In vitro and in vivo suppression of growth of rat liver epithelial tumor cells by antisense oligonucleotide against protein kinase C-alpha

Background/Aims: It has been hypothesized that liver stem cells may be acti vated and proliferate upon liver injury and may participate in the developm ent of liver cancer. GP7TB, a rat liver epithelial tumor cell line, possess es characteristics of liver stem-like cells and can develop into a tumor in syngeneic Fischer 344 rat. We found that protein kinase C-alpha (PKC-alpha ) is overexpressed in GP7TB cells. The importance of PKC-alpha for this liv er tumor cell was elucidated. Methods: Antisense oligonucleotide (ODN) was applied to suppress the produc tion of PKC-alpha in GP7TB cells in vitro and in vivo. Cell viability was m easured by acid phosphatase assay. The cellular levels of PKC-alpha and Bcl -2 were monitored by Western-blot analysis. Activation of nuclear factor NF -kappa B was analyzed by electrophoretic mobility shift assay. Cell cycle p hase distribution was monitored by FACScan. Cell apoptosis was detected by TUNEL assay and histochemical staining of tumor tissue sections. The in viv o experiment was conducted by implanting tumor mass of GP7TB in the liver o f F-344 rat and continuous delivery of the ODN by a mini-osmotic pump. Results: Antisense ODN effectively suppressed the level of PKC-alpha that r esulted in the decrease of Bcl-2 and nuclear NF-kappa B. The cumulative via ble cells also decreased dramatically for the antisense-treated group. FACS can showed that the cells were arrested at early S-phase. These cells in tu rn went into apoptosis without completing a cell cycle. It was found that g rowth of the tumor was suppressed efficiently by antisense ODN. Cell apopto sis was found in the orthotopic tumor. The normal liver cells were not affe cted. Conclusions: A lethal effect of depressing the level of PKC-alpha in GP7TB cells and success in suppressing orthotopic tumor growth in vivo suggests t hat PKC-alpha antisense ODN would be a promising therapeutic agent for some liver cancers.