Cytotoxic and pro-apoptotic effects of selected indigenous medicinal plants on cervical cancer cell line (Hela cells)
A Thesis Submitted to the School of Postgraduate Studies, University of Lagos
Carcinoma of the cervix is the second most common cancer among women worldwide especially in the developing world. Due to severe reported cytotoxic tendenciesby anticancer drugs, newer therapies from natural products are desirable. This work therefore aims to partially purify pro-apoptotic agents from indigenous natural products and to investigate the mechanism of action of the selected plant fractions.Sixteen indigenous medicinal plants were collected from South West Nigeria. Plant extracts were fractionated into hexane (HF), chloroform (CF), ethylacetate (EAF), methanol (MF) and water (WF) fractions using solvent-solvent partitioning and polyamide adsorption chromatography. The resultant ninety-six test fractions were screened for lethality potential using brine shrimp lethality (BSL) assay. Fractions with LC50 less than 10 ug/ml were subjected to Water Soluble Tetrazolium (WST-1) cytotoxicity assay and apoptosis evaluations using: morphological assessment, flow cytometric analysis of phosphatidylserine externalization (Annexin V) and propidium iodide (PI) staining, caspase 3 enzyme assay, immunoblot assessment of Poly ADP-Ribose Polymerase-1 (PARP-1) Cleavage, carboxymethyl-2’,7’-dichlorodihydrofluorescein-diacetate (CM-H2DCFDA) fluorescent microscopic/microplate analysis of reactive oxygen species generation, and Terminal deoxynucleotidyl transferase (Tdt) mediated dUTP-biotin Nick End Labelling (TUNEL) assessment of DNA fragmentation. Inhibitory activity of the hit fractions and tannins on Topoisomerase 1 relaxation of supercoiled plasmid pBR322 was assessed using agarose gel electrophoresis. Relative gene expression of estrogen receptor-α (ESR-1), TP53, retinoblastoma (Rb), and NAD(P)H Quinone Oxidoreductase (NQO1) was done using densitometric analysis of reverse-transcription polymerase chain reaction (RT-PCR). Following BSL assay, Costus afer (EAF), Piper guineense (HF), Piper guineense (CF), Amaranthus viridis (EAF), Catharanthus roseus (CF), Zanthoxylum zanthoxyloides (HF) and Zanthoxylum zanthoxyloides (EAF) had LC50 values < 10 µg/ml. WST-1 Cytotoxicity testing of these hit fractions on HeLa (Tumourigenic) and KMST-6 (Non tumourigenic) cell Lines demonstrated cytotoxic effects with the exception of Zanthoxylum zanthoxyloides (HF). Significant (P<0.05) selective cytotoxic preference for HeLa to KMST-6 Cell lines was exhibited by P. guineense (CF) and C. roseus (CF). Membrane blebs were observed in HeLa cells treated with the hit fractions in comparison to 17.42 µg/ml(50µM) Camptothecin. Annexin V-PI analysis reveals that Z. zanthoxyloides (EAF) and P. guineense (HF)showed similar apoptotic effect in comparison to 21.8% apoptotic population in Camptothecin. A significant (P<0.05) increase in caspase-3 enzyme activity was observed in C. roseus (CF) and Z. zanthoxyloides (EAF) in comparison with the negative control. PARP-1 cleavage was confirmed in all the test groups except forA. virides (EAF) treated cells. Purified polyphenols from A. muricata showed inhibitory activity on topoisomerase 1 enzyme due to the presence of acetogenins as predicted by AUTODOCK in-silico analysis. Gene expression analyses showed down-regulation of ESR1 by P. guineense (HF); TP53 gene was up-regulated in all the groups with the exception of C. afer (EAF); NQO1 gene was down-regulated by C. afer (EAF), C. roseus (CF), P. guineense (HF) and A. viridis (HF). Findings in this studysuggest that all the hit test fractions elicited apoptotic effects but at varying degrees with Z. zanthoxyloides(EAF) showing the most prominent effect. WhileA. viridis (EAF) elicited its cytotoxic effect via necrosis, purified polyphenols from A. muricata inhibited topoisomerase I enzyme, a key enzyme inhibited by the commonly used drug Hycamtin (a semi synthetic derivative of camptothecin).