Recombinant Newcastle disease virus (rL-RVG) triggers autophagy and apoptosis in gastric carcinoma cells by inducing ER stress

Abstract

We have reported that the recombinant avirulent Newcastle disease virus (NDV) LaSota strain expressing the rabies virus glycoprotein (rL-RVG) could induce autophagy and apoptosis in gastric carcinoma cells. In the present study, we explored the upstream regulators, endoplasmic reticulum (ER) stress that induce autophagy and apoptosis and the relationships among them. For this purpose, SGC-7901 and HGC cells were infected with rL-RVG. NDV LaSota strain and phosphate-buffered saline (PBS) were treated as the control groups. Western blotting and immunofluorescence microscopy were used to detect the expression of the ER stress-related proteins glucose-regulated protein 78 (GRP78) and the transcription factor GADD153 (CHOP), among others. The expression of beclin-1 and the conversion of light chain (LC) 3-I were used to determine the occurrence of autophagy, and flow cytometry (FCM) and western blotting were used to examine apoptosis-related protein expression. Transmission electron microscopy was also performed to monitor the ultrastructure of the cells. Moreover, small interfering (si) RNA was used to knock down CHOP expression. rL-RVG treatment increased the expression of ER stress-related proteins, such as GRP78, CHOP, activating transcriptional factor 6 (ATF6), X-box-binding protein 1 (XBP-1), and phosphorylated eukaryotic initiation factor 2 (p-eIF2α), in a time- and concentration-dependent manner, and knockdown of CHOP reduced LC3-II conversion and beclin-1 expression. When ER stress was inhibited with 4-PBA, the expression of both autophagy-related proteins and apoptosis-related proteins markedly decreased. Interestingly, inhibition of autophagy with 3-methyladenine (3MA) decreased not only apoptosis-related protein expression but also ER stress-related protein expression. Moreover, we found that downregulation of the c-Jun N-terminal kinase (JNK) pathway by SP600125 reduced LC3-II conversion, beclin-1 expression and caspase-3 activation. Collectively, the results suggest that rL-RVG increased ER stress in three branch pathways (ATF6, inositol-requiring enzyme 1 (IRE1), and PKR-like ER protein kinase (PERK)) that are upstream regulators of autophagy and apoptosis. Moreover, the IRE1-JNK pathway played an important role in switching ER stress to autophagy. These findings will provide molecular bases for developing rL-RVG into a drug candidate for the treatment of gastric carcinoma.

Keywords: Recombinant Newcastle disease virus; apoptosis; autophagy; endoplasmic reticulum stress.

Figure 1

rL-RVG induces the expression of ER stress-related proteins in SGC-7901 and HGC cells in a time-dependent manner. A and D. For SGC; B and E. For HGC. The cells were infected with 10 MOI virus or with vehicle (control) for different amounts of time. Cell lysates were harvested at the same time after infection with virus for different amounts of time, and proteins were detected by western blotting. A and D. The expression of GRP78 and CHOP increased at 12 h after treatment with rL-RVG, peaked at 24 h (**, p<0.01) and was sustained for at least 12 h in SGC cells. The same was observed in B and E. for HGC cells. A transient increase in eIF2α phosphorylation following rL-RVG infection was observed at 24 h in SGC cells (**, p<0.01) and at 12 h in HGC cells (**, p<0.01). C. Effect of rL-RVG on the level of spliced mRNA forms of XBP-1. uXBP1, unspliced XBP-1; sXBP-1, spliced XBP-1. The spliced XBP-1 mRNA appeared with 12 h of pretreatment, but the effect was transient.

Figure 2

Expression of ER stress-related proteins in infected SGC and HGC cells. The nuclei were stained with Hoechst 33342. The cells had been infected with virus for 24 h. ER stress-related protein expression was monitored by immunofluorescence microscopy (x200 magnification). A and B. For SGC, C and D. For HGC. A and C. GRP78 protein (green) was significantly more strongly expressed in the NDV and rL-RVG groups, and the rL-RVG-infected group exhibited the strongest expression. Similarly, in B and D. CHOP protein expression was strongest in the rL-RVG-infected group.

Figure 3

NDV and rL-RVG each induce the expression of ER stress-related proteins in SGC-7901 and HGC cells in a concentration-dependent manner. A and C. For SGC; B and D. For HGC. The cells were infected with 10 MOI or 1 MOI virus for 24 h. NDV and rL-RVG infection increased the amounts of GRP78 and CHOP in SGC and HGC cells at 24 h post-infection, and the rL-RVG-infected group exhibited the strongest expression. Activation of the three branches of ER stress signaling pathway proteins, or XBP-1, ATF6 and p-eIF2α, increased significantly in SGC and HGC cells (*, p<0.05; **, p<0.01).

Figure 4

N+4: NDV+4-PBA; R+4: rL-RVG+4-PBA. The contribution of ER stress downregulation to the expression of autophagy- and apoptosis-associated proteins in SGC and HGC cells after infection. A. For SGC; B. For HGC. The expression of the autophagy-associated proteins beclin-1 and LC3-II/I decreased in cells with 4-PBA treatment compared with cells without 4-PBA treatment. The same was observed for apoptosis-associated proteins. C and D. Early apoptotic (annexin V+/PI-) and late apoptotic (annexin V+/PI+) cells. Prior to fluorescence-activated cell sorting analysis of annexin V/PI staining, SGC cells were pretreated with 4-PBA (10 mM) 2 h before exposure to NDV or rL-RVG (10 MOI). Annexin V/PI staining was then performed to assess apoptosis/necrosis. Annexin V staining showed that pretreatment with 10 mM 4-PBA resulted in a significant decrease in apoptosis in the rL-RVG-infected SGC cells (**, p<0.01).

Figure 5

si+N: siCHOP+NDV; si+4: siCHOP+rL-RVG. Knockdown of CHOP decreased autophagy- and apoptosis-related protein expression. A. Fluorescently labeled siRNA (FAM-siRNA) was transferred into cells according to the manufacturer’s instructions, and the effects were monitored by immunofluorescence microscopy (x200 magnification). B. I expression of CHOP protein was silenced significantly in the group expressing siRNA 3. C. For SGC; D. For HGC. The expression of the autophagy-associated proteins beclin-1 and LC3-II/I decreased in cells after silencing CHOP compared with expression in cells without treatment. The same was observed for apoptosis-associated proteins.

Figure 6

SP+N: SP600125+NDV; SP+R: SP600125+rL-RVG. The autophagy induced by rL-RVG was mediated by the IRE1/JNK/beclin-1 signaling pathway. A. For SGC; B. For HGC. The phosphorylation of JNK and bcl-2 was upregulated in the rL-RVG- or NDV-infected SGC and HGC cells. The expression of the autophagy-associated proteins beclin-1 and LC3-II/I decreased in cells after treatment with a specific inhibitor of JNK, or SP600125, compared with cells without the treatment. The same was observed for apoptosis-associated proteins. C. The ultrastructure of the SGC cells after infection with virus or pretreatment with SP600125. rL-RVG infection increased the number of autophagosomes in SGC cells, but the number of rL-RVG-induced autophagosomes was markedly decreased after pretreatment with SP600125.

Figure 7

N+3: NDV+3MA; R+3: rL-RVG+3MA. The contribution of autophagy downregulation to the expression of ER stress-related proteins in SGC cells after infection. The expression of the ER stress-related proteins GRP78, CHOP and p-eIF2α decreased in cells after 3MA treatment compared with cells without 3MA treatment (*, p<0.05; **, p<0.01).

Figure 8

Proposed mechanism of rL-RVG-induced cell death.