Drimia calcarata Bulb Extracts Deactivate the PI3K Signalling Pathway in Cervical HPV-18 Positive HeLa Cells

Abstract

• D. calcarata extracts promoted proliferation of HPV types 16 and 18 positive Ca-Ski cells through maintaining the expression of STAT3, CLB1 and CDC2 and maintaining the activated levels of PI3K. • Water extract induced apoptosis and G2/M cell cycle arrest of HeLa cells through increased expression of the tumour suppressor p53. • Both methanol and water extracts deactivated the PI3K signalling pathways in HeLa cells. • D. calcarata plant extracts could be useful for therapeutic purpose against human HPV-18 positive gynaecologic cancers. The efficacy of Drimia calcarata against the human cervical cancer remains unexplored and less understood. The present study focused on investigating the cytotoxic effect of D. calcarata bulb extracts on cervical cancer cells. The growth inhibitory effects of D. calcarata extracts were determined using the MTT, Ki67 and PI3K Activation assays. Apoptosis induction was assessed using fluorescence microscopy, the Muse Ⓡ Cell Analyser and gene expression analysis by RT-PCR. The cytotoxicity of the fractions was evaluated using MTT assay and the apoptosis induction using Muse Ⓡ Cell Analyser. Both methanol extract (ME) and water extract (WE) showed safety against noncancerous KMST-6 and HEK-293 cells. The extracts exhibited anticancer activity against HeLa, with no significant cytotoxic effect against the Ca-Ski cells. The WE increased the Ki67 positive Ca-Ski population, while both ME and WE arrested HeLa cells at G2/M phase, and Ca-Ski cells in G0/G1 phase. AO/EB staining, Annexin V and Caspase 3/7 Activation revealed that the extracts significantly induced apoptosis in HeLa cells. In HeLa cells, the ME downregulated TP53 variants, while WE upregulated both TP53 variants in HeLa cells. Both extracts decreased the STAT5A and STAT5B mRNA expression in HeLa cells; however, these extracts upregulated cancer-promoting STAT3 in Ca-Ski cells. Additionally, these extracts inactivated the PI3K signalling pathway in HeLa cells but not in Ca-Ski cells. The resistance of the Ca-Ski cells to the D. calcarata extracts may be due to the upregulation of STAT3 and activated PI3K signalling pathway. The cytotoxicity and apoptosis induction in HeLa cells by D. calcarata extracts may be attributed due to downregulation of STAT5A survival mechanisms. Water fractions 1, 2, 3 and 6 and methanol fractions 1 and 2 reduced cell viability of HeLa cells. Water fractions 2, 3 and 6 and methanol fractions 1 and 3 induced apoptosis, which was preceded by secondary necrosis. However, water fraction 1 and methanol fraction 2 led to most cells undergoing necrotic cell death. There are several compounds that can be credited with the anticancer activities of the ME and WE extracts since several fractions exhibited cytotoxicity against the HeLa cells. These findings suggest that the D. calcarata extracts have anticancer activities, and thus, could be useful for therapeutic purposes against human HPV-18 positive gynaecologic cancers.