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AIM:To evaluate the effects of 3,3’-diethyl-9-methylthia-carbocyanine iodide (DMTCCI) on DNA primase activity and onapoptosis of human hepatocellular carcinoma BEL-7402 cells.METHODS:DNA primase assay was used to investigateDNA primase activity.MTF assay was applied to determinecell proliferation.Flow cytometric analysis,transmissionelectron microscopy,DNA fragmentation assay wereperformed to detect DMTCCI-induced apoptosis.Expressionlevels of p53,Bcl-2,Bcl-xL,Bad,Bax,survivin,Caspase-3and poly (ADP-ribose) polymerase (PARP) were evaluatedby immunoblot analysis.Caspase-3 activity was assessedwith ApoAlert Caspase-3 colorimetric assay kit.RESULTS:DMTCCI had inhibitory effects on eukaryotic DNAprimase activity with ICso value of 162.2 nmol/L.It alsoinhibited proliferation of human hepatocellular carcinomaBEL-7402 cells with IC_(50) value of 2.09μmol/L.Furthermore,DMTCCI-induced BEL-7402 cell apoptosis was confirmed byDNA fragmentation (DNA ladders and sub-G1 formation)and transmission electron microscopy (apoptotic bodiesformation).During the induction of apoptosis,expression ofBcl-2,Bd-xL and survivin was decreased,and that of p53,Bad and Bax was increased.Caspase-3 was activated andpoly (ADP-ribose) polymerase (PARP) was cleaved in BEL-7402 cells treated with DMTCCI.CONCLUSION:The present data suggest that DMTCCI hasinhibitory effects on eukaryotic DNA primase and can induceapoptosis of BEL-7402 cells.The modulation of expressionof p53 and Bcl-2 family proteins,and activation of Caspase-3 might be involved in the induction of apoptosis.
AIM: To evaluate the effects of 3,3’-diethyl-9-methylthia-carbocyanine iodide (DMTCCI) on DNA primase activity and onapoptosis of human hepatocellular carcinoma BEL-7402 cells. METHODS: DNA primase assay was used to investigate DNA primase activity. Flow cytometric analysis, transmission electron microscopy, DNA fragmentation assay were per- formed to detect DMTCCI-induced apoptosis. Expressions of p53, Bcl-2, Bcl-xL, Bad, Bax, survivin, Caspase- 3 and poly -ribose) were evaluatedby immunoblot analysis. Caspase-3 activity was assessed with ApoAlert Caspase-3 colorimetric assay kit .RESULTS: DMTCCI had inhibitory effects on eukaryotic DNAprimase activity with ICso value of 162.2 nmol / L.It alsoinhibited proliferation of human hepatocellular carcinoma BEL-7402 cells with IC 50 value of 2.09 μmol / L.Furthermore, DMTCCI-induced BEL-7402 cell apoptosis was confirmed by DNA fragmentation (DNA ladders and sub-G1 formation) and transmission e lectron microscopy (apoptotic bodiesformation). The induction of apoptosis, expression of Bcl-2, Bd-xL and survivin was decreased, and that of p53, Bad and Bax was increased. Caspase-3 was activated and poly ADP- ribose polymerase PARP) was cleaved in BEL-7402 cells treated with DMTCCI.CONCLUSION: The present data suggest that DMTCCI has inhibitory effects on eukaryotic DNA primase and can induce apoptosis of BEL-7402 cells. The modulation of expression of p53 and Bcl-2 family proteins, and activation of Caspase-3 might be involved in the induction of apoptosis.