Simvastatin Resensitizes Cetuximab-resistant KRAS-mutant Human Colorectal Cancer Cells To Cetuximab

Objectives:Cetuximab,a monoclonal antibody that targets the epidermal growth factor recepter(EGFR),has become a first-line therapy in metastatic colorectal cancer(CRC)with wild-type v-ki-ras2 Kirsten rat sarcoma viral oncogene homolog(KRAS),while KRAS-mutant patients show great resistance to it.The BRAF-mutant state can also be a predictor of treatment of EGFR inhibitors,and mutant ones often show poor response to cetuximab.In order to benefit more patients from cetuximab targeted therapy,there is an urgent need to solve cetuximab-resistance in KRAS-mutant one and improve the cetuximab efficacy in BRAF-mutant one.Recently,statins have been reported to exhibit a trigger role in antitumor activity.Therefore,our study aims to investigate the effect of simvastatin on resensitizing cetuximab-resistant for KRAS-mutant CRC cells and improving cetuximab efficacy for BRAF-mutant cells in vitro,and also detects the underlying mechanism.Methods:The study selected DLD-1(KRAS mutation),LOVO(KRAS mutation),RKO(BRAF mutation)and HT-29(BRAF mutation)cell lines as the research object.Then the cells were treated with control medium,cetuximab,simvastatin and cetuximab plus simvastatin.MTT,flow cytometry analysis,western blot analysis were used to test the effect of simvastatin on reversing cetuximab-resistance and improving its efficacy in vitro.Results:1.Adding simvastatin to cetuximab,KRAS-mutant DLD-1 cells were highly sensitive to cetuximab than treated with cetuximab alone,whereas BRAF-mutant RKO cells were still high-resistant to cetuximab.Simvastatin plus cetuximab contrasting to cetuximab alone,significantly inhibited the proliferation of KRAS-mutant DLD-1 and LOVO cells(P<0.001),while the proliferation of BRAF-mutant RKO and HT-29 cells with two treatments were no significant difference(P>0.05).In contrast to treated with cetuximab alone,the cell proliferation was decreased for 0.8821±0.0041 to 0.6987±0.0054 in KRAS-mutant DLD-1 cells when treated with simvastatin plus cetuximab,which was decreased for 0.9065±0.0074 to 0.7198±0.0069 in LOVO cells.Whereas,treating cetuximab alone contrasting to Simvastatin plus cetuximab,the cell proliferation were 1.039±0.0561 and 1.013±0.0491 for BRAF-mutant RKO cells,which were 0.8742±0.0057 and 0.8585±0.0058 in HT-29 cells.2.Adding simvastatin to treat DLD-1 cells with cetuximab,the total percentage of apoptosis cells increased from 9.59%to 28.22%in KRAS-mutant DLD-1 cells,while the percentage in BRAF-mutant RKO cells were only 7.49%and 5.54%,respectively.Hence,there was significant induction of apoptosis to reverse cetuximab resistance after simvastatin treating for KRAS-mutant DLD-1 cells(P<0.05),but not for BRAF-mutant RKO cells(P>0.05).In contrast to treated with cetuximab alone,the expression level of anti-apoptotic protein Bcl-xl was decreased while the pro-apoptotic protein Bad level was elevated in KRAS-mutant DLD-1 cells when treated with simvastatin plus cetuximab.Whereas,there was no difference of Bcl-xl and Bad expression between treating with cetuximab alone and simvastatin plus cetuximab in RKO cells with BRAF mutation.3.Treated with simvastatin in different concentrations or for different times respectively,the expression level of BRAF and phosphorylation-ERKl/2 were significantly time-dependent and dose-dependent suppressed in DLD-1 cells with KRAS mutation,but no difference in BRAF-mutant RKO cells.Conclusions:1.Simvastatin might be used in combination with cetuximab in hunan KRAS-mutant CRC cell to increase its sensitivity and inhibit cell proliferation.2.simvastatin plus cetuximab markablely induced KRAS-mutant CRC apoptosis,down-modulated the anti-apoptotic protein Bcl-xl and up-modulated pro-apoptotic Bad proteins.3.The underlying mechanism of simvastatin on reversing cetuximab resistance might be through inhibiting RAS/RAF/MEK/ERK signal.