Study Of Selected Indigenous Medicinal Plants Biology Essay

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Cell proliferation is the basis of growth and development, survival, and maintenance of living organisms. Even more critical is the 'regulation' of the process of cell proliferation. Aberrant regulation of this phenomenon can result in diseases like cancer. In fact abnormal cellular proliferation is one of the hallmarks of cancer. Breast cancer comprises of a heterogeneous group of diseases that vary in morphology, biology and response to therapy (Rakha et al., 2008). Chemotherapy, surgery and radiotherapy have remained the mainstay of breast cancer treatment. Even though several chemotherapeutic drugs have been identified to treat breast cancers, yet a convincing cure has remained elusive. Secondary metabolites from plants play a very important role in cancer chemotherapy. Recent reports suggest that a variety of anticancer drugs are derived from plant sources, leading to significant scientific interest in discovery of anticancer drugs from plants (Cragg et al., 2011). India is one of the richest biodiversity centers with respect to medicinal plants. Such plants have been used as an indigenous cure in folklore or traditional system of medicine for treatment of various kinds of illness including cancer (Khare, 2007). Ayurveda, a traditional sect of Indian medicine based on plant drugs has been successful from very early times in using these natural drugs and preventing various tumors. Recently, a greater emphasis has been given towards research on complementary and alternative medicine in cancer management. Sesquiterpene lactones are diverse group of natural compounds present in the medicinal plants. In recent years, the anticancer potential of sesquiterpene lactones has attracted a great deal of interest (Ghantous et al., 2010). For example, sesquiterpene lactones isolated from medicinal plants such as parthenolide, costunolide, Arteminolide C and Helenalin were found to have the ability to induce apoptosis in various cancer cell lines in vitro and effectively inhibited tumor cell growth in vivo (Kreuger et al., 2012). Apoptosis or programmed cell death defines a genetically encoded cell death program, which is morphologically and biochemically distinct from the necrosis (Cotter, 2009). Induction of apoptosis in cancer cells is considered as one of the strategy for the development of anticancer drugs (Portt et al., 2011). Recent study also suggests that sesquiterpene lactone, parthenolide and its derivatives have the ability to induce apoptosis specifically in acute myelogenous leukemia stem cells and breast cancer stem cells (Guzman et al., 2005, Zhou et al., 2008).

Therefore, the aim of the current work was to study selected indigenous medicinal plants bearing sesquiterpene lactones for anticancer activity in human breast cancer cells. The major objective of the work was: 1) To study cytotoxicity and apoptosis induced by selected indigenous medicinal plants extracts on human breast cancer cells; 2) To study the cancer stem cell inhibitory activities of the selected indigenous medicinal plant extracts and fractions against human breast cancer cells; 3) To undertake bio-activity guided fractionation of the selected medicinal plant extracts for isolation of the active (anticancer) compounds; 4) Phyto-chemical characterization and deciphering the structure of the active compounds; and 5) To study the anticancer effects of isolated compounds on human breast cancer cells.

The current study investigated cytotoxicity and apoptotic activities of two selected indigenous medicinal plant extracts belonging to Withania somnifera of the family Solanacea and Tinospora cordifolia of the family Menispermacea in human breast cancer cells. A methodical evaluation of cytotoxicity effects by MTT assay in human breast cancer cells revealed that ethanolic extracts of Tinospora cordifolia and Withania somnifera imparted dose-dependent cytotoxicity activity. Acridine orange/ethidium bromide assay by fluorescent microscopy revealed that extracts induced apoptosis but not the necrosis in human breast cancer cells. Hoechst 33342 assay and DNA fragmentation assay corroborated hallmark properties of apoptosis such as membrane blebbing, nuclear condensation and DNA fragmentation in breast cancer cells. Moreover, cell cycle analysis using flow cytometry revealed that treatment of breast cancer cells with the Withania somnifera extract caused significant arrest of cells at G2/M phase, significantly increased Sub-G0 phase and diminished S phase indicating mitotic arrest, induction of apoptosis and inhibition of DNA replication. On the other hand, Tinospora cordifolia extract significantly increased Sub-G0 phase and diminished S phase indicating induction of apoptosis and inhibition of DNA replication. The agents that are proficient in inducing selective apoptosis of cancer cells without harming normal cells have received considerable interest towards the development of novel cancer chemotherapeutic drugs (Ocker and Höpfner, 2012). Hence, the current study also addressed the question of whether Withania somnifera and Tinospora cordifolia extract mediated suppression of cell viability and growth was selective to cancer cells. Interestingly, it was found that extracts did not induce any apoptosis in human normal epithelial cells (HaCaT, immortalized cells) at the concentration that was cytotoxic to the breast cancer cells (MCF7).

Over the past decade, initiation and propagation of cancer are increasingly being linked to stem cells. Support for this link comes from the identification of a small population of 'stem cell-like cells' within tumors of multiple organs, which both functionally and phenotypically resemble the normal tissue-specific stem cells (Visvader and Lindeman, 2008). This small population of cells within tumors has been termed as 'cancer stem cells' (CSCs). These cells have self renewal and differentiation capability, like normal stem cells, and only these cells have been proposed to possess the ability to initiate and maintain the tumor (Dalerba et al., 2007). CSCs were first documented in hematological malignancies where only small subsets of leukemic cells were found to be capable of forming new tumors (Lapidot et al., 1994). Following 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, suggesting that the presence of these small subpopulations of cells may be more common to several types of cancers. CSCs have differential ability to efflux a wide variety of chemotherapeutic drugs owing to the elevated expression of multidrug resistant transporters (ABCB1, ABCG2 and ABCC1). Therefore, CSCs are highly resistant to chemotherapy (Greaves, 2011). Traditionally, chemotherapy has been based on the ability of cytotoxic drugs to destroy rapidly proliferating cancer cells. Now, it has reported that only rare population of cells having stem cell characteristics drive tumor initiation/maintenance, while the rest of cancer cells are non-tumorigenic (Wicha et al., 2006). Hence, chemotherapy directed against the rest of the cancer cells may cause tumor regression but leaves the rare CSCs which may lead to relapse. Therefore, it is hypothesized that frequent relapse of cancer may be due to the inability of current drugs to target CSCs (Nguyen et al., 2012). Hence, the aim of cancer therapy should be specific to cancer stem cells for a complete elimination of tumorigenic cells (Rasheed et al., 2011). Stem cells from multiple tissues have been identified based on their ability to efflux the lipophilic dye Hoechst 33342 using flow cytometry which forms the basis of side population assay (Goodell et al., 1996). Side population (SP) phenotype is mediated by the ABC family of drug transporter proteins (ABCB1, ABCG2 and ABCC1) that can actively pump several drugs, including chemotherapeutic drugs, out of cells (Zhou et al., 2002). SP assay has become a critical assay for the identification and isolation of stem cells (Willan and Farnie, 2011). SP has been found to be highly enriched in cancer stem cells. Purified side population cells were found to be more tumorigenic than the corresponding non side population cells (Golebiewska et al., 2011). SP cells have been identified in a number of cancers where they have been shown to display the increased self renewal and tumorigenicity when transplanted into immune-compromised mice (Bleau et al., 2009, Harris et al., 2008, Haraguchi et al., 2006, Szotek et al., 2006). SP also preferentially express stemness genes, including molecules of the Wnt, Notch, and Hedgehog pathways (Shi et al., 2012).

The present study investigated the existence of SP phenotype in different type of cancer cells. Among the various cell types evaluated, it was found that human cervical cancer line (HeLa), human breast cancer lines (MCF7, MDA MB 231), human lung adenocarcinoma (A549), human colon carcinoma (CaCo2) and rat glioma (C6) cells harbor a detectable and well resolved 'side population', which is distinct from the bulk population of cells. When the cancer cells were cultured in presence of sub-lethal concentrations of doxorubicin, a widely used chemotherapeutic drug for treatment of breast cancer, the SP phenotype was significantly enriched. Moreover, it was found that SP cells were highly resistant to doxorubicin. The current study investigated the inhibitory effects of Withania somnifera and Tinospora cordifolia extracts on the SP and found that the latter significantly inhibited SP by inducing SP-specific cell death. Bio-activity guided (based on anti-SP activity) fractionation of Tinospora cordifolia extracts further revealed that the petroleum ether fraction and dichloromethane fractions imparted dose dependent cytotoxicity, effectively inhibited SP phenotype and the multidrug resistant drug transporter (ABCB1, P-glycoprotein), which underlies the 'SP' phenotype.

The current study adopted the strategy of bioactivity or mechanism of action directed isolation and characterization of active compounds from the selected plant Tinospora cordifolia. In order to isolate and characterize the active compound, the petroleum ether and dichloromethane fractions of Tinospora cordifolia were further sub-fractioned into a total of 34 fractions (F1 to F34) by column chromatography and each fraction was screened for anticancer activity. Results indicated that among the 34 sub-fractions, the dichloromethane sub-fractions F4, F5, F6S, F7 and F8 sub-fractions of Tinospora cordifolia significantly inhibited SP and ABCB1 (MDR1) drug transporter. These sub-fractions were isolated by column chromatography of the dichloromethane fraction of Tinospora cordifolia by 10% step wise gradient elution with a solvent system of petroleum ether - ethyl acetate - methanol. Thin layer chromatography of these sub-fractions (F4, F5, F6S, F7 and F8) revealed that each sub-fraction contains more than a single spot, indicating a 'mixture' of compounds. Therefore, most biologically active sub-fractions, F4 (TC-D3), F5 (TC-D4), F6S (TC-D5) were selected for the further isolation of anticancer compounds. Flash chromatography and subsequent purification by column chromatography led to the final purified compounds, TCD5-F2-C (TC-A), TCD5-F3-B (TC-B), TC-D4-A2 (TC-C) and TC-D3-A2 (TC-D). Chemical characterization and structural analysis of the compounds were carried out by NMR and Mass spectroscopy.

The current study investigated detailed mechanism of action of the isolated compounds TC-A, TC-B, TC-C and TC-D for anticancer activity in a panel of human epithelial cancer cells with special emphases towards breast cancer. We found that TC-A, TC-B, TC-C and TC-D possess cancer cell specific cytotoxicity and growth inhibitory activities against human breast cancer cells. The compounds imparted less cytotoxic effects against normal human mammary epithelial cells (MCF10A) compared to breast cancer cells (MCF7). Anticancer drug, doxorubicin showed dose dependent inhibition of cell proliferation in both cancer and normal breast cells. Flow cytometric analysis indicated that treatment with the isolated compounds induced apoptosis in cancer cells (determined by Annexin-V-FITC assay). Similarly, cell cycle analysis revealed elevated sub-Go phase, diminished S phase or G2/M cell cycle arrest. Interestingly, the compounds possessed less cytotoxicity on normal cells as they did not induce apoptosis of normal mammary epithelial cells (MCF10A) at concentrations that effectively inhibited cancer cell proliferation (MCF7). Further, the results also revealed that TC-A, TC-B, TC-C and TC-D significantly imparted cytotoxic effects in SP of breast, lung and colon cancer cells.

Moreover, these compounds also inhibited the quiescent phase of cell cycle phenotype, which is a typical property of stem cells, and possibly cancer stem cells. Cell surface marker (CD44 and CD24) based analysis using flow cytometry provides a valuable technique for the isolation of putative breast cancer stem cells (Krishan et al., 2011). It has been reported that only the breast cancer cells with a CD44 high/CD24 low phenotype demonstrate tumorigenic property and only this fraction possesses stem cell characteristics (Al-Hajj et al., 2003). We demonstrated that TC-A, TC-B, TC-C and TC-D significantly inhibits CD44 high/CD24 low population in breast cancer cells. The cytotoxicity studies in FACS sorted CD44 high /CD24 low cells and the remaining population revealed that the compounds TC-A, TC-B, TC-C and TC-D impart specific cell death in CD44 high /CD24 low cells. However, the CD44 high /CD24 low population was resistant to the chemotherapeutic drug, mitoxantrone. In fact, this drug enriched the CD44 high /CD24 low cells. Three dimensional suspension culture is considered to be one of the strategies to isolate and enrich stem cells and cancer cells. Suspension cultures of cancer cells are termed as 'cancer spheres' (Ponti et al., 2005). It has been reported that cancer spheres have self renewal ability and express stemness markers. The current study also investigated the ability of the compounds, TC-A, TC-B, TC-C and TC-D to inhibit breast cancer spheres cultured in methyl cellulose slurry. Interestingly, it was found that the compounds significantly inhibited cancer sphere formation. Further, multidrug resistance plays a major role in the poor outcome of cancer chemotherapy. Among the various mechanisms identified for the MDR in cancer, the role of ABC transporters, ABCB1 (MDR1), ABCG2 (BCRP) and ABCC1 (MRP1) draws considerable attention (Gillet and Gottesman, 2010). The present study investigated the MDR modulating activity of the isolated compounds in human epithelial cancer cells by flow cytometry based functional assays. Rhodamine 123 efflux assay in cervical cancer cells indicated that TC-A, TC-B, TC-C and TC-D inhibit ABCB1 or P-glycoprotein activity. Similarly, the inhibitory effects on other drug transporters, ABCG2 and MRP1 were investigated by mitoxantrone and calcein AM based assay. The compounds, TC-A, TC-B, TC-C and TC-D were able to inhibit ABCG2 and ABCC1 mediated drug transport in the breast and lung cancer cells.

A recent study led to the generation of immortalized breast epithelial cells (NBLE) by overexpression of SV40ER and hTERT into stem cell enriched mammosphere-derived single cells from normal primary breast tissue (Paranjape et al., 2011). The CD44+/CD24- fraction of NBLE cells was isolated by FACS sorting and cultured in DMEM-F12 with growth factors to derive a cancer stem cell line, the NBLE CD44+/CD24- population. The derived cells possessed stemness property, reduced differentiation markers, retained the ability to differentiate in vitro and imparted tumorigenic potential. We have also investigated the cancer stem cell specific effects of the isolates on NBLE CD44+/CD24- cells. The results revealed that TC-A, TC-B, TC-C and TC-D imparted dose dependent cytotoxicity, induced cell death and significantly inhibited CD44 high CD24 low population of NBLE CD44+/CD24- cells. Among all the four compounds investigated for elucidation of mechanism of action for anticancer activity, TC-B was found to have the most potent activity.

To summarize, bio-activity guided isolation of medicinal plant, Tinospora cordifolia, for anticancer activity yielded four compounds TC-A, TC-B, TC-C and TC-D. Elucidation of mechanisms of action revealed that these compounds inhibited cancer cell proliferation, imparted cytotoxicity against human breast cancer cells without harming the normal cells, inhibited cell cycle progression and induced apoptosis in cancer cells. The compounds also found to have cancer stem cell specific anticancer activity mediated via inhibition of side population, CD44 high CD24 low population, breast cancer spheres and cancer stem cell enriched cell line (NBLE-CD44+ /CD24-). Moreover, these compounds imparted inhibitory effects on MDR transporters, ABCB1, ABCG2 and ABCC1 suggesting it may work as MDR modulators. Among all the four compounds investigated for anticancer activity, TC-B was found to have most potent activity. These compounds will however have to be subjected to preclinical evaluations (in vivo mouse models), clinical trials, and toxicological studies for future therapeutic use as a potential chemotherapeutic drug for the effective treatment of breast cancer.