Mechanism Of Nitric Oxide Induced Apoptosis In Human Hepatoma Cells

Objective: To investigate the effects of sodium nitroprusside(SNP) on viability > cytoplasmic free calcium concentration[Ca2+]k cytosolic cytochrome C and mitochondrial transmembrane potentialm) in human hepatoma cell lines SMMC-7721 and HepG2 and explore the mechanism of NO-mediated apoptosis.Methods: NO-mediated apoptosis in human hepatoma cells SMMC-7721 and HepG2 were investigated by MTT assay, fluorescent imaging, ultrostructural analysis, DNA fragmentation and flow cytometry. Fura-2/AM flourescein loading techniquee was used to detect cytosolic free calcium concentration[Ca2+]i. Cytoplasmic cytochrome C was measured by Western blot and mitochondrial transmembrane potential(A ^m) was analyzed by FCM with Rhl23 and PI.Results:1. NO donor, sodium nitroprusside (SNP) inhibited growth and proliferation in human hepatoma cells SMMC-7721 and HepG2 and induced apoptosis in a time-and dose-dependent manner.2. SMMC-7721 was more sensitive to NO than that of HepG2.3. Voltage-dependent calium channel blocker verapamil and extracellular Ca2* chelatorEGTA partially inhibited the increase [Ca2+]i in both cell lines by SNP. The intracellular Ca2* chelator BAPTA/AM decreased the [Ca2+]i in HepG2 but did not prevent the increase of [Ca2*]i in SMMC-7721.4. The decrease of mitochondria! transmembrane potential(A ^m) and release of cytochrome C from mitochondria in two cell lines were significant positively related to the time course of SNP.5. It were partially blocked the decrease of AWm and release cytochrome C by specific inhibitor of the mitochondrial permeability transition pore-cyclosporin A (CsA), GSH synthesis blocker BSO promoted both by cellular protein thiol oxidation.Conclusion:1. NO donor SNP is able to induce apoptosis in human hepatoma cell lines SMMC-7721 and HepG2 according to characteristic morphological changes and biochemical features, but their sensitivities were different.2. SNP initiated apoptosis in human hepatoma cells via elevating cytosolic free calcium concentration [Ca2+]i, which were mainly come from the extracellular calcium and partially from the inner cellular calcium pool.3. The apoptotic signal transduction pathways involved in mitochondria including the decrease of mitochondrial transmembrane potentialtA^m), open the mitochondrial permeability transition pore and release the cytochrome C.It has been shown that mitochondria plays a key role in NO- mediated apoptosis which allows us to approach the molecular mechanism of apoptosis so that proper therapies on human hepatoma can be given to.