| Background:Gallbladder cancer is the fifth most common malignant tumor with a high mortality rate.In recent years,the incidence of gallbladder cancer in China has been increasing,with about50,000 new cases each year.Radical surgical resection is the first choice for the treatment of gallbladder cancer.However,the disease is often found to be late with liver and lymph node metastases and then advanced rapidly because it has no specific clinical manifestations in the early stages of the disease,which the prognosis is very poor.With the extensive use of chemotherapy drugs in solid tumors,it has played a crucial role in the treatment of advanced gallbladder cancer.The current chemotherapy regimen for gallbladder cancer is mainly gemcitabine combined with platinum-based chemotherapy.Although chemotherapy can increase the resectability and survival rate of the tumor,the median survival time is still less than 15 months.Therefore,looking for new treatments for gallbladder cancer Treatment has become an important clinical issue.In recent years,molecular targeted therapies have used specific blockers to interfere with certain signaling molecules expressed by tumor cells to inhibit tumor growth and invasion,thereby achieving tumor treatment effects,such as apatinib used to treat gastric cancer and thyroid cancer.Lenvatinib has achieved good results in clinical trials.At present,the targets related to gallbladder cancer are known as the vascular endothelial growth factor（VEGF）and epidermal growth factor receptor（EGFR）family.For these targets,there are gefitinib,erlotinib,and bevacizumab.Other targeted drugs have entered clinical research,but the therapeutic effects have been mixed.Pyrotinib is a newly approved targeted chemotherapeutic drug for the treatment of ErbB2/HER2 positive breast cancer in China.This drug further binds and inhibits the target of human epidermal growth factor receptor 2（ErbB2/HER2）.Changes in downstream signaling pathways can inhibit the growth of tumor cells.At present,there is no domestic or foreign research report on the application of this drug in gallbladder cancer.With the development of second-generation gene sequencing technology,a large number of studies have reported the abnormal expression of ErbB2 gene in gallbladder cancer tissue.On this basis,there have been experimental studies on the silent expression of this gene in cell lines such as NOZ and SGC-996,The results showed that the growth of tumor cells was inhibited.These data show the carcinogenic potential of ErbB2.Targeting to reduce the expression of this gene at the cellular experimental level can initially inhibit the growth of tumor cells.Therefore,this study intends to investigate the effect of pirotinib on gallbladder The killing effect of cancer cell lines,and the possible mechanism of action for further study.Objective:To investigate the effect of Pyrotinib on the proliferation,invasion,migration and apoptosis of gallbladder cancer NOZ and SGC-996 cell lines.Methods:The CCK-8 assay was performed to measure the OD values of gallbladder cancer cells NOZ and SGC-996 at 24h,48h,and 72h treated with different concentrations of Pyrotinib.The 50%inhibitory concentration values（IC50）were calculated.Then the concentration of 25%,50%and 75%inhibition rates for 48 hours were selected as the following treated concentration of Pyrotinib.Colony formation experiments,transwell assay and flow cytometry analysis were performed to investigated the ability of cell colony formation,migration,invasion,and apoptotic rate of cancer cells respectively.Moreover,western blot analysis examined the apoptotic related protein expression.Results:1.Pyrotinib inhibits the proliferation of gallbladder cancer cells:CCK-8 experiment was used to detect the effect of pyrotinib on cell viability of NOZ and SGC-996 cells lines.The viability of NOZ and SGC-996 cells treated with pyrotinib gradually decreased with the increasing of the treatment concentration,and as the prolonged period of action of pyrotinib,the cell viability showed a significant downward trend.48 hours after dosing,the cell suppression rate increased from 20%to 70%with the increase of drug concentration;IC50 of NOZ and SGC-996 cells at 24h,48h and72h were:11.5μmol/L,3.6μmol/L,1.4μmol/L and 5.5μmol/L,5.2μmol/L,2.4μmol/L separately.The difference was statistically significant（P<0.05）.2.The effect of pyrotinib on the clonal formation ability of gallbladder cancer cells and the influence on cell proliferation and metastasis:clone formation assay were conducted to determine the single cell proliferation ability.pyrotinib treatment caused a dose-dependent decrease in colony formation ability of gallbladder cancer cells.In addition,the number and size of colonies formed in the treatment group were significantly smaller than those in the control group.The above results prove that pyrotinib can inhibit the viability and proliferation ability of gallbladder cancer cells（P<0.05）.3.Inhibitory effect of pyrotinib on the invasion and migration ability of gallbladder cancer cells:Transwell chamber experiment analysis showed that compared with the control group,after 12 hours of treatment with pyrotinib,the number of cancer cells which passed through the Transwell chamber that with and without Matrigel decreased with increasing treatment concentration.The above results proved that pyrotinib inhibits the invasion and migration of gallbladder carcinoma NOZ and SGC-996 cells（P<0.05）.4.Pyrotinib induces apoptosis of gallbladder cancer cells:To investigate the effect of pyrotinib on the apoptosis of gallbladder cancer cells,the NOZ and SGC-996 cells were treated with the aforementioned concentration for 48 hours,and then analyzed by flow cytometry Apoptosis rate.In a two-parameter scatterplot,the LL quadrant represents live cells,and the LR and UR quadrants represent early and late apoptotic cells,respectively.As the concentration of pyrotinib increased,the percentage of surviving cells decreased,while the number of early and late apoptosis cells both increased.These data indicate that pyrotinib induces apoptosis in gallbladder cancer cells.In order to further study the mechanism of pyrotinib-induced apoptosis,we detected the expression of caspase family,Bcl-2 family and other important proteins that played a key role in the middle of the apoptosis process by Western blot,and showed that pyrotinib increased the expressions of Cleaved-caspase 3,Cleaved-caspase 9,Cleaved-PARP and Bax also reduced the expression of anti-apoptotic protein Bcl-2.In addition,the ratio of Bcl-2 to Bax showed a dose-dependent decreasing trend（P<0.05）.Conclusion:Pyrotinib significantly inhibited the proliferation,colony formation,migration and invasion of gallbladder carcinoma in vitro,and induced the protein expression related to apoptosis and decrease the anti-apoptotic protein expression,which provides a new sights for the treatment of gallbladder cancer. |
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