Yong Soo Lee

Induction of apoptosis by capsaicin in A172 human glioblastoma cells.

Capsaicin induced apoptosis of A172 human glioblastoma cells in a time- and dose-dependent manner. Neither capsazepine, a vanilloid receptor antagonist, nor bis-(o-aminophenoxy)-ethane-N,N,N, N-tetraacetic acid/acetoxymethyl ester (BAPTA/AM), an intracellular Ca(2+) chelator, significantly inhibited the capsaicin-induced apoptosis, although capsaicin increased intracellular Ca(2+) level. Capsaicin markedly reduced the basal generation of reactive oxygen species (ROS) and lipid peroxidation. Exogenous application of H(2)O(2) significantly prevented the cells from the apoptosis by capsaicin. Treatment with N-acetyl cysteine alone induced both reduction of the basal production of ROS and apoptosis. Taken together, these results suggest that capsaicin induced apoptosis in A172 cells and that vanilloid receptors and intracellular Ca(2+) may not be involved in the apoptotic mechanism of capsaicin. Reduction of the basal generation of ROS may play a role in the induction of apoptosis by capsaicin.

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.

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 reactive oxygen species generated by NADPH oxidase in the mechanism of activation of K+-Cl--cotransport by N-ethylmaleimide in HepG2 human hepatoma cells

K+-Cl--cotransport (KCC) is ubiquitously present in all cells, and plays an essential role in ion and volume regulation. In this study we investigated the role of reactive oxygen species (ROS) in regulation of KCC in HepG2 human hepatoblastoma cells. N-ethylmaleimide (NEM), a KCC activator, induced Cl--dependent K+ efflux, which was markedly prevented by KCC inhibitors (calyculin-A, genistein and BaCl2), indicating that KCC is activated by NEM in the HepG2 cells. Treatment with NEM also induced a sustained increase in the level of intracellular ROS assessed by 2′,7′-dichlorofluorescein flourescence. Antioxidants, N-acetyl cysteine or N,N′-diphenyl-p-phenylenediamine significantly inhibited both ROS generation and KCC activation induced by NEM. The NEM-induced ROS production was significantly suppressed by inhibitors of NADPH oxidase (diphenylene iodonium, apocynin and neopterine). These inhibitors also significantly inhibited the NEM-induced KCC activation. Taken together, these results suggest that ROS generated by NADPH oxidase may mediate the NEM-induced activation of KCC in human hepatoma cells.

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.

Role of reactive oxygen species in apoptosis induced by N-ethylmaleimide in HepG2 human hepatoblastoma cells

We have previously reported that N-ethylmaleimide induces apoptosis through activation of K(+), Cl(-)-cotransport in HepG2 human hepatoblastoma cells. In this study, we investigated the role for reactive oxygen species as a mediator of the apoptosis induced by N-ethylmaleimide. N-ethylmaleimide induced a significant elevation of intracellular level of reactive oxygen species. Treatment with antioxidants (N-acetyl cysteine, N,N-diphenyl-p-phenylenediamine) which markedly suppressed generation of reactive oxygen species, significantly inhibited the N-ethylmaleimide-induced activation of K(+), Cl(-)-cotransport and apoptosis. Inhibitors of NADPH oxidase (diphenylene iodonium, apocynin, D-(+)-neopterine) also significantly blunted the generation of reactive oxygen species, activation of K(+), Cl(-)-cotransport and apoptosis induced by N-ethylmaleimide. These results suggest that reactive oxygen species generated through activation of NADPH oxidase may play a role in the N-ethylmaleimide-induced stimulation of K(+), Cl(-)-cotransport and apoptosis in HepG2 cells.

Bepridil enhances in vitro antitumor activity of antiestrogens in human brain tumor cells

The possible interaction between antiestrogens (tamoxifen, clomiphene and nafoxidine) and bepridil, a known Na+-Ca2+ exchange blocker, in the regulation of cell growth was investigated using U-373 MG human astrocytoma and SK-N-MC human neuroblastoma cells as model cellular systems. The co-treatment of bepridil with antiestrogens significantly enhanced the antiestrogen-induced inhibition of the tumor cell growth. This bepridil-induced enhanced growth inhibition was significantly blocked by the addition of BAPTA/AM, an intracellular Ca2+ chelator, implying that increased free intracellular Ca2+ concentration may be involved in these actions. Other Na+-Ca2+ exchange blockers such as nickel and benzamil, also significantly potentiated the antiestrogen-induced inhibition of the tumor cell growth. Taken together, the blockade of Na+-Ca2+ exchange mechanism by these drugs may cause prolongation of increased intracellular Ca2+ concentration, in turn leading to these potentiated growth inhibitions of the tumor cells. These results suggest that the combined treatment with bepridil and antiestrogens may be a potential strategy for chemotherapy of brain tumors.