The Role Of14-3-3Gamma Protein Expression In The Mechanism Of Triptolide-induced Apoptosis In Pancreatic Cancer Cells

Objective: Our study is to explore the role of14-3-3gamma Protein expression inthe mechanism of Triptolide-induced Apoptosis in Pancreatic Cancer Cells.Methods: The pancreatic cancer cells AsPC-1were cultured in the DMEM with10%FBS as usual, then would be used for experiments when they are growing in thelogarithmic phase. The cell viabilities of human pancreatic cancer cell AsPC-1weredetected by MTT assay, as well as the apoptosis was detected by Flow Cytometry.The expression of14-3-3γ protein in AsPC-1was detected by Western blotting assay,the difference between the TPL-induced groups and the normal control was analyzedby relative quantification.Results:1. Compared with the normal control, cells that observed through the microscopebecome less and less following the induction of12.5ng/ml,25ng/ml,50ng/ml TPLrespectively for48h. What was worse, most cells turned to be cell debris suspendingin the culture medium.2. Cells were treated with triptolide in different concentrations (6.25,12.5,25,50,100,150and200ng/ml for48h, MTT assay shows that the proliferation(%) ofAsPC-1cells were inhibited inordinately (F=136.00， p=0.00<0.01).The cellsviabilities of pancreatic cancer AsPC-1cells induced by12.5ng/ml of TPL for48hwas43.94±2.58%which showed significant difference compared to the88.22±4.73%of24h and the20.84±2.63%of72h (F=293.54， p=0.00<0.01). Itdemonstrates that the triptolide could significantly inhibit the proliferation andinduce the apoptosis of pancreatic cancer AsPC-1cells in a concentration-andtime-dependent manner.3. To determine the effect of triptolide on the apoptosis of AsPC-1cells, cells weretreated with triptolide in different concentrations (12.5,25and100ng/ml) for48h.Flow Cytometry demonstrated that the apoptosis of AsPC-1cells were respectively 2.40±0.48%,15.14±2.56%,29.56±2.73%and58.27±2.84%with significantdifference (F=309.657，p=0.00<0.01).4. Protein levels of14-3-3gamma were measured by Western blotting. Comparedwith the normal control, the relative expression of14-3-3gamma in the threeTPL-treated groups (12.5ng/ml,25ng/ml,50ng/ml of TPL for48h) were inhibitedrespectively with significant difference (χ2=10.769, p=0.013<0.05). Moreover,following with25ng/ml TPL-induction, the24h,48h and72h groups all exhibitedsignificantly lower relative expression of14-3-3γ protein than the12h group withsignificant difference(χ2=11.205, p=0.011<0.05).Conclusion:1. It was demonstrated on cell level that TPL could effectively suppress theproliferation and induce the apoptosis of pancreatic cancer cells.2. This study discovered and verified for the first time that the expression of14-3-3γprotein in human pancreatic cancer cells could be down-regulated by Triptolide.3.14-3-3γ should be used as a significant target molecule for inducing apoptosis ofpancreatic cancer cells by triptolide. The expression of14-3-3γ may play asignificant role in the inhibited-proliferation and induced-apoptosis of pancreaticcancer cells by triptolide, though the specific mechanism remains to bedemonstrated through further experimental research.