The Role Of ROS-dependent Endoplasmic Reticulum Stress And Autophagy Pathway In The Apoptosis Induced By Withaferin A In Human Pancreatic Cancer Cells

Part I Withaferin A induced apoptosis of human pancreatic cancer cells through oxidative stress damageObjective To study the effect of withaferin A (WA), an effective component extracted from Withania somnifera Dunal, on proliferation and apoptosis of human pancreatic cancer cells and its possible mechanism.Methods CCK-8assay was performed to detect cell viability; Inverted microscope to observe the influence of WA for cell morphology; Hoechst33258fluorescence staining and Annexin V/PI double staining flow cytometry to detect cell apoptosis; JC-1fluorescent probe were used to detect cell mitochondria membrane potential; the cell cycle distribution of cells was were detected by PI staining flow cytometry; The expressions of apoptosis related proteins were detected by Western blotting; CM-H2DCFDA fluorescent probe were used to detect reactive oxygen species (ROS) production; Finally, using general caspase inhibitor Z-VAD-FMK and various antioxidants interfere cell, then observe the changes of cell apoptosis.Results WA significantly inhibited the proliferation and induced apoptosis of many kinds of human pancreatic cancer cell lines in a dose-and time-dependent manner in vitro, but the inhibitory effect is small for human normal cell lines; WA can induce human pancreatic cancer cells mitochondrial membrane potential decreased, and caused the cell cycle arrested at G2/M phase; Western blotting analysis showed that WA could significantly increased the ratio of Bax/Bcl-2, and enhanced the expression of cleaved caspase-3, cleaved caspase-9and cleaved PARP1in pancreatic cancer cell lines; Z-VAD-FMK could partially reversed the apoptosis effect induced by WA; Furthermore, WA also significantly increased the intracellular ROS level, but when the use of antioxidants NAC and Catalase clear cell ROS, apoptosis rate is significantly reduced.Conclusion WA could significantly inhibit the proliferation of human pancreatic cancer cells in vitro, and caused the cell cycle arrest at G2/M phase; WA also induced cell apoptosis, and the apoptosis is dependent on the activation of the caspase family; Furthermore, WA also significantly increased the intracellular ROS level, and WA induction of cell apoptosis is dependent on the production of ROS. Part II Study of withaferin A induce protective autophagy in vivo and in vitroObjective Study on whether WA induces autophagy in human pancreatic cancer cells and its biological significance through vitro and vivo experiments.Methods The super-microstructural changes were observed by transmission electron microscopy after WA treatment; the expressions of cleavage of microtubule-associated protein1light chain3isoform B (LC3B-Ⅱ) was detected by Western blotting; Construction of pEGFP-LC3plasmid stable transfected cell lines, and the fluorescent autophagy marker GFP-LC3was observed under a confocal microscope; the fusion of lysosomes and GFP-LC3was detected by staining with LysoTracker Red DND-99lysosomal Probe under a confocal microscope; the autophagic activity was inhibited by using variety of autophagy inhibitors or silencing Atg5(a critical component of the autophagic machinery), and then the proliferation and apoptosis induced by WA was assessed; Finally, xenograft tumor model in nude mice was established to observe the effect of using WA alone or combining use of autophagy inhibitor chloroquine (CQ) on the biological characteristics of pancreatic cancer cells about angiogenesis, proliferation, apoptosis, autophagy and so on.Results Transmission electron microscope showed that a large number of double membrane vacuolar autophagosomes were observed in WA-treated cells; simultaneously, Western Blot results show that WA also could significantly increased the expression of LC3B-Ⅱ; confocal microscope show that WA could induce pEGFP-LC3plasmid stable transfected cell lines appeared a large number of GFP-LC3punctate aggregates, and the green GFP-LC3and red LysoTracker Red is colocalized; a combined effect of using autophagic lysosome inhibitors and WA caused GFP-LC3punctate aggregates further increased, and Western Blot showed LC3B-Ⅱ expression is further up-regulation; nevertheless, another autophagy inhibitor3-methyladenine (3-MA) and the generic Caspase family kinase inhibitor Z-VAD had no significant effect on LC3B-Ⅱ expression; Moreover, it could enhanced the effect of inhibition of cell proliferation and induction of apoptosis when WA combined with autophagic lysosome inhibitors; similarly, when the autophagic activity was inhibited by silencing Atg5, apoptosis index also increased after WA interference; xenograft tumor model show that:21days later, single WA could inhibited the tumor growth and reduced the tumor volume compared with control group (P<0.05); when combined with autophagy inhibitor CQ, tumor volume and weight were further reduced compared with WA alone (P<0.05); Immunohistochemical analysis showed, in WA alone group, CD34and Ki67expression had a certain degree of reduction than control group, while p62/SQSTM1and LC3B expression were increased significantly (P<0.05); when combined with CQ, the expression of CD34and Ki67were further reduced compared with WA alone (P<0.05), and the expression of p62/SQSTM1and LC3B were further up-regulation (P<0.05);.Tunel staining showed that WA alone may induce apoptosis compared with control group (P<0.05), when combined with CQ, the apoptosis rate was increased significantly compared with control group (P<0.01).Conclusion WA could induce autophagy in human pancreatic cancer cells in vivo and vitro, and autophagy may serve as a protective response to stress; WA induced autophagy were not blocked by the Caspase kinase inhibitor Z-VAD-FMK, and it also does not depend on the Class Ⅲ P13K pathway; A single WA could partially inhibit the growth of xenograft tumor in nude mice, while combined with autophagy inhibitor CQ could inhibit tumor growth and promote apoptosis significantly. Part III Studies on the effects and molecular mechanism of ER stress response on withaferin A-induced apoptosis and autophagy in human pancreatic cancer cellsObjective To explore the effect and molecular mechanism of endoplasmic reticulum stress response on WA-induced apoptosis and autophagy in human pancreatic cancer cells.Methods The expressions of related proteins were detected by Western blotting; to observe the changes of WA-induced apoptosis and autophagy after inhibition of mTOR pathway; the change of endoplasmic reticulum structure was observed by using indirect immunofluorescence and transmission electron microscopy; colocalization of p62/SQSTM1, ubiquitin and GFP-LC3were observed by using indirect immunofluorescence; The interaction relationship between p62/SQSTM1and LC3B protein were detected by co-immunoprecipitation; observed the effects of ER stress protective agent Salubrinal and ER stress inducer tunicamycin on WA induced apoptosis and autophagy; to observe the changes of apoptosis and autophagy after silencing related gene CHOP, Atg5and p62/SQSTM1, then the expression of ubiquitin and ER stress related protein were also detected by Western blot; Finally, using antioxidants NAC interfere cell, then observe the changes of ER stress and autophagy.Results WA treatment increased the expression of p62/SQSTM1, but had little effect on other autophagy-related proteins; WA could inhibit the PI3K-AKT-mTOR signaling pathway, and inhibition of mTOR pathway can partially reverse the effect of cell apoptosis induced by WA; WA treatment caused ubiquitinated protein accumulated, and the accumulated ubiquitin colocalized with GFP-LC3; WA was able to induce ER stress, and Salubrinal or CHOP siRNA pretreatment inhibiting ER stress caused the suppression of WA-induced apoptosis and autophagy; on the contrary, promoted the ER stress could increase WA-induced cell apoptosis and autophagy; Moreover, inhibition of autophagy could enhance the aggregation of ubiquitined protein and ER stress reaction induced by WA; WA caused p62/SQSTM1gene transcription level, but had no effect on the protein degradation; furthermore, p62/SQSTM1interacts with LC3B; down-regulation of p62/SQSTM1by small interfering RNA could enhance WA-induced apoptosis, autophagy and ER stress; Finally, autophagy and ER stress response which were induced by WA could be blocked by the antioxidant NAC.Conclusion ER stress response mediated by ROS is involved in the induction of apoptosis and autophagy by WA; Autophagy is dependent on PI3K/AKT/mTOR signaling pathway induced by WA; inhibition of autophagy or increased ER stress could significantly enhanced the cytotoxic effect of WA on pancreatic cancer cells; p62/SQSTM1involved in WA resistance in human pancreatic cancer cells by clearing ubiquitinated proteins through the autophagic lysosome.