Abl Kinase Inhibitor STI-571 Affects The Proliferation And Apoptosis Of A375 Cells Through The PTEN-PI3K/AKT Signal Pathway

Malignant melanoma(MM)is one of the most aggressive tumors,mortality of which accounts for the first place in skin malignant tumors.The incidence of MM in white people is higher,but the incidence of the disease in China has increased exponentially in recent years,and about 20 thousand new cases occur annually.Because of its early and wide metastasis and the short median survival time of advanced metastatic MM,MM is rather intractable in clinical treatment.At present,with the continuous research on the cause of MM,some molecular targeted drugs have been approved to be listed.However,it is still urgent to find new targets and effective therapeutic drugs.It has been found that in some primary melanomas and malignant melanoma cell lines,such as A375 cell line,Abl kinases were highly activated,and the activation of Abl kinases is associated with the occurrence and development of a variety of tumors.As a inhibitor of Abl kinases,STI-571 can reverse the drug resistance of some tumor cells and inhibit the proliferation of breast cancer cells and neuroblastoma cells,but the effect on the proliferation and apoptosis of A375 cells and the related signal transduction mechanism have not been reported.Therefore,we will study the effects of STI-571 on the proliferation and apoptosis of A375 cells and the related mechanisms.Objects:In the research,we will study the impacts of STI-571 on proliferation and apoptosis of malignant melanoma cells A375 and the related mechanisms,in order to provide some basis for further studying the functionary mechanism of high activity Abl kinases in melanoma cells and for STI-571 to enter the clinical treatment of MM.Methods:The cell viability was determined by CCK8 assay and flat clones formation assay at different concentrations of STI-571.For proliferation,flow cytometry and Western-blot were used to study the effects of STI-571 on the cell cycle of A357 cells and on the intracellular levels of cell cycle-related protein p27 and SKP2,respectively.Then,the effects of STI-571 on cytoplasm and nuclear distribution of p27 were performed by using nuclear and cytoplasmic protein extraction method and immunofluorescence assay.Flow cytometry operation and DAPI staining were used to detect the effects of STI-571 on A375 cells apoptosis,and Western-blot was used to detect the effects of STI-571 on the intracellular levels of clevead-caspase3 and clevead-PARP as indicator protein of apoptosis.The expression of PTEN was detected by Western-blot as well as RT-qPCR,and the activation of its' downstream signaling molecule AKT was detected by Western-blot.At last,the interaction of p-AKT and p27 was judged by coimmunoprecipitation.Results:It was found that STI-571 could inhibit the cell viability of A357 cells in a concentration-dependent manner.For proliferation,STI-571 increased the intracellular content of cell cycle's negative regulatory protein p27 and its nuclear distribution(but did not affect the content of SKP2),so that induced G1 arrest of A375 cells.As for apoptosis,the use of STI-571 increased the activation of caspase3 significantly,and promoted the apoptosis of A375 cells.Subsequently,it was found that STI-571 could inhibit the activation of AKT by promoting the expression of PTEN in A375 cells.And we further confirmed that p-AKT and p27 were located in the same signaling pathway.Conclusions:(1)STI-571 can inhibit the proliferation and promote the apoptosis of A375 cells.(2)STI-571 may play a role in inhibiting proliferation and promoting apoptosis of A375 cells by up-regulating the expression of PTEN and inhibiting the PI3K/AKT signaling pathway in A375 cells.