Study of inhibit the proliferation and induce apoptosis of human colon adenocarcinoma cell line HCT116

Abstract

AIM: To explore the anti tumor effect of indomethacin (IN) on human colon adenocarcinoma cells and determine the influence of indomethacin on cancer cell proliferation and apoptosis and elucidate the anti-tumor mechanism of Indomethacin.

METHODS: Human colon adenocarcinoma HCT116 cell line were cultured separately in vitro. Indomethacin (final concentration 100-800 μm) was administered alone or altogether with 5-Fu (50 μm). Agarose gel electrophoresis, MTT, and Flow cytometry were used to study cell proliferation and apoptosis in human colon carcinoma cell RT-PCR, western blot were used to detect the expression level of Bcl-2, bax gene and cdk4 protein expression in HCT116 cell lines after treated with IN for 24 h.

RESULTS: Indomethacin can inhibit significantly the proliferation of HCT116 cell, change the morphology, and cause the cells to accumulate in the G0/G1 phase of the cell cycle, and induce apoptosis. The apoptosis of tumor cells was confirmed by DNA ladder formation on gel electrophoresis and sub-G1 peak on flow cytometry. These responses were time-and concentration dependent. A synergic effect of inhibiting cancer cell proliferation was observed when combined with Indomethacin and 5-Fu. RT-PCR results showed that IN down-regulated Bcl-2 mRNA expression, and did not change Bax mRNA expression. Western blot results confirmed that IN inhibited Bcl-2 protein expression. No influence was found in the translation of Bax protein. In inhibited cdk4 protein expression.

CONCLUSION: Our study results indicate that IN induce apoptosis of HCT116 cell by down-regulating Bcl-2 expression and inhibiting cdk4 protein expression partially. This explains the mechanisms of antitumor activity of the Indomethacin.

Key Words: Colonic neoplasms; Indomethacin; Apoptosis; Flow cytometry; Polgmerase chain reaction