Synergistic Interaction Between Sorafcnib And Gemcitabine In Sequence Dependent In Human Non-small Lung Cancer

Sorafenib is a highly selective multi-targeted agent and has been reported to have potentantitumor effects against various tumors, including human non-small cell lung cancer（NSCLC）. In the present study, we explored the antitumor effect and associatedmolecular mechanisms of sorafenib against human lung cancer cell lines in vitro. Wealso investigated the efficacy and related mechanism of concurrent and sequentialadministration of sorafenib and gemcitabine in epidermal growth factor receptor（EGFR）-tyrosine kinase inhibitor （TKI）-sensitive and EGFR-TKI-resistant NSCLC celllines, the PC-9and A549cell lines.After a period of cell culture of PC9and A549cells lines, MTT method to detect theproliferation inhibition effect of different concentration of sorafenib or gemcitabine,make the dose-response curve, and calculate the half inhibition rate （IC₅0） of each drug;Than calculate the inhibition rate and the combination index of sorafenib andgemcitabine in combination or with different schedules. Flow cytometry detected thecell cycle distribution of the cells after using sorafenib and gemcitabine, alone, incombination or with different schedules. Western blot method detected thep-PDGFR, p-AKT, p-ERK and Bcl-2expression after using sorafenib and gemcitabinesingle-agent and in combination or with different schedules.We found that sorafenib exhibited dose-dependent growth inhibition in theEGFR-TKI-sensitive and EGFR-TKI-resistant NSCLC cell lines. At72h the IC₅0values of sorafenib and gemcitabine for A549cells were5.91±0.22μmol/L and 10.38±0.80nmol/L. At72h the IC₅0values of sorafenib and gemcitabine for PC9cellswere6.13±0.14μmol/L and8.38±0.64nmol/L, respectively. And we found that thesequential administration of gemcitabine followed by sorafenib exhibited the strongestsynergism, then the sequential administration of sorafenib followed by gemcitabineexhibited the antagonism. Cell cycle analysis showed that sorafenib arrested the cellcycle at G1phase, whereas gemcitabine caused arrest at S phase. We found that thesequential administration of sorafenib followed by gemcitabine reduce the proportion ofcells in G0/G1phase; then the sequential administration of sorafenib followed bygemcitabine decreased the proportion of cells in S phase; The two drugs interfere witheach other on the cell cycle in different sequential mode of administration. Inwesternblot experiments, we found that gemcitabine up-regulated p-AKT, p-ERK, andBcl-2expression, while sorafenib significantly inhibited p-AKT and p-ERK and theexpression of Bcl-2. The molecular mechanism of synergism effect is that thedownstream signaling pathways which activated by gemcitabine were then suppressedby sorafenib. By contrast, the reverse sequential administration resulted in antagonism,which may be due to that the downstream signaling pathways which suppressed bysorafenib were then activated by subsequent gemcitabine.This study demonstrated that sorafenib can inhibit the growth of non-small cell lungcancer A549and PC9cell lines，and the the sequential administration of sorafenibfollowed by gemcitabine exhibited the strongest synergistic anti-proliferative effect.