去甲泽拉木醛(ZST93)诱导人胰腺癌细胞自噬的分子机制

The mechanism of Demethylzeylasteral (ZST93) on autophagy in human pancreatic cancer cells

目的探讨新型雷公藤提取物去甲泽拉木醛(ZST93)诱导人胰腺癌细胞自噬的分子机制。方法免疫荧光和透射电镜检测ZST93对胰腺癌MIA PaCa-2和PANC-1细胞自噬的影响,Western blot检测ZST93对胰腺癌细胞自噬相关信号通路细胞外信号调节激酶1/2(ERK1/2)和蛋白激酶B/雷帕霉素靶蛋白(Akt/mTOR)活性的影响,抑制ERK1/2通路活性后,免疫荧光和透射电镜检测胰腺癌细胞自噬水平的变化。结果低浓度ZST93可诱导胰腺癌细胞自噬增加,MIA PaCa-2和PANC-1细胞中LC3荧光强度分别增加至3.4倍和19.0倍,自噬泡数目分别增加至13倍和7倍;抑制自噬相关基因1(Beclin1)表达可逆转ZST93诱导的胰腺癌细胞自噬,MIA PaCa-2和PANC-1细胞中LC3荧光强度分别下降至1.9倍(P<0.05)和2倍(P<0.01),自噬泡数目分别下降至2倍(P<0.01)和1倍(P<0.01)。低浓度ZST93诱导胰腺癌自噬与经典自噬通路中磷酸化细胞外信号调节激酶1/2(p-ERK1/2)表达水平显著上调、磷酸化蛋白激酶B(p-Akt)和磷酸化雷帕霉素靶蛋白(p-mTOR)表达水平显著下调相关,低浓度ZST93诱导后,在MIA PaCa-2细胞中,p-ERK/ERK由1.03±0.11增加至1.84±0.13(P<0.01),p-Akt/Akt由0.98±0.07降低至0.51±0.05(P<0.01),p-mTOR/mTOR由1.00±0.03降低至0.63±0.02(P<0.01);PANC-1细胞中p-ERK/ERK由1.15±0.13增加至1.86±0.09(P<0.01),p-Akt/Akt由1.02±0.06降低至0.43±0.05(P<0.01),p-mTOR/mTOR由0.99±0.02降低至0.61±0.01(P<0.01);而高浓度ZST93无此作用。ERK1/2通路抑制剂PD 98059可逆转ZST93诱导的胰腺癌细胞自噬水平增加。结论低浓度ZST93通过激活ERK1/2通路并抑制Akt/mTOR通路活性,诱导胰腺癌细胞Beclin1依赖性细胞自噬水平增加。

Objective To explore the mechanism of the novel tripterygium wilfordii hook F(TWHF) extract Demethylzeylasteral (ZST93) on autophagy of human pancreatic cancer (PC) cells. Methods Autophagy levels induced by ZST93 were evaluated by immunofluorescence and transmission electron microscopy in human pancreatic cancer cell lines. The activity levels of extracellular signal-regulated kinase1/2 (ERK1/2) and protein kinase B/mammalian target of rapamycin (Akt/mTOR) pathways were determined by Western blot in PC cells treated with different concentrations of ZST93. Immunofluorescence and transmission electron microscopy were used to evaluate the autophagy levels induced by ZST93 after inhibition of ERK1/2 pathway with PD 98059. Results Low concentrations of ZST93 could induce autophagy in PC cells. The LC3 expression were elevated to 3.4 and 19 times to control groups in MIA PaCa-2 and PANC-1 cells, respectively. And the number of autophagic vacuole were increased to 13 and 7 times to control groups, respectively. The knock-down of Beclin1 effectively inhibited the autophagy process induced by ZST93, with that LC3 expression reduced to 1.9 (P<0.05) and 2 (P<0.01) times to control groups in MIA PaCa-2 and PANC-1 cells, respectively, and the number of autophagic vacuole reduced to 2 (P<0.01) and 1 time (P<0.01) to control groups, respectively. The autophagy induced by low concentrations of ZST93 on PC cells was associated with the significant up-regulation of phospho-ERK1/2 (p-ERK1/2) and down-regulation of phospho-Akt (p-Akt) and phospho-mTOR (p-mTOR) expression. After introduction of low concentrations of ZST93, p-ERK/ERK was increased from 1.03±0.11 to 1.84±0.13 (P<0.01), while p-Akt/Akt and p-mTOR/mTOR were reduced from 0.98±0.07 to 0.51±0.05 (P<0.01) and from 1.00±0.03 to 0.63±0.02 (P<0.01), respectively, in MIA PaCa-2 cells. In PANC-1 cells, p-ERK/ERK was increased from 1.15±0.13 to 1.86±0.09 (P<0.01), while p-Akt/Akt and p-mTOR/mTOR were reduced from 1.02±0.06 to 0.43±0.05 (P<0.01) and from 0.99±0.02 to 0.61±0.01 (P<0.01), respectively. Treatment with high concentrations of ZST93 did not show such effects. Moreover, ERK1/2 pathway inhibitor PD 98059 could effectively inhibit the elevated level of autophagy induced by low-dose ZST93. Conclusion Low concentrations of ZST93 could induce Beclin1-dependent autophagy by activating ERK1/2 pathway and inhibiting Akt/mTOR pathway.