The Combined Effect Of Genistein With 5-FU On Human Gastric Cancer Cell Lines In Vitro And Its Related Mechanism Of Action

OBJECTIVE: To study genistein in combination with 5-fluoro- uracil (5-FU) exhibited an additive or synergistic inhibitory effect on human gastric carcinoma cells in vitro, and their mechanisms of action.METHODS: Two human gastric carcinoma cell lines (BGC-823 and SGC-7901) were treated with genistein (18.5~296.6μM) and 5-fluorouracil (48~768μM) alone or in combination for 24 to 72 hours. MTT assay was used to investigate its inhibitory effect on the proliferation of cancer cells. The method of Chou and Talalay was utilized to assess the type of drug interaction, i.e. 50% inhibited concentration (IC50) and combined index (CI). BGC-823 and SGC-7901 cells when exposed to genistein (20μM) or 5-FU (10μM) for 72 hours, TEM, FCM, IHC were employed to observe the apoptosis, cell cycle arrest and expression of Bax, Bcl-2, cyclin A, cyclin B, PCNA, mtP53 and P21 proteins in these cancer cells.RESULTS: Genistein or 5-FU alone or in combination inhibited the proliferation of BGC-823 and SGC-7901 cells significantly in a time- and dose-dependent manner. The combination of genistein with 5-FU had synergistic effect on the BGC-823 cell line when the IR exceeds 80% and on the SGC-7901 cell line when the IR is 20%-80%. 20μM genistein arrested the cell cycle progression of BGC-823 and SGC-7901 cells in G2/M phase and decreased the expression of cyclin A,cyclin B,PCNA,mtp53 protein and increased the expression of P21 protein. While 10μM 5-FU arrested the cell cycle of BGC-823 cells in G0/G1 and S phrase and SGC-7901 cells in S phrase which was accompanied with decreased expression of mtP53 and PCNA as well as increased expression of cyclin A, but the expression of P21 and cyclin B have no significant difference as compared with control. Genistein and 5-FU both induced apoptosis in these two cancer cell lines. The protein level of Bcl-2 was down-regulated and the protein level of Bax was up-regulated after treatment with genistein or 5-FU for 72 hours.CONCLUSIONS: Combination therapy of genistein with 5-FU shows more remarkable inhibition on the proliferation of gastric cancer cells at a certain range of concentration. Inhibition of proliferation of human gastric carcinoma cells after treatment with genistein may be caused by enhanced expression of Bax, P21 protein and decreased expression of Bcl-2, cyclin A,cyclin B,PCNA and mtP53, they resulted in cell cycle arrest and apoptosis. Genistein and 5-FU both can down- regulate the expression of Bcl-2, mtP53, PCNA and up-regulate the expression of Bax may be is one of the mechanism that they exert a synergistic action.