combined and analyzed using
principal component analysis technique for discrimination of different patterns. The ATR
spectra of cisplatin and KF01-01 samples treated at IC-30 alongwith their PCA scores
corresponding to raw data and normalized second derivative data are given in Fig. 17- 20.
In the PCA score plots no clear pattern is observed and score points data is highly
scattered. Scattering in samples treated with cisplatin is more in comparisons to samples
treated with KF01-01. These results indicate that drug treated cells are in their different
apoptosis stages.

The ATR spectra and PCA scores of samples treated with cisplatin and KF01-01 at IC-50
corresponding to raw data and normalized second derivative data are given in Fig. 21- 34.
The spectra for cisplatin and KF01-01 at IC-50 are shown in Fig 21 and 22 respectively.
The absorbance intensity in KF01-01 treated samples is high in comparison to cisplatin
samples. In the raw data PCA score plot shown in Fig. 24 clear pattern is observed and
data corresponding to cisplatin scores and KF01-01 scores is lying in different clusters .
Scattering of scores points is also less. In the loading plots ( Fig. 25 and 26) Amide -I
and Amide II bands corresponding to KF01-01 are appearing at 1643 and 1517
respectively. Strong peak is observed at 1137 cm-1 corresponding to cisplatin. This band
is assigned to Oligosaccharide C-OH stretching band 2 – methylmannoside. These results
indicates that drug treated cells are in their similar apoptosis stages and mode of action for
both drugs is different. It is further inferred that IC-50 concentration appears optimum for
both drugs. In second derivative data (fig. 31 & 32) also a similar pattern is observed.
However some points are overlapping.

The ATR spectra of samples treated with cisplatin and KF01-01 at IC-70 alongwith their
PCA scores corresponding to raw data and normalized second derivative data are given
in Fig. 35- 38. In the PCA score plots no clear pattern is observed and score points are
highly scattered. These results indicate that drug treated cells are in their different
apoptosis stages. The score points corresponding to cisplatin and KF01-01 at IC-50
concentration are clearly observed in different clusters (fig.24). These results show that
both drugs are inducing chemical changes at different bands indicating different working
mechanism of both drugs.

- 47 -
PCA was performed on combined spectrum data of all samples treated with cisplatin at
different concentrations (IC-30, IC-50 & IC-70). The spectrum and PCA score plots are
shown in Fig. 39-44. It is observed that score points corresponding to IC-50 are in one
cluster with less scattering score points corresponding to IC-30 and IC-70 are overlapping
and highly scattered. Loading plots of score points are shown in figure (47-48). Peaks are
observed at 1124, 1130, 1246, 1545, 1537, 1643 & at 2918cm-1. These results further
indicate that IC-50 appears optimum dose and drugs effect is not linear as per
concentrations.

PCA was also performed on combined spectral data of all corresponding to samples treated
with KF01-01 drug. The spectrum, PCA scores and loading plots are shown in fig. (45 -
52 ). In the score plots it is observed that all points, corresponding to IC-70 are in one
cluster with overlapping of few points. Score points, corresponding to IC-30 & IC – 50 do
not form any cluster. These results indicate that KF01-01 inducing its cytotoxic effect at
all concentrations. In the loading plots peak are observed at 1050, 1137, 1517 , 1537, 1637
& 1657. These peaks are different in comparison to peaks observed on cisplatin samples.
Most of the peaks are observed in carbohydrate & nucleic acid region (950-1300 cm-1) &
in protein region (1500-1600 cm-1).

4.1.2. Cisplatin and KF01-01 (IC-50) treated samples data analysis for Lipid, Protein ,
Carbohydrates & Nucleic acid Region.

The ATR spectra of combined samples of cisplatin and KF01-1 was analyzed for lipid,
protein , and carbohydrates and nucleic acid at IC-50. The PCA was performed separately
for each region.

 Lipid Region

Spectra , score plot and loading plots for lipid region are shown in Fig. 53-60. PCA score
plot for raw data and second derivative are shown in Fig. 54 & 58 respectively. Two
different clusters of PCA scores are observed with overlapping of some data points. Points
corresponding to cisplatin and KF01-1 are clearly separable. In the loading plots ( Fig. 55
and 56) C-H stretching band is appearing at 2918, 2928, 2925 & at 2930. Other peaks
are observed at 2838 and at 2853.

 Protein Region

Spectra , score plot and and loading plots for protein region are shown in Fig. 51-58. PCA
score plot for raw data and second derivative are shown in Fig. 62 & 66 respectively. Two
different clusters of PCA scores are observed with overlapping of some data
- 48 -
points. In the loading plots ( Fig. 63 and 64) peaks are observed at 1571cm-1 and 1537
cm-1 with respect to cisplatin . Other peak observed at 1589 cm-1 assigned to ring C-C
stretch of phenyl correspondence to KF01-01. These results indicate that mode of action of
both drugs is different in this region.

 Carbohydrate an Nucleic Acid Region

Spectra , score plot and and loading plots for protein region are shown in Fig. 59-66. PCA
score plot for raw data and second derivative are shown in Fig. 70 & 74 respectively.
Two different clear clusters of PCA scores are observed with few overlapping points.
In the loading plots ( Fig. 71 and 72) peak observed at 1137 cm-1 corresponds to cisplatin
pertaining to C-OH band. Amide III and C-O stretching peaks corresponding to KF01-01
are appearing at 1235 cm-1 and at 1050 cm-1 respectively.

In this region separation of cisplatin and KF01-01 on the basis of score points is much
clear in comparison to the data of liquid and protein region. These results indicate that
mode of action of both drugs is clearly different in this region.

- 49 -
Table No. 3.

Details of Observed Peaks and their assignment in Cisplatin and KF01-01 treated
Samples in Nucleic Acid & Carbohydrate and Lipid Region

S. Peak/ Wave Assignment Region Observed


No. number in cm-1 in Sample

1 1050 C-O stretching coupled with C-O Nucleic acid & KF01-01
bending of C-OH of carbohydrate carbohydrates

2 1056 Stretching C-O deoxyribose -do- KF01-01

3 1137 Oligosaccharide C-OH stretching -do- KF01-01


band 2- Methylmannoside &
Cisplatin

4 1180 Amide III band region -do- KF01-01

5 1188 Deoxyribose -do- KF01-01

6 1235 Composed of Amide III as well as -do- KF01-01


phosphate vibration of nucleic acid

7 1236 Amide III and asymmetric -do- KF01-01


phosphodister stretching mode
mainly from nucleic acids

8 1246 PO- 2 asymmetric -do- Cisplatin

9 2838 Stretching C-H Lipid region KF01-01

10 2846 Symmetric stretching of methoxy -do- KF01-01


(4)

11 2853 CH2 of lipids -do- KF01-01

12 2860 Stretching C-H -do- KF01-01

13 2916 Cholestrol, phospholipids and -do- KF01-01


creatine (higher in normal tissue)

14 2918 Stretching C-H -do- Cisplatin

15 2925 C-H stretching bend in normal -do- KF01-01


tissue

16 2928 Stretching C-H -do- KF01-01

17 2930 C-H stretching -do- KF01-01

- 50 -
Table No. 4.

Details of Observed Peaks and their assignment in Cisplatin


and KF01-01 treated Samples in Protein Region

S. Peak/ Wave Assignment Region Observed


No. number in cm-1 in Sample

1 1517 Amide II Protein KF01-01


region

2 1532 Stretching C=N, C=C Protein Cisplatin


region

3 1537 Stretching C=N, C=C Protein Cisplatin


region

4 1541 Amide II absorption ( primarily an -do- KF01-01 &


N-H bending coupled to a C-N Cisplatin
stretching vibrational mode)

5 1545 Protein band Amide II or Peptide -do- KF01-01 &


amide II Cisplatin

6 1549 Amide II -do- KF01-01

7 1552 Ring base -do- KF01-01

8 1571 C=N, Adenine -do- Cisplatin

9 1589 Ring C-C stretch of Phenyl -do- KF01-01

10 1635 β- sheet structure of amide I -do- KF01-01

11 1637 C=C uracil, C=O -do- KF01-01

12 1643 Amide I band of protein and H-O- -do- KF01-01


H deformation of water

13 1653 C=O, C=N , N-H of adenine , -do- KF01-01


thymine, guanine, cytosine

14 1657 α helical structure of Amide I -do- KF01-01

- 51 -
0.12 PCA RAW DATA Plot
0.3
CIS
0.1
KF
0.2
0.08
0.1
Absorbance

0.06

PC2 (11.6%)
0.04 0

0.02 -0.1

0
-0.2

-0.02
-0.3
-0.04
4000 3500 3000 2500 2000 1500 1000
Wavenumber -1 -1 -0.4
Wavenumber /in
cmcm -0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4
PC1 (74.0% )

Fig 17: Normalised ATR spectra (raw data) of Fig 18: ATR spectra scores plot (raw data) of
A2780 cells treated with CIS & KF(IC-30). A2780 cells treated with CIS & KF(IC-30).

PCA Second derivative Plot


0.3 0.6
CIS 30
KF 30
0.2 0.4

0.1 0.2
Absorbance

PC2 (20.6%)

0 0

-0.1 -0.2

-0.2 -0.4

-0.3 -0.6

-0.4 -0.8
3500 3000 2500 2000 1500 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6
Wavenumber cm-1-1
Wavenumberin/ cm PC1 (33.2% )

Fig 19: Normalised Second derivative of A2780 Fig 20: PCA plot of second derivative of A2780
cells treated with CIS & KF(IC-30). cells treated with CIS & KF(IC-30).

0.1 0.12

0.1
0.08
0.08

0.06
Absorbance
Absorbance

0.06

0.04
0.04

0.02
0.02
0

0 -0.02
4000 3500 3000 2500 2000 1500 1000 4000 3500 3000 2500 2000 1500 1000
Wavenumber cm-1-1
in / cm
Wavenumber Wavenumber cm-1-1
Wavenumberin/ cm

Fig 21: ATR spectra of A2780 cells treated with Fig 22: ATR spectra of A2780 cells treated with
CIS(IC-50). KF(IC-50).

- 52 -
PCA RAW DATA Plot
0.3
0.12
CIS
KF
0.1 0.2

0.08 0.1

0.06

PC2 (13.2%)
Absorbance

0.04
-0.1
0.02
-0.2
0

-0.02 -0.3

-0.04 -0.4
4000 3500 3000 2500 2000 1500 1000 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8
-1 -1
Wavenumber in cm
Wavenumber / cm PC1 (71.7% )

Fig 23: Normalised ATR spectra (raw data) of Fig 24: ATR spectra scores plot (raw data) of
A2780 cells treated with CIS(IC-50) & A2780 cells treated with CIS(IC-50) &
KF(IC-50). KF(IC-50).

Loadings for PC 1 Loadings for PC 2


0.1 0.1
1643 1137
1517 0.08

0.05 0.06

0.04

0 0.02

-0.05 -0.02
16531532
1137 -0.04

-0.1 -0.06
4000 3500 3000 2500 2000 1500 1000 4000 3500 3000 2500 2000 1500 1000
Wavenumber -1-1 -1 -1
Wavenumber in
/ cm cm Wavenumber in cm
Wavenumber / cm

Fig 25: Loading plot of PC1 (raw data) of Fig 26: Loading plot of PC2 (raw data) of
A2780 cells treated with CIS(IC-50) & A2780 cells treated with CIS(IC-50) &
KF(IC-50) KF(IC-50)

PCA FIRST DERIVATIVE PLOT


0.2 0.6
CIS
0.5
KF

0.1 0.4

0.3

0
Absorbance

PC2 (24.1%)

0.2

0.1

-0.1 0

-0.1

-0.2 -0.2

-0.3
-0.3
3500 3000 2500 2000 1500 -0.4
-1 -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8
Wavenumber in cm
Wavenumber / cm PC1 (56.2% )

Fig 27: Normalised first derivative plot of ATR Fig 28: PCA plot of first derivative of A2780 cells
spectra of A2780 cells treated with CIS(IC- treated with CIS(IC-50) & KF(IC-50).
50) & KF(IC-50).

- 53 -
Loadings for PC 1 Loadings for PC 2
0.15 0.2
2846
0.1 0.15 2916
2930 1657 1246
0.1
0.05 2853
0.05
0
Absorbance

0
-0.05
2955
-0.05
-0.1
-0.1
-0.15 2860
-0.15
2930
-0.2 -0.2
3500 3000 2500 2000 1500 3500 3000 2500 2000 1500
-1
Wavenumber
Wavenumberin/ cm
cm
-1
Wavenumber cm-1-1
Wavenumberin/ cm

Fig 29: Loading plot of PC1 for first derivative of Fig 30: Loading plot of PC2 for first derivative of
A2780 cells treated with CIS(IC-50) & A2780 cells treated with CIS(IC-50) &
KF(IC-50). KF(IC-50).

PCA SECOND DERIVATIVE Plot


0.3 0.6
CIS 50
KF 50
0.2 0.4

0.1 0.2
Absorbance

PC2 (21.1%)

0 0

-0.1 -0.2

-0.2 -0.4

-0.3 -0.6

-0.4 -0.8
3500 3000 2500 2000 1500 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1
Wavenumber / -1 -1
cm PC1 (32.0% )
Wavenumber in cm
Fig 31: Normalised second derivative plot of Fig 32: PCA plot of second derivative data of
A2780 cells treated with CIS(IC-50) & A2780 cells treated with CIS(IC-50) &
KF(IC-50). KF(IC-50).

Loadings for PC 1 Loadings for PC 2 1653


0.2 0.15
2925
1517
2853 0.1
0.1 1637 1236
0.05

0
0

-0.05
-0.1
2860 -0.1 2918
2853
-0.2 1549
-0.15
1246
-0.3 -0.2
3500 3000 2500 2000 1500 3500 3000 2500 2000 1500
-1
-1 -1-1
Wavenumber
Wavenumberin/ cm
cm Wavenumber in /cm
Wavenumber cm

Fig 33: Loading plot of PC1 component of Fig 34: Loading plot of PC2 component of
second derivative. second derivative.
- 54 -
PCA RAW DATA Plot
0.15 0.4
CIS 70
0.3 KF 70

0.2
0.1
Absorbance 0.1

PC2 (13.7%)
0
0.05
-0.1

-0.2
0
-0.3

-0.4

-0.05
4000 3500 3000 2500 2000 1500 1000 -0.5
-0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8
-1
Wavenumberin/ cm
Wavenumber cm-1 PC1 (77.7% )

Fig 35: Normalised ATR spectra (raw data) of Fig 36: ATR spectra scores plot (raw data) of
A2780 cells treated with CIS & KF(IC-70). A2780 cells treated with CIS & KF(IC-70).

PCA SECOND DERIVATIVE Plot


0.3 0.8
CIS 70
KF 70
0.2 0.6

0.1 PC2 (23.5%) 0.4

0 0.2

-0.1 0

-0.2 -0.2
Absorbance

-0.3 -0.4

-0.4 -0.6
3500 3000 2500 2000 1500 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 1.2
-1 -1
Wavenumber
Wavenumberin cm
/ cm PC1 (40.3% )

Fig 37: Normalised second derivative of A2780 Fig 38: PCA plot of second derivative of A2780
cells treated with CIS & KF(IC-70). cells treated with CIS & KF(IC-70).

PCA CISPLATIN Plot


0.12 0.4
cis 30
0.1 0.3
cis 50
cis 70

0.08
0.2

0.06
Absorbance

PC2 (13.1%)

0.1
0.04
0
0.02

0 -0.1

-0.02 -0.2

-0.04
4000 3500 3000 2500 2000 1500 1000 -0.3
-0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6
Wavenumber cm-1-1
Wavenumberin/ cm PC1 (71.1% )

Fig 39: Combined Normalised ATR spectra (raw Fig 40: Combined ATR spectra scores plot (raw
data) of A2780 cells treated with CIS (IC- data) of A2780 cells treated with CIS (IC-
30, IC-50 & IC-70). 30, IC-50 & IC-70).

- 55 -
Loadings for PC 1 Loadings for PC 2
0.06 0.08
1137
0.04 0.06
0.02
0.04
0
0.02
-0.02
0
-0.04
1246
-0.02
-0.06
1537 1545
-0.08 -0.04 2918 1646
1657
-0.1 -0.06
4000 3500 3000 2500 6572000 1500 1000 4000 3500 3000 2500 2000 1500 1000
Wavenumber in cm-1 / cm -1
Wavenumber Wavenumber cm/-1cm -1
Wavenumber

Fig 41: Loading plot of PC1, (raw data) of A2780 cells Fig 42: Loading plot of PC2 (raw data) of A2780 cells
treated with CIS (IC-30, IC-50 & IC-70). treated with CIS (IC-30, IC-50 & IC-70).

PCA Second derivative Plot


0.6
0.3 CIS 30
CIS 50
0.4
0.2 CIS 70

0.2
0.1
Absorbance

PC2 (22.0%)

0 0

-0.1 -0.2

-0.2 -0.4

-0.3 -0.6

-0.4 -0.8
3500 3000 2500 2000 1500 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8
-1-1 PC1 (28.9% )
Wavenumber
Wavenumber /
in cm cm

Fig 43: Combined Normalised second derivative Fig 44: Combined PCA plot of second derivative
of A2780 cells treated with CIS (IC-30, of A2780 cells treated with CIS (IC-30,
IC-50 & IC-70). IC-50 & IC-70).

PCA RAW Plot


0.12 0.3
KF 30
0.1 0.2 KF 50
KF 70
0.08 0.1

0.06 0
PC2 (12.1%)

0.04 -0.1

0.02 -0.2

0 -0.3

-0.02 -0.4

-0.04 -0.5
4000 3500 3000 2500 2000 1500 1000 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6
-1 PC1 (75.2% )
Wavenumber / cm-1
Wavenumber in cm
Fig 45: Combined ATR spectra (raw data) of A2780 Fig 46: PCA score plot of A2780 cells treated with
cells treated with KF(IC-30, IC-50, IC-70 ). KF(IC-30, IC-50, IC-70).

of A2780 cells treated with CIS & KF(IC-30).

- 56 -
Loadings for PC 1 Loadings for PC 2
0.1 0.06
1637 1137
0.08 0.04
0.06
1517 0.02
0.04

0.02 0
1050
0 -0.02 1537
-0.02
-0.04
-0.04
1657
1137
-0.06 -0.06
4000 3500 3000 2500 2000 1500 1000 4000 3500 3000 2500 2000 1500 1000
Wavenumber / cm
-1 Wavenumber / cm -1

Fig 47: PC1 loading plot of cells treated with Fig 48: PC2 loading plot of cells treated with
KF (IC-30, IC-50, IC-70), Raw Data KF(IC-30, IC-50, IC-70), Raw Data

0.3 PCA SECOND DERIVATIVE Plot


0.8
KF 30
0.2 0.6 KF 50
KF 70

0.1 0.4

0.2
PC2 (25.6%)

0
0
-0.1
-0.2

-0.2
-0.4

-0.3 -0.6

-0.4 -0.8
-0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 1.2
3500 3000 2500 2000 1500 PC1 (32.5% )
-1 -1
Wavenumber
Wavenumber cm / cm
Fig 50: PCA score plot cells treated with KF (IC-30,
FIG49: Normalised Second Derivative Plot of cells treated IC-50, IC-70), Second derivative
with KF (IC-30, IC-50 & IC-70)

Loadings for PC 1 Loadings for PC 2


0.25 0.3

0.2
0.2
0.15

0.1 0.1
0.05
0
0

-0.05
-0.1
-0.1

-0.15 -0.2
3500 3000 2500 2000 1500 3500 3000 2500 2000 1500
Wavenumber
Wavenumber cm/-1cm
-1 Wavenumber / cm -1
Wavenumber cm-1
Fig 51: PCA Loading plot of cells treated with (IC-30, IC-50 & Fig 52: score plot of cells treated with (IC-30, IC-
IC-70), second derivation 50 & IC-70), Second derivative

- 57 -
PCA LIPID IC50 Plot
0.3 0.2
CIS 50
KF 50
0.1
0.2

Absorbance
0

0.1

PC2 (11.1%)
-0.1

0 -0.2

-0.3
-0.1
-0.4

-0.2
2950 2900 2850 -0.5
-0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6
-1
Wavenumber /in
Wavenumber cmcm-1 PC1 (78.2%)
Fig 54: PCA score plot of A2780 cells
Fig.53: ATR spectra of A2780 cells treated
treated with CIS & KF (IC-50), raw data
with CIS & KF (IC-50), raw data (Lipid
(Lipid Region)
Region)

Loadings for PC 2 Loadings for PC 1


0.3 0.2
2925 2945
0.15
0.2
0.1
2853 2980
0.1 0.05
2961
0
0 -0.05
2978
2947
2838 -0.1
-0.1
-0.15
2920
2853
-0.2 -0.2
2950 2900 -1 2850 2950 2900 2850
Wavenumber in cm
-1 Wavenumber
-1
Wavenumber / cm Wavenumber in /cm
cm-1
Fig.55: PC2 loading plot of A2780 cells
Fig.56: PC1 loading plot of A2780 cells
treated with CIS & KF (IC-50), raw data
treated with CIS & KF (IC-50), raw data
(lipid region)
(lipid region)

PCA SECOND DERIVATIVE Plot


0.3 0.6
CIS 50
KF 50
0.2 0.4

0.1 0.2
Absorbance

PC2 (23.9%)

0 0

-0.1 -0.2

-0.2 -0.4

-0.3 -0.6

-0.8
-0.4 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1
2950 2900 2850 PC1 (63.4% )
-1
cm-1
Wavenumberin/ cm
Wavenumber
Fig.58: PCA score plot of A2780 cells
Fig 57: ATR spectra of A2780 cells
treated with CIS & KF (IC-50), normalised
treated with CIS & KF (IC-50),
second derivative (Lipid Region)
normalised second derivative (Lipid
Region)
- 58 -
Loadings for PC 1 Loadings for PC 2
0.4 0.4
2925 2860
0.3 0.3

0.2 2853 0.2 2930


2959
0.1 0.1

0 0

-0.1 -0.1 2955

-0.2 -0.2
2846
-0.3 -0.3
2950 2900 2850 2950 2900 2850
-1 -1
Wavenumber
Wavenumber cm-1
in /cm cm-1
Wavenumberin/ cm
Wavenumber
Fig.59: PC1 loading plot of A2780 cells Fig.60: PC2 loading plot of A2780 cells
treated with CIS & KF (IC-50), normalized treated with CIS & KF (IC-50), normalized
second derivative (lipid region) second derivative (lipid region)

PCA PROTEIN IC50 Plot


0.4 1.6
CIS 50
1.4 KF 50
0.3
1.2

0.2 1
Absorbance

PC2 (26.8%)

0.8
0.1
0.6

0 0.4

0.2
-0.1
0

-0.2 -0.2

-0.4
-0.3 -0.5 0 0.5 1
1600 1580 1560 1540 1520 PC1 (61.4% )
-1
-1
Wavenumberin/ cm
Wavenumber cm
Fig.62: PCA score plot of A2780 cells
Fig.61: ATR spectra of A2780 cells treated
treated with CIS & KF (IC-50), raw data
with CIS & KF (IC-50), raw data (Protein
(Protein Region)
Region)

Loadings for PC 2 Loadings for PC 1


0.3 0.15
1541
0.1
0.2
0.05
0.1
0
0
-0.05
-0.1
-0.1
-0.2
1545 -0.15
-0.3
-0.2
1517
-0.4 -0.25
1600 1580 1560 1540 1520 1600 1580 1560 1540 1520
-1
Wavenumberin/ cm
Wavenumber cm -1 -1-1
Wavenumber
Wavenumber in cm
/ cm
Fig.63: PC2 loading plot of A2780 cells Fig.64: PC1 loading plot of A2780 cells
treated with CIS & KF (IC-50), raw data treated with CIS & KF (IC-50), raw data
(Protein Region) (Protein Region)
- 59 -
PCA SECOND DERIVATIVE Plot
0.4 0.6
CIS 50
KF 50
0.4
0.2
0.2
Absorbance

PC2 (31.4%)
0
0

-0.2
-0.2
-0.4

-0.4
-0.6

-0.8
-0.6 -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8
1600 1580 1560 1540 1520 PC1 (44.8% )
-1
Wavenumber
Wavenumber cm-1
in/ cm
Fig.66: PCA score plot of A2780 cells
Fig.65: ATR spectra of A2780 cells treated
treated with CIS & KF (IC-50), normalised
with CIS & KF (IC-50), normalised second
second derivative (Protein Region)
derivative (Protein Region)

Loadings for PC 2 Loadings for PC 1


0.3 0.3

0.2 0.2

0.1 0.1

0 0

-0.1 -0.1

-0.2 -0.2

-0.3 -0.3

-0.4 -0.4
1600 1580 1560 1540 1520 1600 1580 1560 1540 1520
-1 -1
Wavenumber
Wavenumber in cm
/ cm Wavenumber
Wavenumber / in -1 -1
cmcm
Fig.67: PC2 loading plot of A2780 cells Fig.68: PC1 loading plot of A2780 cells
treated with CIS & KF (IC-50), normalised treated with CIS & KF (IC-50), normalised
second derivative (Protein Region) second derivative (Protein Region)

PCA CARBO & NA Plot


0.2 0.6
CIS 50
KF 50
0.15 0.4

0.1 0.2
Absorbance

PC2 (40.2%)

0.05 0

0 -0.2

-0.05 -0.4

-0.1 -0.6

-0.15 -0.8
-0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6
1300 1200 1100 1000 900
PC1 (49.8% )
-1
Wavenumber
Wavenumber in/ cm
cm-1

Fig.69: ATR spectra of A2780 cells treated Fig.70: PCA score plot of A2780 cells
with CIS & KF (IC-50), raw data treated with CIS & KF (IC-50), raw data
(Carbohydrate & nucleic acid Region) (Carbohydrate & nucleic acid Region)

- 60 -
Loadings for PC 2
0.15 Loadings for PC 1
0.15
852 1137
0.1
0.1
0.05
0.05
0

0 -0.05

-0.1
1235 1050
-0.05
-0.15
1222
-0.1 -0.2
1300 1200 1100 1000 900 1300 1200 1100 1000 900
-1 -1
Wavenumber / cm -1 Wavenumber / cm
Wavenumber in cm Wavenumber in cm -1

Fig.71: PC2 loading plot of A2780 cells Fig.72: PC1 loading plot of A2780 cells
treated with CIS & KF (IC-50), raw data treated with CIS & KF (IC-50), raw data
(Carbohydrate & nucleic acid Region) (Carbohydrate & nucleic acid Region)
0.5 PCA SECOND DERIVATIVE Plot
0.8
CIS 50
KF 50
0.6

0.4
Absorbance

PC2 (23.0%)

0.2
0
0

-0.2

-0.4

-0.5 -0.6
1300 1200 1100 1000 900 -0.5 0 0.5 1
PC1 (41.5% )
Wavenumber / cm -1
Fig.74: PCA score plot of A2780 cells treated
Fig.73: ATR spectra of A2780 cells treated with
with CIS & KF (IC-50), normalised second
CIS & KF (IC-50), normalised second derivative
derivative (Carbohydrate & nucleic acid Region)
(Carbohydrate & nucleic acid Region)
Loadings for PC 2 Loadings for PC 1
0.3 0.15
1137
1050 981
0.2 0.1 941
1246 1180
0.1 0.05

0 0

-0.1 -0.05
1265
-0.2 -0.1
1236
-0.3 -0.15
1056
-0.4 -0.2
1300 1200 1100 1000 900 1300 1200 1100 1000 900
-1 -1 -1
Wavenumber
Wavenumber /in
cmcm Wavenumber / cm

Fig.75: PC2 loading plot of A2780 cells treated Fig.76: PC1 loading plot of A2780 cells treated
with CIS & KF (IC-50), normalized second with CIS & KF (IC-50), normalized second
derivative (Carbohydrate & nucleic acid Region) derivative (Carbohydrate & nucleic acid Region)

- 61 -
4.2 FTIR Data Results

FTIR spectroscopy was also applied to investigate the effect of cisplatin and KF01-01 on
A2780 ovarian cell lines at varying concentrations. In FTIR data RMieS corrections
were applied for removal of distortions from all the measured spectra before applying the
PCA analysis. Different combinations of spectral data were tried for getting the relevant
information from the recorded spectra. PCA allowed the separation between spectra of
cisplatin and KF01-01 treated samples. Initially RMieS and PCA applied on spectral data
from 1000-2200 cm-1 range. Both cisplatin and KF01-01 treated combined sample data
was analyzed for different concentrations (IC-30, IC-50 and IC-70) separately by applying
iteration 1 to iteration 8. It is observed that at IC-50 both cisplatin and KF01-01 treated
samples are in distinct clusters. Further all cisplatin treated sample data at concentrations
(IC-30, IC-50 and IC-70) was combined and analyzed with PCA by applying iteration 1 to
iteration 8. The score plot data indicates that all samples treated at IC-50 are in one cluster
. KF01-01 data with concentrations (IC-30, IC-50 and IC-70) was also analyzed similarly
with RMies &PCA. In order to study the effect of drugs in the lipid area, cisplatin and
KF01-01 data at ((IC-30, IC-50 and IC-70) was also analyzed separately from 950 -4000
cm-1 range . The relevant spectrum and score plots of sample data given in fig. 77 to fig.
100 are discussed below.

4.2.1 Cisplatin and KF01-01 treated samples at IC-30, IC-50 and IC-70
concentrations covering spectral range from 1000 to 2200 cm-1.

The spectral data of cisplatin and KF01-01 drug treated samples at different
concentrations (IC-30, IC-50 & IC-70) covering spectral range from 1000 to 2200 cm-1
was combined and analyzed using principal component analysis after applying RMeiS
correction technique for pattern recognition. The FTIR spectral data of samples treated at
IC-30 is given in fig. 77 . Peaks recorded in the protein region are large and narrow. The
peaks observed in the nucleic acid & carbohydrate region are small and wide. The PCA
scores and spectra of these samples corresponding to iteration 1 and iteration 8 are given
in Fig. 78- 80. In the PCA score plots , points corresponding to both drugs are observed in
different clusters except overlapping of few points. Score plots corresponding to cisplatin
drug treated points are scattered. However score plots corresponding to KF01-01 are
confined in a very narrow cluster.

The FTIR spectral data of samples treated at IC-50 is given in fig. 81 . Peaks recorded in

- 62 -
the protein region are large and narrow. The peaks observed in the nucleic acid &
carbohydrate region are small and wide. The FTIR spectra of samples treated at IC-50 and
their PCA scores corresponding to iteration 1 and iteration 8 data are given in Fig. 82- 84.
In the PCA score plots there is a remarkable discrimination between samples treated with
KF01-01 and cisplatin and points corresponding to both drugs are observed in different
clusters indicating different drug action mechanism (fig. 82 & fig. 84). In case of KF01-
01 points are falling in a narrow band which indicates that all samples are in similar stage
of apoptosis . However points corresponding to cisplatin are scattered in a larger band
area. These results indicate that all samples treated with KF01-01 are almost in similar
stage of apoptosis .

The FTIR spectra of cisplatin and KF01-01 treated samples at IC-70 and their PCA
scores corresponding to iteration 1 and iteration 8 are given in Fig. 85- 88. In the PCA
score plots points corresponding to both drugs are again in different clusters except
overlapping of some points. These results indicate that both KF01-01 and cisplatin induce
different chemical changes at all concentrations suggesting different mode of action
induced by KF01-01 with respect to cisplatin.

PCA was also performed on combined spectrum data of all samples treated with cisplatin
at different concentrations (IC-30, IC-50 & IC-70). The spectrum and PCA score plots
corresponding to iteration 1 and iteration 8 data are given in Fig. 89- 92 . A clear cluster
of sample points corresponding to IC-50 is observed ( Fig. 92 ). Score plot points
corresponding to IC-30 and IC-70 are overlapping and scattered. Score points
corresponding to IC-50 are in one cluster in a narrow band.

PCA was also performed on combined spectrum data of all samples treated with KF01-01
at different concentrations (IC-30, IC-50 & IC-70). The spectrum and PCA score plots
corresponding to iteration 1 and iteration 8 data are given in Fig. 93- 96 . Score points
corresponding to IC-50 are in a very narrow and small cluster. Score points corresponding
to IC-30 and IC-70 are overlapping and scattered ( fig. 94) .

It is observed that score points corresponding to IC-50 are in one cluster and in a narrow
for both ciplatin and KF01-01 treated samples. In case of KF01-01 drug the score points
are in a small cluster . On the other hand points corresponding to cisplatin are in a bigger
cluster.

- 63 -
4.2.1 Cisplatin and KF01-01 at IC-30, IC-50 and IC-70 ( 950-4000 cm-1)

In order to study the effect of drugs covering the lipid area also, combined cisplatin at
((IC-30, IC-50 and IC-70) and KF01-01 at ((IC-30, IC-50 and IC-70) was also analyzed
separately from 950 -4000 cm-1 range . The spectrum data of Cisplatin and KF01-01 drug
treated samples at different concentrations (IC-30, IC-50 & IC-70) from 950 to 4000 cm-
1
was combined and analyzed using principal component analysis after applying RMeiS
correction technique for discrimination of patterns. The FTIR spectra of samples treated
with cisplatin alongwith their PCA scores corresponding to iteration 8 data are given in
Fig. 97- 98. In the PCA score plots points corresponding to IC-50 concentration are
observed in one cluster. However score points corresponding to IC-30 & IC-70 are
scattered. Results of similar analysis corresponding to KF01-01 are shown in Fig. 99-100.
In score plot, points corresponding to IC-50 are observed in one cluster having a very
narrow band . Score points coreponding to IC-30 and IC-70 are also in different cluster
with so highly scattered. These results confirm that samples treated with KF01-01 at
different concentration are in different stage of apoptosis.

In all the results the samples treated with both drugs at IC-50 concentrations are clearly
separated in one cluster. The similar results also observed with spectrum data of ATR
study. These results indicate that that IC-50 appears optimum dose for both cisplatin and
KF01-01 drugs.

- 64 -
PCA IC 30 Plot
1.2 0.07
CIS 30
0.06 KF 30
1
0.05

0.8 0.04
Absorbance

PC2 (31.9%)
0.03
0.6
0.02

0.4 0.01

0
0.2
-0.01

0 -0.02

-0.03
-0.2 -0.06 -0.04 -0.02 0 0.02 0.04 0.06
2200 2000 1800 1600 1400 1200 1000 PC1 (50.1% )
-1
Wavenumber in cm-1
Wavenumber/ cm
Fig. 77: FTIR spectra of A2780 cells treated Fig. 78: PCA score plot of A2780 cells
with CIS & KF (IC-30), RMIES (itr1) treated with CIS & KF (IC-30),
RMIES (itr 1)

1.2 PCA IC30 IT8 Plot


0.15
CIS 30
1 0.1
KF 30

0.8 0.05
Absorbance

PC2 (25.4%)

0.6 0

0.4 -0.05

0.2 -0.1

0 -0.15

-0.2 -0.2
2200 2000 1800 1600 1400 1200 1000 -0.1 -0.05 0 0.05 0.1 0.15 0.2 0.25
-1 PC1 (50.0% )
cmcm-1
Wavenumber / in
Wavenumber
Fig.69: FTIR spectra of A2780 cells treated Fig.80: PCA score plot of A2780 cells
with CIS & KF (IC-30), RMIES (itr8) treated with CIS & KF (IC-30),
RMIES (itr 8)

PCA IC 50 Plot
1.2 0.12
CIS 50
0.1 KF 50
1
0.08
0.8
0.06
PC2 (19.0%)
Absorbance

0.6 0.04

0.4 0.02

0
0.2
-0.02
0
-0.04

-0.2 -0.06
2200 2000 1800 1600 1400 1200 1000 -0.1 -0.05 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35
-1 -1
Wavenumber in cm
Wavenumber / cm PC1 (72.7% )

Fig.81: FTIR spectra of A2780 cells treated Fig.82: PCA score plot of A2780 cells treated
with CIS & KF (IC-50), RMIES (itr1) with CIS & KF (IC-50), RMIES (itr1)

- 65 -
PCA IC50 IT8 Plot
1.2 0.1
CIS 50
1 KF 50

0.05
Absorbance 0.8

PC2 (18.2%)
0.6 0

0.4
-0.05

0.2

-0.1
0

-0.2
2200 2000 1800 1600 1400 1200 1000 -0.15
-0.15 -0.1 -0.05 0 0.05 0.1 0.15 0.2
-1 -1
Wavenumber
Wavenumber /in
cmcm PC1 (47.6% )

Fig.83: FTIR spectra of A2780 cells treated Fig.84: PCA score plot of A2780 cells treated
with CIS & KF (IC-50), RMIES (itr8) with CIS & KF (IC-50), RMIES (itr8)

PCA IC70 IT1 Plot


1.4 0.06
CIS 70
1.2 KF 70
0.04

1
0.02
0.8
PC2 (10.8%)
Absorbance

0
0.6
-0.02
0.4

0.2 -0.04

0 -0.06

-0.2
2200 2000 1800 1600 1400 1200 1000 -0.08
-0.25 -0.2 -0.15 -0.1 -0.05 0 0.05 0.1
-1 -1
Wavenumber
Wavenumber /in
cmcm PC1 (80.0% )

Fig.85: FTIR spectra of A2780 cells treated Fig.86: PCA score plot of A2780 cells treated
with CIS & KF (IC-70), RMIES (itr1) with CIS & KF (IC70), RMIES (itr1)

PCA IC70 IT8 Plot


1.2 0.15
CIS 70
KF 70
1
0.1

0.8
PC2 (14.4%)
Absorbance

0.05
0.6

0.4
0

0.2
-0.05
0

-0.2 -0.1
2200 2000 1800 1600 1400 1200 1000 -0.35 -0.3 -0.25 -0.2 -0.15 -0.1 -0.05 0 0.05 0.1 0.15
Wavenumber -1 -1
Wavenumber /in
cmcm PC1 (55.9% )

Fig.87: FTIR spectra of A2780 cells treated Fig.88: PCA score plot of A2780 cells treated
with CIS & KF (IC-70), RMIES (itr8) with CIS & KF (IC-70), RMIES (itr8)

- 66 -
PCA CISPLATIN FTIR Plot
1.4 0.16
CIS 30
0.14 CIS 50
1.2
CIS 70
0.12
1
0.1
0.8
Absorbance

PC2 (17.9%)
0.08

0.6 0.06

0.4 0.04

0.02
0.2
0
0
-0.02

-0.2 -0.04
2200 2000 1800 1600 1400 1200 1000 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3
-1 -1 PC1 (67.6% )
Wavenumber
Wavenumber /in
cmcm

Fig.89: FTIR spectra of A2780 cells treated Fig.90: PCA score plot of A2780 cells treated
with CISPLATIN (IC-30, IC-50 & with CISPLATIN (IC-30, IC-50 &
IC-70), RMIES (itr1) IC-70), RMIES (itr1)
PCA CISPLATIN IT8 Plot
1.2 0.25
CIS 30
CIS 50
1 0.2
CIS 70
Absorbance

0.8 0.15
PC2 (15.0%)

0.6 0.1

0.4 0.05

0.2 0

0 -0.05

-0.2 -0.1
2200 2000 1800 1600 1400 1200 1000 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3
Wavenumber -1-1
Wavenumberin cm
/ cm PC1 (58.9% )

Fig.91: FTIR spectra of A2780 cells treated Fig.92: PCA score plot of A2780 cells treated
with CISPLATIN (IC-30, IC-50 & with CISPLATIN (IC-30, IC-50 &
IC-70), RMIES (itr8) IC-70), RMIES (itr8)
PCA GOLD IT1 Plot
1.4 0.03
KF 30
1.2 0.02 KF 50
KF 70
0.01
1
0
0.8
PC2 (9.5%)
Absorbance

-0.01
0.6
-0.02
0.4
-0.03
0.2
-0.04

0
-0.05

-0.2 -0.06
2200 2000 1800 1600 1400 1200 1000 -0.05 0 0.05 0.1 0.15 0.2 0.25 0.3
Wavenumber -1 -1
Wavenumber /in
cmcm PC1 (75.6% )

Fig.93: FTIR spectra of A2780 cells treated Fig.94: PCA score plot of A2780 cells treated
with KF (IC-30, IC-50 & IC-70), with KF (IC-30, IC-50 & IC-70),
RMIES (itr1) RMIES (itr1)

- 67 -
PCA GOLD IT8 Plot
1.2 0.15
KF 30
KF 50
1 0.1
KF 70

0.8 0.05
Absorbance

PC2 (20.1%)
0.6 0

0.4 -0.05

0.2 -0.1

0 -0.15

-0.2 -0.2
2200 2000 1800 1600 1400 1200 1000 -0.1 -0.05 0 0.05 0.1 0.15 0.2 0.25
Wavenumber / cm -1-1 PC1 (38.7% )
Wavenumber in cm
Fig.95: FTIR spectra of A2780 cells treated Fig.96: PCA score plot of A2780 cells treated
with KF (IC-30, IC-50 & IC-70), with KF (IC-30, IC-50 & IC-70),
RMIES (itr8) RMIES (itr8)
PCA CIS IT8 Plot
0.3
1.2
CIS 30
0.25 CIS 50
1 CIS 70
0.2
0.8
0.15
PC2 (18.4%)
Absorbance

0.6 0.1

0.05
0.4
0
0.2
-0.05

0
-0.1

-0.2 -0.15
4000 3500 3000 2500 2000 1500 1000 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3
-1 -1 PC1 (48.8% )
Wavenumber
Wavenumber /in
cmcm
Fig.97: FTIR spectra of A2780 cells treated Fig.98: PCA score plot of A2780 cells treated
with CIS (IC-30, IC-50 & IC-70) with CIS (IC-30, IC-50 & IC-70)
(itr8), 950 - 4000 (itr8), 950 - 4000
PCA GOLD IT8 Plot
1.2 0.25
KF 30
0.2 KF 50
1 KF 70

0.15
0.8
Absorbance

0.1
PC2 (15.9%)

0.6
0.05
0.4
0

0.2
-0.05

0
-0.1

-0.2 -0.15
4000 3500 3000 2500 2000 1500 1000 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4
Wavenumber -1 -1
Wavenumber /in
cmcm PC1 (61.5% )

Fig.99: FTIR spectra of A2780 cells treated Fig.100: PCA score plot of A2780 cells treated
with KF (IC-30, IC-50 & IC-70) with KF (IC-30, IC-50 & IC-70)
(itr8), 950 - 4000 (itr8), 950 - 4000

- 68 -
CHAPTER 5

5. CONCLUSIONS

Attenuated Total Reflectance (ATR) and FTIR spectroscopy has been applied to
investigate the effect of increasing concentration of cisplatin and KF01-01 drugs on
ovarian cancer cell lines as well as for studying the mechanism of these drugs. The A2780
ovarian cancer cells treated with cisplatin and KF01-01 drugs at different concentrations
i.e. IC-30, IC-50 and IC-70 were utilized for ATR & FTIR measurements. Total 15
samples of cisplatin and KF01-01 were used in the analysis and 150 spectra were
measured. ATR-FTIR measured data provided clean spectra without any distortions and
data was analyzed directly with PCA. However FTIR spectral data indicated some
distortions and therefore Resonant Mie Scattering (RMieS) corrections were applied
before principal component analysis. PCA was performed for both ATR and FIR by taking
several combinations of spectral data of both drugs .

PCA analysis of ATR data of combined full range spectrum of Cisplatin and KF01-01
drugs revealed that drug samples at IC-50 concentration are lying in different clusters than
the samples of IC-30 and IC-70. Further analysis of spectral data of both drugs
corresponding to lipid, protein and carbohydrate & nucleic acid regions indicate that
different clusters are seen pertaining to these drugs. The separation of clusters is more
clear in carbohydrate & nucleic acid region in comparison to lipid and protein region.
These results indicate that both drug induce different chemical changes in the samples.
This means that working mechanism of both drugs is different.

Combined data analysis of cisplatin samples taken at IC-30, IC-50 & IC-70 concentrations
revealed that samples corresponding to IC-50 are lying in separate cluster. Samples
corresponding to IC-30 and IC-70 do not fall in separate cluster and lot of scattering
observed in their score points. Data analysis of KF01-01 samples also revealed similar
results. These results suggest that both drugs induce different chemical changes at different
concentrations. This means that changes observed in A2780 cells as a result of drug action
are not linear with the concentration. Points corresponding to IC-50 are seen in separate
cluster in both drug treated samples. This indicates that IC-50 appears optimum dose for
both drugs.

- 69 -
PCA analysis of FTIR data done at concentrations (IC-30, IC-50 & IC-70) also revealed
the clear separation of score points of IC-50 sample data from the IC-30 & IC-70
concentrations corresponding to both cisplatin and KF01-1 treated samples. Combined
analysis of cisplatin and KF01-01 sample data corresponding to different concentrations
also revealed that sample data are clearly separable in case of sample data of IC-50
concentration. A clear separation is not observed in samples corresponding to IC-30 & IC-
70 concentrations. These results also indicate that working mechanism of both drugs is
different and drugs action is not linear with their concentrations.

These results of both ATR and FTIR are in agreement . However the spectrum measured
with ATR are clear and provide fine details in comparison to spectral data measured
with FTIR system. Moreover no corrections like RMieS were applied before PCA. In
FTIR spectral data distortions were observed and RMieS correction was applied before
PCA analysis. These correction techniques sometimes can remove the important bands
from the spectra. Accordingly ATR spectroscopy technique appears better than FTIR.

ATR & FTIR spectroscopy can distinguish between normal and cancerous tissues & also
between different grade of malignancies. It is possible to calculate the extent of spread of
cancer / pre-malignancy using biopsies at a regular distance from the foci of the cancer
utilizing these techniques . ATR & FTIR can be used for identification of pre-malignancy
and to predict relapse of cancer or on set of pre-malignancy. IR spectroscopy can be
applied successfully for assessment of effects of drugs on cancer cells by monitoring
biochemical changes in cells before and after treatment with drugs. Sensitivity or
resistance to drugs can also be investigated by these techniques. Apoptosis process induced
by anticancer drugs can also be studied by analyzing the DNA, protein and lipid and this
can be helpful in development of anticancer drugs.

- 70 -
References:

1. Anderus, G P., Robert, D., and Strickl, R D. (1998). Cancer grading by Fourier
Transform Infrared spectroscopy. .Biospectroscopy, 4, 37-46.
2. Argov, S., Ramesh, J., Salman, A., Sinelnikov, I., Goldstein, J., Guterman, H., and
Mordechai, S. (2002). Diagnostic potential of Fourier-transform infrared
microspectroscopy and advanced computational methods in colon cancer patients.
J Biomed Opt, 7, 248-254.
3. Barbacid, M. (1987). Ras genes. Annu Rev Biochem, 56, 779-827.
4. Barnard, P J. and Berners –Price, S J.( 2007). Targeting the mitochondrial cell
death path way with gold complexes. Coordination chemistry reviews, 251, 1889-
1902.
5. Bhargava, R., Fernandez, D C., Hewitt, S M,. and Levin, I W. (2006).
Biochamica et Biophysica Acta (BBA). Biomembranes, 1758, 813-974.
6. Binoy, J., Abraham, J.P., Joe, I.H., Jayakumar, V.S., Petit, G.R. and Nielsen, O.F.
(2004). NIR-FT Raman and FT-IR spectral studies and ab initio calculations of
the anti-cancer drug combretastatin-A4. Journal of Raman Spectroscopy,
35, 939 – 946.
7. Bruijnincx, P. and Sadler, P.(2008). New trends for metal complexes with
anticancer activity. Science Direct Current. Option in Chemistry Biology, 12, 197-
206.
8. Cara, A. and Rabik, M E.b Leen. (2007). Molecular mechanisms of resistance
and toxicity associated with platinating agents. Cancer treatment Review, 333, 9-
23.
9. Cohenford, M A., Godwin, T ., Cahn, F., Bhandare, P., Caputo, T A., and Rigas, B.
(1997). Infrared spectroscopy of normal and abnormal cervical smears, evaluation
by principal component analysis. Gynecol Onco, 66(1), 59-65.
10. Deleris, G., and Petibois, C. (2003). Vib Spectrosc, 32, 129-136.
11. Diem, M., Chiriboga, L., Lasch, P. and Pacifico, A. (2002). IR spectra and IR
spectra maps of individual normal and cancerous cell. Biopolymers 67(4-5), 349-
353.
12. Dukor, R., (2002). Vibrational spectroscopy in the detection of cancer, in
Handbook of Vibrational Spectroscopy, edited by J M Chalmers and P R Griffiths,
Vol. 5, Wiley, chichester, pp. 3335-3361.
13. Dumas, P., Jamin, N., Teilland, J I., Miller, L M. and Beccard, B. (2004). Imaging
capabilities of synchrotrom infrared microscopy, Faraday Discuss, 126, 289-302.
14. Dumas, P., Sockalingum, G D. and Sule-Suso J., (2007). Adding synchrotron
radiation to infrared microspectroscopy: What‟s new in biomedical applications?
Trends Biotechnol, 25, 40-44.
15. Fabian, H., Jackson, M., Murphy, L., Watson, P.H., Fichtner, I. and Mantsch, H.H.
(1995). A comparative infrared spectroscopic study of human breast tumors and
breast tumor cell xenografts. Biospectroscopy, 1 (1), 37 – 45.

- 71 -
16. Faolain, O E., Hunter, B M., Byrne, M J., Kelehan, P., Byrne, J H., and Lyng, M
F. (2005). The potential of vibrational spectroscopy in the early detection of
cervical cancer: an exciting emerging field. Proc of SPIE, 5826.
17. Gasparri, F. and Muzio, M. (2003). Monitoring of apoptosis of HL 60 cells by
Fourier –transform infrared spectroscopy. Biochem J, 369, 239-248
18. Gery, K., Schwartz. and Manish, A.S. (2005). Targeting the cell cycle: A new
approach to cancer therapy. J.of clinical oncology; 23, 36.
19. Gazi, E., Dwyer, J., Lockyer, N P. and et al. (2005). Fixation protocols for
subcellular imaging by synchrotron-based. Biopolymers, 77, 18-30.
20. German, M J., Hammiche, A., Ragavan, N., and et al. (2006). Infrared
spectroscopy with multivariate analysis potentially facilitates the segregation of
different types of prostate cell, Biophys J, 90, 3786-3795.
21. Haitao, L., Matthew, K., Daddysman., Garry, O., Rankin. and et al (2010).
Kaempferol enhances Cisplatin‟s effect on ovarian cancer cells through promoting
apoptosis caused by down regulation of cMyc . Cancer Cell interracial, 10.
22. Heri, R. and Sugiyama, J A.( 2003). Combined FTIR microscopy and principle
component analysis on softwood cell walls Carbohydrate Polymers, 53, 449-453.
23. Jackson, M. and Mantsch, H.H. (1996). Biomedical spectroscopy in: Infrared
spectroscopy of biomolecules (Ed) by H.H Mantsch and D.Champan. Wiley Liss
Inc .New York, NY.
24. Jackson, M. and Mantsch, H H. (1996). Biomedical infrared spectroscopy. In
Mantsch HH, Chapman D, eds. Infrared Spectroscopy of Biomolecules. New
York: Wiley-Liss, Inc, 311-40.
25. Jagannathan, R., Salman, A., Hammody, Z., Cohen, B., Gopas, J., Grossman, N.
and Mordechai, S. (200). FT-IR Microscopic studies on normal and H-Ras
oncogene transfected cultured mouse fibroblasts .Eur .Journal of Biophys, 30,
(4)250-255.
26. Jamin, N., Dumas, P., Moncuit, J., Fridman, WH., Teillaud, J L., Carr, G L. and
Williams, G P. (1998). Chemical imaging of nucleic acids, proteins and lipids of a
single living cell. Application of synchrotron infrared microspectrometry in cell
biology. Cell Mol Biol (Noisy-le-Grand), 44, 9-13.
27. Jamin, N., Dumas, P., Moncuit, J., Fridman, W H., Teillaud, J L., Carr, G L. and
Williams, G P. (1998). High resolved chemical imaging of living cells by using
synchrotron infrared micro- spectrometry. Proc Natl Acad Sci USA, 95, 4837-80.
28. Kartalon,M. and Essigmann, J.( 2001). Mechanisms of resistance to cisplatian.
Mutat Res . 478, (1-2) 23-43.
29. Kelland, L.(2007). The resurgence of platinum based cancer chemotherapy.
Nature Reviews, 7, 573 – 583.
30. Kerr, J F R., Winterford, C M. and Harmon, B V. (1994). Apoptosis –its
significance in cancer and cancer Therapy. Cancer,. 73, 2013-2026.
31. Lasch, P., Haensch, W., Naumann, D. and Diem, M. (2004). Imaging of colorectal
adenocarcinoma using FT-IR microspectroscopy and cluster analysis. Biochim
biophys Acta, 1688, 176-86.
32. Lasch, P., Pacifico A. and Diem, M. (2002). Biopolymers, 67, 335-338.
33. Leedham, S J., Brittan, M., McDonal, S A C. and Wright, N A. (2005). 9, 11-
24.

- 72 -
34. Liu, K-Z., Xul, M. and Scott, A D.(2007). Britch journal of haematology, 136,
713-722.
35. Lohr, M., Maisonneuve, P. and Lowenfels, A B. (2000). K-ras mutations and
benign pancreatic disease, Int J Pancreatol. 27, 93-103.
36. Lucaroui, M E., Habib, N A., Kelly, S B., Rochnie, N D., Nelson, O., Lindholm,
L., Cooper, M J., Wood, C B. and Williamson, R C. Clinical evaluation of
combined use of CE, CA19-9 and CA50 in the serum of patients with pancreatic
carcinoma.
37. Malins, D C., Polissar, N L., Schaefer, S., Su, Y. and Vinson, M. (1998). Pro
Natl Acad Sci USA, 95, 7637-7642.
38. Mahadevan-Jansen, A. and Richards-Kortum, R. (1997). Raman spectroscopy for
cancer detection, 19th Int. Conf. IEEE EMBS, Chicago, Oct 30 – Nov 2.
39. Martin M C., Tevetkova, N M., Crowe, J H. and McKinney, W R. (2001).
Negligible sample heating from synchrotron infrared beam, Appl Spectros, 55, 111-
113.
40. Mcguire, W P., Rowinsky, E K., Rosenshein, N B., Grumbine, F C., Ettinger, D
S., Armstrong, Donehower, D K., and Taxol R C., (1989). A unique antineoplastic
agent with activity in advanced ovarian epithelial neoplasms. Ann, Intern Med,
111, 273-279.
41. Messori, L. and Marcon, G. (2004). Gold complexes as antitumor agents. Met
Ions Biol Syst, 42, 385-424.
42. Murli Krishna, C., Sockalingum, G D. Jacob, Kurien., Lakshmi, Rao., Venteo L.,
Pluot, M., Manfait, M. and Kartha V B. (2004) appl. Spectrose, 58, 107-114.
43. Murli Krishna, C., Sockalingum, G D., Venteo L., Bhat, R A., Pralhad, Kustagi.,
Pluot, M. and Manfait, M. (2006). Bioploymers, 79, 269-276.
44. Morris, B J., Lee, C. and Nightingale, B N. (1995). Fourier transform Infrared
spectroscopy dysplastic, papillomavirus-positive cervicovaginal lavage specimens.
Gynecol Oncol, 56(2), 245-249.
45. Novasaghi, Z., Rehman, S. and Rehaman, I. (2008). Fourier Transform Infrared
(FTIR) spectroscopy of biological tissues. Applied Spectroscopy Reviews,
43, 134-179.
46. Reed, J C.( 2002). Apoptosis-based therapies. Nat Rev Drug Disco, 1, 111-121.
47. Rockett, J C., Larkin, K., Darnton, S J., Morris, A G. and Matthews, H R.
(1997). Br. J. Cancer, 75, 258-261.
48. Rohit, B. (2007). Towards a practical fourier transform infrared chemical imaging
tool. Anal. Bioanal Chem., 1155-69.

- 73 -
49. Romeo, M., Burden, F., Quinn, M., Wood, B. and McNaughton, D. (1998).
Infrared microspectroscopy and artificial neural networks in the diagnosis of
cervical cancer. Cell. Mol. Biol, 44(1), 179-187.
50. Sahu, P.K. and Mordechai, S. (2005). Fourier transform infrared spectroscopy in
cancer detection. Future Oncology, 1, 635 – 647.
51. Salman, A., Agrov, S., Ramesh, J., Goldstein, J., Sinelnikov, I., Guterman, H. and
Mordechai, S. (2001). FT-IR Microspectroscopy characterization of normal and
malignant human colonic tissues. Journal of Cell .Mol .Biol; 47(22).
52. Spring Harbor Laboratory Press, Cold Spring Harbor, New York. pp 475-585.
53. Stoner, G D., Kaighn, M E., Reddel, R R., Resau, J H., Bowman, D., Naito, Z.,
Matsukura, N., You M., Galati, A J. and Harris, C C. (1991). 51, 365-371.
54. Sukuta, S. and Bruch, R. (1999). Factor analysis of cancer Fourier transform
infrared evanescent wave fiberoptical (FTIR-FEW) spectra. Lasers in Surgery
and Medicine. 24, 382 – 388.
55. Tachimori, Y. and Kato, H., (1998). Diagnosis and surgery of esophageal cancer.
Crit Rev Oncol/ Hemato, 28, 57-71.
56. Tobin, M., Chesters, M.A., Chalmers, J.M. and et al. (2003). Infrared microscopy
of epithelial cancer cells in whole tissues and in tissue culture using synchrotron
radiation, Faraday Discuss, 126, 27-39.
57. Wang, J C Y., and Dick, J E. (2005). Trends Cell Biol, 15, 494-501.
58. Walsh, J M., Singh, N M. and Stringfellow, F H., et al. (2008). FTIR
microspectroscopy coupled with two-class discriminate segregate markers
responsible for inter- and intra category nuance in Exfoliate cervical cytology
Biomarker insights, 3, 179- 189.
59. Wang, H.P., Wang, H.-C. and Huang, Y.-J. (1997). Microscopic FTIR studies of
lung cancer cells in pleural fluid. Science of the Total Environment. 204, 283 – 287.
60. Wang, J. S., Shi J S, Xu Y Z., Duan, X Y., Zhang, L., Wang, J., Yang, L M.,
Weng, S F. and Wu, J G. ( 2003). FTIR Spectroscopic analysis of normal and
cancerous tissues of esophagus, World journal of Gastroenterology. 9, 1897-1899.
61. Wong, P T T. (1994). In High-pressure science and technology edited by C
Schmidt., J W Shaner, G A Samra and M Ross. ATP Press, New York, pp.
1431-1434.
62. Wood, B.R., Chiriboga L., Yee, H., Quinn, M.A., McNaughton, D. and Diem, M.
(2004). Fourier transform Infrared (FTIR) spectral mapping of the cervical
transformation zone and dysplastic squamous epithelium Gynecologic Oncology,
93(1), 59-68.
63. Wood, B.R., Quinn, M.A., Burden, F.R., and McNaughton, D. (1996). FT-IR
spectroscopy as a biodiagnostic tool for cervical cancer. Biospectroscopy, 2(3)
145 – 155.
64. Wood, B.R., Quinn, M.A., Burden, F.R., and McNaughton, D. (1996). An
investigation into FT-IR spectroscopy as a bio-diagnostic tool for cervical cancer.
Biospectroscopy, 2, 143 – 153.

- 74 -
65. Wood, B.R., Quinn, M.A., Tait, B., Ashdown, M., Hislop, T.,
Romeo, M., and McNaughton, D. (1998). FTIR microspectroscopic study of
cell types and potential confounding variables in screening for cervical
malignancies. Biospec- troscopy, 4, 75 – 91.
66. Wood, R B., Chiriboga, L., Yee, H., Quinn, A M., McNaughton, D., Diem,
M.(2004). Fourier transform Infrared (FTIR) spectral mapping of the cervical
transformation zone and dysplastic squamous epithelium Gynecologic Oncolog,
993, 59-68.
67. Wood, R B., Quin, M A.., Borden, R F. and McNaughton, D. (1996). An
investigation into FTIR spectroscopy as a biodignosic tool for cervical cancer.
Biospecroscopy, 2, 143-153.
68. Wong, P.T.T., Lacelle, S., Fung, M.F.K., Senterman, M. and Mikhael, N.Z. (1995).
Characterization of exfoliated cells and tissues from human endocervix and ecto-
cervix by FTIR and ATR/FTIR spectroscopy. Biospectroscopy, 1 (5), 357 – 364.
69. www. visualsonline.cancer.gov (12 August, 2010)
70. www.nutritiondelight.com (14 August, 2010)
71. http://en.wikipedia.org/wiki/Cisplatin (14 August, 2010)
72. http://www.drugs.com/taxol.html (14 August,2010)
73.http://chem4513.pbworks.com/The-Evolution-of-Cancer-Treatments (23 July, 2010)
74. www.macmillan.org.uk (17August, 2010)
75. http://wikipedia.org (17 August, 2010)
76. www.chem.ucla.edu (18August, 2010)
77. www.orgchem.colorado.edu (26July, 2010)
78. www.en.wikipedia.org (22 August, 2010)
79. www.thermonicolet.com (23 August, 2010)
80. www.thermonicolet.com (24 August, 2010)
81. http://www4.uwn.edu (28 July, 2010)
82. www.thermonicolet.com (22 August, 2010)
83. www.forensicscience.ie/index.asp (27 August, 2010)
84. www.acanas.co.uk (24 August, 2010)

- 75 -