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DOI: 10.1055/s-0031-1295995
Induction of endoplasmic reticulum-mediated stress pathways in liver cancer cell lines after overexpression of Hepatitis B virus envelope proteins
Background. Hepatocellular carcinoma (HCC) is the 5th most common cancer worldwide. More than 90% of HCCs develop in cirrhotic livers on a background of chronic liver disease, esp. infection with the Hepatitis B virus (HBV). Although HBV has been documented to cause HCC, the exact role of HBV remains enigmatic. HBV is a hepatotropic virus with an outer lipoprotein envelope that consists of 3 proteins, the small (SHBs), the middle (MHBs) and the large surface protein (LHBs) that are also secreted as non-infectious subviral particles via the secretory pathway. The LHBs cannot build viral particles by itself, but needs SHBs or MHBs for proper propagation. A massive storage of HBV envelope proteins leads to cell stress and sustained inflammatory responses. Here we investigate if overexpression of L and S proteins in liver cancer cells can induce ER-stress and trigger the expression of genes involved in this pathway.
Methods. Human HCC cells HuH–7 was cultured under standard conditions and transfected with expression-plasmids, overexpressing only LHBs and SHBs, respectively. Impedance based real-time cell analysis was performed to monitor cell viability. ER-stress factors were evaluated by RT-PCR, FACS and IF analysis. ER-staining was performed by IF analysis.
Results. Cell based impedance analysis of HuH–7 cells showed that the plasmid encoding LHBs caused a reduction of cell growth comparable with the effect of 10 nM thapsigargin, an ER-stress inducer. Interestingly, the SHBs (over)-expression was cytostatic. The expression of both viral proteins induced an increase of BiP and CHOP mRNAs after 24h–96h of transfection, while the protein level was stable. ER detection by IF revealed an increase of the selective fluorescent dye in cells transfected with LHBs.
Conclusions. Overexpression of HBV envelope-proteins L and S activate ER-stress markers in HuH–7 cells. Further investigations are needed to better clarify their mechanism of action.