Triptolide regulates nSMase2-Cer sphingolipid metabolism signal pathway against-pancreatic cancer and its related mechanism
Objective To determine the mechanism of triptolide inhibiting the growth and proliferation of pancreatic cancer(PAC)cell line PANC-1,and to reveal the interaction between triptolide anti-PAC effect and neurosphingolipids.Methods Different concentrations of gradients of triptolide were used to treat PANC-1 cells,and the proliferation,migration and apoptosis of cells were detected after drug treatment.The changes of neurosphingolipid genes in PANC-1 cells after the treatment were screened by PCR array,and the transcription and translation of the genes screened by PCR array were verified by q-PCR and Western blot.The overexpression model of related genes in PANC-1 cells was constructed and verified by cell function experiment.The changes of nSMase2 protein level and Caspase-3 protein level in transiently transfected PANC-1 cells before and after the treatment and at different drug concentrations were detected by Western blot.The changes of ceramide(Cer)with different drug gradients and in the overexpression model were detected by immunofluorescence test.The subcutaneous tumorigenic model of PANC-1 in BALB/C nude mice was established,and the effect of triptolide on the tumor size in nude mice was verified in vivo.The tumor tissue was extracted for Western blot,and the liver tissue was stained with HE.Results Triptolide inhibited the proliferation,and migration and promoted the apoptosis of PANC-1 cells in a concentration gradient manner.The inhibition of triptolide on PANC-1 cell viability was most related to SMPD3 gene,and triptolide down-regulated its expression in PANC-1 cells.After overexpression of SMPD3,the proliferation and migration of PANC-1 cells were significantly enhanced,and the apoptosis was decreased.Triptolide promoted the increase of Cer ceramide content in PANC-1 cells,while overexpression of SMPD3 reduced the content of Cer in PANC-1 cells.In addition,triptolide up-regulated Caspase-3 expression in PANC-1 cells.In vivo experiments showed that triptolide down-regulated the expression of SMPD3 protein to promote the activation of Caspase-3 and further promote tumorigenesis and apoptosis to inhibit the tumor growth in nude mice.Conclusion In vivo and in vitro experiments show that triptolide inhibits the growth of PANC-1 cells by down-regulating the expression of SMPD3 and affecting the neutral sphingomyelin enzyme 2(nSMase2-Cer)signaling pathway.
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