Plants have always been a source of medicine and are still being used by traditional healers in the rural part of Africa, Asia and India to treat a range of illnesses including cancer. The in vitro anticancer activity of an 80 percent ethanol extract of Cyathula prostrata, an annual branching shrub used by traditional healers in Nigeria to treat cancer was investigated. No previous studies have outlined the possible pathways and mechanisms used by cancer cells when treated with C. prostrata. Dose response analysis was performed to determine the effective cytotoxic concentrations of C. prostrata on HeLa (cervical cancer cell line) and U937 (myelo-monocytic cell line). The IC50 values were 100.8 μg/ml and 64.4 μg/ml for HeLa and U937 cells, respectively. All further experiments were performed using 125 μg/ml C. prostrata extract and 50 μM cisplatin as positive control. With the use of the fluorescent DNA binding dye propidium iodide, the induction of tumour cell death by C. prostrata extract has been linked to cell cycle arrest in the G1 phase at 24 and 48 h. In both cell lines, more than 80 percent of the C. prostrata treated cells were found in the G1 phase after 48 hours of treatment. The annexin V-FITC/PI assay revealed an increase in the percentage apoptotic cells from 4.9 percent to 53.1 percent at 24 h and 8.3 percent to 50.3 percent at 48 h. Since apoptosis induction can occur via a number of different pathways, distinct features were used as markers to investigate the mode of action of this C. prostrata extract. Markers such as activated caspase-8, p21 and cyt-c, were investigated with the aid of fluorescently labelled (FITC) antibodies with analysis using flow cytometry. No change in p21 levels was observed in response to treatment with the extract for up to 48 h. Cell cycle arrest in G1 was therefore not induced by this cyclin-CDK inhibitor. Increase in caspase-8 activation was observed in response to treatment with the extract with no cyt-c release from the mitochondria. The lack of cyt-c release was due to no change in mitochondrial membrane potential, which was investigated with the aid of fluorescent mitochondrial dyes and flow cytometric techniques. Caspase-8 activation is unique to the extrinsic apoptotic pathway. The results from this study therefore show that C. prostrata extract induces apoptosis via the extrinsic pathway and that this activation in independent of the mitochondria. The levels of hTERT, the catalytic subunit of telomerase, were investigated as an additional molecular target for C. prostrata. This was also investigated using FITC labelled antibodies and flow cytometry. A decrease in hTERT levels was observed following C. prostrata treatment. The findings from this study suggest that the extract acts through multiple targets, by inducing: cell cycle arrest in the G1 phase through an unknown mechanism; apoptosis through an extrinsic death receptor pathway and replicative senescence through inhibition of telomerase.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:nmmu/vital:10309 |
| Date | January 2010 |
| Creators | Schnablegger, Gerald |
| Publisher | Nelson Mandela Metropolitan University, Faculty of Science |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis, Masters, MSc |
| Format | viii, 62 leaves, pdf |
| Rights | Nelson Mandela Metropolitan University |
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