Chapter 5 Anticancer and Immunomodulatory...
Transcript of Chapter 5 Anticancer and Immunomodulatory...
Chapter 5 Anticancer and Immunomodulatory Studies
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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), 2g/ml N-p-Tosyl-
L-phenylalanine chloromethyl ketone
(TPCK),2g/ml Nα-p-tosyl-L-lysine chloromethyl
ketone (TLCK), 5g/ml Leupeptin, 5g/ml
Aprotinin, 5g/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
(Santa Cruz Biotech., USA) at 1: 500
Rabbit antigoat IgG- AP (Sigma, USA)
at 1: 10000
Caspase- 9 rabbit polyclonal antibody
(Cell Signaling, Germany) at 1: 1000
Goat antirabbit IgG- AP (Sigma, USA)
at 1: 10000
Bad rabbit polyclonal antibody (Cell
Signalling, Germany) at 1: 1000
Goat antirabbit IgG- AP (Sigma, USA)
at 1: 10000
Bax mouse monoclonal antibody
(Oncogene) at 1: 300
Goat antimouse IgG- AP (Sigma, USA)
at 1: 10000
Bcl2 rabbit polyclonal antibody (Santa
Cruz Biotech, USA) at 1: 300
Goat antirabbit IgG- AP (Sigma, USA)
at 1: 10000
18. Substrate solution.
Enzyme conjugated
to secondary
antibody
Reaction buffer Substrate Co substrate
Alkaline
phosphatase (AP)
100 mM Tris
HCl (pH 9.5),
containing
100mM NaCl
and 50 mM
MgCl2
Nitrotetrazolium
blue (NBT) at a
final concentration
of 33 µg/ ml
5- bromo- 4-
chloro- 3- indolyl
phosphate (BCIP)
added to a final
concentration of
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, 100g/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 4C 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 37
0C 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)
Chapter 5 Anticancer and Immunomodulatory Studies
Page 20
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
Chapter 5 Anticancer and Immunomodulatory Studies
Page 21
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).
Chapter 5 Anticancer and Immunomodulatory Studies
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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.
Chapter 5 Anticancer and Immunomodulatory Studies
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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
Chapter 5 Anticancer and Immunomodulatory Studies
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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
Chapter 5 Anticancer and Immunomodulatory Studies
Page 25
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.
Chapter 5 Anticancer and Immunomodulatory Studies
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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.
Cell line Dose
(μg/ml)
Mean counts per minute (cpm)
± SD
% decrease in
cell count
K562
0 35896.5 ± 120.03
1 34101.68 ± 55.3*** 5%
5 27999.27 ± 142*** 22%
10 16334.7 ± 45.7*** 54 %
25 13750 ± 119.15*** 61.8 %
50 10009 ± 209.02*** 72 %
Chinese Hamster
Ovary (CHO)
0 38415.5 ± 286.59
1 35918.49 ± 23.78** 6.5%
5 29579.94 ± 175** 23%
10 16839 ± 336.81*** 56 %
25 11176 ± 85.73*** 70.9 %
50 10935 ± 36.76*** 71.5 %
U937
0 35853.6 ± 87.6
1 35136.53 ± 55*** 2%
5 33343.85 ± 83*** 7%
10 24526.67± 186.25*** 31.6 %
25 23581.4 ± 49.8*** 34.2 %
50 27823.5 ± 103.38*** 35.4 %
CEM/ADR5000
0 48615.2 ± 238.42
1 36461.4 ± 58.75*** 25%
5 20418.38 ± 84*** 58%
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.
Chapter 5 Anticancer and Immunomodulatory Studies
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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
(μg/ml)
Mean counts per minute (cpm) ±
SD
% decrease
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
Ovary (CHO)
0 39108.35 ± 65.71
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.
Chapter 5 Anticancer and Immunomodulatory Studies
Page 28
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
ascetic fluid
(ml)
Total number of
EAC/ Dox cells
(x106)
% Protection
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
b.w.)
Dose not
tolerated
Dose not tolerated Dose not
tolerated
MEPJ (500 mg/kg
b.w.)
Dose not
tolerated
Dose not tolerated Dose not
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
ascetic fluid (ml)
Total number of
EAC/ Dox cells
(x106)
% Protection
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%
Chapter 5 Anticancer and Immunomodulatory Studies
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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
OVA peptide)
0 3245 ± 113
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.
Chapter 5 Anticancer and Immunomodulatory Studies
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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
albino (N)
EAC/ Dox bearing
mice (RD)
P. javanica treated
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.
Chapter 5 Anticancer and Immunomodulatory Studies
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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.
Chapter 5 Anticancer and Immunomodulatory Studies
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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.
Chapter 5 Anticancer and Immunomodulatory Studies
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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).
Chapter 5 Anticancer and Immunomodulatory Studies
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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
Chapter 5 Anticancer and Immunomodulatory Studies
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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).
Chapter 5 Anticancer and Immunomodulatory Studies
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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.
Chapter 5 Anticancer and Immunomodulatory Studies
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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.
Chapter 5 Anticancer and Immunomodulatory Studies
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Fig. 5.10. P. javanica and E. nummularius methanol extract enhances MHC
class II mediated antigen presentation.
Chapter 5 Anticancer and Immunomodulatory Studies
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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).
Chapter 5 Anticancer and Immunomodulatory Studies
Page 40
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.
Chapter 5 Anticancer and Immunomodulatory Studies
Page 41
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
Chapter 5 Anticancer and Immunomodulatory Studies
Page 42
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.
Chapter 5 Anticancer and Immunomodulatory Studies
Page 43
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.