aIn vitro Cytotoxic and Apoptosis Effects of Vatica diospyroides Symington Type SS Fruit Extracts on Cervical Cancer Cells

Abstract

Cervical cancer is the fourth most common cancer diagnosed in woman worldwide in 2018. Astoundingly, the data base of GLOBOCAN 2018 has been reported that Thailand was ranked as fourth for most common woman cancer diagnosed of South-Eastern Asia. In Thailand, cervical cancer is the second of cancer incidence rate and fourth rank of mortality rate in female. The reports show that cervical cancers until remains a crucial also in Thailand and global public health problem. Vatica diospyroides Symington (VDS) has been used as a Thai medicinal herb for cardiac and haematonic therapy and shown greatly cytotoxic response on human breast cancer cells as showed in previous reports. In this study, fruit extracts (cotyledon and pericarp) of VDS type SS were obtained using acetone and methanol. The aim of this research is to determine the cytotoxic activity and mode of cell death of VDS type SS fruit extracts on cervical cancer cell lines. The cytotoxicity was performed by MTT assay and the 50% inhibitory concentration (IC50) was used to indicate the cytotoxic effect. In order to investigate the mode of cell death including cell cycle arrest and apoptosis induction were subsequently measured using flow cytometry analysis. Protein expression involved with mode of cell death was elucidated by western blotting. The MTT result showed that the crude extracts exhibited highly cytotoxicity on HeLa with IC50 rank at 7.69-17.93 μg/mL and SiHa with IC50 rank at 9.81-19.00μg/mL, respectively. Surprisingly, the cotyledon-acetone extract notably showed the highest cytotoxicity on both cell lines. Furthermore, the selectivity index or SI was used to determine the harmless response of VDS extracts on normal cells. The result exhibited that only cotyledon-acetone extract shows high safety response on L929 cell with SI value more than 3. Remarkable feature of anticancer agent as cell cycle arrest was performed on cervical cancer cells and result showed that the extract could induce G2/M arrest in HeLa and SiHa with 2-fold IC50 at 48h through accumulation of cyclin-B1 expression and increasing of p-cdc2 CDK inhibitor in time-dependent manner. Additionally, the mode of cell death which effect by cotyledon-acetone extract was found that HeLa and SiHa treated cells were shown apoptosis induction in a dose-dependent manner. In addition, the expression of Bax was increased at 48 h and caspase-8 was declined together with increasing of cleaved caspase-8 expression. In conclusion, the anti- proliferation effect of cotyledon-acetone extract may be due to cell cycle arrest at G2/M phase via cyclin-B1 accumulation and apoptosis induction by stimulation of Bax and cleaved caspase-8 expression on cervical cancer cells. Based on our study, the cotyledon-acetone extract would be a candidate anticancer agent with promise anticancer properties and might be lead to alternative approach for cancer therapies.