The Effect And Mechanism Of Lycorine On Proliferation And Apoptosis Of Pancreatic Cancer

Object: Pancreatic cancer is a highly malignant tumor of the gastrointestinal tract,and the anatomical location of the pancreas is hidden;therefore,early diagnosis of pancreatic cancer is difficult,and 80% of patients are diagnosed at an advanced stage at the first time.Chemotherapy as the mainstay of late treatment.However,due to the high drug resistance of pancreatic cancer,it is extremely urgent to find more effective anti-pancreatic cancer drugs and explore the key targets to inhibit the development of pancreatic cancer.Lycorine is an alkaloid derived from the natural plant garlic.Previous studies have shown that Lycorine has good inhibitory effects on lung,colorectal and breast cancers,but its effects and mechanisms of action on pancreatic cancer are still unclear.This study aims to investigate the antitumor effect of Lycorine on pancreatic cancer by combining transcriptomics and basic tumor research,and to find the key targets and pathways against pancreatic cancer,so as to provide new ideas for the treatment of pancreatic cancer.Methods:Cell experiment: Human pancreatic cancer cell lines PANC-1 and Bx PC-3 were selected and divided into control and different concentrations of Lycorine intervention groups.CCK-8 was used to detect the viability of pancreatic cancer cells.Colony formation and Ed U incorporation assays were performed to observe the changes in the proliferative ability of pancreatic cancer cells after Lycorine treatment.Flow cytometry was used to determine the cell cycle changes.The apoptosis level of cell lines was detected by Annexin V-FITC/PI assay.Transcriptomic sequencing of cell samples from Control and 20 μM Lycorine groups was performed to screen the differential genes,and Gene set enrichment analysis(GSEA)of the differential genes was performed.Observation of lipid deposition in cells by Oil Red O staining.The changes of free fatty acid content were measured by the Free Fatty Acid Assay Kit.Western blot was used to detect changes in the expression levels of proliferation,cell cycle,apoptosis-related proteins,key genes aldehyde dehydrogenase,family 3,subfamily A,member 1(ALDH3A1)and key pathway enzymes.Real-time quantitative PCR(q RT-PCR)to detect the effect of Lycorine on the m RNA expression level of ALDH3A1;knockdown of ALDH3A1 by small interfering RNA(si RNA)transfected cells.Animal experiments: The C57 BL/6 mouse pancreatic cancer axillary transplantation tumor model was established.They were divided into Control group,5 mg/kg·d and 10 mg/kg·d Lycorine group.The in vitro tumor suppressive effect of Lycorine on pancreatic cancer was assessed by observing tumor size,measuring tumor weight and volume in tumor-bearing mice.The toxic effects of Lycorine on mouse liver and kidney were evaluated by recording the changes in body weight during drug administration,measuring the serum levels of glutamic pyruvic transaminase(ALT),glutamic oxaloacetic transaminase(AST)and creatinine(Cr).The morphological changes of tumor,liver and kidney tissues were observed by H&E staining.Expression of Ki67 was detected by immunohistochemistry.The expression of key proteins in tumor tissues of different treatment groups was detected by Western blot.Results:Cell experiment:(1)Lycorine significantly inhibited the cell viability of PANC-1 and Bx PC-3 cells in a time-and concentration-dependent manner(P < 0.05 or P < 0.01).(2)The number of cell colonies was significantly decreased and the size was remarkably decreased(P < 0.05 or P < 0.01),and the proportion of Ed U incorporation gradually decreased(P < 0.05 or P < 0.01).(3)Flow cytometry results showed a significant increase in the percentage of G2/M phase cells and a decrease in the percentage of G0/G1 phase cells(P < 0.05 or P < 0.01)after Lycorine intervention.(4)Annexin V-FITC/PI results showed a gradual increase in apoptotic cells with increasing Lycorine concentration(P < 0.05 or P < 0.01).(5)A total of 17,542 genes were altered in expression after Lycorine intervention in Bx PC-3 cells.(6)Gene set enrichment analysis(GSEA)enrichment analysis showed that fatty acid metabolism was significantly inhibited and that ALDH3A1 was a key target in the fatty acid metabolism process.(7)The effect of Lycorine on pancreatic cancer cells resulted in a gradual increase in intracellular red lipid droplets(P < 0.001).At the same time,the content of intracellular free fatty acids increased significantly(P < 0.001).(8)Lycorine down-regulated proliferating cell nuclear antigen(PCNA)expression(P < 0.05 or P < 0.01),down-regulated G0/G1 phase and G2/M phase related protein expression(P < 0.05 or P < 0.01),down-regulated B-cell lymphoma-2(Bcl-2)protein expression(P < 0.05 or P < 0.01),had no significant effect on BCL-2-associated X protein(Bax)expression(P > 0.05),inhibited the expression of ALDH3A1 and key pathway enzymes,and had no significant effect on fatty acid synthase(FASN)expression(P > 0.05).(9)Lycorine significantly reduced the m RNA level of the key gene ALDH3A1(P < 0.001).(10)After knockdown of ALDH3A1,the viability of pancreatic cancer cells was significantly decreased(P < 0.05),the levels of PCNA,Cyclin B1,Cyclindependent kinase 1(CDK1),Bcl-2 were downregulated(P < 0.05),and there was no significant change in the expression of Bax(P > 0.05).Animal experiments:(1)The tumor-bearing mice in the Lycorine-treated group were in better condition,with smaller tumor size and lighter tumor weight than those in the Control group(P < 0.01).(2)There was no significant fluctuation in the body weight of the Lycorine-treated mice during the administration period(P > 0.05).Compared with the Control group,there was no significant difference in the organ indexes of the treated mice(P > 0.05),and there was no significant difference in the levels of ALT,AST and Cr in the serum(P > 0.05).(3)The number of cells decreased in the Lycorine-treated group compared with the tumor tissues in the Control group.(4)The expression of Ki 67,a proliferation-associated marker,was reduced by Lycorine inhibition.(5)The protein expression of the key gene ALDH3A1 was down-regulated in tumor tissues of animals after Lycorine intervention(P < 0.05 or P < 0.01).Conclusion: Lycorine can exert an inhibitory effect on the proliferation of pancreatic cancer cells by inhibiting ALDH3A1 and thus block the cells in G2/M phase and promote apoptosis of pancreatic cancer cells.At the same time,Lycorine inhibits the fatty acid oxidation process in pancreatic cancer cells,causing intracellular lipid deposition and increasing the amount of free fatty acids in the cells,leading to cell death.