Mechanism of apoptosis induced by diazoxide, a K+ channel opener, in HepG2 human hepatoma cells.
The effect of diazoxide, a K+ channel opener, on apoptotic cell death was investigated in HepG2 human hepatoblastoma cells. Diazoxide induced apoptosis in a dose-dependent manner and this was evaluated by flow cytometric assays of annexin-V binding and hypodiploid nuclei stained with propidium iodide. Diazoxide did not alter intracellular K+ concentration, and various inhibitors of K+ channels had no influence on the diazoxide-induced apoptosis; this implies that K+ channels activated by diazoxide may be absent in the HepG2 cells. However, diazoxide induced a rapid and sustained increase in intracellular Ca(2+) concentration, and this was completely inhibited by the extracellular Ca(2+) chelation with EGTA, but not by blockers of intracellular Ca(2+) release (dantrolene and TMB-8). This result indicated that the diazoxide-induced increase of intracellular Ca(2+) might be due to the activation of a Ca(2+) influx pathway. Diazoxide-induced Ca(2+) influx was not significantly inhibited by either voltage-operative Ca(2+) channel blockers (nifedipine or verapamil), or by inhibitors of Na+, Ca(2+)-exchanger (bepridil and benzamil), but it was inhibited by flufenamic acid (FA), a Ca(2+)-permeable nonselective cation channel blocker. A quantitative analysis of apoptosis by flow cytometry revealed that a treatment with either FA or BAPTA, an intracellular Ca(2+) chelator, significantly inhibited the diazoxide-induced apoptosis. Taken together, these results suggest that the observed diazoxide-induced apoptosis in the HepG2 cells may result from a Ca(2+) influx through the activation of Ca(2+)-permeable non-selective cation channels. These results are very significant, and they lead us to further suggest that diazoxide may be valuable for the therapeutic intervention of human hepatomas.
Lee
Yong Soo
YS
College of Pharmacy, Duksung Women's University, Seoul 132-714, Korea. yongslee@duksung.ac.kr
D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D015220:Calcium Channels / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D003981:Diazoxide / Q000494:pharmacology*; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology*; D015221:Potassium Channels / Q000378:metabolism*
D015220:Calcium Channels; D015221:Potassium Channels; D003981:Diazoxide