EFFECTS OF WITHANIA SOMNIFERA AND TINOSPORA CORDIFOLIA EXTRACTS ON SIDE POPULATION PHENOTYPE

4 CHAPTER FOUR EFFECTS OF WITHANIA SOMNIFERA AND TINOORA CORDIFOLIA EXTRACTS ON SIDE POPULATION PHENOTYPE 68

4.1 INTRODUCTION Recent studies suggest that cancer is a stem cell disorder. Indeed a small population of cancer stem cells have been identified within several malignancies that both functionally and pehnotypically resemble normal stem cells. Cancer stem cells have self renewal and differentiation ability, like normal stem cells and only these cells have been proposed to possess the ability to initiate and maintain the tumor. Cancer stem cells were first documented in hematological malignancies where only small subsets of leukemic cells were capable of forming new tumors (Lapidot et al., 1994). Following the suit, such a small population was reported in breast cancer (Al-Hajj et al., 2003), brain tumor (Singh et al., 2003), multiple myeloma (Matsui et al., 2004), pancreatic cancer (Li et al., 2007), colon cancer (O Brien et al., 2006), head and neck squamous cell carcinoma (Prince et al., 2007), melanoma (Fang et al., 2005), prostate cancer (Collins et al., 2005) etc. One of the properties of stem cells commonly exploited to isolate them is the overexpression of drug efflux pumps on their cell membrane. These pumps refer to the group of ATP Binding Cassette (ABC) transporters. Stem cells from multiple tissues have been identified based on their ability to efflux the lipophilic dye Hoechst 33342 utilizing the ABC transporters (Goodell et al., 1996). Hoechst 33342 is a fluorescent dye which binds to the AT regions of DNA. Typically, this molecule is excited at 352 nm and emits at 440-460 nm with a weak emission in the red range. When cells are stained with Hoechst 33342 and analyzed by a flow cytometer equipped with a UV laser, while most of the cells show high emission in a flow cytometry plot (with red emission on x- axis and blue emission on y-axis), stem cells appear as a hypodiploid, low fluorescing population. The low florescent population, which appears on the far left side of a cytometry dot plot has been termed as the side population, or (Goodell et al., 1997). Side population technique was first used for the isolation and characterization of hematopoietic stem cells from C57BL/6 mouse bone marrow and demonstrated that the is highly enriched for stem cells, as it has the ability to reconstitute both lymphoid and myeloid lineages and also enriched at least 1,000-fold for in vivo reconstitution in lethally irradiated mice (Goodell et al., 1996). The Hoechst 33342 based side population () assay has become a critical assay for the identification and isolation of mammalian stem cells (Willan and Farnie, 2011). has been found to be highly enriched in normal stem cells and cancer stem cells (Golebiewska et al., 69

2011). One of the major mediators of side population phenotype seems to be ABCG2 or BCRP, which was initially identified in drug selected MCF7 breast cancer cells and later found to efflux multiple chemotherapeutic drugs and xenobiotics (Zhou et al., 2002). The strongest evidence linking ABCG2 and the side population phenotype comes from the nearly complete loss of the bone marrow side population phenotype in ABCG2 knockout mice (Lechner et al., 2002). Both side population and known stem or progenitor cells also express other ABC transporters such as MDR-1 (ABCB1 or P-glycoprotein), MRP-1 (ABCC1), and ABCA2 suggesting that these molecules may also be involved in mediating the side population phenotype (Britton et al., 2012). It has been reported that cultured human cancer cells and xenograft tumors possess a detectable side population (Christgen et al., 2012). Purified side population cells were found to be more tumorigenic than the corresponding non side population cells (Golebiewska et al., 2011). These side population cells also possess some intrinsic stem cell properties as they were found to generate non side population cells which form the bulk of cancer cells in vivo. These could also be further transplanted, and were found to preferentially express some stemness genes, including molecules of the Wnt, Notch, and Hedgehog pathways (Shi et al., 2012). Therefore, Hoechst 33342 based flow cytometry assay (or assay ) serves as a unique tool to isolate cancer stem cells. In the current study, we have first investigated whether the efflux phenomenon is applicable to other human cancer cells, specifically human epithelial cancer cell lines in longterm cultures. In this direction, a panel of cancer cell lines was critically evaluated for existence of side population. As part of this study, we have investigated the cancer stem cell inhibitory effects of two indigenous medicinal plants, Withania somnifera of the family Solanacea and Tinospora cordifolia of the family Menispermacea. These medicinal plants are extensively used in the Indian system of medicine for various ailments including cancer. Moreover, these medicinal plants are widely cultivated in various parts of India as a medicinal plant and the process for collection and identification of these plants is simple. Preliminary pharmacological characterization of Withania somnifera and Tinospora cordifolia extracts for potential anticancer activity revealed that ethanolic extracts of Withania somnifera and Tinospora cordifolia imparted cytotoxicity, induced apoptosis and possessed cell cycle specific inhibitory activities in human breast cancer cells (MDA MB 231, MCF7) and cervical cancer cells (HeLa) with less cytotoxicity in normal human epithelial cells (HaCaT). We have 70

raised the question whether ethanolic extracts of Withania somnifera and Tinospora cordifolia have any specific inhibitory activity against or cancer stem cells. In this direction, we have evaluated the pharmacological effects of ethanolic extracts of Withania somnifera and Tinospora cordifolia on the side population phenotype of human breast cancer cells. 4.2 MATERIALS AND METHODS 4.2.1 Drugs and chemicals Doxorubicin, Verapamil, Cyclosporin A, Fumetrimorgin C, Dulbecco s modified eagle medium (DMEM), Trypsin-EDTA, Hank s balanced salt solution (HBSS), HEPES buffer, Hoechst 33342 (Bisbenzimide Trihydrochloride), Propidium iodide and Thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased from Gibco (Invitrogen, USA). Dimethyl sulfoxide (DMSO) was purchased from Calbiochem. 4.2.2 Identification, collection and preparation of crude extracts of selected medicinal plants The stem part of Tinospora cordifolia and the roots of Withania somnifera were collected, authentified and the crude ethanolic extracts were prepared as described earlier (Section, 3.2.1). For biological studies, crude ethanolic extracts were dissolved in DMSO (Calbiochem) at 20 mg/ml. 4.2.3 Cell culture HeLa (human cervical carcinoma), A549 (human lung adenocarcinoma), MCF7, BT549, MDA MB231, MDA MB453, T47D (human breast carcinoma), CaCo2 (human colon carcinoma), G361, B6 (Melanoma-mouse origin), WM 266.4 (Melanoma-human), HBL-100 (human normal breast epithelial cells, immortalized) and rat C6 glioma cells were cultured in DMEM with high glucose containing 10% FBS and penicillin/streptomycin in a humidified CO 2 incubator at 37 C. 4.2.4 Side population analysis of C57BL/6 mouse bone marrow C57BL/6 mice (6-8 weeks of age) were sacrificed and bone marrow cells were isolated from femurs and tibias in pre-warmed HBSS. Nucleated cells were counted in an inverted microscope. Briefly, 1x10 6 cells/ml nucleated cells were incubated in pre-warmed DMEM 71

containing 2% FBS and 10mM HEPES with freshly added Hoechst 33342 (5 μg/ml) for 90 minutes at 37 C with intermittent mixing in a circulating water bath. In control set, cells were also incubated with Hoechst dye in the presence of verapamil (50 M) or FTC (10 M). At the end of incubation, cells were washed twice in ice-cold HBSS (by centrifugation at 1200 rpm for 4 minutes in 4 C) and pellet was resuspended in HBSS containing 2% FBS and10 mm HEPES. Propidium Iodide (PI), 2 g/ml was added just before FACS analysis, which allows the discrimination of live and dead cells. Cells were analyzed in a flow cytometer (MoFlo-Beckmen coulter, USA) equipped with ultra violet (UV) laser. Hoechst 33342 was excited with the UV laser at 355 nm and emission was detected with a 450/40 band pass filter (Hoechst Blue) and a 670/30 band pass filter (Hoechst red). 1 x 10 5 events were recorded and the data was analyzed in Summit v4.3 sofware (Beckmen Coulter, USA). Dead cells were excluded from the analysis based on PI gating (PI positive cells indicate dead cells). Live cells were gated back on Hoechst Blue (Y-axis) and Hoechst Red (X-axis) channel to detect the Side population (). defined as a distinct and small population of cells on the left corner of the dot plot as compared to bulk cells. was gated based on complete inhibition in presence of ABC transporter inhibitor (Verapamil or FTC). 4.2.5 Side population analysis in human cancer cells HeLa, A549, MCF7, BT549, MDA MB 231, MDA MB 453, T47D, CaCo2, G361, B6, WM 266.4, HBL-100 and rat C6 glioma cells at log phase were trypsinised to generate single cells. Single cell suspensions of above mentioned cell type (1x10 6 cells/ml) were incubated with fluorescent dye Hoechst 33342 (5.00 μg/ml) at 37 C in a circulating water bath for 90 minutes in DMEM containing 2% FBS and 10 mm HEPES. In control set, cells were also incubated with Hoechst dye in the presence of verapamil (50 M) or FTC (10 M) or verapamil and cyclosporine A (10 M respectively). At the end of incubation, cells were washed twice with ice-cold HBSS containing 2% FBS. Propidium iodide, 2 g/ml was added just before FACS analysis, as described above (Section, 4.2.4). 4.2.6 Side population analysis in human cancer cells after treatment with drugs and plant extracts Briefly, HeLa and MDA MB231 were treated with sub-lethal concentration of doxorubicin (0.8 μm and 0.2 μm respectively) for 96 hours. Similarly, cells were also treated 72

with sub-lethal concentration of ethanolic extracts of Withania somnifera and Tinospora cordifolia (20 μg/ml and 100 μg/ml respectively, in HeLa cells; 20 μg/ml and 50 μg/ml respectively, in MDA MB 231 cells) for 96 hours. At the end of treatment period, cells were trypsinized and stained with the Hoechst 33342 with or without verapamil or both verapamil and cyclosporine A in a assay as described above (Section, 4.2.4). Cells were then analyzed by flow cytometry equipped with UV laser to detect phenotype as described above (Section, 4.2.4). Level of inhibition and p-values were determined by one way ANOVA using Dunnett's Multiple Comparison Test. 4.2.7 FACS sorting of For sorting experiments, HeLa and MDA MB 231 cells were cultured in 140 mm tissue culture dish as described above (Section, 4.2.3). The exponentially growing cells were trypsinised to generate single cells. assay was performed and analyzed in a flow cytometer as described above (Section, 4.2.4). and non- were gated on Hoechst blue (Y-axis) and Hoechst red (X-axis) channel based on inhibition with verapamil (in HeLa cells) or verapamil and cyclosporine (MDA MB 231 cells) as live cells (PI negative cells). Both and N cells (5,000 cells each) were sorted under sterile conditions into 96 well microtiter tissue culture plates using sort master in MoFlo high speed cell sorter (Beckman Coulter, USA). 4.2.8 Cytotoxicity assay in FACS sorted and N cells were cultured in DMEM containing 10% FBS for 10-12 hours. Defined concentrations of the extracts in culture media were freshly prepared by serial dilution to get final concentration of 50, 100 μg/ml (For Tinospora cordifolia ethanolic extract); 20 μg/ml (For Withania somnifera ethanolic extract). Serial dilution was carried out in cell culture media in such a way that the final concentration of DMSO in the well did not exceed 0.5% (v/v). After overnight incubation, and N cells were treated with 100 μg/ml (in HeLa) and 50 μg/ml (in MDA MB 231) of Tinospora cordifolia ethanolic extract in triplicates for 48 hours. Similarly, and N cells were treated with 20 μg/ml Withania somnifera ethanolic extract. Doxorubicin 0.8 μm (in HeLa) 0.2 μm (in MDA MB 231) was used as a positive control. Equal volume of DMSO was used as vehicle control. At the end of treatment, MTT assay was performed as described earlier (Section, 3.2.4.2). Level of inhibition and p-values were determined by two way ANOVA using Bonferroni post tests. 73

4.3 RESULTS 4.3.1 Isolation of haematopoietic stem cells by side population analysis using flow cytometry First, side population assay was established using C57BL/6 mouse bone marrow cells as an experimental control. Bone marrow cells were isolated from C57BL/6 mouse and stained with Hoechst 33342 for identification of cells by flow cytometry. As shown in Fig. 4.1, using the experimental conditions 0.09-0.12% side population cells in mouse bone marrow cells were detected. defined as a distinct and small population of cells as compared to rest of the cells and has low fluorescence emission characteristics. Hoechst binds with A-T regions of DNA; hence the distinct regions of the profile also identify different phases of the cell cycle (G 0 /G 1, S, and G 2 /M). is enriched in the G 0 phase or quiescent phase of cell cycle. It has been identified that ATP transporters like ABCB1 (MDR1) or ABCG2 which are preferentially expressed in stem cells underlies the efflux of Hoechst, mediating the phenotype of Side Population (). In order to understand the effects of ABC transporters on phenotype, bone marrow cells were isolated from C57/BL6 mouse and stained with Hoechst 33342 with or without verapamil (ABCB1 inhibitor) or fumetrimorgin C (ABCG2 inhibitor). After the staining, cells were analyzed by flow cytometry to detect cells. As shown the Fig. 4.2; cells in the mouse bone marrow cells were 0.10 ± 0.05% and completely inhibited when cells are incubated with verapamil (0.00 ± 0.003%) or fumetrimorgin C (0.01 ± 0.006%). These results demonstrated the existence of a well defined and distinct in mouse bone marrow cells validate the protocol. 4.3.2 Human breast cancer cells harbor side population phenotype Several studies have recently reported the presence of stem cell-enriched side population in cultured cancer cell lines. Therefore, the current study investigated whether this phenomenon is applicable to human epithelial cancer cell lines especially breast cancer cells in long term cultures. In order to study this, a panel of human epithelial cancer cell lines such as, HeLa (human cervical carcinoma), A549 (human lung adenocarcinoma), MCF7, BT549, MDA MB 231, MDA MB 453, T47D (human breast cancer cell lines), CaCo2 (human colon carcinoma), G361, B6 (melanoma-mouse origin), WM 266.4 (melanoma-human), HBL-100 74

(human normal breast epithelial cells, immortalized) and rat C6 glioma were analyzed for phenotype. ABC transporter inhibitors like verapamil (ABCB1 inhibitor), cyclosporine A (ABCB1 inhibitor) and fumetrimorgin C (ABCG2 inhibitor) were used as experimental controls to validate distinct phenotype. More interestingly, our result indicates that among various cell types studied, human cervical carcinoma (HeLa), lung adenocarcinoma (A549), breast carcinoma (MCF7, MDA MB 231), colon carcinoma (CaCo2) and rat C6 glioma cells contain well resolved cells distinct from the bulk cells (N cells). As shown in Fig. 4.3; percentage of in various cell types were; HeLa (1.43 ± 0.03%), A549 (9.08 ± 0.75%), MCF7 (2.38 ± 0.58%), MDA MB 231 (0.32 ± 0.05%), CaCo2 (1.66 ± 0.11%) and C6 glioma (0.45 ± 0.03%). We were unable to detect distinct cells in other cancer cell type such as, BT-549, T47D, MDA-MB-453, G361, B6, WM 266.4 and HBL-100. In each experiment, the side population was eliminated by treatment with verapamil in HeLa, CaCo2 and C6 glioma cells, thereby validating the function of the ABC transporters. In A549, MCF7 and MDA MB 231 cells was not sensitive to verapamil or cyclosporine A. However, the synergistic effect of verapamil and cyclosporine-a effectively blocked the phenotype more prominently in MDA MB 231. in A549 and MCF7 cells were also completely eliminated in the presence of fumetrimorgin C indicating a valid side population phenotype (Fig. 4.3). A comparative data of percentage in various types of cancer cell lines analyzed is shown in Fig. 4.4.A. These results reveal that cultured human epithelial cancer cells, like human cervical cancer (HeLa), breast cancer (MCF7, MDA MB 231), lung adenocarcinoma (A549), colon carcinoma (CaCo2) and rat glioma (C6) cells harbor a detectable and well resolved side population phenotype distinct from bulk population of cells (Fig. 4.4.B). 4.3.3 Enrichment of side population phenotype upon treatment with anticancer drug It has been reported that the side population phenotype is mediated by ABC transporters predominantly, ABCB1 (MDR1) and ABCG2 (BCRP). These drug transporters are primarily located across the cell plasma membrane and are able to efflux many lipophilic agents including anticancer drugs. Therefore, ABC transporters play a significant role in reducing the intracellular concentration of an anticancer drug which is a leading cause of multidrug resistance in cancer. Recent reports also suggest that side population cells are highly resistant to chemotherapy or radiation therapy as they over-express multidrug resistant transporters or ABC transporters. Doxorubicin is an anthracycline antibiotic and a routinely used 75

chemotherapeutic drug for treatment of various types of epithelial cancers. Since most of the anticancer drugs target rapidly proliferating cancer cells, the current study investigated whether the side population was resistant to such anticancer drugs. This would be evaluated by analyzing an enrichment of side population cells, if any, after treatment with doxorubicin. As shown in Fig. 4.5; in HeLa cells, the average percentage of in the control (cells without any drug treatment) was found to be 1.37 ± 0.10% and was completely vanished with verapamil (0.00 ± 0.00%). Similarly, in MDA MB 231 cells, the average percentage of cells in the control (cells without any drug treatment) was found to be 0.23 ± 0.02% and was significantly inhibited with verapamil+cyclosporine A (0.03 ± 0.00%). In both these cell types, significant increase in the phenotype was observed upon doxorubicin treatment in comparison with untreated control (1.37 ± 0.10% to 6.27 ± 0.27% in case of HeLa cells, 0.23 ± 0.02% to 0.66 ± 0.04% in case of MDA MB 231 cells). The phenotype in both HeLa and MDA MB 231 doxorubicin treated cells was significantly inhibited in the presence of verapamil or cyclosporine A thereby indicating a valid. All values are mean ± SD of three independent experiments, **p<0.01, ***p<0.001, determined by one way ANOVA using Dunnett s Multiple Comparison Test. These results reveal that the anticancer drug doxorubicin significantly enriches the side population in both human cervical cancer (HeLa) and breast cancer (MDA MB 231) cells. 4.3.4 Side population inhibitory effects of Withania somnifera and Tinospora cordifolia extracts in human cancer cells Preliminary pharmacological characterization had revealed that the ethanolic extracts of Withania somnifera and Tinospora cordifolia possess potential anticancer activity by increasing cytotoxicity, inducing apoptosis and cell cycle specific inhibitory activities in human breast cancer cells (MDA MB 231, MCF7) and cervical cancer cells (HeLa), with less cytotoxicity in normal human epithelial cells (HaCaT). Side population assay by flow cytometry on human cervical cancer cells (HeLa) and breast cancer cells (MDA MB 231) indicated that these cells do contain a rare and distinct population of side population () cells. It is evident that cells have cancer stem like characteristics with the ability to initiate tumor and are highly resistant to chemotherapeutic drugs. Hence, the current study investigated whether ethanolic extracts of Withania somnifera and Tinospora cordifolia have any specific inhibitory activities on cultured human cancer cells. In this direction, human 76

cervical (HeLa) and breast cancer cells (MDA MB 231) were treated with ethanolic extracts of Withania somnifera and Tinospora cordifolia for a period of 96 hours at sub-lethal concentration. The anticancer drug, doxorubicin was used as positive control and DMSO (0.5%) was used as solvent control. At the end of treatment period, assay was performed. Results indicated that in untreated HeLa cells was 1.37 ± 0.07%. Verapamil, completely inhibited the (0.00%) indicating valid cells. Upon treatment with ethanolic extract of Withania somnifera and Tinospora cordifolia, was found to be 0.97 ± 0.12% and 0.00 ± 0.00% respectively. Vehicle control (DMSO) did not have any effects on (1.22 ± 0.14%). However, in the presence of the chemotherapeutic drug, doxorubicin was found to be 6.27 ± 0.27%. Similarly, in untreated MDA MB 231 cells was 0.23 ± 0.02%. Using a combination of verapamil and cyclosporine A, the (0.03 ±0.00%) completely disappeared, indicating a valid transporter based function. Upon treatment with ethanolic extract of Withania somnifera, Tinospora cordifolia and doxorubicin were found to be 0.21 ± 0.03%, 0.01 ± 0.00% and 0.66 ± 0.04% respectively (Fig. 4.6). All values are mean ± SD of three independent experiments, *p<0.05, **p<0.01, ***p<0.001, determined by one way ANOVA using Dunnett s Multiple Comparison Test. This data suggested that ethanolic extract of Tinospora cordifolia significantly inhibited cells in both HeLa and MDA MB 231 cells (p<0.001) and level of inhibition was quite comparable with standard inhibitors of such as verapamil or cylosporin A (p<0.001); whereas, Withania somnifera extract did not show any significant inhibitory activity (Fig. 4.7). On the other hand, the currently used anticancer drug, doxorubicin, significantly enriches in both the cell types studied (p<0.001). The inhibition of phenotype in the human cancer cells upon treatment with ethanolic extract of Tinospora cordifolia suggests a tremendous potential of this plant in cancer chemotherapy. 4.3.5 Cytotoxicty of Withania somnifera and Tinospora cordifolia extracts in In order to understand the cellular mechanism underlying the inhibition of phenotype in presence of Withania somnifera and Tinospora cordifolia extracts, we tested if these extracts specifically mediate cell death in side population cells. This question was addressed by performing a cytotoxicity assay (MTT assay) in both and N cells. In this direction, and N cells in both human cervical cancer (HeLa) and breast cancer (MDA MB 231) cells were isolated by FACS sorting. In sorting experiments, in HeLa cells was found to be 77

1.68% and was completely blocked (0.00%) in presence of verapamil. Similarly, in MDA MB 231 cells, was found to be 0.13% and was effectively blocked in presence of both verapamil and cyclosporine (Fig. 4.8 A). Both gated and N cells were sorted in 96 well plates and treated with ethanolic extracts of Withania somnifera, Tinospora cordifolia and doxorubicin for a period of 48 hours. Results indicate that in HeLa cells, ethanolic extract of Tinospora cordifolia impart significantly high cytotoxicity againt (Cell viability=38.98 ± 4.66%) as compared to N (Cell viability=76.36 ± 5.52%, p<0.001). Similarly, in MDA MB 231 cells, ethanolic extract of Tinospora cordifolia imparts significantly high cytotoxicity in cells (Cell viability=34.27 ± 2.65%) as compared to N cells (Cell viability=64.05 ± 2.15%, p<0.001). However, ethanolic extract of Withania somnifera and doxorubicin did not have any increased cytotoxicity against as compared to N in both HeLa and MDA MB 231 cells. In contrast, ethanolic extract of Withania somnifera and doxorubicin show an increased cell death in N as compared to cells. Vehicle control DMSO did not alter viability of either or N (Fig. 4.8 B). All the experiments were done three times independently, ***p<0.001, determined by two way ANOVA using Bonferroni posttests. These data suggested that Tinospora cordifolia ethanolic extract do possess or cancer stem cell-specific cell death in human cancer cells, though ethanolic extract of Withania somnifera and doxorubicin exert cell death predominantly in differentiated non-side population or bulk cells. 4.4 DISCUSSION Although the concept of stem cell origin of cancer has existed for long, this concept and the cancer stem cell hypothesis have drawn considerable attention only in recent years. Recent reports suggest that cancer is a heterogeneous population in which a rare population of cells within a tumor, with stem cell characteristics is largely responsible for the initiation and progression of the tumor. Existence of the so called cancer stem cells was first documented in acute myeloid leukemia. Following the suit, multiple researchers reported the existence of such distinct tumor initiating cells in breast cancer, brain tumor, multiple myeloma, pancreatic cancer, colon cancer, head and neck squamous cell carcinoma, melanoma and prostate cancer. If cancer growth and metastases are predominantly mediated by cancer stem cells, as the recent work suggests, then getting rid of these cells should eliminate the cancer at its root, since the remaining cells do not have the potential of maintaining the tumor. However, the 78

majority of cancer treatments that exist today have been directed at rapidly dividing cells that constitutes the bulk of the tumor. Hence, currently available chemotherapeutic drugs reduce the tumor mass but do not eliminate the cancer stem cells. This leads to recurrence of the disease which occurs frequently with current chemotherapy. Recent evidences suggest that cancer stem cells have increased expression of ABC transporters (Multi drug resistant transporters) especially, ABCB1 and ABCG2 which enable stem cells to pump out lipophilic agents including anticancer drugs. Cancer stem cells are relatively quiescent, tend to divide slowly, have elevated expression of drug efflux pumps and are thereby resistant to drugs, and have an active DNA-repair capacity. All these properties make them resistance to anticancer drugs. Among the various types of cancer cells investigated for, breast, lung, cervical, colon and glioma cells harbor distinct phenotype. Our results indicate that an enrichment of in cancer cells upon treatment with doxorubicin. This may be due to increased overexpression of multidrug transporters like ABCB1 or ABCG2 which enrich compartment, the enrichment of cancer stem cells which are highly resistant to anticancer drugs, death of cells lacking or have reduced number of ABC transporters (the non-) and the side population cells possessing high number of these transporters efficiently efflux the drug and remain viable. The enrichment seen with the drugs may be because of over expression of multidrug resistant transporters (ABC family of membrane transporters) which include ABCB1, encoded by the MDR1 gene, ABCG2 a half membrane transporter and the multidrug resistance-associated protein (MRP) gene. Among this, ABCB1 and ABCG2 predominantly contributes to the generation of phenotype in cancer cells. The enrichment of compartment with doxorubicin treatment also suggests that doxorubicin might be excluded from cells by the same mechanism as the rapid efflux of Hoechst 33342. In each comparison, cells within the side population excluded the drug more efficiently than their non- counterparts (bulk of cancer cells). This increased drug efflux capacity of rare and distinct population of cells () and enrichment of upon treatment with anticancer drug doxorubicin might influence the outcome of current chemotherapy for the treatment of cancer. Side population assay on human cervical cancer cells (HeLa) and breast cancer cells (MDA MB 231) indicated that these cells do contain a rare and distinct population of side population () cells. It is evident that cells have cancer stem cell like characteristics with 79

the ability to initiate tumor and are highly resistant to chemotherapeutic drugs. Hence for effective cancer therapy, novel anticancer drugs should be designed to eliminate the bulk of cancer cells as well as rare cancer stem cells, which underlie the initiation and maintenance of a tumor. Towards this direction, we have investigated here the cancer stem cell inhibitory effects of the ethanolic extracts of two plants, Withania somnifera and Tinospora cordifolia on phenotype of human cancer cells. The inhibition by Tinospora cordifolia may be mediated by blockade of one or few of the ABC transporters or MDR proteins in cancer cells that are responsible for the efflux of Hoechst 33342 out of the cells. This remarkable property of the medicinal plant, Tinospora cordifolia to inhibit the highly resistant stem like cells within a cancer is an encouragement to intensify further research on cancer stem cellinhibition guided isolation and characterization of biologically active anticancer compounds. 80

Hoechst blue emission (450/20 nm) G 0 /G 1 phase Side Population () G 2 /M phase S phase Hoechst red emission (675/40 nm) Figure 4.1 Side population analysis in mouse bone marrow cells Stem cells have the ability to efflux, the lipohilic DNA binding dye, Hoechst 33342. Typically, Hoechst 33342 is excited in a flow cytometer equipped with a UV laser, and emits in two wavelengths, Hoechst blue (450/20nm) and Hoechst red (670/40nm). defined as a distinct and small population of cells as compared to rest of the cells and has low Hoechst emission characteristics. Hoechst binds with A-T regions of DNA; hence the distinct regions of the profile also identify different phases of the cell cycle (G 0 /G 1, S, G 2 /M). bears in G 0 phase or quiescent phase of the cell cycle. Control- 0.10 ± 0.05% +Verapamil - 0.00 ± 0.003% + Fumetrimorgin C - 0.01 ± 0.006% Hoechst blue (450/40 nm) Hoechst red (675/30nm) Figure 4.2 analysis in mouse bone marrow cells with ABC transporter inhibitors Bone marrow cells were isolated from C57/BL6 mouse and stained with Hoechst 33342 for identification of cells by flow cytometry. 0.10% of the total population of bone marrow cells was found to be the (the boxed blue population). When in a control set, the cells were incubated with ABC transporter inhibitors, verapamil (ABCB1) and fumetrimorgin C (ABCG2 inhibitor) the dye efflux was completely blocked. As a consequence, the eliminated completely. All values are mean ± SD of three independent experiments (n=3). 81

HeLa -1.43±0.03% HeLa + Verapamil -0.00±0.01 % Cervical A549-9.08±0.75% A549 + FTC - 0.01±0.01% Lung MCF7-2.38±0.58% MCF7+ FTC- 0.02±0.01% Breast CaCo2-1.66±0.11% CaCo2 + Verapamil - 0.01±0.01% Colon C6-0.45±0.03% C6 + Verapamil - 0.00±0. 01 % Brain glioma MDA-MB-231-0.32±0.05% MDA-MB-231 - Ver+ CsA 0.01±0.01% Breast BT-549-0.01±0.01% BT549 +Verapamil - 0.00±0. 00 % Breast B6-0.11±0.01% B6 + Verapamil - 0.02±0.01% Melanoma-mouse WM 266.4-0.03±0. 01% WM 266.4 + Verapamil -0.01±0. 01% Melanoma G 361- Undetected Melanoma-mouse G 361 + Verapamil T47D - Undetected T47D + Verapamil HBL 100 - Undetected HBL 100 +Verapamil Breast Normal epithelial cells Figure 4.3 analysis in cancer cell lines analysis of cultured cancer cell lines indicating human breast cancer cells (MCF7, MDA MB 231), lung cancer cells (A549), cervical cancer (HeLa), colon cancer (CaCo2) and rat glioma (C6) cells harbor distinct and rare populations of phenotype. When in a control set, the cells were incubated with verapamil, cyclosporin or fumetrimorgin C, eliminated completely indicating valid. These figures are representative of three independent experiments (n=3). 82

A 10 8 %Sidepopulation() B HeLa = 1.43 ± 0.027% 6 4 2 0 HeLa A549 MCF7 MDAMB231 C6GLIOMA B6MELANOMA BT549 MDAMB453 MDA MB 231 = 0.32 ± 0.08% CaCo2 G361 WM266.4 HBL100 T47D A549 = 9.08 ± 0.75% CaCo2 = 1.67 ± 0.12% MCF7 = 2.34 ± 0.56 % C6 glioma = 0.46 ± 0.04% Figure 4.4 profile of breast, lung, cervical, colon and glioma cancer cells A. analysis in various types of cancer cells. B. profile of human breast cancer cells (MCF7, MDA MB 231),) lung cancer cells (A549) cervical cancer (HeLa), colon cancer (CaCo2) and rat glioma (C6) indicating distinct and well resolved phenotype. All values are mean ± SD of three independent experiments (n=3). 83

A-HeLa B- MDA MB 231 Untreated control - 1.37 ± 0. 10% Verapamil - 0.00 ± 0.00% Untreated control - 0.23 ± 0.02% Verapamil+CsA 0.03 ± 0.00% Doxorubicin - 6.27 ± 0. 27% Doxorubicin+verpamil - 0.04 ± 0. 01% Doxorubicin - 0.66 ± 0.04% Doxorubicin+ver+CsA - 0.02 ± 0. 00% A B %Sidepopulation() 8 6 4 2 *** %Sidepopulation() 0.8 0.6 0.4 0.2 *** 0 Untreatedcontrol *** Verapamil Doxorubicin ** Doxorubin+Verapamil 0.0 Untreatedcontrol Verapamil+CsA ** ** Doxorubicin Doxorubin+Verapamil+CsA Figure 4.5 Enrichment of phenotype upon treatment with doxorubin Human cervical cancer (HeLa) and breast cancer (MDA MB 231) ells were treated with doxorubicin for 96 hours. At the end of the incubation period, assay was performed. Verapamil or verapamil+csa were used as a standard inhibitor of cells. Doxorubicin significantly enriched cells in both cell types studied. All values are mean ± SD of three independent experiments, **p<0.01, ***p<0.001, ns=not significant, determined by one way ANOVA by using Dunnett s Multiple Comparison Test. 84

A-HeLa Untreated control - 1.3 ± 0.07% Verapamil - 0.00 ± 0. 00% WS-A - 0.97 ± 0.12% TC-A - 0.00 ± 0. 00% Doxorubicin - 6.27 ± 0.27% DMSO - 1.22 ± 0.14% B-MDA MB 231 Untreated control 0.23 ± 0.02% Verapamil+CsA - 0.03 ± 0.00% WS-A - 0.21 ± 0.03% TC-A - 0.01 ± 0.00% Doxorubicin - 0.66 ± 0.04% DMSO - 0.24 ± 0.01% Figure 4.6 Screening of Withania somnifera extract and Tinospora cordifolia extract against phenotype The pharmacological screening of Withania somnifera extract, Tinospora cordifolia extract and doxorubicin against side population phenotype. These figures are representative of three independent experiments (n=3). 85

A B %Sidepopulation() 8 6 4 2 *** ns ns %Sidepopulation() 0.8 0.6 0.4 0.2 *** ns ns 0 Untreatedcontrol *** Verapamil Doxorubicn WSA *** TCA Solventcontrol(DMSO) 0.0 Untreatedcontrol Verapamil+CsA ** *** Doxorubicn WSA TCA Solventcontrol(DMSO) Figure 4.7 The pharmacological effects of Withania somnifera extract, Tinospora cordifolia extract in HeLa (A) and MDA MB 231 (B) cells were treated with the plant extracts, doxorubicin, and vehicle control (DMSO) for 96 hours. At the end of the incubation period, assay was performed. Verapamil or verapamil+ CsA was used as a standard inhibitor of. Tinospora cordifolia extract significantly inhibited in both cell types. However, Withania somnifera did not impart any significant inhibition of. Doxorubicin significantly enriched. All values are mean ± SD of three independent experiments, **p<0.01, ***p<0.001, ns=not significant, determined by one way ANOVA by using Dunnett s Multiple Comparison Test. 86

HeLa cells A PRE-SORT (-1. 68%) +VERAPAMIL ( 0.00%) /N SORT N N N B 120 Untreated control 100 Vehicle control (DMSO) % Cell viability 80 60 40 *** *** Doxorubicin WS-A TC-A 20 *** 0 N N N N N MDA MB 231 cells A PRESORT (0.13%) +VERAPAMIL+CsA (0.01%) /NSORT B % Cell viability 120 100 80 60 40 20 *** *** *** Untreated control Vehicle control (DMSO) Doxorubicin WS-A TC-A 0 N N N N N Figure 4.8 specific cytotoxicity of Withania somnifera and Tinospora cordifolia extracts A. and N cells of human cervical cancer (HeLa) and breast cancer (MDA MB 231) cells were sorted by FACS sorter. B. Cytotoxicity of Withania somnifera extract, Tinospora cordifolia extract and doxorubicin in sorted and N cells. Tinospora cordifolia extract show an increased cytotoxicity against as compared to N. However, Withania somnifera extract and doxorubicin did not show any -specific cytotoxicity. All values are mean ± SD of three independent experiments, ***p<0.001, determined by two way ANOVA using Bonferroni post test. 87

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