桦木酸通过抑制STAT3的活化提高胰腺癌细胞对吉非替尼的敏感性

Betulinic acid enhances gefitinib-sensitivity of pancreatic cancer cells via inhibition of STAT3 activation

目的:探讨桦木酸(BEA)提高胰腺癌Panc-1、Miapaca-2细胞对吉非替尼的敏感性及其潜在的作用机制。方法:细胞培养完成后,将对吉非替尼不敏感的Panc-1、Miapaca-2细胞随机分为4组:对照组、BEA组、吉非替尼组及BEA联合吉非替尼组,分别予以不处理、BEA、吉非替尼及BEA联合吉非替尼处理。MTS法检测BEA对2种细胞的增敏效果,集落形成实验检测BEA协同吉非替尼的治疗效果,WB实验检测BEA对Panc-1细胞凋亡相关蛋白的影响,流式细胞术检测BEA对Panc-1细胞凋亡的影响,表面等离子体共振(SPR)实验验证信号转导子和转录激活子3(STAT3)和BEA的直接结合,分子对接和分子动力学模拟实验预测STAT3和BEA的结合模式。结果:BEA协同增强Panc-1、Miapaca-2细胞对吉非替尼的敏感性(P<0.05),使其对两种细胞的IC50值均降低至原值的50%以下。吉非替尼联合BEA较单用吉非替尼或BEA促进Panc-1细胞的凋亡以及凋亡相关蛋白cleaved-PARP和Bax的表达,减少对凋亡抑制蛋白Bcl-2的表达(均P<0.05或P<0.01)。BEA对Panc-1细胞中STAT3的活化有剂量依赖性抑制作用(P<0.01)。BEA通过与STAT3的Lys-591、Ser-613形成氢键而稳定BEA与STAT3的结合作用,同时BEA稳定在STAT3的蛋白结合位点内,以此阻断STAT3二聚发挥增敏作用。结论:联用BEA和吉非替尼显著抑制胰腺癌Panc-1、Miapaca-2细胞的增殖并促进其凋亡,这种增敏作用可能是由BEA对STAT3抑制作用所介导。

Objective:To investigate the effects and the underlying mechanisms of betulinic acid(BEA)on sensitizing pancreatic cancer cell lines Panc-1 and Miapaca-2 to gefitinib.Methods:After the cell culture was completed,Panc-1 and Miapaca-2 cells were randomly divided into 4 groups:control group(without treatment),BEA group,gefitinib group and BEA combined with gefitinib group,respectively.The sensitization effect of BEA on gefitinib-insensitive pancreatic cancer cells was detected by MTS assay.The treatment effects of combined treatment of gefitinib and BEA against Panc-1 and Miapaca-2 cells were evaluated by colony formation assay.Flow cytometry was used to examine the effect of BEA on apoptosis of Panc-1 cells while WB was applied to determine the effect of BEA onapoptosis-related proteins.Surface plasmon resonance(SPR)experiment was used to detect the direct combination between signal transducer and activator of transcription 3(STAT3)and BEA;Molecular docking and molecular dynamics simulation experiments were adopted topredict the combining mode between STAT3 and BEA.Results:BEA synergistically enhanced the gefitinib-sensitivity of pancreatic cancer Panc-1 and Miapaca-2 cells(P<0.05),and IC50 of gefitinib on two cells were reduced by over 50%.Compared with single treatment,the combined treatment of BEA and gefitinib promoted the apoptosis and up-regulated the expressions of apoptosis-relatedproteins(cleaved-PARP and Bax),but reduced the apoptosis-inhibitory protein Bcl-2(all P<0.05 or P<0.01).Moreover,the inhibitory effect of BEA on STAT3 activation in Panc-1 cells was in a dose-dependent mannar(P<0.01).BEA stabilizes its binding to STAT3 by forming hydrogen bonds with Lys-591 and Ser-613 of STAT3;in the meanwhile,BEA stabilized inthebinding site of STAT3,there by blocking STAT3 dimerization to enhance the drug sensitivity.Conclusion:Combined use of BEA and gefitinib could significantly inhibit the proliferation and induce apoptosis of Panc-1 and Miapaca-2 cells,which might be mediated by the inhibition of BEA on STST3.