Anti-proliferation effect of sorafenib in combination with 5-FU for hepatocellular carcinoma in vitro: antagonistic performance and mechanism

Abstract

To investigate the anti-cancer efficacy and mechanism of sorafenib and 5-fluorouracil (5-FU) therapy in vitro using the HCC cell line MHCCLM3. The effects of sorafenib and 5-FU, alone or in combination, on the proliferation of MHCCLM3 cells were evaluated by cell viability assays. Combined-effects analyses were conducted according to the median-effect principle established by Chou and Talalay. Effects on cell cycle distributions were tested by flow cytometry and expression of proteins related to the RAF/MEK/ERK and STAT3 signaling pathways and cyclinD1 were tested by western blotting. Sorafenib and 5-FU alone or in combination displayed significant efficacy in inhibiting proliferation of the MHCCLM3 cells, with the following inhibition rates: sorafenib: 46.16% +/- 2.52%, 5-FU: 28.67% +/- 6.16%, and sorafenib + 5-FU: 22.59% +/- 6.89%. The sorafenib + 5-FU combination did not provide better results than treatment with either drug alone. The combination index values of the sorafenib and 5-FU treatments were mainly greater than 1, indicating that the two agents induced antagonistic, instead of synergistic, effects on the MHCCLM3 cells. In addition, the MHCCLM3 cells were less sensitive to 5-FU when administrated in combination with sorafenib, as evidenced by the half inhibitory concentration (IC50) significantly increasing from (102.86 +/- 27.84) mg/L to (178.61 +/- 20.73) mg/L (P = 0.003). Sorafenib alone induced G1 phase arrest (increasing from 44.73% +/- 1.63% to 65.80% +/- 0.56%; P less than 0.001) and significantly decreased the proportion of cells in S phase (decreasing from 46.63% +/- 0.65% to 22.83% +/- 1.75%; P less than 0.01), as well as down-regulated cyclinD1 expression (0.57 +/- 0.03-fold change vs. untreated control group; P less than 0.01). 5-FU alone up-regulated cyclinD1 expression (1.45 +/- 0.12-fold change vs. untreated control group; P less than 0.01). Moreover, sorafenib alone significantly inhibited the RAF/MEK/ERK and STAT3 pathways, with the fold-changes of p-C-RAF, p-ERK1/2 and p-STAT3 being 0.56 +/- 0.05, 0.54 +/- 0.02 and 0.36 +/- 0.02, respectively (all P less than 0.01); 5-FU alone produced no significant effects on these pathways. Administered alone, both sorafenib and 5-FU exert anti-tumoral activity on in vitro cultured HCC cells. The sorafenib + 5-FU combination treatment produces antagonistic, rather than synergistic, effects. Sorafenib-inhibited RAF/MEK/ERK and STAT3 signaling and cyclinD1 expression may have induced the observed G1phase arrest and S phase reduction, thereby reducing the cells' sensitivity to 5-FU.