Résumé
Objective:To investigate the synergistic antitumor effect of pyrotinib in combination with 5-fluorouracil(5-Fu)on human epidermal growth factor receptor 2(HER2)positive breast cancer cells and its underlying molecular mechanism. Methods:HER2 positive breast cancer cells were screened by Western blotting.HER2 positive SKBR-3 and BT474 cells were treated with pyrotinib and 5-Fu individually or in combination for the following experiments.MTT assay was used to assess the effect of different drugs on the proliferation of the treated cells,and the combination index(CI)values were calculated using Combidrug software.Colony formation assay was used to evaluate the effect of different drugs on the colony-forming ability of the treated cells.FCM assay was used to analyze the effect of different drugs on the apoptosis rate and cell cycle of the treated cells.Western blotting was used to examine the effect of different drugs on the expression levels of proteins in the proliferation-and apoptosis-related signaling pathways.SKBR-3-cell-based tumor xenograft model was established using BALB/c nude mice.After treatment with pyrotinib and 5-Fu individually or in combination,the growth profiles of the xenograft tumors were recorded and the expression levels of proteins in the proliferation-and apoptosis-related signaling pathways were examined in the tumor tissues. Results:HER2 positive breast cancer cell lines SKBR-3 and BT474 were selected for further experiments after screening.The proliferation SKBR-3 and BT474 cells could be inhibited after treatment with pyrotinib and 5-Fu individually or in combination(all P＜0.05).Compared with pyrotinib or 5-Fu single drug treatment,pyrotinib in combination with 5-Fu had higher inhibition rate on the proliferation of SKBR-3 and BT474 cells with a Cl value of＜1,indicating the synergistic effect of pyrotinib and 5-Fu.In addition,in contrast to pyrotinib or 5-Fu single drug treatment,there was a further decrease in the number of colonies formed,increase in apoptosis rate,and increase in the percentage of G0/G,cells in SKBR-3 and BT474 cells after treatment with pyrotinib in combination with 5-Fu(all P＜0.01).Animal experiment results showed that the growth rate of xenograft tumors in mice treated with pyrotinib in combination with 5-Fu was significantly slower than that of the single-drug treated mice(P＜0.05).Western blotting analysis showed that the expression levels of HER2,HER4,AKT and phosphorylated ERK were significantly decreased after treatment with pyrotinib in combination with 5-Fu both in vitro and in vivo(all P＜0.01),indicative of the blockage of proliferation-related signaling pathways.Meanwhile,analysis of the apoptosis-related proteins revealed a decrease in the expression levels of caspase 3,poly ADP-ribose polymerase(PARP),and Bcl-2(all P＜0.01),while an increase in the expression levels of cleaved-caspase 3,cleaved-PARP,and p21(all P＜0.01). Conclusion:Pyrotinib and 5-Fu had synergistical antitumor effect on HER2 positive breast cancer cells,and the underlying mechanism may be related to the blockage of proliferation-associated signaling pathways and the induction of apoptosis and cell cycle arrest.