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Studying The Effects of Newly Synthesized Pyrazoline

Compounds As Anti-Cancer Reagents in A549 cell line




Bachelor Degree Project of Biotechnology


Yara Adnan Hassan Al-Saadawi



Work was done under supervision of:
Dr. Mohammad Abdel-Halim Abdel-Naby Abdel-Latif


Lab Supervisor:
Yasmin Adel ElSayed ElMaghloob



June 2014

2
Index


Table of Contents
Acknowledgment ................................................................................................................ 4
Abstract .................................................................................................................................. 5
I. Introduction ................................................................................................................. 6
Protein Kinases Enzymes .......................................................................................................... 6
Structure ......................................................................................................................................... 6
Protein Kinase C Family ............................................................................................................ 7
Regulation of PKCs ...................................................................................................................... 8
Protein Kinase C zeta PKC ................................................................................................... 8
Physiological Activity of PKC in Cells.................................................................................. 9
Protein Kinase Inhibitors .......................................................................................................12
Classification of Inhibitors .....................................................................................................12
PIF Pocket as Allosteric Regulatory Site............................................................................13
PIF Pocket As A Target for Kinase Inhibition By Drugs ...............................................13
P53 for Tumor Suppression ...................................................................................................14
P53-Mdm2 and Cancer Therapy ...........................................................................................14
Aim ..................................................................................................................................................16
II. Materials and Methods .......................................................................................... 17
Materials and equipment ........................................................................................................17
Methods .........................................................................................................................................22
III. Results: ....................................................................................................................... 28
IV. Discussion and Future Recommendation ...................................................... 29
V. References ................................................................................................................. 30










3






Declaration


This thesis was proposed as my bachelor degree graduation project and it was only a
continuation for the PhD work of Dr. Mohammad Abdel-Halim Abdel-Naby
Abdel-Latif under his knowledge and supervision.

































4
Acknowledgment

This thesis could not be done without Allahs welling and blessing among us. Only
He could help me and my colleagues to finish this work and hopefully the results we
got could be helpful for further studies in cancer therapy.

I would like also to thank our dear prof. Khaled Abou Aisha for accepting us to gain
some of his knowledge and practical experience in our field, no one could help us the
same way he did to us.


Not to forget to thank Dr. Mohammad Abdel-Halim for giving us his permission and
the opportunity to continue his work and to be a part of his work and to be helpful and
a kind person to us.

And of course, our (awesome) lab supervisor, Dr. Yasmin Adel ElSayed ElMaghloob
who was nothing but a great, generous and patient person to us who handled all our
questions, even the most smartest ones without complaining and was more than a
supervisor to us, she was a real friend.

And finally and most importantly, my family and friends who supported me all the
way during this project. I could not be more thankful to all of them and no words can
be spoken nor written to describe how thankful I am for having you all beside me.

Simply, thank you all!
5





Abstract

PKC is an enzyme that belongs to the PKC family. It has a significant regulatory role
in cellular growth activity since it controls NF- B transcription factor that is
important to initiate transcription of genes required for cellular survival and
proliferation. Many studies proved a linkage between PKC and cancer cells whereas
it was found that PKC present in high intracellular concentration in cancer cells.
Another important protein in the cell is Mdm2 that also regulates P53 transcription
factor that initiate transcription of genes important for the programmed cell apoptosis.
In this thesis, the effects of newly synthesized pyrazoline drugs were tested on lung
cancer cells (A549 cell line, ATCC) transfected with plasmids containing NF- B and
P53 promoters controlling luciferase gene using PEI transfection reagent, the effects
were then measured by luciferase assay after treatment with MA72, MA114 and
MA117 pyrazoline compounds. The findings for the drugs MA114, MA117 effects
were promising to be used in cancer therapy where the drug MA72 showed slight
effects. Future recommendations include the study of effects of these drugs in
different concentrations than the used concentration (10 M), changing of some
residues in the drugs could enhance the activity of the drug binding to its targeted site
and testing them using different cancer cell lines and with addition of cytokines that
are naturally involved in activation signal in the cells.

6
I. Introduction:

Protein Kinases Enzymes

Cellular activity of proteins is mainly controlled by the level of its phosphorylation
that is controlled by a family of enzymes known as kinases enzymes. This process
depends on transferring a phosphor group from an ATP molecule to a protein by the
action of a kinase enzyme. The result of phosphorylation might activate or inhibit the
protein functioning that will lead to a cascade effect for other cellular mechanisms
and functions. Protein phosphorylation either occurs on tyrosine residue or
serine/threonine residues of the protein; based on that, kinases are generally described
as Ser/Thr kinases, Tyr kinases or dual-specificity kinases (phosphorylate both of
them).
[1][2]

Structure

For a given kinase, the structure of the catalytic domain is the same among
different kinases enzymes but with different regulatory mechanism, it consists of
two loops, the small N-terminal loop that has conserved regulatory helix C and
C-terminal large loop, both of them linked by a hinge region, with the protein-
binding site facing the ATP-binding site in-between the two loops. The ATP-
binding site that is conserved in all protein kinases enzymes. The catalytic
activity is regulated by the active site that contains highly conserved ATP/Mg
+2

binding motifs and an activation loop.
[4][5]

In a kinase family known as AGC family, the C-terminal (large loop) of the
catalytic domain folds back onto a hydrophobic motif within the N-terminal
(small loop) known as PIF pocket (PDK1-interacting fragment).
The AGC family structure involves two regulatory motifs that are highly
conserved which are the activation loop presents in the catalytic domain at the
C-terminal loop, and the hydrophobic motif (HM) presents in the non-catalytic
domain. In addition, a turn motif site is found in several AGC kinases, which is
important in regulation and activation mechanisms of the AGC enzymes.
[6]











7











Figure 1. Illustration of protein kinases structure.


Protein Kinase C Family

A member of ACG family (subgroup of Ser/Thr kinases) that represents 2% of
total human kinome. There are 10 subtypes for PKCs enzymes that all share a
conserved catalytic domain containing motifs for ATP/substrate binding and
more divergent regulatory domain at the N-terminal; both catalytic and
regulatory domains linked through hinge regions. The regulatory domains of
PKCs contain an auto-inhibitory isoform-specific sequence known as
pseudosubstrate that contains an alanine residue as phosphoacceptor instead of
serine/threonine residues. Moreover, the regulatory domains contain structural
domains known as C1 and C2 that are responsible for the sensitivity of each PKC
to different stimuli, functions and mechanism of regulation.
[7]

According to the structural differences in the regulatory domains of PKCs, it can
be classified into three groups:
The classical PKCs (cPKCs): consists of PKC, PKCI, PKCII and PKC.
Members of this group have a conserved C1 domain containing a double
zinc-finger and a pocket for diacylglycerol (DAG) and phospholipid
binding resulting its activation, and a C2 domain containing a calcium-
binding site responsible for calcium sensitivity.
The novel PKCs (nPKcs): consists of PKC, PKC, PKC, and PKC. As
cPKCs, nPKCs are activated by DAG and phospholipids binding to their
pocket found in the C1 domain, however, nPKCs lack the calcium binding site
that is found in cPKCs and thus, nPKCs are not sensitive to calcium.
The atypical PKCs (aPKCs): consists of PKC and PKC. They lack the
structural domains for DAG and phospholipid binding in C1 domain in both
cPKCs and nPKCs, and the calcium-binding site in the C2 domain of cPKCs
and they contain only one zinc-finger structure in the C1 domain. At the N-
terminus, a distinct structural domain which makes the aPKCs unique known
as Phox/Bem 1 (PB1) which is a protein-protein interaction domain mediating
8
interactions with other proteins containing the PB1 domain thus, making them
linked in a network form.

Regulation of PKCs

PKCs are inactivated by the auto-inhibitory sequence, the pseudosubstrate that blocks
the substrate-binding site in the catalytic domain of the kinase.
[8]
In-order to get
activated, the PKCs Ser or Thr residues must get phosphorylated at three locations in
cPKCs and nPKCs or two locations at aPKCs, this phosphorylation stabilize the
active form of the kinase enzyme. The sites to be phosphorylated were mentioned
before in the structure of the AGC enzymes, all the three sites (HM, activation loop
and turn motif) are required for activation of cPKCs and nPKCs, while for the aPKCs
only requires phosphorylation of the activation loop and the turn motif. The activation
loop is phosphorylated by phosphoinositide-dependent kinase-1 (PDK1) that requires
the HM to be phosphorylated (at Ser/Thr residues) in cPKCs and nPKCs,
[9][10]
while
in aPKCs, an acidic phosphomimetic Asp or Glu is found in the HM instead of the Ser
of Thr residues which can replace the phosphate.
[10][11]
The HM (phosphorylated in
cPKCs and nPKCs, Asp/Glu in aPKCs) is docked to the PIF pocket of the PDK1. The
turn motif is phosphorylated by the mammalian target of rapamycin 2 complex
(mTORC2). After the activation of the PKCs activation loop and turn motif, the
pseudosubstrate must be released from the substrate-binding site, which is only
achieved by a second lipid messenger or an allosteric stimulation and thus, the kinase
will reach the fully active state.
[6]


Protein Kinase C zeta PKC

PKC is an isotype of the atypical PKC (aPKC) subfamily with a size of 79 KDa. Is
has an important rule in cellular functions regulation. PKC regulates cells polarity in
earlier development stages. PKC is involved in several immune responses, where it
regulates the apoptosis and controlling T-dependent responses.
[12]

9

Figure 2. (A) Computed 3D model structure of PKC. (B) PKC active site.
(C) PKC ATP-binding site. (D) PKC substrate-binding site. (E) Modified
residue threonine residue (phosphorylated) by the action of PDK1. (Ref.
Q05513; The Protein Model Portal).


Physiological Activity of PKC in Cells

PKC and NF- B signaling:
NF- B is a transcription factor plays an important role in different functions in
growth process, inflammatory and immune responses of the cell under regulated
activation, where an uncontrolled activation of NF- B would result in uncontrolled
growth of the cell and thus, cancerous cells will be the result. It also might result
autoimmune and chronic inflammatory diseases due to aggravated lymphocyte
function.
[13]

NF- B is kept in the cytosol and inhibited from translocation into the nucleus by
IB protein and only released when tumor necrosis factor- (TNF- ) and
interleukin-1 (IL-1), thus, allowing initiation of transcription of the targeted genes.
NF- B is released when IB is phosphorylated (which initiates ubiquitination and
degradation of IB) by a complex kinase called IKK, which is a heterodimer of three
subunits; two catalytic subunit (IKK and IKK) and one regulatory subunit (IKK).
[14]

PKC shows an important role in NF- B transcriptional activity and IKK activation
by the following:
10
PKC acts as a kinase for the IKK complex.
[15]

PKC phosphorylate a Ser residue in NF- B (a subunit known as RelA or
p65) and thus, NF- B is being turned to its full transcriptional activity form.
[16][17]

In lung cells (during inflammatory infections), PKC is activated in response to TNF-
, IL-2 or LPS (lipopolysaccharide).
[15]


PKC, Secondary Lymphoid and B Cells:
PKC has significant role in the differentiation, maturation and survival of B-cells.
Previous experimental results showed that the maturation and formation of secondary
lymphoid organs was not efficient in young mice with inactive/deficiency of PKC in
comparison with normal mice; but it was overcame in adults animal that put in
consideration of probability of presence of alternative molecule that initiated the
maturation of secondary lymphoid organs in adults only while in young animals
PKC has the role of B-cells and secondary lymphoid organs maturation.
[15]

After an antigen is being recognized by a B-cell receptor (BCR), PKC got activated
and it mediates the activation of extracellular signal-regulated kinase (ERK) and both
of them together increase the transcriptional activity of NF- B which will initiate
transcription of important genes required for B-cell to differentiate and proliferate.
[18]


PKC and T-helper Cells (CD4
+
):
T-helper cells play important role in immune responses to pathogens, infectious
agents and inflammatory diseases. Several experiments were carried to understand the
role of PKC in T-helper cells functioning.
T-helper 2 cells: It was proved experimentally that PKC is important for Th2 proper
functioning and T-dependent humoral immune response. In comparison between two
nave CD4
+
cell-lines were taken from two animal models, one has deficient PKC
and the other one has wild type PKC, the cell lines were allowed to differentiate into
Th2 cells in vitro; the animal with PKC deficiency showed weak immune response
to T-dependent antigens, in addition to low levels of Th2 cytokines (IL-4, IL-5, IL-10
and IL-13) secretion, this was later explained by two factors; the first is the impaired
NF- B translocation into nucleus and thus, low transcription activity of important
genes required for Th2 cells maturation and response, however, that was not the key
factor. The second factor was the importance of PKC in IL-4 signaling which is an
essential factor for Th2 cells maturation process along with activation to T-cell
receptors TCR-. It was found that PKC interact and phosphorylate other proteins
(Jak1/Stat6) in Th2 cells in response to IL-4 signaling that would activate Th2 cells
differentiation either in vivo or in vitro.
[19][20]

T-helper 1 cells: when PKC-deficient nave CD4
+
cells were differentiated in vitro
into Th1 cells, the function appeared not to be effected by the lack of PKC since Th1
IFN- cytokine secretion was not effected by PKC.



11


PKC and Asthma:
Asthma is a chronic inflammatory disease that infects lung cells. Based on what was
explained before, T-helper cells have a critical role in immune response to asthma,
where the Th2 secreted cytokines have important role as the following:
[21][22]

Immunoglobulin E (IgE) production which, in turn, function against
inflammatory diseases
Enhancing mast cells growth that have enzymes against inflammatory agents
Eosinophil accumulation
Mucus hypersecretion and airway hyperreactivity (AHR).
As mentioned before, PKC has critical role in Th cells differentiation and
functioning, and thus, it is considered as an important factor in immune response to
asthma. Where at first, PKC activates T-helper cells to secrete its cytokines that will
have the previous functions and will attach to lung receptors that will in turn will
activate PKC in lung cells leading to translocation of NF- B into nucleus and
initiating transcription of important genes needed for survival function and
inflammatory response against asthma.
[15]


PKC and Liver Inflammation:
When liver got infected with infectious reagent or a pathogen, T-helper cells got
activated (which was as explained, by the aim of PKC) and will secrete cytokines
that are important for recruiting of eosinophil cells and natural killer cells NKC that
will react against the infectious reagent.
[20]


PKC and Cancer:
PKC has important role in cell growth and differentiation under regulated conditions
of PKC activation. When activation of PKC become unregulated, it would produce
uncontrolled cellular growth that will turn into cancerous cells eventually and PKC
will mediate the proliferation and survival of these cells. PKC is involved in several
cancer types:
Epidermal growth factor (EGF)-induced migration in breast and lung
cancer.
[23][24]

Cytoskeleton rearrangement, cell adhesion and matrix metalloprotease-9
expression in glioblastoma.
[25]

Examining different cancerous cells showed high expression levels of PKC. When
PKC was inhibited in head and neck tumors, it was found that the level of
proliferation and squamous cell carcinomas of the head and neck (SCCHN) viability
were reduced.
[26]

When testing cancer cells with high level of PKC with chemotherapeutic drugs, it
was found that some samples were resistant to chemotherapeutics drugs, whereas
inhibition of PKC allowed the sample to become sensitive to the drug and thus,
treatment was more effective.
[27]

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Protein Kinase Inhibitors

It is important to keep kinases under control due to their importance in several cellular
regulatory mechanism which will be unregulated if kinases were not controlled
properly, which might cause different diseases including cancer, inflammatory
disorders, diabetes and central nervous system diseases.
[28]

The recent focus for cancerous diseases studies was to control the protein kinases,
(more specifically PKC); thus, it was important to know what molecules are able to
inhibit a certain kinase from functioning and to classify them properly. The main
reason for this new focus was because of inhibitors off-target effects due to their
poor selectivity. The main difficulty in these inhibitors is that they mainly target ATP-
binding pocket (which is considered as a highly conserved region) that might be
directed to other proteins than the main targeted kinase that might affect other cellular
processes. Thus, studies are mainly about synthesizing inhibitors or altering the
existing ones to target other sites of the kinase rather than the ATP-binding site.
[28]


Classification of Inhibitors:

Type I inhibitors: inhibitors that bind to the ATP-binding site in active kinase.
This type of inhibitors is competitive with the ATP molecules with high
intracellular concentration. These inhibitors are not widely used in treatment
since targeting the highly conserved ATP-binding site for a specific kinase
rather than the others cannot be done.
[29][30]

Type II inhibitors: these inhibitors target the ATP-binding cleft and an
adjacent hydrophobic pocket created by the activation loop when Phe side-
chain of the conserved DFG motif moves out of the hydrophobic pocket.
Type III inhibitors: also known as allosteric inhibitors. It targets the regulatory
(allosteric) sites of the kinase. In comparison with types I and II inhibitors, the
allosteric inhibitors are widely used in recent researches due to their targeting
to the regulatory region of the kinase which is considered unique for each
particular kinase. These allosteric inhibitors are the ones used by cells to
control and regulate the catalytic activity of kinases within the cell.
[5][31]

Allosteric inhibitors have the following advantages:
- High selectivity for a particular kinase
- Avoid the difficulties faced with types I and II inhibitors to reach their
targeted site within a particular kinase.
- No need for high concentration of the inhibitor molecules within the cell
compared with type I inhibitor in case for high ATP-binding affinity for a
given kinase.
[32]

- Some allosteric inhibitors result prolonged inhibition for a given kinase
since the inhibitor might dephosphorylate the activation loop and thus,
inactivate the kinase; whereas in case of type I inhibitors, it might cause
13
further stabilization of the phosphorylated kinase since the inhibitor targets
the phosphorylated kinase (which is in active form) and covers the
phosphorylated residues, which can be considered as protection for these
residues.
[31][33]


PIF Pocket as Allosteric Regulatory Site:

An enzyme known as PDK1 as mentioned before does functions to phosphorylate a
kinase. The PIF pocket plays a significant role in regulation of the kinase activation.
The PDK1 enzyme contains PIF pocket; yet, it lacks the hydrophobic motif that
usually folds back into the PIF motif in AGC enzymes, it was found that PIF pocket
of PDK1 enzymes act as a docking site. Thus, during the activation process of a
kinase, the HM moves from PIF pocket of the enzyme and it docks in PDK1 PIF
pocket docking site. Once the enzyme got phosphorylated at its activation loop by
the PDK1 enzyme, the HM folds back into the PIF pocket of the enzyme. This
phosphorylation of the enzyme results the stabilization of the turn motif (T-loop) and
thus, stabilizing the enzyme in its active form in-order to be ready to function
properly.
[10][34]

To prove the regulatory function of PIF pocket, tests were done to examine the
conformational changes in PDK1 enzyme (which has PIF pocket similar to AGCs)
[10]
using PIF pocket ligand activators. It was found that local changes occurred to PIF
binding pocket, ATP-binding site and activation loop when that activator was bound
to its site. When a type II inhibitor was applied to the enzyme in another sample,
crystallization was done to study the structural relation between both PIF pocket and
ATP-binding site, which proved that there was an intimate coupling between them;
the regulatory importance of PIF pocket was indicated from the previous results.
[31][35][36]


PIF Pocket As A Target for Kinase Inhibition By Drugs:

Since the ATP-binding site is highly conserved in many kinases (and other proteins as
well), while regulatory sites show less conservation; it was more focused to develop
inhibitors that that target the regulatory sites rather than targeting ATP-binding sites
to avoid the unwanted consequences that might result as previously explained. Since
it was proved that PIF pocket acts as regulatory site, first trials to develop inhibitors
were toward PIF pocket of PDK1 enzyme. The results were synthesizing activators
for PDK1 but at the same time, they inhibited the phosphorylation of kinases.
Synthesizing drugs that acts as small inhibitory molecules was only successful for
PKC enzyme.
[34][36][37][38][39][40]

First discovery of inhibitory drugs was while screening of PDK1 activators that was
found to act as inhibitors for AGC kinases.
[40]
The compounds were then optimized
by involving benzimidazole analogs (which replaces other analogs in the original
compounds); it was found that the efficiency had increased with the new scaffold of
14
the compounds. Following trials involved synthesizing compounds with pyrazoline
analogs (one or more per compound) and some involved substitutions of different
atoms (including halogens) at the aryl rings presenting in the compounds.
[37][41]


P53 for Tumor Suppression:

A transcription factor known as the guardian of the genome. It has a critical role in
programmed cell death (or apoptosis),
[42][43]
where it prevents the survival and
proliferation of abnormal cells, including the cancerous ones by initiating the
transcription of genes that encode proteins involved in cell death. Activity of P53 was
examined in several cancerous tissues; the results indicated that in several samples,
P53 was not functioning properly. Thus, it was important to understand the regulation
of P53 factor.
[44]
A protein known as Mdm2 (murine double minute 2) acts as an
inhibitory protein for P53 whereas it has ubiquitin ligase activity (recruitment of
ubiquitin molecules and enhance its attachment to P53) and thus, it down-regulates
the activity of P53 and causes P53 degradation.
[45][46]
Once P53 is interacted with
Mdm2, it cannot bind to other proteins nor gets phosphorylated (meaning it remains
in its inactive form), beside that, Mdm2 causes P53 to be exported out of the nucleus
decreasing its interactions with the targeted genes in the DNA.
[45][46]
In cancerous
cells with wild type P53, it was found that Mdm2 has high cellular concentrations and
effectively inhibited the function of P53 factors, thus, programmed cell death process
was not initiated resulting the survival of these cancerous cells.
[47]
It is worth
mentioning that expression of Mdm2 gene is initiated by P53 itself, thus, Mdm2
protein is actually a product of P53 activation. During cell cycle, growing cell
undergoes several check points; if the cell was abnormal, regulatory proteins at that
check point acts to phosphorylate P53 protein, thus, affinity of P53 to Mdm2 is
reduced and less P53-Mdm2 complexes are formed but with weak interaction between
P53 and Mdm2 protein.
[48]


P53-Mdm2 and Cancer Therapy:

Since P53 has important role in elimination of cancerous cells, and hence P53 activity
is controlled by Mdm2; recent studies aimed to synthesize that can act as
replacements and interact with Mdm2 proteins instead of P53 factor.
Structural analysis of P53-Mdm2 was important to understand the complex formation.
It was found that a well-defined hydrophobic surface pocket in Mdm2 and three key
hydrophobic residues in P53 factor, the Phe19, Trp23 and Leu26, mediate the
interaction between P53 and Mdm2. The interactions between these residues in P53
and the hydrophobic pocket in Mdm2 can be pictured as three fingers (P53) inserting
themselves into three complementary pockets (Mdm2).
[49]

This structural analysis helped in synthesizing small non-peptide molecules that have
the ability to mimic these fingers and interact with the hydrophobic pocket in
15
Mdm2 protein, allowing more P53 factors to escape from Mdm2 and function
properly to initiate programmed cell death.

16


Aim

This project aimed to study the effects of newly synthesized pyrazoline compounds in
cancer therapy. The drugs were constructed to have dual-inhibitory function as anti-
cancer reagents. The drugs were designed to act as both, PKC and Mdm2 inhibitors.
The experiment was done on cancerous lung cell line (A549) to measure the activity
of NF- B and P53 after the cells were treated with the drugs in comparison with
control non-treated cells.



17
II. Materials and Methods:

Materials and equipment:


A) Plasmids

Plasmid Provider Catalog No.
pNL3.2.NF- B -RE
[NlucP/NF- B -RE/Hygro]
Vector
Promega 9PIN111
pp53-TA-Luc Clontech 631914
pEGFP-C3 BD Biosciences Clontech 6082-1
pSV--Galactosidase
Control Vector
Promega E1081




Figure 3. pNL3.2.NF- B -RE [NlucP/NF-B-RE/Hygro] Vector map

18


Figure 4. Restriction map of pp53-TA-Luc Vector



Figure 5. Restriction map of pEGFP-C3 Vector


19


Figure 6. pSV--Galactosidase Control Vector map



B) Cells:

Cell type Provider
A549 cell line American Type Culture Committee
(ATCC); Vacsera, Cairo, Egypt.
E.coli XL1 MRF cells Stratagene; Catalog No. 200230

C) Materials and Kits:

Materials/Kits Provider Catalog No.
QIAprep Spin Miniprep Kit (50) Qiagen 27104
Steady-Glo Luciferase Assay System Promega E2520
MassRuler DNA Ladder, Mix, ready-to-
use
Fermentas SM0403
6x Mass Ruler Loading Dye Fermentas R0621
HindIII Restriction Enzyme New
England
BioLabs
R0104S
BamHI Restriction Enzyme New
England
BioLabs
R3136S
BglII Restriction Enzyme New
England
BioLabs
R0144S
20
NEBuffer2 New
England
BioLabs
B7002S
NEBuffer 3.1 New
England
BioLabs
B7203S
Polyethylenimine (PEI) Transfection
Reagent
Polysciences 23966-2
BioWhittaker DMEM Lonza BE12-604F
Fetal Bovine Serum (FBS) SIGMA F7524
BioWhittaker Trypan Blue Stain Lonza 17-942E


























Figure 7. MassRuler DNA Ladder, Mix, ready-to-use marker guide


D) Equipment:

Equipment Provider
miniSpin centrifuge eppendorf
Thermomixer compact eppendorf
Laboratory Oven Ecocell MMM-group
HERAcell 240i CO
2
incubator Thermo scientific
Mupid-exU, a submarine type electrophoresis system Mupid
Geldocumentation Uvitec
Micromaster Inverted Microscope Fisher Scientific
21
Axiostar Plus Fluorescence Microscope ZEISS
Water bath Clifton
Centrifuge 5810 R eppendorf
Victor 3V Multilabel Counter Perkinelmer
Aura vertical S.D.4 laminar flow BIOAIR
HERAsafe KS biosafety (level 2) cabinet Thermo scientific


E) Software:

Wallac 1420 Workstation
AxioCam ERc5s Centrifugation Tool
UVIproMW



F) Media Preparation:

a) LB media for E.coli cells:

NaCl 10 g/l
Trypton 10 g/l
Yeast Extract (YE) 5 g/l
pH adjusted to 7.5; autoclave.


b) LB/Amp agar plates:

Agar 20 g/l
NaCl 10 g/l
Trypton 10 g/l
YE 5 g/l
pH adjusted to 7.5 then autoclaved and left to cool to 55
o
C. Under sterile conditions
(laminar flow), 10 ml of filter-sterilized ampicillin (10 mg/ml) stock solution is
added, mixed by swirling then poured into sterile petri dish, kept open at room
temperature till solidification.

c) LB/Kan agar plates:

Agar 20 g/l
NaCl 10 g/l
Trypton 10 g/l
YE 5 g/l
pH adjusted to 7.5 then autoclaved and left to cool to 55
o
C. Under sterile conditions
(laminar flow), 2 ml of filter-sterilized kanamycin (25 mg/ml) stock solution is added,
mixed by swirling then poured into sterile petri dish, kept open at room temperature
till solidification.

22

d) 1x PBS:

NaCl 137 mM
KCl 2.7 mM
Na
2
HPO
4
10 mM
KH
2
PO
4
1.76 mM
pH adjusted to 7.2 with HCl, sterilization by using 0.22 m filer.

e) Complete media (10%FBS) - 10 ml:

DMEM 9 ml
FBS 1 ml
Under sterile conditions (using biosafety cabinet level 2)


f) TAE buffer (50x):

Tris-HCL 242 g/l
EDTA 18.6 g/l
pH adjusted to 7.7

To prepare 1000 ml of 1x TAE:
MV = MV
50V = 1*1000
V= 20 ml, the final volume is completed by adding 980 ml of dH
2
O

Methods:

A) Plasmid preparation:

Cloning:
Each plasmid of pNL3.2.NF- B RE, pp53-TA-Luc and pEGFP-C3 was transformed
separately into E.coli XL1 competent cells. This was done by mixing 2 l of the
plasmid with 18 l dH
2
O; the mixture was then added to an appindorf containing
competent E.coli XL1 cells (about 200 l) placed in icebox and left for 30 minutes;
the eppindorf was then placed in thermomixer at 40
o
C for 3 minutes (heat shock) and
left in room temperature (RT) for 10 minutes. 10 ml of LB media (does not contain
antibiotic) was added to the eppindorf, placed on thermomixer at 37
o
C. After 1 hour,
the eppindorf was placed in miniSpin centrifuge at the maximum speed for 3 minutes,
1 ml of the supernatant (S/N) was discarded and the pellet was resuspended in the
remaining media (around 220 l). After the pellet was completely resuspended, it was
then spread on LB-agar plate containing antibiotic and left in the incubator at 37
o
C for
16 hours.
According the plasmid maps (available in material section), both pNL3.2.NF- B RE
and pp53-TA-Luc plasmids have ampicillin resistant gene, thus, the plates were LB-
23
agar-Amp100. For pEGFP-C3 plasmid, it contains kanamycin resistant gene, thus, the
plate was LB-agar-Kan50.


Extraction:
After the plates were left overnight, only the cells containing the plasmid would be
able to grow and form colonies (due to the antibiotic resistant gene in the plasmid).
One colony was picked from the plate and transferred into a tube containing 5 ml of
LB-antibiotic (Kan50/Amp100) and left to grow overnight (O/N) in a shaker at 200
rpm and 37
o
C. 2 ml of the culture was then transferred into an eppindorf and
centrifuged at maximum speed for 5 minutes, S/N was discarded and another 2 ml of
the culture media was then added to the eppindorf and the pellet was resuspended
followed by another 5 minutes of centrifugation at the maximum speed. S/N was then
discarded. The extraction kit, QIAprep spin miniprep (50) was used to extract the
plasmid from the cell. 250 l of P1 (resuspension buffer + RNase A) was added to the
eppindorf and the pellet was resuspended in it. After the pellet was completely
resuspended, 250 l of P2 (lysis buffer) was added and left for 5 minutes at RT,
following by addition of 350 l of NB (neutralizing buffer) and left for another 5
minutes at RT, then for 10 minutes on ice. After 5 minutes were done, the eppindorf
was then centrifuged for 20 minutes and the maximum speed; the S/N (containing the
plasmid) was then transferred into a spin column placed inside an open centrifugation
tube, then it was placed in the centrifuge for 30 seconds at the maximum speed. 500
l of PB (binding buffer) was added, centrifuged at maximum speed for 30 seconds,
750 l of PE (wash buffer) was added and centrifuged at maximum speed for 30
seconds. Another centrifugation for 30 seconds at the maximum speed was done
without the addition of any buffer. Finally, 50 l of TE (elution buffer, pre-warmed at
60
o
C) was added to the spin column, left for 1 minute at RT and then centrifuged at
the maximum speed for 1 minute. After extraction was done, the eppindorf was stored
at -20
o
C.
At each step, starting from which the spin column was used, the centrifugation tube
was changed. At the final step (addition of TE), pre-labeled 1.5 ml eppindorf was
used instead of the centrifugation tube.

Gel electrophoresis:
This step was done to make sure the extraction protocol was done successfully. 25 ml
of 1% agarose was prepared by dissolving 0.25 g of agarose in 25 ml TAE (1x) (flask
was weighed), flask was then heated and swirled continuously to dissolve the agarose
(without boiling it), and the final volume was then adjusted again by adding sterile
H
2
O. After the agarose had been completely dissolved and the final volume was
adjusted, 2 l of ethidoum bromide was added to the flask carefully, the mixture was
then poured into the electrophoresis chamber, covered with aluminum foil (to avoid
any particles to land on it) and allowed to solidify for 1 hour. During the time of
agarose solidifying, the plasmids were treated with restriction enzymes to prepare it
24
for the run. Each plasmid was treated according to its restriction sites (available in
plasmid map, materials).
Plasmid

Restriction Enzyme
(1l each)
Buffer
(2 l)
pNL3.2.NF- B RE
(8 l)
BamHI NEBuffer 2
pp53-TA-Luc
(2 l)
BglII NEBuffer 3.1
pEGFP-C3
(8 l)
(Double Digest) (Single Digest) NEBuffer 2
HindIII
BamHI
HindIII

For each eppindorf, the final volume was adjusted to 20 l using sterile H
2
O; it was
then centrifuged just get the droplets to the bottom of the eppindorf. The eppindorfs
were then placed in the thermomixer for 1 hour at 37
o
C and 350 rpm. After one hour,
4 l of loading dye was added to the mixture and then loaded in the gel. The running
continued for 45 minutes and the applied voltage was 70 V. After the running was
done, the gel was then examined under Geldocumented-UV detector chamber and
UVIproMW software to check the plasmid fragments.

Gelelectrophoresis images:




















Figure 8. Gelelectrophoresis results using Uvitec geldocumented cabinet and
UVIproMW software. The used marker was MassRuler DNA Ladder, Mix,
ready-to-use (A) pp53-TA-Luc plasmid (2 l) and 3 l marker. (B)
pNL3.2.NF- B RE plasmid (8 l) and 6 l marker. (C) pEGFP-C3 plasmid
(8 l), marker was too concentrated (20 l), results were compared to the
marker ladder in the catalog (available in materials).


A
B
C
M
P53
treated
P53
Non-
treated
M
NF- B pEGFP
SD
pEGFP
DD
25

B) Cell Culturing:

Thawing:
The A549 cells were thawed after being stored in liquid nitrogen and allowed to
proliferate at 37
o
C and 5% CO
2
. This was done as the following; the screw-cap tube
containing the cells was transferred from liquid nitrogen to an icebox. After a while,
cells were transferred into 15 ml falcon tube and 5 ml of DMEM (pre-warmed at
37
o
C) was added, the tube was then centrifuged at 1200g for 5 minutes, S/N was
discarded and 10 ml complete medium was added and the pellet was resuspended in
it. The media was then transferred into a T-25 flask and incubated at 37
o
C and 5%
CO
2
using HERAcell 240i CO
2
incubator. When needed, media was changed (when it
turned yellow) by removing the old media and adding 10 ml of new complete media.

Splitting cells into a 24-well plate:
After cells were allowed to proliferate in the T-flask, the old media was removed and
the cells were washed for 30 seconds with 4 ml 1x T/E (trypsin) (diluting from 10x
T/E using PBS) and then the 4 mls were discarded. Another 4 ml 1x T/E was added to
the flask and then it was incubated at 37
o
C for 15 minutes (or until cells were fully
detached). 4 ml of complete media was added to stop the effect of trypsin. To count
the number of cells, 20 l of the solution was then transferred into an eppindorf and
20 l of trypan blue stain was added and mixed, counting was done using Thomas
chamber and Micromaster inverted microscope. 2x10
4
cells (around 200 l) were then
transferred into each well in the 24-well plate, incubated at 37
o
C and 5% CO
2
.

C) Transfection with PEI reagent:

Transfection reagent preparation:
1 mg of the transfection reagent PEI (Polyethylenimine 25 KDa linear) was first
dissolved in 1 ml endotoxin-free dH
2
O (pre-warmed at 80
o
C), and then cooled to RT.
The pH was then adjusted to 7.0 and filtered with 0.22m filter sterilize into a 2 ml
screw-cap tube and stored at -20
o
C till used.

Transfection mixture preparation:
24 l of pNL3.2.NF- B RE and pp53-TA-Luc plasmids was first diluted in 600 l
serum-free DMEM media, while 4 l of pSV--Galactosidase plasmid was diluted in
50 l serum-free DMEM media, then, the prepared PEI reagent was added in ratio of
3:1 (PEI:DNA). The mixture was incubated at RT for 15 minutes and then 55 l was
added to the cell culture in the 24-well plate. The plate was then incubated for 48
hours at 37
o
C (5% CO
2
for cells from ATCC, CO
2
-free for cells taken from Vacsera).
Before transfection with the plasmids to be tested, the transfection efficiency was first
examined using the pEGFP-C3 plasmid. The fluorescence from the pEGFP-C3
expression was examined using Axiostar Plus Fluorescence Microscope and
26
AxioCam ERc5s Centrifugation Tool software. Around 2.5310
4
cells were
successfully transfected with the pEGFP plasmid.

Figure 9. Fluorescence detected from A549 cells due to the expression of
pEGFP plasmid to examine transfection efficiency by PEI reagent using
Axiostar Plus Fluorescence Microscope and AxioCam ERc5s Centrifugation
Tool software.

Cells treatment:
Once transfection was tested and proved the success of the PEI, the cells were
cultured in a 24-well plate a day before transfection and treatment with the desired
drugs (final concentration 10 M). In total, 9 drugs were used to examine their
efficiency as anti-cancer reagents.
Of the 24-well plate:
1 well was used as a normalizer: cells transfected with pSV--Galactosidase
plasmid + DMSO
1 well was used as a control for pNL3.2.NF- B RE plasmid + 3 l DMSO
1 well was used a control for pp53-TA-Luc plasmid + 3 l DMSO
9 wells were transfected with pNL3.2.NF- B RE plasmid, each well treated
with different drug
9 wells were transfected with pp53-TA-Luc plasmid, each well treated with
different drug.
After all components were added to the cells (in each well 300 l cells, 55 l
transfection mixture, 100 l fresh media), the plate was incubated for 48 hours at
37
o
C (CO
2
-free atmosphere, Vacsera).

27

D) Luciferase Assay:

Luciferase assay aims to measure the luciferase gene expression under the control of
the promoter of the gene to be tested. Steady-Glo Luciferase Assay System protocol
was used to measure the luciferase expression after treatment with the drugs. In this
thesis, the luciferase gene was under control of P53 promoter and NF- B promoter.
Luciferase substrate was first prepared by addition of equal volume of substrate buffer
to the substrate screw-cap tube. The cells were prepared first by transfecting them
with pNL3.2.NF- B RE and pp53-TA-Luc plasmids (each separately) in the 24-well
plate as explained before.
Once 48 hours were done, the old media was removed from each well of the 24-well
plate, and the cells were lysed by adding 100 l of lysis buffer and left for 5 minutes.
100 l of the substrate was then added to each well, mixed by pipetting and left for 30
minutes approximately (this was done in dark room to avoid quenching effect of the
substrate). To measure the luciferase, the solution in each well was then transferred
into a clear-bottom 96-well plate, each well separated into duplicates (since the well
contained 200 l, 100 l was transferred into two wells in the clear-bottom 96-well
plate). Measurement was done using Victor 3V Multilabel Counter and Wallac 1420
Workstation software.





Figure 10. Detailed diagram of the 96-well plate after transferring the mixture
from the 24-well plate.


28
III. Results:

The following results were obtained from the Wallac 1420 Workstation software, the
values of average, normalized average, (control-treated) and percentage of inhibition
were calculated using Microsoft Excel.


Plate Well CPS Avg.
Normalized
Avg.
Control -
treated
%Inhibition
Control
Normalizer C01-02 1209 1083 1146 - -
C (NF-kB) C03-04 247 199 223 0.194589878 - -
C (p53) C05-06 173 197 185 0.161431065 - -
pNL3.2.
NF- B
RE
plasmid
MA7 D01-02 200 199 200 0.17408377 0.020506108 10.53811658
MA72 D03-04 185 217 201 0.17539267 0.019197208 9.865470844
MA114 D05-06 181 176 179 0.155759162 0.038830716 19.95515693
MA117 D07-08 176 166 171 0.14921466 0.045375218 23.31838563
MA119 D09-10 166 201 184 0.160122164 0.034467714 17.71300447
MA120 D11-12 207 205 206 0.179755672 0.014834206 7.623318379
MA121 E01-02 185 184 185 0.160994764 0.033595113 17.26457398
MA122 E03-04 182 199 191 0.166230366 0.028359511 14.57399102
MA130 E05-06 166 194 180 0.157068063 0.037521815 19.28251119
pp53-
TA-Luc
plasmid
MA7 E07-08 189 188 189 0.164485166 -0.003054101 -1.891891887
MA72 E09-10 203 190 197 0.171465969 -0.010034904 -6.2162162
MA114 E11-12 156 197 177 0.154013962 0.007417103 4.594594582
MA117 F01-02 192 185 189 0.164485166 -0.003054101 -1.891891887
MA119 F03-04 176 204 190 0.165794066 -0.004363002 -2.702702696
MA120 F05-06 191 191 191 0.166666667 -0.005235602 -3.243243235
MA121 F07-08 194 200 197 0.171902269 -0.010471204 -6.486486469
MA122 F09-10 203 204 204 0.177574171 -0.016143106 -9.999999974
MA130 F11-12 178 203 191 0.166230366 -0.004799302 -2.972972965

As this thesis is only concerned about MA72, MA114 and MA117 drugs, their results
are only to be discussed.

Values of percentage of inhibition of pNL3.2.NF- B RE plasmid for both MA114
and MA117 drugs indicated that the drugs have successfully inhibited the activity
PKC and thus, the expression of NF- B had been down regulated. Meaning that the
cancer cell will not be able to survive and proliferate as long as the NF- B
transcription factor will not be able to function properly and thus, would not be able
to initiate transcription of genes needed for cell survival.
As for MA72 drug, percentage of inhibition was not significant in comparison with
the results of remaining drugs.
As for values of percentage of inhibition of pp53-TA-Luc plasmid, it indicated that
there was excess expression of P53 factor, which is important to initiate programmed
cell death. Yet, the values for the given drugs MA72 and MA117 were not significant
as well. The inhibition of Mdm2 activity was not successful enough to activate p53
expression. The drug MA114 showed a positive value of percentage of inhibition,
which means that the drug has not inhibit Mdm2 successfully.


29
IV. Discussion and Future Recommendation:

Scientists have tried to understand cancer development and treatment methods for it
for many years and many studies were carried on to understand cellular regulatory
mechanism and the cellular pathways of avoiding cancerous cells development.
Recent studies were focused on an enzyme known as PKC. It was found that this
enzyme plays important role in regulation of cellular molecules and proteins, like NF-
B that in turn, are important for cell growth, proliferation and survival by activating
transcription of genes for survival process; thus, presence of regulatory molecules for
PKC would lead to regulation of cell growth and survival.

Another protein known as p53 (a transcription factor) was also found to have
significant role in cell growth regulation, whereas it initiates programmed cell death
(also called apoptosis) in case of damaged DNA that cellular repairing mechanism
could not repair it where it initiates transcription of genes that are involved in
apoptosis process, preventing development of possible cancer cells. The p53 protein
was found to be regulated by another protein named Mdm2 that enhances
ubiquitiliation of p53 in case of normal cellular growth.

Recent researches aimed to synthesize small molecules that could fit into allosteric
site (regulatory site) of the mentioned protein and stop its function, thus, it would lead
to inhibit survival of cancer cells if PKC was inhibited and would initiate
programmed cell death if Mdm2 was inhibited.

The aim of this study was to study the effects of newly synthesized pyrazoline
compounds as anti-cancer reagents in A549 lung cancer cell line. For the drugs that
were examined in this thesis, MA72, MA114 and MA117, the experiment was done
to test its activity as inhibitors for both PKC and Mdm2 (dual inhibition). In this
work, A549 cell line was transfected using PEI transfecting reagent with pNL3.2.NF-
B RE plasmid and pp53-TA-Luc plasmid and luminesce was measured for each
plasmid when cells were treated with 10 M of each drug, the expression of luciferase
gene under p53 and NF- B promoters was measured using Steady-Glo Luciferase
Assay System. The results indicated that the tested drugs MA114 and MA117 showed
promising results as anticancer reagents for NF- B inhibition where the drug MA72
showed weak inhibition. Where for the inhibition of p53, drugs MA72 and MA117
showed only weak inhibition of Mdm2 protein (thus activation of p53), where the
drug MA114 showed no inhibition for the Mdm2.

As for future recommendation, it is recommended to examine the effects of these
drugs at different concentrations than 10 M and on different cancer cell lines. In this
experiment, the effects were studied without activation of signaling pathway, thus, it
recommended, especially for NF- B, studying the effects when cytokines involved in
activation pathway are added to the experiment.

30


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