Ca2+ influx mediates apoptosis induced by 4-aminopyridine, a K+ channel blocker, in HepG2 human hepatoblastoma cells

Abstract

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 Ca(2+) concentration, which was completely inhibited by the extracellular Ca(2+) chelation with EGTA. 4-AP also induced Mn(2+) influx, indicating that the 4-AP-induced increased intracellular Ca(2+) levels were due to activation of Ca(2+) influx pathway. 4-AP also depolarized membrane potential that was measured by using di-O-C(5)(3), a voltage-sensitive fluorescent dye. 4-AP-induced Ca(2+) influx was significantly inhibited not by voltage-operative Ca(2+) 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 Ca(2+) 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 Ca(2+) influx through the activation of voltage-sensitive Ca(2+)-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.