Apoptin causes apoptosis in HepG-2 cells via Ca2+ imbalance- triggered mitochondrial apoptotic pathway

doi:10.21203/rs.3.rs-1619281/v1

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Apoptin causes apoptosis in HepG-2 cells via Ca2+ imbalance- triggered mitochondrial apoptotic pathway

https://doi.org/10.21203/rs.3.rs-1619281/v1

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Apoptin is derived from the chicken anemia virus and exhibits specific cytotoxic effects against tumor cells. Herein, we found that Apoptin induced a strong and lasting endoplasmic reticulum (ER) stress response and Ca²⁺ imbalance, and triggered the mitochondrial apoptotic pathway. The aim of this study was to explore the mechanisms by which Apoptin exhibited anti-tumor effects on the HepG-2 cells. The intracellular level of calcium (Ca²⁺) was induced by ER stress and determined by electron microscopy, flow cytometry and fluorescence staining. Mitochondrial injury was determined by mitochondrial membrane potential, electron microscopy. Western blotting was used to investigate the levels of key proteins in the ER stress and the apoptotic pathway in the mitochondria. The relationship between Ca²⁺ level and apoptosis on Apoptin-treating cells was analyzed using Ca²⁺ chelator (BAPTA-AM), flow cytometry and fluorescence staining. We also investigated the in vivo effects of Ca²⁺ imbalance on the mitochondrial apoptotic pathway using the tumor tissues xenografted on nude mice. In vitro and in vivo experiments showed that Apoptin caused an imbalance in Ca²⁺, and increased the expression levels of Smac/Diablo and Cyto-C. In summary, Apoptin induced apoptosis in HepG-2 cells via Ca²⁺ imbalance-triggered mitochondrial apoptotic pathway. This study provided a new direction for antitumor research in Apoptin.

Apoptin

endoplasmic reticulum stress

Ca2+ imbalance

mitochondria structural injury

mitochondria apoptosis pathway

Apoptin (GenBank: AY171617.1) is derived from chicken anemia virus, which exhibits cytotoxicity that is specific to tumor cells [12, 20]. In most of the cell lines investigated thus far, Apoptin has been shown to induce apoptosis in tumor cells via the intrinsic mitochondrial pathway. Apoptin reduces the mitochondrial membrane potential in tumor cells and induces the mitochondria to release cytochrome c (Cyto-C) and apoptosis inducing factor (AIF) [1, 11, 19], thus resulting in apoptosis. Apoptin-induced apoptosis is known to occur independently of p53 [35]. In our research, we found that Apoptin induced a strong and long-lasting endoplasmic reticulum stress. However, as yet, the specific mechanism that underlying the association between ER stress and Apoptin has yet to be elucidated.

The endoplasmic reticulum (ER) is an important intracellular organelle and participates in a number of cellular processes in vivo. The ER is also the main site of calcium (Ca²⁺) storage. Ca²⁺ can be stored in the ER in concentrations of 10–100 mmol/L, but is present at concentrations of 100–300 nmol/L in the cytoplasm. A strong and lasting ER stress response creates an imbalance in Ca²⁺. ER stress is known to involve the Endoplasmic Reticulum Kinase (PERK) pathway [9, 15, 17], the Inositol-Requiring Enzyme1 (IRE1) pathway [7, 24, 30], and the Activating Transcription Factor 6 (ATF-6) pathway [31, 32].

As the key site of energy transformation, the mitochondria also represent the main regulatory site for apoptosis and also play a key role in most of the regulatory processes related to apoptosis. An abnormal change in the mitochondrial membrane potential triggers the release of apoptosis-related proteins from the mitochondria, including AIF, High-Temperature Requirement Protein A2 (HtrA2), Endonuclease G (Endo G), Apoptosis-Related Proteins (ARTs), Second Mitochondria-Derived Activator of Caspase/Direct IAP Binding Protein with Low pl (Smac/Diablo), and Cyto-C. Ca²⁺ imbalance caused by ER stress can affect the structure and function of the mitochondria. Despite that some research has indicated that Apoptin induces mitochondria to release Cyto-C and AIF, no previous study has investigated how Apoptin-induced ER stress caused Ca²⁺ imbalance and influenced the structure and function of the mitochondria. Moreover, previous studies have not investigated the specific effect of Apoptin on the expression of key apoptosis-related proteins in the mitochondria, such as HtrA2, Smac/Diablo, Endo G, and ATRS.

Therefore, it is of great significance to investigate the specific effect of ER stress on the ER and apoptosis-related pathways in the mitochondria with regards to the induction of apoptosis. In the present study, we demonstrated that Apoptin can induce ER stress, cause Ca²⁺ imbalance, affect the structure and function of the mitochondria, and induce significant changes in the expression of key proteins related to the ER stress and the mitochondria. Our research involved the assessment of dynamic changes in the ER and mitochondria, the determination of Ca²⁺ levels, the Western blotting and immunohistochemical analysis of tumor tissue from a tumor-bearing nude mouse model.

2.1 Materials

Human hepatoma cell line HepG-2 was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). We also used a recombinant type 5 adenovirus (Ad-Apoptin; hereafter referred to as Ad-Vp3) that contained a CMV promoter and the Apoptin gene from chicken anemia virus. We used the recombinant type 5 adenovirus (hereafter referred to as Ad-Mock) without the Apoptin gene as a blank control. Ad-Vp3 and Ad-Mock were constructed previously in our laboratory.

Six-week-old male BALB/c nude mice were housed in light, comfortable temperature and humidity room (22 ± 2°C, 50 ± 5% relative humidity), given solid diet (Changchun billion Adams Laboratory Animal Technology Co., Ltd.) and sterilized tap water. All mice were obtained from Beijing vital River Laboratory Animal Technology Co., Ltd., and the animal experiment protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the Changchun University of Chinese Medicine. All mice surgery was performed under 40 mg/kg 1% sodium pentobarbital via intraperitoneal injection.

Dihydrorhodamine 123 (DHR) (No. D632), JC-1 (No. T3168), MitoTracker™ Red CMXRos (TMRM) (No. M7512), Oregon Green™ 488 BAPTA-1, AM, and Cell-permeant Special Packaging (No. O6807) were purchased from ThermoFisher Scientific (Shanghai, China). We also purchased an FITC Annexin V Apoptosis Detection Kit I (No. 556547) from BD Biosciences (Beijing, China). We also acquired a Minute™ Total Protein Extraction Kit (No. SD-001/SN-002) and a Mitochondria Isolation Kit For Mammalian Cells And Tissues (No. MP-007) from Invent (Beijing, China). We purchased a range of reagents from Cell Signaling Technology (Shanghai, China), including a PARP (46D11) Rabbit mAb (No. 9532), Caspase-3 (D3R6Y) Rabbit mAb (No. 14220), Cytochrome c (D18C7) Rabbit mAb (No. 11940), Smac/Diablo (D5S3R) Rabbit mAb (No. 15108), Endonuclease G Antibody (No. 4969). We also purchased a monoclonal anti-ARTS antibody mAb (No. 106M4840) from Sigma-Aldrich (Shanghai, China). We purchased an anti-AIF antibody (No. ab1998), anti-HtrA2/Omi antibody (No. ab75982), anti-PERK antibody (No. ab65142), anti-ATF-6 (ab37149), anti-IRE1 (ab37073) from Abcam (Shanghai, China).

2.2 Methods

2.2.1 Annexin V analysis

HepG-2 cells were seeded at a density of 6 × 10⁵ cells per well in a 6-well plate and incubated for 24 h at 37°C in 5% CO₂. Cells were then infected with Ad-Vp3 (MOI: 50) for 12, 24, 36, 48, and 72 h. Ad-Vp3-infected HepG-2 cells were incubated in the dark for 15 min at room temperature in the presence of 5 µL Annexin V-FITC and 5 µL of PI in 100 µL of 1 × binding buffer. We then quantified the extent of apoptosis in HepG-2 cells by flow cytometry. Ad-Mock-infected cells and non-infected HepG-2 cells were used as negative controls.

2.2.2 ROS levels analysis

HepG-2 cells were seeded at 6× 10⁶ cells per well in a 6-well plate and incubated for 24 h at 37°C with 5% CO₂. The cells were then infected with 50 MOI Ad-Vp3 for 12, 24, 36, 48, and 72 h. HepG-2 cells infected with Ad-Vp3 were incubated in the dark for 15 min at room temperature in the presence of 1 µL DHR in 1 mL of DMEM. Flow cytometry was used to detect the level of ROS in HepG-2 cells infected with Ad-Vp3. Ad-Mock-infected cells and non-infected HepG-2 cells were used as negative controls.

2.2.3 Electron microscopy

HepG-2 cells were seeded at a density of 6 × 10⁵ cells/well into a 6-well plate, cultured for 24 h at 37°C in 5% CO₂, and infected with Ad-Vp3 (MOI: 50) for 48 h. Cells were then harvested, washed with PBS, and sections prepared using ultramicrotomy. Cells were analyzed by electron microscopy. Ad-Mock-infected cells and non-infected HepG-2 cells were used as negative controls.

2.2.4 Ca²⁺ levels analysis

HepG-2 cells were seeded at a density of 6 × 10⁵ cells/well into a 6-well plate, cultured for 24 h at 37°C in 5% CO₂, and infected with Ad-Vp3 (MOI: 50) for 12, 24, 36, 48, and 72 h. Cells were then incubated in the dark for 20 min at 37°C, in the presence of 1 µg Oregon Green™ 488 BAPTA-1. We then quantified Ca²⁺ levels in the HepG-2 cells by fluorescence microscopy and flow cytometry, in accordance with the manufacturer’s instructions. Ad-Mock-infected cells and non-infected HepG-2 cells were used as negative controls.

2.2.5 Mitochondrial analysis: structure and function

HepG-2 cells were seeded at a density of 6 × 10⁵ cells/well into a 6-well plate, cultured for 24 h at 37°C in 5% CO₂ and infected with Ad-Vp3 (MOI: 50) for 12, 24, 36, 48, and 72 h. Cells were then incubated in the dark for 20 min at 37°C in the presence of 1 µL JC-1 in 1 mL of DMEM. We then determined the mitochondrial membrane potential of HepG-2 cells by flow cytometry. Ad-Mock-infected cells and non-infected HepG-2 cells were used as negative controls.

For structural analysis, HepG-2 cells were seeded at a density of 1 × 10⁷ cells into a 100 mm cell culture dish, cultured for 24 h at 37°C in 5% CO₂ and infected with Ad-Vp3 (MOI: 50) for 48 and 72 h. We then collected mitochondria by use of a Mitochondria Isolation Kit for Mammalian Cells and Tissues, in accordance with the manufacturer’s guidelines. We then prepared sections by ultramicrotomy and investigated the structure of the mitochondria by electron microscopy Ad-Mock-infected cells and non-infected HepG-2 cells were used as negative controls.

2.2.6 Western blotting

HepG-2 cells were seeded at a density of 6 × 10⁵ cells/well into a 6-well plate, cultured for 24 h at 37°C in 5% CO₂ and infected with Ad-Vp3 (MOI: 50) for 12, 24, 36, 48, and 72 h. Ad-Mock-infected cells were used as negative controls. Total protein was extracted from cells using the Minute™ Total Protein Extraction Kit, separated by SDS-PAGE, and transferred to membranes for Western blotting so that we could determine the expression levels of proteins related to apoptotic proteins, ER stress pathway and apoptotic pathways in the mitochondria.

2.2.7 Ca²⁺ imbalance analysis

HepG-2 cells were seeded at a density of 6 × 10⁵ cells/well into a 6-well plate, cultured for 24 h at 37°C in 5% CO₂ and treated with Ad-Vp3 (MOI: 50) and 10 µM BAPTA-AM for 72 h. For Annexin V analysis, the cells were incubated in the dark for 15 min at room temperature in the presence of 5 µL Annexin V-FITC and 5 µL of PI in 100 µL of 1 × binding buffer. We then quantified the extent of apoptosis in HepG-2 cells by a fluorescence microscope and low cytometry. For Mitochondrial analysis, Cells were then incubated in the dark for 20 min at 37°C in the presence of 1 µL JC-1 in 1 mL of DMEM. We then determined the mitochondrial membrane potential of HepG-2 cells by a fluorescence microscope and low cytometry.

2.2.8 In vivo studies

The xenograft models were established via subcutaneous injection of HepG-2 cells (3 × 10⁶/100 µL) with Matrigel® Matrix Basement Membrane (yielding a 1:1 ratio) into mice left legs. When the tumors had formed clearly (usually 14 days), the mice were divided randomly into five groups (9 mice per group). Group 1 was injected with 1 × 10⁸ plaque forming units (PFU) of Ad-Vp3 and 5 mg/kg BAPTA-AM, group 2 was injected with Ad-Vp3 (1x10⁸ PFU). Group 3 was injected with 5 mg/kg BAPTA-AM. Group 4 was injected with Ad-Mock (1 × 10⁸ PFU) and group 5 was injected with the same volume of the vehicle. All groups were infected with Ad-Vp3 or Ad-Mock via intratumoral injection and treated with BAPTA-AM via intraperitoneal injection from the day 14. The injections were given every 3 days for 15 days. Tumor volumes (length × width² × 0.5) and survival rates were calculated every 7 days for 35 days. During the experiment, tumors from 3 mice and from each group were harvested. We then used Western-blot and immunohistochemical staining to investigate the apoptotic pathway in the mitochondria, including AIF, HtrA2, Smac/Diablo, and Cyto-C. All immunohistochemical detection was performed by Servicebio (Wuhan, China). All animals’ experimental procedures were approved by the Institutional Animal Care and Use Committee of the Changchun University of Chinese Medicine.

2.3 Statistics

All statistical analyses were performed by using the Statistical Package for the Social Sciences (SPSS) statistical software package (version 15.0; SPSS Inc., Chicago, IL, USA). Data were presented using GraphPad Prism version 7.0 (GraphPad Software Inc., La Jolla, CA, USA). For analysis, we used the Student’s t-test or one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test; p < 0.05, p < 0.01, or P < 0.001, were considered to be statistically significant. Data are presented as the mean ± standard deviation (SD).

Immunohistochemical analyses were performed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA). At least 3 fields of vision were randomly selected for each slice (and for each group). When acquiring images, we organized the entire field of view to ensure that the background illumination was consistent. The method was used to define positive staining in cells for other types of proteins; a brown/yellow stain indicated positive immunostaining. We used Image-Pro Plus 6.0 software to determine the integrated optical density (IOD) and the area occupied by the entire tissue (AREA). The Areal density was then defined by IOD/AREA. A larger areal density indicated a higher level of positive expression.

3.1 Apoptosis of HepG-2 cell induced by Apoptin

The apoptosis of HepG-2 cell can be significantly induced by Apoptin (Figure. 1A). The apoptotic rate in Ad-Vp3-infected cells was significantly higher than that in Ad-Mock-infected cells and non-infected HepG-2 cells at 48 and 72 h post-infection (p < 0.05). The results of apoptosis-related proteins in Ad-Vp3-infected HepG-2 cells are shown in Figure. 1B. The expression levels of Cleaved-PARP and Cleaved-Caspase-3 in Ad-Vp3-infected HepG-2 cells were significantly higher at 48 and 72 h post-infection, when compared with HepG-2 cells infected with Ad-Mock (p < 0.01). The ROS levels of HepG-2 cells can be significantly induced by Apoptin (Figure. 1C). ROS levels in Ad-Vp3-infected cells were significantly higher than that in Ad-Mock-infected cells and HepG-2 cells at all time points (p < 0.05).

3.2 Apoptin induction of endoplasmic reticulum stress

Our analysis showed that some of the proteins related to ER stress pathway were significantly affected by Apoptin (Figure. 2A and B). For example, the expression of PERK and ATF-6 in Ad-Vp3-infected cells were significantly higher at 12 and 24 h than cells infected with Ad-Mock, but were significantly lower at 48 and 72 h post-infection (p < 0.01). The expression level of IRE1 in Ad-Vp3-infected cells was significantly higher than that in Ad-Mock-infected cells at all time points (p < 0.01).

The effect of Apoptin on the structure of the ER was evaluated by electron microscopy. Electron microscopy revealed clear structural damage in the ER and mitochondria of infected cells at 48 h (Figure. 2C). The structure of ER and the crest of the mitochondria in Ad-Vp3-infected cells had incurred significant damage by 48 h post-infection. This result indicated that the strong and lasting ER stress response can damage the structure of the ER.

The intensity of green fluorescence (as an indicator of Ca²⁺ level) in HepG-2 cells infected with Ad-Vp3 was significantly higher than cells infected with Ad-Mock at 24, 36, and 48 h, post-infection. At 72 h post-infection, the level of green fluorescence in the HepG-2 cells infected with Ad-Vp3 was significantly lower than that in the control group (Figure. 2D and E) (p < 0.05). The trend for green fluorescence over time was similar to that shown by flow cytometry (Figure. 2F). These results showed that ER stress increased the levels of Ca²⁺, leading to an imbalance in Ca²⁺. Therefore, Apoptin can induce a strong and lasting ER stress that ultimately leads to Ca²⁺ imbalance.

3.3 Effect of Apoptin on mitochondria

The mitochondrial membrane potential of HepG-2 cells was significantly reduced by Apoptin. The relative fluorescence (R2/R3; Red/Green) associated with mitochondrial membrane potential in Ad-Vp3-infected HepG-2 cells was significantly lower than that in Ad-Mock-infected cells and non-infected cells at 24, 36, 48 and 72 h post-infection (Figure. 3A) (p < 0.05).

It was also evident that Apoptin caused damage to the mitochondria of HepG-2 cells (Figure. 3B); the crest of the mitochondria in Ad-Vp3-infected cells appeared to be broken and dissolved at 48 h post-infection. At 72 h post-infection, it was evident that the mitochondria had been completely destroyed.

The expression levels of AIF, HtrA2, Smac/Diablo, and Cyto-C, in Ad-Vp3-infected cells were significantly higher than those in Ad-Mock-infected cells at all time points (Figure. 3C and D) (p < 0.05). However, there were no significant differences with regards to the expression of ARTs and Endo G in HepG-2 cells infected with Ad-Vp3 and Ad-Mock (p > 0.05). It was evident that Apoptin can trigger the apoptotic pathway in the mitochondria by increasing the expression levels of AIF, HtrA2, Smac/Diablo, and Cyto-C.

Collectively, these results indicated that Apoptin significantly reduced mitochondrial membrane potential, caused damage to the mitochondrial structure and triggered the apoptotic pathway in the mitochondria of HepG-2 cells.

3.4 BAPTA-AM reduced the apoptosis of HepG-2 cell induced by Apoptin

In order to verify whether Ca²⁺ imbalance has an effect in HepG-2 cells, we performed Annexin V and JC-1 analysis on Ad-Vp3-infected HepG-2 cells (Fig. 4A and B). The apoptotic rate in Ad-Vp3 + BAPTA-AM-treated cells and Ad-Vp3-infected cells was significantly higher than that in BAPTA-AM-treated cells, Ad-Mock-infected cells and HepG-2 cells, 72 h post-infection (p < 0.001), while that in Ad-Vp3 + BAPTA-AM-treated cells was significantly lower than that in Ad-Vp3-infected cells, 72 h post-infection (Figure. 4A) (p < 0.01). The mitochondrial membrane potential of HepG-2 cells in Ad-Vp3 + BAPTA-AM group and Ad-Vp3 group was significantly higher than that in BAPTA-AM group, Ad-Mock group and HepG-2 group at 72 h post-infection (p < 0.001), while that in Ad-Vp3 + BAPTA-AM group was significantly higher than that in Ad-Vp3 group at 72 h post-infection (Figure. 4B) (p < 0.01). These results showed that apoptotic rate and mitochondrial membrane potential were significantly affected by Ca²⁺ imbalance.

3.5 Ca²⁺ level affected the antitumor ability of Apoptin in vivo

The antitumor effects of Ad-Vp3 can be significantly reduced by BAPTA-AM in the HepG2-xenografted nude mice. The tumor volume in the Ad-Vp3 + BAPTA-AM group was significantly lower than that in the Ad-Vp3 group, at 28 and 35 days after tumor xenografting (Figure.5A and B) (p < 0.01). The survival rate of the Ad-Vp3 group was 100%, while that of the Ad-Vp3 + BAPTA-AM group was 70%, 35 days after tumor xenografting, respectively. The survival rate in the Ad-Vp3 group was significantly longer than that in the Ad-Vp3 + BAPTA-AM group. (Figure. 5C) (p < 0.01). The KI67 positivity rate in the Ad-Vp3 + BAPTA-AM and Ad-Vp3 group tumors was significantly lower than that in the BAPTA-AM, Ad-Mock and PBS groups (p < 0.0001), while that in Ad-Vp3 + BAPTA-AM group was significantly higher than that in Ad-Vp3 group (p < 0.01). The Tunel positivity rate in the Ad-Vp3 + BAPTA-AM and Ad-Vp3 group tumors was significantly higher than that in the BAPTA-AM, Ad-Mock and PBS groups (p < 0.0001), while that in Ad-Vp3 + BAPTA-AM group was significantly lower than that in Ad-Vp3 group (Figure. 5D) (p < 0.01). These results showed that BAPTA-AM could significantly inhibit the antitumor effect of Apoptin.

3.6 Western-blot and immunohistochemical detection of mitochondrial apoptotic pathway related proteins in tumor tissues

Western-blot analysis of the tumor tissues showed that the expression levels of AIF, HtrA2, Smac/Diablo and Cyto-C in Ad-Vp3 + BAPTA-AM and Ad-Vp3 groups were significantly higher, when compared with BAPTA-AM, Ad-Mock and PBS groups (p < 0.05). While the expression levels of Smac/Diablo and Cyto-C in the Ad-Vp3 + BAPTA-AM group were significantly lower than that in the Ad-Vp3 group (Figure. 6A) (p < 0.01). The variation trend of expression levels of AIF, HtrA2, Smac/Diablo and Cyto-C in immunohistochemical data (Figure. 6B) was similar to that shown by Western-blot. Collectively, these in vivo results showed that Apoptin caused significant changes in the expression of AIF, HtrA2, Smac/Diablo and Cyto-C, while BAPTA-AM could reduce the expression of Smac/Diablo and Cyto-C in Apoptin-treated tumor tissues.

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 [18] and subsequently induce the release of pro-apoptotic proteins from the mitochondrial membrane mesenchyme [21]. 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.

DATA AVAILABILITY

All datasets generated or analyzed during this current study are included in this published article.

Author contributions

XYY, TXW, YL and YLZ conceived and performed the experiments and analysed the data. YQL, BB, JBF, JCH, SZL, ZRX, XY, ZRL, YRL, GZZ and NYJ conducted the mouse experiments. XL and CS overall supervised the experiments and revised the manuscript. All authors read and approved the final manuscript.

Acknowledgment

The authors would like to express their gratitude to EditSprings (https://www.editsprings.com/) for the expert linguistic services provided.

Funding

This study was sponsored by the Changchun Science and Technology Development Plan Project (NO. 21QC01) and the Science and Technology Research Planning Project of Jilin Provincial Department of Education (NO. JJKH20220887KJ), and the National Natural Science Foundation of China (Grant no. 82151221).

Ethics approval and consent to participate The animal experiment protocols were approved by the Institutional Animal Care and Use Committee of the Changchun University of Chinese Medicine (Approval No. 2021078).

Informed consent Informed consent was obtained from all individual participants included in the study.

Consent for publication Not applicable.

Research involving human participants and/or animals The animal experimental protocols were approved by the IACUC of the Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Changchun, China) (10ZDGG007). The manuscript does not contain human clinical studies or patient data.

Conflict of interests The authors declare that they have no competing interests.

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Apoptin causes apoptosis in HepG-2 cells via Ca2+ imbalance- triggered mitochondrial apoptotic pathway

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Version 1

Abstract

Figures

1 Introduction

2 Materials And Methods

2.1 Materials

2.2 Methods

2.2.1 Annexin V analysis

2.2.2 ROS levels analysis

2.2.3 Electron microscopy

2.2.4 Ca²⁺ levels analysis

2.2.5 Mitochondrial analysis: structure and function

2.2.6 Western blotting

2.2.7 Ca²⁺ imbalance analysis

2.2.8 In vivo studies

2.3 Statistics

3 Results

3.1 Apoptosis of HepG-2 cell induced by Apoptin

3.2 Apoptin induction of endoplasmic reticulum stress

3.3 Effect of Apoptin on mitochondria

3.4 BAPTA-AM reduced the apoptosis of HepG-2 cell induced by Apoptin

3.5 Ca²⁺ level affected the antitumor ability of Apoptin in vivo

3.6 Western-blot and immunohistochemical detection of mitochondrial apoptotic pathway related proteins in tumor tissues

4. Discussion

Declarations

References

Additional Declarations

Status:

Version 1

Apoptin causes apoptosis in HepG-2 cells via Ca2+ imbalance- triggered mitochondrial apoptotic pathway

Status:

Version 1

Abstract

Figures

1 Introduction

2 Materials And Methods

2.1 Materials

2.2 Methods

2.2.1 Annexin V analysis

2.2.2 ROS levels analysis

2.2.3 Electron microscopy

2.2.4 Ca2+ levels analysis

2.2.5 Mitochondrial analysis: structure and function

2.2.6 Western blotting

2.2.7 Ca2+ imbalance analysis

2.2.8 In vivo studies

2.3 Statistics

3 Results

3.1 Apoptosis of HepG-2 cell induced by Apoptin

3.2 Apoptin induction of endoplasmic reticulum stress

3.3 Effect of Apoptin on mitochondria

3.4 BAPTA-AM reduced the apoptosis of HepG-2 cell induced by Apoptin

3.5 Ca2+ level affected the antitumor ability of Apoptin in vivo

3.6 Western-blot and immunohistochemical detection of mitochondrial apoptotic pathway related proteins in tumor tissues

4. Discussion

Declarations

References

Additional Declarations

Status:

Version 1

2.2.4 Ca²⁺ levels analysis

2.2.7 Ca²⁺ imbalance analysis

3.5 Ca²⁺ level affected the antitumor ability of Apoptin in vivo