Kim J A, Kang Y S, Jung M W, Kang G H, Lee S H, Lee Y S
Department of Physiology, College of Pharmacy, Yeungnam University, Kyongsan, Korea.
Pharmacology. 2000 Feb;60(2):74-81. doi: 10.1159/000028350.
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.
细胞凋亡似乎与癌症的发病机制和治疗应用有关。在本研究中，我们研究了钾通道阻滞剂4-氨基吡啶（4-AP）诱导人肝癌HepG2细胞凋亡的作用及其机制。4-AP以剂量依赖的方式降低细胞活力并诱导DNA片段化，这是细胞凋亡的一个标志。此外，4-AP诱导细胞内Ca(2+)浓度持续升高，而细胞外Ca(2+)与EGTA螯合可完全抑制这种升高。4-AP还诱导Mn(2+)内流，表明4-AP诱导的细胞内Ca(2+)水平升高是由于Ca(2+)内流途径的激活。4-AP还使使用电压敏感荧光染料di-O-C(5)(3)测量的膜电位去极化。4-AP诱导的Ca(2+)内流不受电压依赖性Ca(2+)通道阻滞剂（硝苯地平或维拉帕米）的显著抑制，而是受已知的非选择性阳离子通道阻滞剂氟芬那酸（FA）的显著抑制。通过流式细胞术对细胞凋亡进行定量分析表明，用FA或细胞内Ca(2+)螯合剂BAPTA处理可显著抑制4-AP诱导的细胞凋亡。综上所述，这些结果表明，在HepG2细胞中观察到的4-AP诱导的细胞凋亡可能是由于电压敏感的Ca(2+)通透非选择性阳离子通道激活导致Ca(2+)内流所致。这些结果进一步表明，通过调节钾通道活性引起的膜电位变化可能参与了人肝癌细胞的凋亡机制。
