The mechanism of Demethy1zeylasteral (ZST93) on autophagy in human pancreatic cancer cells

Abstract

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. Key words: Demethylzeylasteral; Autophagy; Pancreatic cancer; Extracellular signal-regulated kinase1/2 pathway; Protein kinase B/mammalian target of rapamycin pathway