Exogenous hepatitis B virus envelope proteins induce endoplasmic reticulum stress: involvement of cannabinoid axis in liver cancer cells

HBV represents the most common chronic viral infection and major cause of hepatocellular carcinoma (HCC), although its exact role in liver tumorigenesis is unclear. Massive storage of the small (SHBs), middle (MHBs) and large surface (LHBs) HBV envelope proteins leads to cell stress and sustained inflammatory responses. Cannabinoid (CB) system is involved in the pathogenesis of liver diseases, stimulating acute and chronic inflammation, liver damage and fibrogenesis; it triggers endoplasmic reticulum (ER) stress response. The aim of our work was to investigate the activation of ER stress pathway after ectopic HBV envelope proteins expression, in liver cancer cells, and the role exerted by CB receptors. PCR, immunofluorescence and western blotting showed that exogenous LHBs and MHBs induce a clear ER stress response in Huh-7 cells expressing CB1 receptor. Up-regulation of the chaperone BiP/GRP78 (Binding Immunoglobulin Protein/Glucose-Regulated Protein 78) and of the transcription factor CHOP/GADD153 (C/EBP Homologous Protein/Growth Arrest and DNA Damage inducible gene 153), phosphorylation of PERK (PKR-like ER Kinase) and eIF2α (Eukaryotic Initiation Factor 2α) and splicing of XBP1 (X-box binding protein 1) was observed. CB1−/− HepG2 cells did not show any ER stress activation. Inhibition of CB1 receptor counteracted BiP expression in transfected Huh-7 and in HBV+ PLC/PRF/5 cells; whereas no effect was observed in HBV− HLF cells. These results suggest that HBV envelope proteins are able to induce the ER stress pathway. CB1 expression is directly correlated with ER stress function. Further investigations are needed to clarify the involvement of cannabinoid in HCC progression after HBV infection.


Flow cytometry
Flow cytometry was employed for transfection efficiency establishment in Huh-7 and HepG2 cells transfected for 72 hours with pSVL and pSVM plasmids. MA 18/07 and HB01 antibodies were used to detect HBV envelope proteins. Secondary AlexaFluor488-conjugated antibody was purchased from Life Technologies and used as negative control to perform the fluorescence setting also. Flow cytometry was employed for cell cycle analysis also in plasmid transfected or thapsigargin treated cell lines for 72 hours after staining with propidium iodide as described previously (Di Fazio et al., 2010). Analysis of labeled cells and nuclei was performed on an Attune acoustic focusing cytometer (Applied Biosystems, Carlsbad, USA) and results were analyzed with the Attune ® Cytometric Software 1.2.5.3891. Twenty thousand events were collected for each sample for transfection efficiency analysis. Ten thousand events were collected for each sample for cell death analysis. All experiments were performed in triplicates.

Quantitative RT-PCR
For real time PCR, total cellular RNA was extracted using the RNeasy Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions and reverse transcription (RT) was performed with Quantitect Reverse Transcription Kit (QIAGEN). QuantiTect Primers for IRE1α, BiP, ATF4, CHOP, CB1 and GAPDH were purchased from QIAGEN and run with the QuantiFast SYBR Green PCR Kit (QIAGEN) on a CFX96 Real Time PCR Detection System (BioRad, Munich, Germany). Results were analyzed with the CFX Manager v2.0 and Rest 2008 software and normalized to GAPDH mRNA content for each sample.

Cell cycle analysis distribution
To assess the effects of pSVL and pSVM plasmids transfection on cell cell cycle distribution, we performed flow cytometry analyses after 72 hours transfection in Huh-7 and HepG2 cell lines. Both plasmids showed any significant variation of cell cycle phases in both cell lines, and only a small increase of the percentage of sub-G1 events in Huh-7 cells (Supplementary Figure S1A and S1B).

Transfection efficiency evaluation
In order to establish the highest transfection efficiency, Huh-7 and HepG2 cells have been transfected with pSVL and pSVM plasmids for 96 hours and Immunofluorescence and Flow cytometry analysis have been performed. Here we show the results after 72 hours transfection, time in which the highest transfection efficiency has been achieved (Supplementary Figure  S1C and S1D). In Huh-7 cells, around 40% of cells were positive to MA18/07 antibody, specific for LHBs protein, and to HB01 antibody, specific for MHBs protein (Supplementary Figure S1C); in HepG2 cells, around 30% of cells were positive to MA18/07 antibody, and around 15% were positive to HB01 antibody (Supplementary Figure S1D).

The expression of both HBV envelope proteins upregulates ER stress-related factors
We analyzed the expression levels of the ER stressrelated factor IRE1α, of the chaperone BiP and of the transcription factors ATF4 and CHOP in both cell lines after transfection with pSVL and pSVM plasmids or after treatment with 10 nM TG for 72 hours. The expression of BiP and CHOP was significantly induced already after 48 hours transfection with both plamids in Huh-7 cell line (Supplementary Figure S2A) and it increased significantly after 72 hours transfection ( Figure 2C); while its level was stable in HepG2 cells ( Figure 2D and Supplementary Figure S2B). IRE1α and ATF4 levels were almost stable in both cell lines after transfection with both plasmids (Supplementary Figure S2A and S2B). ER stress markers were confirmed to be strongly upregulated after 10 nM TG treatment, which was used as positive control of ER stress induction ( Figure 2C and 2D; Supplementary Figure S2A and S2B).

Analysis of CHOP protein level
The PCR results showed that both pSVL and pSVM transfection were able to strongly and significantly upregulate CHOP in Huh7 cells (Figure 2A). CHOP protein level was analyzed in Huh7 cells by Immunofluorescence, showing an increase and nuclei localization after transfection with both plasmids comparable to the effects of 10 nM TG (Supplementary Figure S2).

HBV envelope proteins activated PERK/eIF2α arm of ER stress
In order to further confirm ER stress pathway induction in Huh-7 cells, we analyzed by Immunofluorescence the status of PERK and phospho-eIF2α after transfection with pSVL and pSVM plasmids or treatment with TG. The results showed a strong increase of both PERK and Ser51-eIF2α after expression of both HBV envelope proteins. Treatment with 10 nM TG induced also a high increase of PERK and Ser51-eIF2α (Supplementary Figure S4A and S4B).