|dc.description.abstract||Cancer has become a major health problem due to its high rate of morbidity and mortality. Severe side effects associated with most of the available anticancer drugs and the development of the drug resistant cells are the major hurdles limiting their application and therapeutic success. Much attention has thus been focused on natural compounds with minimal or no toxicity in humans and having capacity to suppress proliferation and/or induce apoptosis in cancer cells. Therefore, it is imperative to discover and develop new anticancer drugs. Endophytic fungi though underexploited have remained a rich source of structurally novel and biologically active secondary metabolites. That they are a prolific resource for new compounds in drug discovery is evident from a plethora of reported research findings of the recent past. Those of endophytic fungi especially isolated from medicinal plants with known therapeutic value have especially caught the attention of several research groups worldwide for the production of host associated or novel lead molecules. In this backdrop, the focus of my research work has been centered on exploring the endophytic fungal population associated with Catharanthus roseus in search of production of novel anticancer compounds.
Twenty endophytic fungi were isolated from different parts of Catharanthus roseus plant. The ethyl acetate (EA) extracts of three week grown liquid cultures of the individual endophytes were assessed in vitro for their cytotoxic activities in HeLa and HepG2 cancer cells using MTT assay. Of all the isolates thus screened, the EA extract from an isolate identified as Chaetomium nigricolor exhibited significant cytotoxicity in the tested cell lines with a better activity profile against HeLa cells. To understand if the liquid culture filtrate or the mycelium harbored the active cytotoxic principle, the C. nigricolor culture filtrate and mycelia was extracted separately using organic solvents with varying polarity namely ethyl acetate, dichloromethane, chloroform and hexane. These extracts were further tested for cytotoxicity induction in HeLa and MCF-7 cells by MTT assay. The ethyl acetate culture filtrate and mycelial extracts showed best cytotoxic activity on HeLa cells compared to other extracts; therefore, the mycelial and culture filtrate ethyl acetate extracts of C. nigricolor were pooled and was used for further work. Hexane culture filtrate extract of C. nigricolor showed best antiproliferative activity against MCF-7 cells. Thus, the compounds with anticancer potential from both ethyl acetate pooled extracts and hexane culture filtrate extract of C. nigricolor were purified using bioassay guided isolation method. This finally resulted in the isolation of two different anticancer compounds, one each from EA pooled extract and Hexane culture filtrate extracts of C. nigricolor named as compound 1 and compound 2, respectively. Based on various analyses including mass spectrometry (MS and MS-MS) and UV-visible, Fourier transform infrared (FT-IR) and nuclear magnetic resonance (NMR) spectroscopy, the compounds 1 and 2 were identified as 1, 2 Bis (diethylamino) ethene-1, 2 diol (BDED) and Nonacos-4-enoic acid (NA), respectively
An improved understanding of the cellular responses to chemotherapeutic agents in cancer cells and the underlying molecular mechanisms gains particular relevance in the efforts to improve the clinical outcome of chemotherapeutic agents. Arresting the growth of cancer cells in one way or the other and induction of apoptosis in a drug specific manner is the hallmark of almost all the clinically used anticancer drugs. Towards this end, evaluation of the cytotoxic effects of the newly identified compounds on various human cancer cells was carried out. BDED was examined for in vitro cytotoxicity of against a panel of five human cancer cell lines namely HeLa, A-431, A549, COLO 205, and MCF-7. Among the cell lines screened, HeLa cells were most vulnerable to BDED treatment with an IC50 value of 27 μM. This cytotoxicity was later identified as resulting from apoptosis induction by BDED which was scored by the characteristic events of membrane externalization of phosphatidylserine, cytoplasmic shrinkage and chromatin condensation which were observed in a large majority of the BDED-treated cells. Further detailed studies were performed to delineate the cellular mechanisms of BDED-induced apoptosis in HeLa cells. Analysis of cell cycle progression by propidium iodide (PI) staining revealed BDED-induced cell cycle arrest in the G1 phase of cell cycle. The results indicated that BDED induced a significant ROS generation and a significant loss of mitochondrial membrane potential which were abrogated upon pretreatment of the cells with an antioxidant, N-acetyl cysteine (NAC). These observations suggested the probable involvement of the initial events of BDED-induced ROS production in causing cell death via mitochondria-based intrinsic pathway of apoptosis. In addition, the results from the expression profile of pro- and anti- apoptotic proteins by western blot analysis in the BDED-treated HeLa cells further corroborated this hypothesis. These included an abnormally deregulated cellular abundance of BAX 1 and BCL 2 proteins, elevated levels of APAF-1, and activated cleaved species of procaspases 9 and 3. In addition, a pronounced abundance of cleaved PARP protein was observed in these experiments. Thus, the results suggest that BDED induced apoptosis in HeLa cells via ROS mediated mitochondrial dependent pathway.
In parallel, the in vitro cytotoxic effects of NA was also studied, the second antiproliferative compound identified from hexane extracts of C. nigricolor using a panel of four human cancer cell lines - A-431, A549, COLO 205, and MCF 7. NA was thus found to be most potent against MCF 7 (breast cancer) cells. PI staining-based viability assays and microscopic observations showed a dose dependent cytotoxicity of NA on MCF 7 cells. Our data reveled an IC 50 of 40 μM for NA The NA-induced apoptosis was confirmed by flow cytometric detection of membrane externalization of phosphatidylserine using Annexin V FITC/PI dual staining. NA was also observed to induce cytotoxicity best at 72 h this could be attributed to the fact that it is a derivative of a Nonocosane-(a naturally prevalent molecule found in several vegetables). Similar to our results from BDED-treated HeLa cells, ROS generation and loss of mitochondrial membrane potential which were abrogated on pretreatment with NAC, were also observed in NA-treated MCF-7cells. Similar to several other fatty acids, NA was also observed to induce cell cycle arrest in the G1 phase of cell cycle. Further, apoptotic signature of an altered expression of anti-apoptotic BCL-2 and pro-apoptotic BAX, APAF-1 and procaspase 9 and 3 and a cleaved PARP were observed in NA-treated MCF 7 cells. In conclusion, the results indicate that BDED and NA have cytotoxic and apoptotic effects on HeLa (cervical cancer) and MCF 7 (breast cancer) cell lines, respectively. This leaves open further avenues to evaluate their potential application as anticancer agents for treatment of human cervical and breast cancers.||en_US