Young Shin Kang

Involvement of Ca2+ influx in the mechanism of tamoxifen-induced apoptosis in HepG2 human hepatoblastoma cells

The signaling mechanism of tamoxifen (TAM)-induced apoptosis was investigated in HepG2 human hepatoblastoma cells which do not express the estrogen receptor (ER). TAM induced cytotoxicity and DNA fragmentation, a hallmark of apoptosis, in a dose-dependent manner. TAM increased the intracellular concentration of Ca2+. This effect was completely inhibited by the extracellular Ca2+ chelation with EGTA. TAM also induced a Mn2+ influx, indicating that TAM activated Ca2+ influx pathways. This action of TAM was significantly inhibited by flufenamic acid (FA), a known non-selective cation channel blocker. Quantitative analysis of apoptosis by flow cytometry revealed that treatment with either FA or BAPTA, an intracellular Ca2+ chelator, significantly inhibited TAM-induced apoptosis. These results suggest that intracellular Ca2+ signals may play a central role in the mechanism of the TAM-induced apoptotic cell death in ER-negative HepG2 cells.

Inhibitory effects of constituents ofGastrodia elata Bl. on glutamate-induced apoptosis in IMR-32 human neuroblastoma cells

The inhibitory effects of the constituents ofGastrodia elata Bl. (GE) on glutamate-induced apoptosis in human neuronal cells were investigated using IMR32 human neuroblastoma cells. Glutamate (GLU) induced DNA fragmentation, a hallmark of apoptosis, in a dose-dependent manner. GLU also induced a slow and sustained increase in intracellular Ca2+ concentration. Treatment with EGTA, an extracellular Ca2+ chelator, in a nominal Ca2+-free buffer solution abolished the GLU-induced intracellular Ca2+ increase, indicating that GLU stimulated Ca2+ influx pathway in the IMR32 cells. BAPTA, an intracellular Ca2+ chelator, significantly inhibited the GLU-induced apoptosis assessed by the flow cytometry measuring hypodiploid DNA content indicative of apoptosis, implying that intracellular Ca2+ rise may mediate the apoptotic action of GLU. Vanillin (VAN) and p-hydroxybenzaldehyde(p-HB), known constituents of GE, significantly inhibited both intracellular Ca2+ rise and apoptosis induced by GLU. These results suggest that the apoptosis-inhibitory actions of the constituents of GE may account, at least in part, for the basis of their antiepileptic activities. These results further suggest that intracellular Ca2+ signaling pathway may be a molecular target of the constituents of GE.

Ca2+ Influx Mediates Apoptosis Induced by 4-Aminopyridine, a K+ Channel Blocker, in HepG2 Human Hepatoblastoma Cells

Apoptosis appears to be implicated in the pathogenesis and therapeutic applications of cancer. In this study we investigated the induction of apoptosis by 4-aminopyridine (4-AP), a K+ channel blocker, and its mechanism in HepG2 human hepatoblastoma cells. 4-AP reduced cell viability and induced DNA fragmentation, a hallmark of apoptosis, in a dose-dependent manner. In addition, 4-AP induced a sustained increase in intracellular Ca2+ concentration, which was completely inhibited by the extracellular Ca2+ chelation with EGTA. 4-AP also induced Mn2+ influx, indicating that the 4-AP-induced increased intracellular Ca2+ levels were due to activation of Ca2+ influx pathway. 4-AP also depolarized membrane potential that was measured by using di-O-C5(3), a voltage-sensitive fluorescent dye. 4-AP-induced Ca2+ influx was significantly inhibited not by voltage-operative Ca2+ channel blockers (nifedipine or verapamil), but by flufenamic acid (FA), a known nonselective cation channel blocker. Quantitative analysis of apoptosis by the flow cytometry revealed that treatment with either FA or BAPTA, an intracellular Ca2+ chelator, significantly inhibited the 4-AP-induced apoptosis. Taken together, these results suggest that the observed 4-AP-induced apoptosis in the HepG2 cells may result from Ca2+ influx through the activation of voltage-sensitive Ca2+-permeable non-selective cation channels. These results further suggest that membrane potential change by modulation of K+ channel activity may be involved in the mechanism of apoptosis in human hepatoma cells.

Role of Ca 2+ influx in the tert-butyl hydroperoxide-induced apoptosis of HepG2 human hepatoblastoma cells

Oxidative stress appears to be implicated in the pathogenesis of various diseases including alcoholic liver injury. In this study we investigated the mechanism of apoptosis induced by tert-butyl hydroperoxide (TBHP) in HepG2 human hepatoblastoma cells. Treatment with TBHP significantly reduced glutathione content and glutathione reductase activity, and increased glutathione peroxidase activity, indicating that TBHP induced oxidative stress in the HepG2 cells. TBHP also induced reduction of cell viability and DNA fragmentation, a hallmark of apoptosis, in a dose-dependent manner. In addition, TBHP induced a sustained increase in intracellular Ca2+ concentration, which was completely prevented by the extracellular Ca2+ chelation with EGTA. TBHP also induced Mn2+ influx. These results indicate that the intracellular Ca2+ increase by TBHP is exclusively due to Ca2+ influx from the extracellular site. Treatment with either an extracellular (EGTA) or an intracellular Ca2+ chelator (BAPTA/AM) significantly suppressed the TBHP-induced apoptosis. Taken together, these results suggest that TBHP induced the apoptotic cell death in the HepG2 cells and that Ca2+ influx may play an important role in the apoptosis induced by TBHP.

Inhibitors of Na+/Ca2+ exchanger prevent oxidant-induced intracellular Ca2+ increase and apoptosis in a human hepatoma cell line

Oxidative stress appears to be implicated in the pathogenesis of various diseases including hepatotoxicity. Although intracellular Ca2+ signals have been suggested to play a role in the oxidative damage of hepatocytes, the sources and effects of oxidant-induced intracellular Ca2+ increases are currently debatable. Thus, in this study we investigated the exact source and mechanism of oxidant-induced liver cell damage using HepG2 human hepatoma cells as a model liver cellular system. Treatment with 200 μM of tert-butyl hydroperoxide (tBOOH) induced a sustained increase in the level of intracellular reactive oxygen intermediates (ROI) and apoptosis, assessed by 2′,7′-dichlorofluorescein fluorescence and flow cytometry, respectively. Antioxidants, N-acetyl cysteine (NAC) or N,N′-diphenyl-p-phenylenediamine significantly inhibited both the ROI generation and apoptosis. In addition, tBOOH induced a slow and sustained increase in intracellular Ca2+ concentration, which was completely prevented by the antioxidants. An intracellular Ca2+ chelator, bis-(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid/cetoxymethyl ester significantly suppressed the tBOOH-induced apoptosis. These results imply that activation of an intracellular Ca2+ signal triggered by increased ROI may mediate the tBOOH-induced apoptosis. Both intracellular Ca2+ increase and induction of apoptosis were significantly inhibited by an extracellular Ca2+ chelator or Na+/Ca2+ exchanger blockers (bepridil and benzamil), whereas neither Ca2+ channel antagonists (verapamil and nifedipine) nor a nonselective cation channel blocker (flufenamic acid) had an effect. These results suggest that tBOOH may increase intracellular Ca2+ through the activation of reverse mode of Na+/Ca2+ exchanger. However, tBOOH decreased intracellular Na+ concentration, which was completely prevented by NAC. These results indicate that ROI generated by tBOOH may increase intracellular Ca2+ concentration by direct activation of the reverse mode of Na+/Ca>2+ exchanger, rather than indirect elevation of intracellular Na+ levels. Taken together, these results suggest that the oxidant, tBOOH induced apoptosis in human HepG2 cells and that intracellular Ca2+ may mediate this action of tBOOH. These results further suggest that Na+/Ca2+ exchanger may be a target for the management of oxidative hepatotoxicity.

Role of pertussis toxin‐sensitive G‐proteins in intracellular Ca2+ release and apoptosis induced by inhibiting cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channels in HepG2 human hepatoblastoma cells

Previously, we have reported that inhibition of cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channels by glibenclamide induced intracellular Ca2+ release from IP3‐sensitive stores and apoptosis in HepG2 human hepatoblastoma cells (Kim JA, Kang YS, Lee SH, Lee EH, Yoo BH, Lee YS. 1999 . Biochem Biophys Res Commun 261:682–688). In this study we investigated the upstream signals involved in the mechanism of these actions of glibenclamide. Treatment with glibenclamide initiated production of inositol 1,4,5‐trisphosphate (IP3) in a dose‐ and time‐dependent manner. The glibenclamide‐induced formation of IP3 was significantly inhibited by CFTR activators (levamisole and bromotetramisole). The intracellular Ca2+ release and apoptosis induced by glibenclamide were significantly suppressed by treatment with phospholipase C (PLC) inhibitors (U‐73122 and manoalide) or by pretreatment with pertussis toxin (PTx). In addition, PTx‐catalyzed ADP‐ribosylation of GTP‐binding proteins (G‐proteins) was markedly enhanced by treatment with glibenclamide in a time‐dependent manner. Taken together, these results suggest that PTx‐sensitive G‐proteins coupled to PLCβ may mediate the intracellular Ca2+ release and apoptosis induced by inhibiting CFTR Cl− channels in HepG2 cells. These results further suggest that the PTx‐sensitive G‐proteins may be a valuable target for the therapeutic intervention of human hepatomas. J. Cell. Biochem. 81:93–101, 2001.

Involvement of K+-Cl--cotransport in the apoptosis induced by N-ethylmaleimide in HepG2 human hepatoblastoma cells

The role of K(+)-Cl(-)-cotransport in apoptosis in human cancer cells was investigated. N-Ethylmaleimide, a K(+)-Cl(-)-cotransport activator, induced apoptosis in a dose-dependent manner in HepG2 human hepatoblastoma cells. N-Ethylmaleimide induced Cl(-)-dependent K(+) efflux, indicating that K(+)-Cl(-)-cotransport is functionally present in HepG2 cells. Calyculin-A and genistein, inhibitors of K(+)-Cl(-)-cotransport, significantly prevented both K(+)-Cl(-)-cotransport activation and apoptosis induced by N-ethylmaleimide. These results demonstrate, for the first time, a novel role for K(+)-Cl(-)-cotransport in apoptosis in human hepatoma cells. These results further suggest that K(+)-Cl(-)-cotransport may be a valuable target for therapeutic interventions for human hepatoma.