Cytotoxic And Pro-Apoptotic Effects Of Selected Indigenous Medicinal Plants On Cervical Cancer Cell Line (Hela Cells)

ABSTRACT

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 solventsolvent 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