Apoptosis of human BEL-7402 hepatocellular carcinoma cells released by antisense H-ras DNA--in vitro and in vivo studies.

ABSTRACT

Recent findings suggest that over-expression of activated H-ras inhibited apoptotic cell death by blocking the activity of apoptotic endonuclease(s). This study was designed using antisense H-ras oligodeoxynucleotides (ODN) to evaluate whether alterations of H-ras expression in BEL-7402 human hepatocellular carcinoma cells could influence the induction of apoptosis in vitro and in vivo. We found that, in vitro, continuous suppression of H-ras expression could decrease the proliferation of BEL-7402 cells and inhibit H-ras-induced entry into S phase. In situ end labeling showed that a large number of cells underwent apoptotic cell death after treatment with antisense H-ras ODN (P < 0.01), and gel electrophoresis of DNA extracted from these cells demonstrated a typical DNA ladder, characteristic of apoptosis. In vivo study indicated that pretreatment with antisense H-ras significantly retarded tumor growth in comparison with the untreated controls or tumors treated with non-specific ODN (P < 0.01, P < 0.01). In situ end-labeling revealed that pronounced apoptotic nuclei were also present in the tissue treated with antisense H-ras ODN (P < 0.01). Immunocyto-histochemical study showed that expression of p21H-ras was significantly decreased after treatment with antisense H-ras. These results indicate that suppression of H-ras over-expression by antisense ODN could effectively inhibit tumor growth and revive the apoptotic pathway by releasing the activity of apoptotic endonuclease(s). The data also suggest the need for further studies to elucidate molecular events involved in antisense H-ras-released apoptosis and evaluate its therapeutic implications.