Research On The Function And Mechanism Of GSK343 In Pancreatic Cancer

ObjectivesBeing one of the most common digestive tract malignant tumor,the incidence of pancreatic cancer is ascending year after year.Due to the lack of typical clinical symptoms in early-stage,most patients often go to hospital after the tumor invades surrounding tissues or metastases to distant organs,and the prognosis is extremely poor.According to predictions by the American Cancer Society,pancreatic cancer will be the third leading cause of cancer death in the USA and lead to an estimated 227 000 deaths per year worldwide.With the deeper recognition of tumors and advance of medical technology,comprehensive therapeutic strategies based on the operations have acquired better effect for cancer treatment.Nevertheless,patients' five-year survival rate is still only 8%.Faced with such grim situation,effective and innovative treatment strategies are extremely urgentEnhancer of zeste homolog 2(EZH2),which is the enzymatically active subunit of polycomb repressive complex 2(PRC2),can catalyze trimethylation of lysine 27 on histone 3(H3K27),leading to transcriptional silencing of target genes.A large number of studies have reported that EZH2 plays a key role in the progression of various cancers,such as prostate cancer,breast cancer,endometrial cancer,melanoma,and hematological malignancies.GSK343 acts as a SAM-competitive inhibitor to exert an enzymatically repressive function on EZH2.The high expression of EZH2 in cancer is closely related to the prognosis and metastasis of cancer.Inhibition of EZH2 is regarded as a novel and potential therapeutic target for carcinomas.In this study,we selected two pancreatic cancer cell lines AsPC-1 and PANC-1 to explore the function and mechanism of EZH2 inhibitor GSK343,and may provide a new strategy and method for the treatment of pancreatic tumors.Methods1.Determination of IC50 in GSK343 in different pancreatic cancer cell linesAfter treated with different concentrations of GSK343,the IC50 concentration of GSK343 in pancreatic cancer cell lines AsPC-1 and PANC-1 was detected by MTT assay,and then draw the cell viability-drug concentration curve.2.The effects of GSK343 on cell biological behavior of pancreatic cancer cells1)The effect of GSK343 on the proliferation of pancreatic cancer cells was determined by CCK-8 and EdU assay.2)Select appropriate concentration and analyze the effect of GSK343 on apoptosis of pancreatic cancer cells by flow cytometry.3)Select the suitable concentration and use flow cytometry to analyze the effect of3.The effects of GSK343 on autophagyGSK343 on pancreatic cancer cell cycle.in pancreatic cancer cells and its mechanism1)Using immunofluorescence technique,the puncta of LC3B-? was observed under a fluorescence microscope to examine the effect of GSK343 on autophagy in pancreatic cancer cells.2)Select the appropriate concentration and detect the ATG5 and LC3B by Western-blotting assay to confirm the ability of GSK343 on autophagy in pancreatic cancer cells.3)After adding appropriate concentration of GSK343,Western blotting was used to detect AKT,p-AKT(ser473),mTOR and p-mTOR(ser2448),and explore the possible mechanism of GSK343-induced autophagy in pancreatic cancer cells.Results1.IC50 values of GSK343 in different pancreatic cancer cell linesThe MTT method showed that the IC50 value of GSK343 in the pancreatic cancer cell line AsPC-1 was 12.71±0.41?mol/l,while the IC50 value in the pancreatic cancer cell line PANC-1 was 12.04±1.10?mol/l.There were no significant statistical differences.2.The effect of GSK343 on the biological behavior of pancreatic cancer cellsCCK-8 and EdU assay showed that the proliferation of the two pancreatic cancer cell lines was significantly decreased after the addition of GSK343.Flow cytometry analysis showed that GSK343 could increase the apoptosis rate of pancreatic cancer cells and arrest the cell cycle in G0/G1 phase.3.GSK343 induces autophagy in pancreatic cancer cells and its mechanism1)Immunofluorescence technique showed that the puncta of LC3B-? significantly increased after adding appropriate concentration of GSK343,meanwhile Western-blotting showed that the expression of ATG5 did not change,LC3B-? decreased,and LC3B-? increased,and the ratio of LC3B-?/LC3B-? augmented.2)After treatment of pancreatic cancer cells at a suitable concentration,Western-blotting showed that the expression of AKT and mTOR unchanged,while the expression of p-AKT(ser473)and p-mTOR(ser2448)was significantly decreased.Conclusions1)The IC50 values of GSK343 in pancreatic cancer cell lines AsPC-1 and PANC-1 are no statistically significant difference,which have a general killing effect on pancreatic cancer cells,and have certain application prospects in the clinical treatment of pancreatic cancer.2)GSK343 can effectively inhibit the proliferation of pancreatic cancer cells,induce apoptosis of pancreatic cancer cells,and block the cell cycle in G0/G1 phase.3)GSK343 can induce autophagy in pancreatic cancer cells and down-regulate AKT/mTOR signaling pathway,which may be one of the important mechanisms of GSK343 to play a role in tumor suppression.