The endoplasmic reticulum is a highly dynamic organelle that plays a key role in homeostasis. The homeostatic balance created by the ER can, however, be disrupted by genetic and environmental damage, thus resulting in ER stress. In order to restore dynamic balance, the ER stress pathway is triggered and produces increased levels of several stress-related proteins, including PERK [2, 27, 28], IRE1 [4, 22], and ATF-6 [6, 10, 34]. Collectively, these proteins lighten the load on the ER by reducing misfolding and helping avoid the accumulation of unfolded proteins. In this study, we found that the expression levels of proteins in the ER stress pathway, including PERK and ATF-6, were significantly higher in the HepG-2 cells infected with Ad-Vp3 than cells infected with Ad-Mock when tested at 12 h and 24 h post-infection. These changes were caused by ER stress. However, the expression levels of these proteins in Ad-Vp3-infected cells were significantly lower than those in the Ad-Mock-infected cells, at 48 h and 72 h post-infection. These changes might be caused by ER stress injury. Electron microscopy also revealed that the ER in HepG-2 cells had incurred significant structural damage when assessed at 48 h post-infection. Collectively, these results indicated that the structure and function of the ER were both damaged by strong and persistent ER stress, thus resulting in the increased expression of ER-related proteins; subsequently, these ER-related proteins showed a significant decrease in expression. In conclusion, these data indicate that Apoptin can cause ER stress injury in HepG-2 cells by inducing strong and persistent ER stress.
From one perspective, ER stress and injury can both cause an increase in intracellular Ca2+ levels, thus resulting in Ca2+ imbalance [5, 8, 16]. The levels of Ca2+ in Ad-Vp3-infected cells were significantly higher than those in Ad-Mock-infected cells and non-infected HepG-2 cells at 24, 36, and 48 h post-infection. This result may have been caused by a strong and lasting ER stress, and consequential ER stress injury. However, the Ca2+ levels in the Ad-Vp3-infected cells were significantly lower than that in the control groups at 72 h post-infection. We believe that this result was due to the fact that the Ad-Vp3-infected cells underwent a significant extent of apoptosis. In the present study, Apoptin triggered the ER stress pathway in the ER, thus increasing the expression levels of PERK, IRE1 and ATF-6. Consequently, we believe that Apoptin can induce a strong and lasting ER stress and Ca2+ imbalance.
An imbalance in the intracellular concentration of Ca2+ can affect the mitochondrial membrane potential  and subsequently induce the release of pro-apoptotic proteins from the mitochondrial membrane mesenchyme . Several pro-apoptotic proteins can be released from the mitochondria, including AIF, HtrA2, Endo G, ARTS, Smac/Diablo, and Cyto-C. AIF is a flavoprotein and represents the main protein responsible for Caspase-independent apoptosis. HtrA2 and Caspase are known to competitively bind Inhibitors of Apoptosis Proteins (IAP) [25, 26]. The interaction between these pro-apoptotic proteins and IAP can relieve the inhibition of caspase by IAP, thus allowing caspase to become activated and then induce apoptosis. Endo G is known to migrate to the nucleus and trigger cell apoptosis by causing DNA fragmentation [14, 29, 33]. ARTs are known to mediate various pro-apoptotic stimuli in order to induce apoptosis. The function of Smac/Diablo is the same as HtrA2 [23, 25]. In vitro, we found that the mitochondrial membrane potential of Ad-Vp3-infected cells was significantly lower than that in Ad-Mock-infected cells and non-infected HepG-2 cells at 24 post-infection. Furthermore, compared with all other timepoints, the mitochondrial membrane potential at 36 h post-infection, was also significantly reduced in the cells infected with Ad-Vp3. We speculate that this result might be caused by an abnormal change in intracellular Ca2+ level. In order to analyze whether Ca2+ imbalance affects the abnormal changes of mitochondrial membrane potential mediated by Apoptin in HepG-2 cells, Ad-Vp3 infected cells were further treated with BAPTA-AM. These results showed that the imbalance of Ca2+ induced by Apoptin could reduce the mitochondrial membrane potential and induce the apoptosis. In vivo, the regulation of Ca2+ imbalance reduced the expression levels of Smac/Diablo and Cyto-C in Ad-Vp3 + BAPTA-AM group. This result showed that the Apoptin might increase the expression levels of Smac/Diablo and Cyto-C to inhibit the proliferation of HepG-2 cells in tumor bearing nude mice via inducing Ca2+ imbalance.
In this study, we found that Apoptin induced a strong and lasting ER stress and thus cause ER stress injury. These events subsequently led to an imbalance of intracellular Ca2+, a reduction in mitochondrial membrane potential, a significant extent of damage in the mitochondrial structure, and an increase in the expression levels of Smac/Diablo, and Cyto-C, by triggering the mitochondrial apoptotic pathway in HepG-2 cells. However, it is not clear how Apoptin induces ER stress. We speculate that Apoptin-induced ER stress may be related to Apoptin-induced G2/M phase arrest [3, 13]. G2 phase involves the synthesis of a large number of RNAs and proteins. Therefore, G2/M phase arrest may cause an accumulation of unfolded or misfolding proteins, thus triggering the Unfolded Protein Response (UPR), thus leading to ER stress. Previous studies have not reported the specific relationship between G2/M phase arrest and UPR/ER stress. It is clearly evident that we now need to be able to elucidate the specific relationships between these three factors. By investigating the relationships between G2/M phase arrest, UPR, and ER stress, we may be able to enhance our understanding of the role of Apoptin-induced ER stress in Apoptin-induced apoptosis.