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Cell Biology International (2012) 36, 733–738 (Printed in Great Britain)
EBV up-regulates cytochrome c through VDAC1 regulations and decreases the release of cytoplasmic Ca2+ in the NPC cell line
Xuesong Feng, Chi Bun Ching and Wei Ning Chen1
School of Chemical and Biomedical Engineering, Technological University, 62 Nanyang Drive 637459, Singapore


EBV (Epstein–Barr virus) is considered to be a major factor that causes NPC (nasopharyngeal carcinoma), which is one of the sneakiest cancers frequently occurring in Southeast Asia and Southern China. Apoptosis and pro-apoptotic signals have been studied for decades; however, few have extended the prevailing view of EBV to its impact on NPC in perspective of apoptosis. One of the important proteins named VDAC1 (voltage-dependent anion protein 1) on the mitochondrial outer membrane controls the pro-apoptotic signals in mammalian cells. The impact of EBV infection on VDAC1 and related apoptotic signals remains unclear. In order to study the VDAC1's role in EBV-infected NPC cells, we employ siRNA (small interfering RNA) inhibition to analyse the release of Ca2+ and Cyto c (cytochrome c) signals in the cytoplasm, as they are important pro-apoptotic signals. The results show a decrease of Ca2+ release and up-regulation of Cyto c with EBV infection. After siRNA transfection, the dysregulation of Cyto c is neutralized, which is evidence that the level of Cyto c release in virus-infected NPC cells is the as same as that of non-infected NPC cells. This result indicates that EBV infection changes the cytoplasmic level of Cyto c through regulating VDAC1. In summary, this study reports that EBV changes the release of Ca2+ and Cyto c in the cytoplasm of NPC cells, and that Cyto c changes are mediated by VDAC1 regulation.


Key words: Ca2+, cytochrome c, Epstein–Barr virus nasopharyngeal carcinoma, ELISA, siRNA inhibition

Abbreviations: Cyto c, cytochrome c, EBV, Epstein–Barr virus, NPC, nasopharyngeal carcinoma, VDAC1, voltage-dependent anion channel protein 1

1To whom correspondence should be addressed (email WNChen@ntu.edu.sg).


1. Introduction

NPC (nasopharyngeal carcinoma) is one of the most common types of cancer that occurs frequently in Southeast Asia and Southern China (McDermott et al., 2001; Lo and Huang, 2002; Parkin et al., 2002). The tumour cells often arise from the epithelium of the nasopharynx, where vague symptoms are hard to detect due to the difficulty of physical examination in early stages. NPC is caused by mainly three factors: EBV (Epstein–Barr virus) chronic infection, environmental stimuli and human genetic defects. As EBV gene was found to exist in 96% of NPC patients, this virus has gained attention due to its significant correlation with NPC (Li et al., 2006). Many researchers studied the impact of EBV infection on B-cells on the level of immunochemistry (Li et al., 2007). Some studies were performed on the relationship of NPC with EBV infection, where several potential biomarkers were discovered for prognosis and possible pathways were proposed (Wu et al., 2005; Zhang et al., 2005; Chow et al., 2006). However, the mechanism of NPC occurrence in response to EBV infection remains unclear. Our previous study showed that VDAC1 (voltage-dependent anion channel protein 1) protein was significantly up-regulated in EBV-infected NPC cell lines through iTRAQ-labelled two-dimensional LC-MS/MS (tandem MS) analysis (Feng et al., 2011a, 2011b). Clinical samples from NPC patients were also found to have up-regulated VDAC1 in the serum (Feng et al., 2011a, 2011b). The evidence triggered our interest in VDAC1's role in NPC cells during EBV infection.

VDACs, are the most important proteins in the outer membrane of the mitochondria, where many cellular events take place. VDAC proteins are involved in apoptosis as the gatekeeper of mitochondria metabolites. In programmed cell death, various proteins and molecules are activated and released through mitochondria membranes. Defects of key proteins controlling apoptosis were proved to induce cancers (Hanahan and Weinberg, 2000). VDAC proteins have three isoforms in mammalian cells. VDAC1 is the most abundant one, compared with the other two isoforms VDAC2 and VDAC3, and VDAC1 was studied most extensively out of the three isoforms (De Pinto et al., 2010). As the controller of the convergence point on the outer membrane of mitochondria, VDAC1 has been studied for decades due to its powerful channel activity. The most common method to study the channel activity is the reconstitution of the VDAC1 pore in a planar lipid bilayer (Shoshan-Barmatz et al., 2010). During mitochondria-mediated apoptosis, VDAC1 controls many pro-apoptotic signals, such as Ca2+, Cyto c (cytochrome c) and ROS (reactive oxygen species) (Le Bras et al., 2005; Keeble and Gilmore, 2007; Kroemer et al., 2007). Cyto c located in the inner membrane of the mitochondria serves as an electron shuttle in the respiratory chain. Several models have been proposed for the release of Cyto c by VDAC1 (Bernardi, 1999; Halestrap et al., 2000; Shoshan-Barmatz and Gincel, 2003; Lemasters and Holmuhamedov, 2006; Tan and Colombini, 2007), most of which were from electrochemical perspectives. Ca2+ is an important molecule that modulates key enzymes involved in TCA (tricarboxylic acid) cycle, fatty acid oxidation, amino acid catabolism, F1 ATPase and ANT (adenine nucleotide translocator) (Nichols and Denton, 1995). VDAC mediate Ca2+ transportation from inner membrane to outer membrane of the mitochondria (Gunter et al., 1998; Gincel et al., 2002; Shoshan-Barmatz and Gincel, 2003). Several models have been put forward for the VDAC–Ca2+ binding site (Colombini, 2004; Israelson et al., 2007). The relationship between VDAC1, Cyto c and Ca2+ needs to be deeply explored because of its complexity in cellular events and mitochondrial structure.

We have used siRNA to inhibit VDAC1 expression for both EBV-infected and non-infected NPC cell lines. Changes of Ca2+ and Cyto c in the cytoplasm before and after VDAC1 inhibition were detected. Before siRNA transfection, Ca2+ was down-regulated in infected cells and Cyto c was up-regulated. After siRNA transfection, Cyto c concentration was back to the same level in both cell lines. These findings suggest that EBV infection has a significant impact on pro-apoptotic signals Cyto c and Ca2+. With evidence of changes of Cyto c in response to siRNA inhibition, we conclude that Cyto c is up-regulated in NPC cells in response to EBV's infection through VDAC1 mediation.

2. Materials and methods

2.1. Cell culture and virus infection in vitro

The cell lines used were human NPC squamous epithelial cell line (ATCC: HTB-43) and the normal epithelial cell line (ATCC: CRL-2007). Both were cultured in EMEM (Eagle's minimum essential medium) supplemented with 10% fetal bovine serum and 1% anti-mycotic at 37°C with controlled 5% CO2 in air. The virus (ATCC: VR-1492) used was the human EBV that is shed in the supernatant of persistently infected and transformed B95-8 cells (ATCC: CRL-1612). Virus infection was employed as previously described (Feng et al., 2011a, 2011b). Cells were stored in liquid nitrogen at −80°C and recovered quickly at 37°C prior to harvest. As normal epithelial cell line was unable to be infected using our direct infection protocol, most comparisons are between the EBV-infected cell line and the non-infected cell line.

2.2. Blocking of VDAC1 by siRNA transfection

Two sequences of siRNA to inhibit VDAC1 expression were supplied to perform the gene transfection, using the TransIT-TKO® Transfection Reagent (Mirus). Cells were cultured into a 24-well plate in 0.5 ml of complete growth medium/well until they reached 3×105 per well before collection. siRNA complex was prepared immediately before transfection. Prior to transfection, medium volume was adjusted to 0.25 ml. The siRNA complex was added drop-wise to target wells and gently mixed (25 nM final concentration). Transfected cells and controls were cultured for 48 h before real-time RT–PCR (reverse transcription–PCR) validation.

2.3. RNA extraction and real-time RT–PCR

The sequence of the VDAC1 gene was obtained from NCBI nucleotide database (GI: 14250131; GB: BC008482.1). Primers were carefully designed to produce 150–200 bp PCR products to reduce the non-specific binding of SYBR Green. Primer information is given in Table 1.


Table 1 Primer sequences for real-time RT–PCR

Primers used for real-time RT–PCR using SYBR Green. The amount of VDAC1 mRNA was calculated relative to the expression of β-actin. The primers were synthesized from 1st BASE, Singapore provided with product ID.

Protein name Primer sequences Primer ID (1st BASE)
VDAC1 (human) Sense: 5′-ACTCACCTGAATGGGACTTT-3′Antisense: 5′-ATCACATCCACCTTCTCCAC-3′ 935171-1935172-1
β-actin (human) Sense: 5′-CTTAGTTGCGTTACACCCTTTC-3′Antisense: 5′-ACCTTCACCGTTCCAGTTTT-3′ 182622-1182623-1



Total RNA was isolated from both the transfected cells and non-transfected cells using the RNeasy Mini kit (Qiagen). The concentration and the quality of RNA were checked by detecting D260 (attenuance at 260 nm) and the attenuance ratio of D260 and D280.

Real-time RT–PCR was used to quantify the amount of VDAC1 mRNAs in the RNAi-transfected cells and controls (iScrpt One-step RT–PCR kit; Bio-Rad). An IQ5 multicolour real-time PCR detection system analysed the mRNA amount after incubation under the following thermal cycles: first strand cDNA reverse transcription: 50°C, 10 min; reverse transcriptase inactivation: 95°C, 5 min; 40 cycles of PCR and detection: 95°C, 10 s and 60°C, 30 s; disassociation analysis was carried out by detecting fluorescence signals for 1°C increase each cycle from 55 to 95°C.

Amplification analysis, experimental report melting curve analysis and threshold cycle number were obtained by IQ5 optical system software 2.0 with the formatted Microsoft Excel data (Bio-Rad). The fold changes were calculated as follows: SampleΔCt = Ctsample–Ctβ-actin; ΔΔCt = SampleΔCt–ControlΔCt. The fold changes of sample/control = 2−ΔΔCt. Three independent experiments were performed with duplicate calculations.

2.4. Cytoplasmic Ca2+ assay

Calcium level in the cytoplasm of the cell was detected as one of the important pro-apoptotic signals by using Fluo-4 NW Calcium Assay Kit (Molecular Probes). NPC cells and virus-infected cells (Vir) were harvested in a 96-well plate. Then 50000 cells were grown in each well before calcium assay. Three independent batches of experiment were carried out with the same 50000 cells per well in 96-well plate to make sure that the comparison was on the same level. Reagents were prepared within 6 h before the test. Medium was removed from the well to eliminate sources of baseline fluorescence, particularly esterase activity. After quickly and carefully adding 100 μl of loading solution into each well, the plate was incubated at 37°C for 30 min before an additional 30 min at room temperature, making them ready for assay. Relative fluorescence was measured with λex at 494 nm and λem at 516 nm.

2.5. Cyto c ELISA test

Changes in the amount of Cyto c was detected in NPC cells, virus-infected NPC cells (Vir), VDAC1 siRNA-transfected cells (NPC/T) and siRNA-transfected NPC cells infected with EBV (Vir/T) using an ELISA (Invitrogen). Cells cultured in a 24-well plate were grown to 3×105 per well before being treated with trypsin, and centrifuged at 2000 rev./min for 2 min at room temperature. They were put in lysis solution on ice for 30 min with vortexing at 10 min intervals, following which they were centrifuged at 13000 rev./min for 10 min at 4°C to obtain the required lysate.

Cyto c standard was serially diluted from 5 ng/ml concentration and labelled as 5, 2.5, 1.25, 0.625, 0.312, 0.156 and 0.0078 ng/ml Cyto c. The standard curve is shown in Figure 3. Samples prepared for ELISA were diluted to 1:8 in the standard diluents buffer. Then 100 μl of standards, controls and samples were incubated for 2 h at room temperature with originally seeded primary antibody (Anti-Cyto c). After aspirating and washing four times, 100 μl of biotin conjugate was added into each well and incubated for 1 h at room temperature. After aspirating and washing four times, samples were incubated with 100 μl of Streptavidin-HRP (horseradish peroxidase) working solution for 30 min. The wells went through another four cycles of aspirating and washing before being incubated with 100 μl of stabilized chromogen for 30 min per well. Finally, 100 μl of stop solution was added to each well and the solution read at 450 nm. Three sets of experiments were performed independently for Cyto c estimations. The attenuance of each sample was based on the lysates of the same total cell amount.

2.6. Statistical analysis

Student's t test was employed in the 3 independent experiments in each section. Data were displayed as means±S.D. as calculated from the different batches. Values were only considered as significant when P<0.05.

3. Results

3.1. siRNA transfection validated by real-time RT–PCR

VDAC1 expression was blocked using siRNA transfection. Using NPC cells as the control, the fold changes of VDAC1 mRNA are given in Figure 1. Approximately 80% of the VDAC1 mRNA were inhibited by siRNA transfection in both NPC cells and EBV-infected NPC cells. When compared between NPC cells (NPC) and EBV-infected cells (Vir), the amount of VDAC1 mRNA was much higher in virus-infected cells, consistent with the results of previous proteomics study (Feng et al., 2011a, 2011b). Total RNA was isolated and used as the template for real-time RT–PCR. The results indicate that VDAC1 expression was successfully inhibited via siRNA transfection in NPC cells and EBV-infected NPC cells.

3.2. EBV infection decreases the Ca2+ release into the cytoplasm

As one of the important pro-apoptotic signals, Ca2+ abundance was tested to show the changes of Ca2+ in NPC cells in response to EBV infection. Ca2+ abundance was given by its relative fluorescence at an λex of 494 nm and λem at 516 nm. Table 2 shows the attenuance of the fluorescence for NPC cells, virus-infected NPC cells and the control. All the triplicates showed that cytoplasmic Ca2+ was decreased in response to EBV infection (Figures 2A and 2B). This indicates that EBV can affect NPC cells in perspective of down-regulating the Ca2+ release in the cytoplasm.


Table 2 D (attenuance) of the Ca2+ fluorescence in NPC- and EBV-infected cells

The entries in the centre of the table represent D of Ca2+ fluorescence in NPC cell line (NPC) and EBV-infected NPC cell line (Vir). The control used was the complete medium without cells. The actual Ca2+ D was calculated as the result of sample D minus control D. Std represents the standard deviation of three independent batches’ results.

D Batch 1 Batch 2 Batch 3 Ave Act Ca2+ Std
NPC 2808 2951 2861 2873 1288 72
Vir 2535 2533 2654 2574 989 69
Control 1622 1535 1597 1585



3.3. EBV up-regulates Cyto c release in the cytoplasm through VDAC1

A standard curve was made by measuring the attenuance of absorption at 450 nm with a Cyto c standard solution diluted from 5 to 0.0078 ng/ml. Three independent batches showed similar trends of Cyto c changes in response to virus infection and VDAC1 inhibition (Table 3 and Figure 3). Before VDAC1 blocking, by comparing the Cyto c concentration between the NPC cells and EBV-infected cells, cells with virus had higher concentrations of Cyto c, which means EBV infection increases the level of Cyto c in the cytoplasm. After VDAC1 blocking, Cyto c was down-regulated to the same level as in both NPC cells and virus-infected cells, which indicates the direct control of Cyto c release by VDAC1.


Table 3 Cyto c ELISA assay

The central entries are D of Cyto c in ELISA. The NPC represents non-infected NPC cell line. Vir represents EBV-infected NPC cell line. NPC/T represents NPC cell line whose VDAC1 expression was inhibited by siRNA. Vir/T represents EBV-infected NPC cell line inhibited by VDAC1 siRNA. Std was calculated as a result of analysing three independent batches. The real Cyto c concentration (the last column) was calculated from the standard curve considering sample dilution.

D Batch 1 Batch 2 Batch 3 Ave Std Cyto c (ng/ml)
NPC 1.541 1.502 1.408 1.484 0.068 17.536
Vir 1.717 1.603 1.662 1.661 0.057 19.752
NPC/T 0.822 0.967 0.989 0.926 0.091 10.584
Vir/T 1.032 0.928 0.935 0.965 0.058 11.072



4. Discussion

VDAC1's important role has been recognized in mammalian cells due to its significance in regulating metabolism across the mitochondrial membrane. For NPC, several groups, including our own, have shown that VDAC1 is up-regulated in cancer cells in response to EBV infection. However, few studies have addressed the relationship of VDAC1's functions and EBV, though some groups have shown VDAC1's relationship with HIV, influenza A virus and hepatitis B virus (Boya et al., 2003; Castedo et al., 2003; Shirakata and Koike, 2003; Boya et al., 2004; Zamarin et al., 2005). Ca2+ was not significantly affected by siRNA transfection, indicating the cytoplasmic Ca2+ concentration was not mainly controlled by VDAC1 (data not shown). The full pathway(s) showing the relationship among Ca2+, Cyto c, VDAC and EBV remain(s) unclear.

Several other groups have studied that the silencing of VDAC1 could efficiently prevent apoptosis by different stimuli (Tajeddine et al., 2008; Yuan et al., 2008). Overexpression of VDAC1 from different species can induce programmed cell death (Godbole et al., 2003; Zaid et al., 2005; Ghosh et al., 2007; Lu et al., 2007a, 2007b). In our study, apoptosis did not occur in EBV-infected NPC cells, evidence that tumour cells were still proliferating. The concentration of Ca2+ impacts the pro-apoptotic effects mediated by various Ca2+ sensitive factors in ER (endoplasmic reticulum), cytoplasm and mitochondria. In normal cells, Ca2+ involves in triggering apoptosis facilitated by other factors, such as Bax and Bak through mitochondrial dysfunction, cyto c release and caspase activation. Down-regulation of Ca2+ by EBV infection indicated that EBV might prevent apoptosis by decreasing Ca2+ release into the cytoplasm. Cyto c also involves in the invitation of apoptosis. Once Cyto c is released into the cytoplasm, the protein binds apoptotic protease activating factors. Although more Cyto c release was related to overexpression of VDAC1, this was insufficient to trigger apoptosis in NPC cell line without specific stimuli.

Upon siRNA inhibition, down-regulation of Cyto c in both EBV-infected cells and non-infected NPC cells further indicated Cyto c interactions with VDAC1, which was consistent with other studies on different cell lines (Szabadkai et al., 2006). This finding indicates the potential application of pro-apoptotic signals Ca2+ and Cyto c in the prognosis of NPC. Furthermore, the finding that Cyto c release up-regulated in response to EBV infection is mediated by VDAC1 indicates its significance in EBV infection. These findings provide new information regarding managing the NPC and EBV infection.

5. Conclusions

VDAC1's role in regulating Ca2+ and Cyto c release in response to EBV infection in the NPC cell line were studied. Cytoplasmic Ca2+ is down-regulated by EBV infection in NPC cells. In EBV-infected NPC cells, Cyto c release into the cytoplasm is up-regulated before siRNA inhibition. After siRNA transfection, the increase was abrogated and the concentration of Cyto c in virus-infected NPC cells returned to the same level as those in non-infected NPC cells. Changes of Cyto c concentration in the cytoplasm in response to siRNA inhibition indicates that, upon EBV infection, Cyto c release is up-regulated in NPC cells, and is mediated by VDAC1. The pro-apoptotic signal Ca2+ and Cyto c release was altered by EBV infection, the latter being mediated by VDAC1 regulation. These findings extend the mechanisms during EBV infection in NPC cells and could contribute to improve the diagnosis and treatments for NPC.

Author contribution

Wei Ning Chen proposed the project, designed the experiment, analysed the results, and prepared the final draft of paper for submission. Xuesong Feng performed experiments, analysed results, and prepared the first draft of paper. Chi Bun Ching contributed to experimental design.

Acknowledgment

We thank Jiang Xu for his help during the test.

Funding

This work was supported by the School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore. X.F. is a recipient of graduate research scholarship supported by Nanyang Technological University, Singapore.

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Received 24 June 2011/16 February 2012; accepted 13 April 2012

Published online 19 June 2012, doi:10.1042/CBI20110368


© The Author(s) Journal compilation © 2012 International Federation for Cell Biology


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)