Chapter 5 Anticancer and Immunomodulatory Studies

5.1. INTRODUCTION

The role of ROS and NO in apoptosis mediated tumor cell death and self defense

mechanisms, as has been discussed in the earlier chapter, and the pro-oxidant nature of the

plants evoked the idea to examine the anticancer and immunomodulatory activities of the

same.

The treatment of many diseases owes much to plants-derived drugs, and the

treatment of cancer is no exception (Hartwell, 1971). More than 120 phamacologically

active plant derived compounds have been isolated. In addition to cytotoxic drugs, the

potentiation of host defense mechanism has been recognized as a possible means of

inhibiting tumor growth (Ameho et al., 1997). Therefore searching for immunomodulatory

materials from natural herbs and characterizing the immune enhancement effects may have

great potential in cancer treatment, based on combination of time honored traditional usage

and ongoing scientific research (Rivera 2003).

5.2. REVIEW OF LITERATURE

5.2.1. Cancer: a global health problem

Cancer is one of the most dreaded diseases of the 20th century and spreading further

continuously with increasingincidence in 21st century (Dashora et al., 2011). As of 2004,

worldwide cancer caused 13% of all deaths (7.4 million). The leading causes were: lung

cancer (1.3 million deaths/year), stomach cancer (803,000 deaths), colorectal cancer

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(639,000 deaths), liver cancer (610,000 deaths), and breast cancer (519,000 deaths) (WHO,

2006).

5.2.2. Cancer status in India

The Indian subcontinent is home to 16.5% of the world’s population and at any one

time it is estimated that there are over 2 million people with cancer. Presently in India, out

of a million newly diagnosed cancer patients each year, more than 50% die within 12

months of diagnosis and another one million cancer survivors show progressive disease

within five years of diagnosis (Pal and Mittal, 2004). In 2005, cancer killed approximately

826,000 people in India; 519,000 under the age of 70 (WHO, 2005). This is predicted to

rise disproportionally compared with cardiovascular and communicable diseases by 2030,

to nearly 1.5 million deaths annually. Based on the cancer registry data it is estimated that

there will be about 800,000 new cancers cases in India every year. At any given point there

is likely to be 3 times this load that about 240,000 cases.

5.2.3. Folk medicine and Plant derived anticancer agents

Medicinal plants – either through systematic screening programs or by serendipity -

possess an important position in the drug discovery and many modern drugs have their

origin in traditional medicine of different cultures (Abu-Dahab and Afifi, 2007).

Ethnopharmacological data are obtained by consulting traditional healers and by

accumulating information on the popular medicinal use of plants, but also from literature

on folk medicine (Mans et al., 2000). A compilation of more than 3,000 reports on the

folkloristic use of plants for treating “cancer” (Hartwell, 1967; Hartwell, 1982). About

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25% of prescribed drugs in the world originate from plants (Rates, 2001) and over 3000

species of plants have been reported to have anticancer properties (Graham et al, 2000).

Medicinal plants have been used in Asian countries and interest in this area of research has

recently increased in all over countries (Arulvasu et al, 2010).

Natural products or related substances or extracts of folk medicine accounted for

30% of the top 35 worldwide natural product-based drugs sold (Butler, 2004) in recent

years. The plant-derived anticancer drugs, or the plant derived cancer chemotherapeutic

agents were responsible for approximately one third of the total anticancer drug sales

worldwide, or just under $4 billion dollars in 2007; namely, the taxanes, paclitaxel and

docetaxel, and the camptothecin derivatives, irinotecan, topotecan, etc. Historical

experiences with plants as therapeutic tools have helped to introduce single chemical

entities in modern medicine. Plants, especially those with ethnopharmacological uses in

tropical countries, have been the primary sources of medicines for early drug discovery. In

fact, a recent analysis by Fabricant and Farnsworth showed that the uses of 80% of 122

plant-derived drugs were related to their original ethnopharmacological purposes

(Fabricant and Farnsworth, 2001). Current drug discovery from folk-medicine plants has

mainly relied on bioactivity-guided isolation methods, also, for example, bioactivity-

guided isolation methods have led to discoveries of the important anticancer agents,

paclitaxel from Taxus brevifolia and camptothecin from Camptotheca acuminate

(Fabricant and Farnsworth, 2001). Medicinal herbs of folk-origin are significant sources of

synthetic and herbal drugs.

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The discovery of paclitaxel from the bark of the Pacific Yew, Taxus brevifolia

Nutt. (Taxaceae), is an evidence of the success in natural product drug discovery. Various

parts of Taxus brevifolia and other Taxus species (e.g., Taxus Canadensis Marshall, Taxus

baccata L.) have been used by several Native American Tribes for the treatment of some

non-cancerous cases (Cragg and Newman, 2005) while Taxus baccata was reported to use

in the Indian Ayurvedic medicine for the treatment of cancer.

5.2.4. Reports on plant extract and anticancer activity

Ali et al., (1996) assessed ethanolic extracts of 61 medicinal plants of Malaysia for

cytotoxicity against HeLa cell line and cytotoxic activity was found in the extracts from

Acalypha indica, Andrographis paniculata, Cerbera manghas, Codiaeum variegatum,

Cosmos caudatus, Elephantopus scaber, Etlingera elatior, Eugenia michelii, Freycinetia

malaccensis, Hibiscus rosa-sinensis, Centella asiatica, Lecythis ollaria, Mentha arvensis,

Mirabilis jalapa, Morinda elliptica, Ocimum tenuiflorum, Piper sarmentosum and

Polygonum minus. Kamuhabwa et al., (2000) studied antiproliferative effect of the

methanol extracts of forty seven Tanzanian traditional medicinal plants on three human

cell lines: HeLa, HT29 and A431 cells. From the nine plants that were used to treat cancer,

two plants (22%) exhibited pronounced cytotoxic effect (<25% cell proliferation) at least

in one of the tested cell lines. Other 38 plants that were used to treat non-cancer diseases,

14 plants (37%) exhibited pronounced cytotoxic effect (<25% cell proliferation). Growth

inhibitory effect of thirteen Indian medicinal plant extracts on prostate cancer cell lines

was tested by Rao et al., (2004) and observed that Withania somnifera, Momordica

charantia, Camellia sinensis (I & II), Curcuma longa and Polygonum cuspidatum were

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effective on highly metastatic PC-3M prostate cancer cell line. Moongkarndi et al., (2004)

screeened ethanolic extracts of selected nine Thai medicinal plants for antiproliferative

activity against SKBR3 human breast adenocarcinoma cell line and Garcinia mangostana

was found to show most potent activity. Scheck et al., (2006) observed anti proliferative

activity of Scutellaria baicalensis extract on malignant glioma cells was also found active

on recurrent and drug resistant brain tumor cell lines. Abu-Dahab and and Afifi, (2007)

screened 76 ethanolic extracts belonging to 67 species of medicinal herbs from the

Jordanian flora, on breast cancer cell line (MCF7) and found that four plant extracts with

antiproliferative activity. Sun et al., (2007) reported anticancer activity of aqueous and

ethanol extracts of fifteen traditional medicinal plants of China against six human digestive

tumor cell lines: human liver carcinoma cell lines (HepG-2 and SMMC- 7721), human

gastric cancer cell line (BGC-823), human colon adenocarcinoma cell lines (LoVo and

SW-116) and esophagus adenocarcinoma cell line (CaEs-17) and observed that most

ethanol extracts demonstrated a more powerful inhibitory effect than aqueous extracts.

Efferth et al., (2007) reported the killing effect of Artesunate (ART) on human acute T cell

leukemia Jurkat cell line J16, the human acute lymphoblastic leukemia cell lines CEM and

Molt-4. Mothana et al., (2008) evaluated thirty four extracts (methanol and hot water

extracts) of traditional medicinal plants of Yemen against FL-cells, a human amniotic

epithel cell line, by using the neutral red uptake assay and remarkable cytotoxic activity

against FL-cells was observed for the methanolic extracts of Acalypha fruticosa, Iris

albicans, Lippia citriodora and Tragia pungens. Kee et al., (2008) reported anticancer

activity of methanol and aqueous extracts of nine plants of Eastern Cape Province of South

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Africa against HT-29, A-549, MCF-7, K562 and HL-60 cells. Gridling et al., (2009)

demonstrated anti- neoplastic potentiality of two traditional medicinal plants of Guatemala

on HL-60 promyeloic leukaemia cells and MCF-7 breast cancer cells. Patel and Suthar

(2009) observed in vitro anticancer activity of methanolic extract of Semecarpus

anacardium nut on the human epidermoid larynx carcinoma cell line (Hep 2) and African

green monkey kidney Normal cell line (Vero). Bhattacharya et al., (2011) repoted

anticancer activity of Coccinia grandis against Ehrlich Ascites Carcinoma (EAC), “ in

vitro” and “in vivo”. Nisa et al., (2011) noted the activity of Debregeasia salicifolia

extracts on MCF- 7 cancer cell line. Patel et al., (2011) observed inhibitory effect of Rubia

cordifolia methanol extract on human cervical cancer cell line and human larynx

carcinoma cell line.

5.2.5. Immunomodulatory activity of crude plant extracts

There are a number of plants that have been reported to have immunomodulatory

activity; (Puri, 2003). Crude extract of Tinospora cordifolia has been reported to contain a

polyclonal B cell mitogen that enhanced immune response in mice. Intra peritoneal

administration of alcoholic extract of Tinospora cordifolia in Dalton's lymphoma bearing

mice was found not only to augment the basic function of macrophages such as

phagocytosis, but also their antigen presenting ability and secretion of IL-1, TNF and RNI.

Slow down the tumor growth and increases the life span of tumor bearing host, were also

observed (Singh et al., 2004). Ethanolic extract of Boerhaavia diffusa, a plant used in

Indian traditional system of medicine, significantly inhibited the cell proliferation

(Mungantiwar et al., 1999). Methanol extract of Eclipta alba and Centella asiatica whole

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plant showed increased phagocytic index and antibody titer in treated animals (Kaul et al.,

2003). The ethanol extract of the root of the plant Cryptolepis buchanani was reported to

cause significant stimulation of the delayed type hypersensitivity reaction and humoral

antibody production in mice. An aqueous extract of Rhodiola imbricata rhizome has been

reported to stimulate production of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-

α) in human PBMCs as well as RAW 264.7 cell lines. Aqueous leaves extract of

biopesticidal plant Nyctanthes arbor-tristis has been found as a potent immunomodulator

(Puri et al., 1994). Immunosuppressive activity of crude aqueous extracts of Emblica

officinalis and Evolvulus alsinoides, two extensively used medicinal herbs in Indian

Ayurvedic medicine has been reported (Ganju et al., 2003).

5.3. MATERIALS AND METHODS

5.3.1. Reagents

Caspase- 3 and Bcl2 antibody were obtained from Santa Cruz Biotech., USA,

Caspase- 9 and Bad antibody from Cell Signaling, Germany, Bax antibody from

Oncogene. Bradford reagent, Ethylene glycol-bis (2-aminoethyl ether)-N,N,N',N'-

tetraacetic acid (EGTA), Ethylenediamine tetra acetic acid (EDTA), Tris base, TritonX-

100, PMSF, Leupeptin, Aprotinin, Antipain, N-p-tosyl-phenylalanine chloromethyl ketone

(TPCK), Nα-p-tosyl-L-lysine chloromethyl ketone (TLCK), Starch, Penicillin,

Streptomycin, Medium 199 (M199), Roswell Parker Memorial Institute (RPMI) 1640 was

obtained from Molecular Probes (Eugene, OR). Nitrotetrazolium blue (NBT), 5- bromo- 4-

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chloro- 3- indolyl phosphate (BCIP) Phorbol ester (PMA) and ionomycin (Io),

concanavalin A (Con A), were obtained from Sigma (St Louis) USA. Ficoll Hypaque was

purchased from Pharmacia (Uppsala, Sweden). Fetal bovine serum (FBS) was obtained

from Invitrogen Corporation (Carlsbad, CA). K562, U937, Chinese Hamster Ovary

(CHO), PgP over expressing adriamycin resistant CEM/ADR 5000 (Mookerjee et al.,

2006) and MRP1 over-expressing doxorubicin resistant Ehrlich Ascites Carcinoma

(EAC/Dox) cells (Mookerjee et al., 2006) were used. RPMI1640 supplemented with 100

IU/ml of penicillin and 100 g/ml of streptomycin containing 10% (v/v) heat inactivated

Fetal Bovine Serum (FBS) was used as culture medium.

5.3.2. Buffers and Solutions

1. Phosphate buffered saline: 20 mM Phosphate buffer, 150 mM NaCl, pH

7.2

2. Phosphate buffer: 136 mM NaCl, 2.7 mM KCl, 6.5 mM Na2HPO4, 1.46mM KH2PO4,

pH 7.2

3. HANKS buffer: 135 mM NaCl, 5 mM KCl, 5 mM D- glucose, 3mM Na2HPO4, 4

mM KH2PO4, 10 mM HEPES, 4 mM NaHCO3, pH 7.2

4. Cell fixation buffer: Phosphate buffered saline, 1% para formaldehyde,

pH 7.2

5. FACS buffer: phosphate buffered saline, 1% FCS, pH 7.2

6. Phosphate Citrate (PC) buffer: 0.2 M Na2HPO4, 0.1 M Citric acid, pH 7.8

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7. Propidium Iodide (PI) stock: 1 mg/ ml in PBS

8. Annexin binding buffer: 10 mM HEPES, 140 mM NaCl, 5 mM KCl, 1 mM

MgCl2, 5 mM CaCl2

9. Tris-EGTA-EDTA- Me (TEEM):

(buffer with protease

Inhibitors) 20mM Tris-HCl, pH 7.5, 0.5mM Ethylene glycol-bis

(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid

(EGTA), 1.0mM Ethylenediamine tetra acetic acid

(EDTA), 0.1% (v/v) 2-mercaptoethanol, 1mM Phenyl

methyl sulfonyl fluoride (PMSF), 2g/ml N-p-Tosyl-

L-phenylalanine chloromethyl ketone

(TPCK),2g/ml Nα-p-tosyl-L-lysine chloromethyl

ketone (TLCK), 5g/ml Leupeptin, 5g/ml

Aprotinin, 5g/ml Antipain.

10. Laemmli buffer (SDS-PAGE 10mM Tris, 2% (w/v) SDS, 10% (v/v)

sample buffer) (Laemmli, 2-mercaptoethanol, 1mM EDTA, 0.002%

1970): (w/v) Bromophenol blue, pH 6.8

11. Electrophoresis buffer: 0.025 M Tris, 0.192 M Glycine, 0.1% (w/v) SDS, pH 8.3

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12. Sample buffer (10X): 0.625 M Tris, pH 6.8, 10% (w/v) SDS, 0.5 mg/ml

Bromophenol blue (BPB)

13. Blotting buffer: 25 mM Tris, 192 mM Glycine, 20% (v/v) methanol,

pH 8.3

14. Wash buffer

Tris buffered saline (TBS): Tris, NaCl, pH 7.4

15. Phosphate buffer for washing: Na2HPO4, NaH2PO4, pH 7.1

16. Bradford Reagent (Bollag et al., 1996)

Ingredients Amounts

Coomassie Blue G 200 (Sigma) 10 mg

Ethanol (Bengal Chemical) 10 ml

Orthophosphoric acid (E. Merck) 10 ml

Double distilled water 80 ml

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17. Primary and secondary antibodies.

Primary antibody used and its dilution Secondary antibody and its dilution

Caspase- 3 goat polyclonal antibody Rabbit antigoat IgG- AP (Sigma, USA)

(Santa Cruz Biotech., USA) at 1: 500 at 1: 10000

Caspase- 9 rabbit polyclonal antibody Goat antirabbit IgG- AP (Sigma, USA)

(Cell Signaling, Germany) at 1: 1000 at 1: 10000

Bad rabbit polyclonal antibody (Cell Goat antirabbit IgG- AP (Sigma, USA)
Signalling, Germany) at 1: 1000 at 1: 10000

Bax mouse monoclonal antibody Goat antimouse IgG- AP (Sigma, USA)

(Oncogene) at 1: 300 at 1: 10000

Bcl2 rabbit polyclonal antibody (Santa Goat antirabbit IgG- AP (Sigma, USA)
Cruz Biotech, USA) at 1: 300 at 1: 10000

18. Substrate solution.

Enzyme conjugated Reaction buffer Substrate Co substrate

to secondary
antibody

Alkaline 100 mM Tris Nitrotetrazolium 5- bromo- 4-

phosphatase (AP) HCl (pH 9.5), blue (NBT) at a chloro- 3- indolyl
containing final concentration phosphate (BCIP)
100mM NaCl of 33 µg/ ml added to a final
and 50 mM concentration of
MgCl2 16.5 µg/ ml.

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5.3.3. Cell lines, Animals and Cell culture

Human pro-monocytic leukemia cells K562 and U937, Chinese Hamster Ovarian

cells (CHO), doxorubicin resistant human acute T- lymphoblastic leukemia cells

CEM/ADR 5000 (Mookerjee et al., 2006) and doxorubicin resistant Ehrlich Ascites

Carcinoma (EAC/Dox) cells (Mookerjee et al., 2006) were used.

OVA257- 264 peptide, Ovalbumin peptide: specific B cell hybridoma LB, class II

restricted T cell hybridoma 7.13 and IL-2-dependent cell line HT-2 maintained in IICB

was used for antigen presentation study.

Swiss albino mice from Chittaranjan National Cancer Institute, Kolkata were used

for experimental purposes with prior approval of the animal ethics committee of the

Institute.

RPMI-1640 buffered with 20 mM HEPES and 0.2 g NaHCO3, supplemented with

10% (v/v) heat inactivated FBS, 500 M ME, 100U/ml penicillin, 100g/ml streptomycin

was used for cell culture.

5.3.4. In Vitro anticancer assay methodology

Various cancer cell lines like- K562, CHO, U937 and Dox resistant CEM/ADR

5000 cells were used for this study. Cells were plated in triplicate at 5x104 cells/ 200μl/

well concentration in a 96 well plate. Cells were then pulsed with 1μCi (6.7 Ci/mole) [ 3H]

thymidine/ well and allowed to proliferate for 48 hr at 370C in 5% CO2 incubator in

presence of different concentrations of extract of two plants along with vehicle control

(0.01% DMSO in PBS) (Bandyopadhyay et al., 2004). After 48 hr they were harvested and

[3H] thymidine uptake, as an index of proliferation was measured by Liquid Scintillation

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Counter (TRI CARB 2100TR, Packard) (Mookerjee et al., 2006). Data of anti-

proliferative index against different cell lines was expressed as percentage decrease in cell

number.

5.3.5. In Vivo anticancer assay methodology

Six weeks old female mice weighing 20- 25g kept in identical laboratory condition

were intraperitoneally inoculated with 1X107 Doxorubicin resistant Ehrlich ascites

carcinoma (EAC/Dox) cells derived from peritoneum of EAC/Dox bearing mice treated

with doxorubicin (1mg/Kg body weight) (Majumder et al., 2005). Experimental animals

were either kept mock treated (control) or treated with methanol extract of Parkia javanica

(MEPJ) at four different concentrations viz., 500, 100, 20 and 2 mg/kg body weight

through three different routes (i.e., oral, intramuscular (im) and intraperitoneal (ip)) 7 days

post inoculation with 1 x 107 EAC/Dox cells (Mookerjee et al., 2006). Treatments

continued for 5 doses, at two days interval between each treatment.

5.3.6. Isolation of EAC/Dox cells from peritoneal cavity of mice

The EAC/ Dox cells, were isolated from the peritoneal cavity of EAC/Dox-bearing

mice (control or treated) at two different time intervals i.e., 13 days and 29 days post

inoculation. Sterile PBS (2-3 ml) was injected into the peritoneal cavity of the mice and the

peritoneal fluid containing the tumor cells was withdrawn, collected in sterile Petri dishes,

and incubated at 370C for 2 hours. The cells of macrophage lineage adhered to the bottom

of the Petri dishes. The nonadherent population was aspirated out gently and washed

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repeatedly with PBS. EAC/Dox cells thus separated were processed for further

experiments.

5.3.7. Apoptosis Assay

To know whether the decrease in cancer cell count observed in response to P. javanica and

E. nummularius methanolic extract in in vitro and P. javanica methanol extract in vivo was

due to apoptosis, the following cellular changes that occur during apoptosis was studied.

5.3.7.1. Flow cytometry

For the determination of DNA degradation in apoptotic cells as sub- G1 peak in

DNA histogram, Cancer cells either treated/ untreated in vitro with P. javanica methanol

extract (MEPJ) were permeabilized and nuclear DNA was labeled with Propidium Iodide

(PI). Degradation of nuclear DNA was determined on fluorescence- activated cell sorting,

fluorescence detector equipped with 488nm argon laser source and 623 nm band pass filter

(linear scale) using Cell Quest software (Becton Dickinson Mountain View, CA

(Mookerjee et al., 2006). A total of 10,000 events were acquired and results were analyzed

using WinMDI 2.8 software. A histogram of DNA content (X axis, PI fluorescence) versus

counts (Y axis) has been displayed. To detect the changes in the plasma membrane that

occur during apoptosis, in both in vivo and in vitro MEPJ treated/ untreated cancer cells a

double-labeling system involving Annexin V and Propidium Iodide (PI) has been used. PI

and Annexin V-fluorescence (flous) was added directly to the culture medium. The

mixture was incubated for 15 minutes at 370C. Excess PI and Annexin V-fluos were then

washed off, and cells were fixed and then analyzed on flow cytometer (equipped with

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488nm argon laser source; 515 nm band pass filter for FITC fluorescence and 623 nm band

pass filter for PI fluorescence) using Cell Quest software. A total of 10,000 events were

acquired and the cells were properly gated for analysis. Annexin V bind specifically to

phosphatidylserine that was translocated to the outer leaflet of the membrane of apoptotic

cells. On the other hand, PI being a large molecular weight DNA binding dye cannot enter

intact cells without permeabilization treatments, do not label apoptotic cells until the final

lysis stage.

5.3.7.2. Confocal Microscopy

To confirm apoptosis by observing changes in nuclear morphology i.e., loss of

DNA integrity in vivo MEPJ treated/ untreated EAC/ Dox cells were harvested, fixed and

nuclear DNA was stained with PI (10 µg/ ml) for 15 minutes at room temperature. The PI

stained cells were observed under confocal laser scanning microscope (LSM 510; Carl

Zeiss, Jena, Germany) (Mookerjee et al., 2006).

5.3.7.3. Immunoblot analysis of apoptotic enzymes and some Bcl- 2 family proteins

involved in apoptosis

(a). Preparation of cell lysates:

Cells from 13 days post inoculated mice along with their untreated or control

counterpart were harvested, resuspended in chilled Tris-EGTA-EDTA-Me (TEEM)

buffer, and rapidly freeze-thawed thrice and passed through a 26-gauge needle (10 times)

for lysis (Braunsonic 1510, Germany). The lysates were centrifuged at (800 x g for 10 min

at 4°C) and subsequently, at 10000xg for 10 min at 4C to remove the debris. The

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supernatants were collected, and the proteins were estimated using Bradford reagent

(Bradford, 1976). The supernatants were mixed with Laemmli buffer, heated in a boiling

water bath for 5 min, and cooled to room temperature. For Western blot analysis, about

20μg of protein was subjected to electrophoresis on each lane of 13.5% sodium dodecyl

sulfate (SDS) poly acrylamide gels as described below.

(b). Western blot analysis:

Protein samples were electrophoresed in SDS polyacrylamide gel by the method of

Laemmli (1970). This technique consists of the following steps:

i) Preparation of gel slab: Gel was cast between glass plates (10cm x 10 cm) to a

height of about 5.5 cm using a spacer of 1 mm thickness. About 9 ml of gel

mixture [containing 13.5% (w/v) acrylamide (Sigma, USA), 0.33% (w/v) N,N'-

methylene- bis- acrylamide (Sigma, USA), 0.17 (w/v) SDS (Sigma, USA),

0.38% (w/v) potassium persulphate (Sigma, USA), 0.06% (v/v) of N,N,N',N'-

tetramethyl ethylene diamine (TEMED) (Sigma, USA) in 1.5 M Tris HCl

buffer, pH 8.8] was used to prepare the separating gel which was overlaid with

a few drops of water. After polymerization, the water layer at the top of the gel

was removed and stacking gel mixture [containing 4.5% (w/v) of acrylamide,

0.12% (w/v) of N,N´-methylene bis- acrylamide, 0.1% (w/v) of SDS, 0.03%,

(w/v) of potassium persulphate, 0.1% (w/v) of potassium persulphate, 0.1%

(v/v) TEMED in 0.5M Tris-HCl buffer, pH 6.8] was applied on the top of the

separating gel. A comb was then inserted into the stacking gel mixture and the

gel was allowed to polymerize. Following polymerization of the stacking gel,

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the comb was removed and slots, thus formed, were washed with

electrophoresis buffer pH 8.3.

ii) Electrophoresis: Protein samples, prepared for SDS- PAGE, were

electrophoresed in a vertical mini slab gel unit (Atto corp. Japan) at room

temperature by applying constant voltage (60V initially for about 10- 15 min

which was subsequently increased to 120 V). Electrophoresis continued for

about 2.5 hr till the tracking dye reached approximately 1 cm above the bottom

of the gel. For each electrophoresis run, one lane contained a mixture of

prestained marker.

iii) Electroblotting: Following electrophoresis, the gel was removed from casting

plates and equilibrated with the blotting buffer for about 10 min. After

equilibration, the electrophoresed gel was transferred to thick scotch- brite pads

which were presoaked with blotting buffer for 20 min. Next, polyvinylidene

difluoride membrane (PVDF) (0.2 µM pore size, Sigma, USA) of appropriate

size (presoaked in methanol followed by distilled water) moistened in buffer,

was carefully placed on the surface of the gel (containing the electrophoresed

material) avoiding entrapment of air bubbles. Several layers of the presoaked

pads (Scotch brite) were then placed over the membrane. The complete

sandwich (keeping the membrane toward the anode) was then put into a

transblot apparatus and the latter was subsequently filled in with the blotting

buffer. Protein components separated in the electrophoresed gel were then

transferred electrophoretically onto the PVDF membrane soaked in methanol

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for 1 minute, washing with 10mM Tris- HCl buffer (pH 7.4) and kept for

immunostaining.

iv) Immunostaining: following electroblottting, blotted membrane was treated with

a solution of 3% (w/v) of BSA (Sigma, USA) in 10 mM Tris- HCl buffer (pH

7.4) for overnight at 40C or for 2h at 370C to block the remaining unbound sites.

The membrane was then washed thrice with TBS (pH 7.4) containing 0.05%

(v/v) Tween 20 (TBS- Tween 20). Next, the membrane was incubated with

specific antibody, appropriately diluted for 3- 4h at room temperature under

mild shaking condition or overnight at 40C. Following incubation, the blotted

membrane was washed thoroughly with TBS- Tween 20 followed by washing

in TBS and, subsequently, treated with appropriately diluted specific secondary

antibody conjugated to alkaline phosphatase (AP) (Table 1) for 2 h at room

temperature under mild shaking condition. Following incubation with the

secondary antibody, the membrane was thoroughly washed with TBS- Tween

20 and the blot was developed subsequently by treatment with the appropriate

substrate solution (Table2).

5.3.8. Determination of Immunomodulatory activity of plant methanolic extracts

Immunomodulatory activity of plant extracts was determined by two methods-

lymphocyte proliferation assay and antigen presentation assay.

5.3.8.1. Antigen presentation assay

To study the antigen presenting function, ovalbumin peptide specific LB cells, was

kept either untreated or treated with Parkia javanica and Evolvulus nummularius methanol
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extract at 1, 5 and 10 µg/ ml concentration for 24 hr. The antigen presenting cells (APCs),

LB at the concentration of 2x 104 cells/ well was incubated for 24 hr with OVA257- 264

peptide (1mg/ ml) and T cell hybridoma 7.13 (1x 105 cells/ well) in complete RPMI 1640

medium in a 37°C incubator. The culture supernatants were analyzed for the presence of

IL-2 by growing an IL-2 dependent cell line, HT-2 at the concentration of 1x 104 cells/

well in the supernatants. HT-2 (104/well) was incubated with a 50% concentration of

culture supernatant for 24 hr. The cells were then pulsed with 1 µCi of [3H] thymidine for

the last 18 hr (Roy et al., 1989). The incorporation of radioactive thymidine was assessed

by a scintillation counter (TRI CARB 2100TR, Packard) (Chakraborty et al., 2005).

5.3.8.2. Lymphocyte proliferation assay

(a). Preparation of mononuclear cell from spleen:

EAC/Dox- bearing mice (untreated or MEPJ treated) were euthanized, and their

spleen was removed. Isolated spleen was macerated between the frosted ends of two sterile

glass slides. Splenocytes suspension in phosphate buffered saline (PBS) was laid over

Ficoll Hypaque and density gradient centrifugation was done at 1600 rpm for 20 min. The

cells in the interface were collected and washed twice in PBS and used as splenic

mononuclear cells.

(b). Assay methodology:

Lymphocyte proliferation experiments were carried out in vitro in 96-well tissue

culture plates, each well of which contained 2 x 105 cells in 200 µl culture. Cells were

stimulated with concanavalin A (2.5 µg/ml) or a combination of PMA (20 ng/ml) and

ionomycin (500 ng/ml) for 48 hours at 370C under 5% CO2. Unstimulated (control)
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cultures did not receive any concanavalin A or PMA plus ionomycin. Next, cell

suspensions were pulsed with [3H] thymidine (0.5 µCi/ well) for another 20 hours

(Mookerjee et al., 2003). Cells were harvested on glass fiber filter papers (Whatman,

Maidstone, United Kingdom) by using a cell harvester (Nunc, Roskilde, Denmark), and

incorporation of [3H] thymidine was measured by a liquid scintillation counter (TRI CARB

2100TR, Packard) (Mookerjee et al., 2006; Mookerjee Basu et al., 2006).

5.4. RESULTS

5.4.1. In vitro antiproliferative activity of methanol extract of P. javanica (MEPJ) and

E. nummularius (MEEN) against four different cancer cell lines

Results of in vitro growth inhibitory effect study of P. javanica methanol extract

(MEPJ) are presented in Table 5.1., Fig. 5.1. and 5.2. The response was dose dependent.

The growth inhibitory effect was more prominent against K562 and CHO (>70%) than

against U937 (~35%) at a dose of 25 μg/ ml (p<0.001). Interestingly, MEPJ inhibited the

proliferation of doxorubicin resistant human lymphoblastic leukaemia CEM/ADR 5000

cells by >94% (p<0.001) at 10 µg/ml dose (Table 5.1). Drug resistant cells were more

sensitive compared to drug sensitive cancer cells and this is an significant observation

since drug resistance is the main problem for cancer chemotherapy and worldwide search

for new drug with minimal toxicity is on the way.

Results of in vitro anticancer study of E. nummularius methanol extract (MEEN)

are presented in Table 5.2., Fig. 5.3. and 5.4. The dose dependent response was also
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observed against MEEN. The growth inhibitory effect The decrease in response to MEEN

was observed to be 54.6% (p= 0.001), 58.59% ( p= 0.001) and 16.3% (p= 0.001) for

K562, U937 and CHO respectively at a dose of 50 µg/ml.

The activity of MEEN was also tested against doxorubicin resistant cell line

CEM/ ADR 5000. Here also, similar to P. javanica extract, maximum anti-proliferative

activity (>99%, p= 0.001) was observed against this cell line. However the response was

achieved at two fold higher dose compared to that of P. javanica.

5.4.2. Induction of apoptosis of cancer cells in-vitro by MEPJ

To study whether the decrease in cancer cells was due to apoptosis or not flow

cytometric analysis was done. Since P. javanica extract showed better results compared to

E. nummularius regarding anti proliferative activities, further studies were carried out with

P. javanica extract.

Results of flow cytometric analysis (Fig. 5.5.A) revealed that MEPJ treatment in

vitro, at 50µg/ml concentration, increased K562 cell population (22.2%), U937 cell

population (17.2%) and doxorubicin resistant CEM/ADR 5000 cell population (31.8%)

showing sub G1 peak compared to 0.8%, 0.4% and 1.8% untreated control respectively.

Which points towards that MEPJ induces apoptosis in these cancer cells when given in

vitro.

Next apoptosis was further confirmed as the mode of cell death in cancer cells on

MEPJ treatment as reflected by increase in number of Annexin-V positive cells in K562

(34.7%), U937 (23.2%) and CEM/ADR 5000 (43.7%) compared to 1.9%, 2.3% and 1.4%

in untreated control reapectively (Fig. 5.5B).

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5.4.3. Anti-cancer activity of MEPJ in vivo

To study anti-cancer activity in vivo Swiss albino mice were taken. One group of

animal was mock treated or MEPJ untreated control. The other groups were trated with

MEPJ at four different concentrations viz., 500, 100, 20 and 2 mg/kg body weight through

three different routes (i.e., oral, intramuscular (im) and intraperitoneal (ip). The in-vivo

treatment results reveal that 20 mg/kg body weight dose was the effective dose Table 5.3 a

and Fig. 5.6. 500 and 100 mg/kg doses were not well tolerated by EAC/Dox bearing mice

as observed feebleness and shedding of hair of the animals within 7 days of treatment as

shown in Table 5.3 a. Intra peritoneal (i.p.) route was found to be more effective than i.m.

or oral routes Interestingly 20 mg/kg of MEPJ administration through i.p. route was

observed to give protection of about 99% as shown in Table 5.3 b and increased survivality

of peritoneal EAC/Dox bearing mice as shown in Fig. 5.7 A. Whereas MEPJ

administration by two other routes i.e., oral and i.m. did not show much protection (Table

5.3 b). The 2mg/kg dose through i.p. route gave negligible protection (~10 %) (Table 5.3

a).

To know whether the protection offered by MEPJ was due to apoptosis of the

tumour cells, double labeling technique involving Annexin V-FITC and PI was used in in

vivo treated animals. Results show much higher increase in percentage (4.3% Vs 59%) of

both Annexin V and PI positive EAC/Dox cells in MEPJ treated mice in comparison to

untreated one. Annexin V positive EAC/Dox cells also increased in MEPJ treated mice

with respect to untreated control (~15% Vs 2.3%) as shown in Table 5.4 and Fig. 5.7 b.

Apoptotic cells were also observed under confocal microscope as shown in Fig. 5.8.
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5.4.4. Western blot analysis of pro and anti-apoptotic protein

Since evidence for induction of apoptosis by MEPJ was observed in the above

experiment, immunoblot analysis was done for apoptosis specific proteins. Lysate of

EAC/Dox cells were prepared from remnant ascites of mice treated with 20mg/kg of MEPJ

(14 days post treatment) and corresponding vehicle control group. Proteins were resolved

on a denaturing PAGE, transferred to PVDF membrane and probed with antibodies of

interest.

Immunoblot analysis showed down-regulation of the anti-apoptotic protein, Bcl2 in

EAC/Dox cells derived from mice treated with 20mg/kg MEPJ when compared to control

(Fig. 5.9). An up regulation of expression of Bad and Bax (pro apoptotic protein) was

observed (Fig. 5.9). These results further confirm the activation of apoptosis upon

treatment with MEPJ.

Since activation of Caspase 3 is a hallmark of activation of apoptotic pathway,

cleavage of Caspase 3 and Caspase 9 was tested by western blot analysis. It was observed

that there is clear increase of cleavage of Caspase 3 and Caspase 9 in EAC/Dox cells from

in vivo treated mice with respect to mock treated control (Fig. 5.9). Re-probing with anti-

beta actin antibody, the loading control, confirmed that the amount of protein loaded was

equal in all the experiments (Fig. 5.9). Therefore, immune blot analysis suggests that in

vivo treatment of MEPJ induces expression of pro-apoptotic proteins and down-regulation

of anti-apoptotic proteins to induce apoptosis in EAC/Dox cell, which might involve

mitochondrial apoptotic pathway involving Caspase 9 activation.

5.4.5. Immunomodulatory study

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5.4.5.1. Antigen presenting function of MEPJ and MEEN

In order to study immunomodulatory property, it was tested whether P. javanica

and E. nummularius methanol extracts could enhance antigen presenting function of

antigen presenting cells LB (OVA peptide specific B cell hybridoma) in vitro. It was

observed that both the extracts enhanced class II restricted antigen presentation

significantly (Table 5.5 and Fig. 5.10). In case of P. javanica maximum enhancement was

observed at 5µg/ ml concentration, but E. nummularius extract enhanced the antigen

presenting function maximum at 1 µg/ ml concentration after which the activity declined.

Maximum enhancement was observed in E. nummularius treated samples.

5.4.5.2. Lymphoproliferation of splenic mononuclear cells isolated from MEPJ in vivo

treated animals

Since MEPJ could induce >99% elimination of EAC/Dox in vivo although it failed

to achieve such spectacular results in vitro (45%), it was tested whether it could reduce

cancer-induced suppression of cellular immune response in vitro. EAC/ Dox- bearing mice

manifested severe suppression of lymphoproliferative response toward stimulation with a

combination of PMA and ionomycin (PMA + ionomycin) or concanavalin A. However in

vivo treatment with MEPJ could induce proliferation of both splenic mononuclear cells

(SPMC) (85.84 % compared with normal values, p=0.001) although it marginally inhibited

lymphoproliferation in response to in vitro treatment with the T-cell mitogen, concanavalin

A but significant (p=0.001, Fig.5.11). Intriguingly, SPMC from MEPJ treated EAC/ Dox

bearing mice failed to proliferate in response to in vitro treatment with a combination of

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phorbol ester and ionomycin. In vivo treatment with MEPJ reverses the suppression of

lymphoproliferation in EAC/ Dox bearing mice.

However, in vivo treatment with MEPJ itself could induce proliferation of splenic

mononuclear cells (SPMC) in EAC/Dox bearing animals (Fig.5.11) although it marginally

inhibited lymphoproliferation in response to in vitro treatment with the T-cell mitogen,

concanavalin A. Intriguingly, SPMC from MEPJ treated EAC/Dox bearing mice failed to

proliferate in response to in vitro treatment with a combination of phorbol ester and

ionomycin.

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Chapter 5 Anticancer and Immunomodulatory Studies

Table 5.1. In vitro Antiproliferative activity of P. javanica methanol extract

expressed as mean cell counts per min against four different cancer cell lines.
Dose Mean counts per minute (cpm) % decrease in
Cell line
(μg/ml) ± SD cell count
0 35896.5 ± 120.03
1 34101.68 ± 55.3*** 5%
5 27999.27 ± 142*** 22%
K562
10 16334.7 ± 45.7*** 54 %
25 13750 ± 119.15*** 61.8 %
50 10009 ± 209.02*** 72 %
0 38415.5 ± 286.59
1 35918.49 ± 23.78** 6.5%
Chinese Hamster 5 29579.94 ± 175** 23%
Ovary (CHO) 10 16839 ± 336.81*** 56 %
25 11176 ± 85.73*** 70.9 %
50 10935 ± 36.76*** 71.5 %
0 35853.6 ± 87.6
1 35136.53 ± 55*** 2%
5 33343.85 ± 83*** 7%
U937
10 24526.67± 186.25*** 31.6 %
25 23581.4 ± 49.8*** 34.2 %
50 27823.5 ± 103.38*** 35.4 %
0 48615.2 ± 238.42
1 36461.4 ± 58.75*** 25%
5 20418.38 ± 84*** 58%
CEM/ADR5000
10 2499.8 ± 87.74*** 94.8 %
25 2904 ± 41.01*** 94 %
50 2056.5 ± 230.66*** 95.77 %
Statistical analysis was done using student’s T- test. *, ** and *** represent significant
differences of the extract treated compared to control at the level of p= 0.05, p= 0.01 and
p= 0.001 respectively.

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Table 5.2. In vitro antiproliferative activity of E. nummularius methanol extract

expressed as mean cell counts per min against four different cancer cell lines
Cell line Dose Mean counts per minute (cpm) ± % decrease
(μg/ml) SD
K562 0 35896.5 ± 335.16
1 35178.57 ± 59.12* 2%
5 33670.92 ± 125** 6.2%
10 32654 ± 110.3** 9%
25 22668 ± 38.37*** 36.8 %
50 16284 ± 83.43*** 54.6 %
Chinese Hamster 0 39108.35 ± 65.71
Ovary (CHO)
1 38521.73 ± 97.6 1.5%
5 37935.1 ± 184.2** 3%
10 35218 ± 267.28** 9.94 %
25 32760 ± 69.29*** 16.23 %
50 32732 ± 82.02*** 16.3 %
U937 0 35853.6 ± 87.6
1 35600 ± 14* 0.7%
5 35136.53 ± 83.69*** 2%
10 33439.13± 204.51*** 6.73 %
25 27390.4 ± 69.51*** 23.6 %
50 14844.4 ± 105.14*** 58.59 %
CEM/ADR5000 0 48603 ± 529
1 33890.87 ± 237.8*** 30.27%
5 20413.26 ± 162*** 58%
10 8508 ± 288.45*** 82.49 %
25 330.93 ± 10.56*** 99.31 %
50 341.6 ± 37.4*** 99.29 %
Statistical analysis was done using student’s T- test. *, ** and *** represent significant
differences of the extract treated compared to control at the level of p= 0.05, p= 0.01 and
p= 0.001 respectively.
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Table 5.3. a. Effect of methanol extract of P. javanica (MEPJ) injected intra

peritoneally (i.p.) at two different doses into EAC/ Dox bearing mice in decreasing

ascetic fluid volume and cell number after 29 days of inoculation.

Animal group Volume of Total number of % Protection

ascetic fluid EAC/ Dox cells
(ml) (x106)

Mock treated 7 ± 1.5 8000 ± 0.05

MEPJ (2 mg/kg b.w.) 6.8 ± 1.25 7140 ± 1 10.75%

MEPJ (20 mg/kg b.w.) 0.1 ± 0.28 10 ± 0.08 99.8%

MEPJ (100 mg/kg Dose not Dose not tolerated Dose not
b.w.) tolerated tolerated

MEPJ (500 mg/kg Dose not Dose not tolerated Dose not
b.w.) tolerated tolerated

Table 5.3 b. Effect of methanol extract of P. javanica (MEPJ) at 20 mg/ kg body

weight administered through three routes on EAC/ Dox bearing mice in

decreasing ascetic fluid volume and cell number after 29 days of inoculation.

Animal group Volume of Total number of % Protection

ascetic fluid (ml) EAC/ Dox cells
(x106)

Mock treated 7 ± 1.5 8000 ± 0.05

MEPJ oral treated 6±1 7800 ± 1 2.5%

MEPJ i.m. treated 3 ± 0.5 4030 ± 5 49.625%

MEPJ i.p. treated 0.1 ± 0.28 10 ± 0.08 99.8%

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Table 5.4. Flow cytometric data of double staining analysis of EAC/ Dox cells

collected from peritoneum of in vivo treated animals (7 days post treatment).

Animal group % Positive cells

FL1-H positive Double positive

Mock treated 2.3 % 4.3 %

Treated (20mg/kg) 15.6 % 59.5 %

Table 5.5. Antigen presentation data of in vitro plant methanol extract treated.

Plant methanol extract Concentration (µg/ ml) Cell count per minute ± SD
Positive control (with 0 3245 ± 113
OVA peptide)
P. javanica 1 7453 ± 235***
5 12091 ± 137***
10 7026 ± 85***
E. nummularius 1 35071 ± 435***
5 9084 ± 119***
10 8180 ± 399**
Statistical analysis was done using student’s T- test. All the values are significant at p=
0.001 compared to the control.

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Table 5.6. Effect of P. javanica methanol extract on proliferation of splenic

mononuclear cells (SPMC) derived from in vivo treated animal 22 days post
treatment. Result expressed as mean cell count per minute (CPM) ± SD.

Stimulant Normal Swiss EAC/ Dox bearing P. javanica treated

albino (N) mice (RD) EAC/ Dox bearing
mice (MEPJ)

Unstimulated 4800 ± 172 6710.67 ± 571* 33892 ± 510***

Con A 45120 ± 525 8606.67 ± 550*** 24759.6 ± 900***

PMA+ Io 35102 ± 255 1952 ± 525.221*** 6839 ± 11**

Statistical analysis was done using student’s T- test. ** and *** represent significant
differences of the extract treated compared to untreated control at the level of p= 0.01 and
p= 0.001 respectively.

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Fig. 5.1. Inhibition of proliferation of various cancer cells

by different in vitro doses of methanol extract of P. javanica.

Fig. 5.2. Dose dependent response of MEPJ on K562,

CHO, U937 and CEM/ ADR 5000 treated in vitro.

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Fig. 5.3. Inhibition of proliferation of various cancercells

by different in vitro doses of methanol extract of E. nummularius.

Fig. 5.4. Dose dependent response of MEEN on K562,

CHO, U937 and CEM/ ADR 5000 treated in vitro.

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Fig. 5.5. Methanol extract of P. javanica induces apoptosis (in vitro) in various human

cancer cell lines. A) Cell cycle analysis (the numbers indicate sub G0/G1 population).

B) Assessment of apoptosis by double staining of unfixed cells with propidium iodide

and annexin-V by flowcytometry (the numbers indicate the percent positive cells in

respective quadrant).

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Fig. 5.6. Intraperitoneal injection of the extract at a dose of 20mg/kg body weight

was observed to achieve >99% protection in mice bearing EAC/Dox cells

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Fig. 5.7. Methanol extract of P. javanica overcomes doxorubicin resistant cancer

(EAC/Dox) in vivo. 7 days post inoculation of EAC/Dox cells mice were treated intra

peritonially with methanol extract of P. javanica at a dose of 20mg/kg as described in

materials and methods A) suvivality of the animals were studied up to 120 days. B)

After 7 days of completion of treatment the apoptosis of EAC/Dox cells were studied

by double staining of unfixed cells with propidium iodide and annexin-V by

flowcytometry (the numbers indicate the percent positive cells in respective

quadrant).
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Fig. 5.8. Confocal microscopic photographs showing induction

of apoptosis in EAC/Dox cells treated (in vivo) with P. javanica extract.

A) Untreated EAC/Dox cells stained with (PI). B) Treated EAC/Dox

cells stained with PI.

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Fig. 5.9. Western blot analysis of Bcl2, Bad, Bax, Caspase 9 and Caspase 3. EAC/Dox

bearing mice were either kept mock treated (Control) or treated with methanol

extract of P. javanica. On 7th day post treatment one group of animal was sacrificed

and isolated EAC/Dox cell from remaining ascites were collected and performed the

western blot analysis for above molecules.

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Fig. 5.10. P. javanica and E. nummularius methanol extract enhances MHC

class II mediated antigen presentation.

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Fig. 5.11. Effect of P. javanica methanol extract (MEPJ) treatment in vivo on

proliferation of splenic mononuclear cells (SPMC) derived from EAC/ Dox – bearing

mice. Cells from MEPJ treated mice were either kept untreated or treated in vitro

with PMA + ionomycin (Io) or concanavalin A (Con A). Results were compared with

normal (N) and in vivo mock treated EAC/Dox-bearing Swiss albino mice (RD).

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5.5. DISCUSSION

A tumor is a disease state characterized by a proliferation disorder and an apoptosis

obstacle. Inducing apoptosis is an efficient method of treating cancers (Hu & Kavanagh,

2003). The present set of investigations was therefore initiated to study the anticancer

potential of Methanolic extract of Parkia javanica (MEPJ) and Methanolic extract of

Evolvulus nummularius (MEEN) in vitro against different cancer cell lines (including

conventional drug resistant cancer line).

The present study showed that both the extracts were effective in imparting growth

inhibition in in vitro against various human cancer cell lines (including conventional drug

resistant lymphoblastic leukemia). Dose dependent response was observed.

The growth inhibitory effect of MEPJ was more prominent against K562 and CHO

(>70%, p<0.001) than against U937 (~35%, p<0.001) at a dose of 25 μg/ ml. Growth

inhibitory effect in response to MEEN was observed to be 54.6% (p< 0.001), 58.59% ( p<

0.001) and 16.3% (p<0.001) for K562, U937 and CHO respectively, at a dose of 50 µg/ml.

Interestingly, both MEPJ and MEEN inhibited the proliferation of doxorubicin

resistant human lymphoblastic leukaemia CEM/ADR 5000 cells by >94% (p<0.001) at 10

µg/ml and 25 µg/ml dose respectively. Regarding dose, MEPJ exhibited two fold higher

response compared to MEEN.

Drug resistant cells were more sensitive compared to drug sensitive cancer cells

and this is a significant observation since drug resistance is the main problem for cancer

chemotherapy and worldwide search for new drug with minimal toxicity is on the way.

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Apoptosis, or programmed cell death, is an essential event that plays an important

role in organism development (Hidalgo & Ffrench-Constant, 2003); (Vaux & Korsmeyer,

1999) and homeostasis (Kucharczak et al., 2003); (Cory et al., 2003); (Reed, 2001).To

study whether the decrease in cancer cells was due to apoptosis, flow cytometric analysis

was done in MEPJ treated cells. Results pointed towards that MEPJ induced apoptosis in

these cancer cells when given in vitro.

In vivo assay was performed in Swiss albino mice and Intra peritoneal (i.p.) route was

found to be more effective compared to other routes. Twenty mg/kg of MEPJ

administration through i.p. route was observed to give protection of about 99% and

increased survivality of EAC/Dox bearing mice. Again, confocal microscopic and flow

cytometric analysis confirmed induction of apoptosis in tumour cells.

Therefore, MEPJ was found to be very effective inducer of apoptosis in doxorubicin

resistant CEM/ADR 5000 and at a dose of 20mg/kg MEPJ could overcome doxorubicin

resistant EAC/Dox ascetic cancer (~99%) by 30 days of post treatment and effectively

induced apoptosis of EAC/Dox cells.

The potential mechanism that directs a cell to undergo apoptosis exists in a balance

between apoptosis induction factors and apoptosis inhibition factors. A search for a safe

agent that enhances the levels of expression of tumor suppressor proteins is a worthwhile

but relatively under-explored approach towards cancer therapy. The present study reveals

that MEPJ enhances expression of pro apoptotic molecules like Bad and Bax on the other

hand down regulated expression of anti-apoptotic protein Bcl2 in EAC/Dox cells. Bax,

being a Bcl-2 family member, not only promotes apoptosis but also counters the protective
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effect of survival molecule Bcl-2 (Lee et al., 2001). In fact, over-expression of Bax, has an

effect that is associated with the formation of Bax/Bax homodimers, has been shown to

accelerate the cell death of murine FL5.12 cells after interleukin-3 withdrawal (Oltvai et

al., 1993).

The intrinsic pathway of apoptosis is tightly controlled at mitochondria by Bcl-2

family proteins. Specifically, these proteins regulate the permeability of the mitochondrial

outer membrane and thereby control the release of multiple apoptogenic molecules from

the intermembrane space (Danial et al., 2004.). Since MEPJ enhances expression of Bad

and Bax on the other hand down regulated expression of Bcl2 in EAC/Dox cells therefore

involvement of mitochondria has been reconfirmed by the Caspase 9 the cleavage in

observed death pathway. Finally western blot analysis of EAC/Dox cells derived from

mice treated with MEPJ suggested activation of hall mark of apoptosis Caspase 3.

The immunomodulatory study is concerned with stimulation in Ag-presenting

ability of LB cells by the plant methanol extracts. The study revealed that both plant

extracts could enhance antigen presentation of LB cells to class II restricted 7.13 T cells.

Enhancement of antigen presentation to T cell is important immunomodulatory property of

any drug/ extract. Out of the two extracts, maximum enhancement was observed in E.

nummularius treated antigen presenting cells.

Methanol extracts of P. javanica was found to induce gradual reversal of

immunosuppression as evidenced by induction of lymphoproliferation indicating the

immunomodulating property of the extract.

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In vivo treatment with MEPJ reversed the suppression of lymphoproliferation in

EAC/ Dox bearing mice. This indicated that MEPJ was not toxic for normal cells.

Moreover, it may also activate the immune cells besides directly killing the cancer cells.

As mentioned earlier, the phytochemical analysis rvealed the presence of ursolic

acid (pentacyclic triterpene acid), iridoid glucosides, beta-sitosterol in MEPJ. All the

compounds are reported to possess anti-tumor and immunomodulatory properties (Han et

al., 1988; Lee et al., 1988; Numata et al., 1990; Huang et al., 1994; Liu, 1995; Es-Saady et

al., 1996; Choi et al., 2000; Law, 2000; Plat et al., 2000; Awad et al., 2000; Konoshima et

al., 2000; Bouic, 2001; Andersson et al., 2003; Salminen et al., 2008). Induction of

apoptosis in cancer by these compounds are also reported. Therefore the plant shown to

possess important anti cancer and immunomodulatory principles with proved anti tumor

and immunomodulatory activities as observed in the present study.

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