The Role of Connexin 43 and 26 Hemichannels In cisplatin-Induced Apoptosis
Kim, Yeon Ju; Tian, Chunjie; Kim, Seung Won; Lim, Hye Jin; Kim, Young Sun; Choung, Yun-Hoon
Department of Otolaryngology, Ajou University School of Medicine
INTRODUCTION : Connexins are assembled into a hemichannel and two identical hemichannels form a gap junction, which play an important role in K+ recycling, metabolic communication in the cochlea. Nonjunctional hemichannels can gate open and pass molecules (<1 kDa) by depolarization, extracellular Ca2+, oxidative stress, and metabolic inhibition. The purpose of this study was to investigate the role of hemichannel, connexin 43 (Cx43) and 26 (Cx26) in cisplatin-induced apoptotic process using Genetransfection techniques.
MATERIALS AND METHODS : We constructed recombinant plasmid of pcDNA 3.1(-) -Cx43, pcDNA 3.1(-) -Cx26. The Cx-pcDNA 3.1 (-) plasmid was transfected in Cx-deficient HeLa cells. The formation of functional hemichannels was tested by the transfer of Lucifer yellow (LY, m.w 457.25) from a Ca2+ free condition with/without hemichannel blockers (25μM Carbenoxolone; CBX, 25μM 18 alpha-glycyrrhetinic acid; 18 AGA). To investigate the mechanism for cisplatin-induced regulation of hemichannels, we analyzed opening of hemichannels under 4 different conditions (Ca2+ condition, Ca2+ Free condition, Hydrogen peroxide (H2O2), potassium chloride (KCl) and cisplatin-induced stress condition).
RESULTS : Cx43 and Cx26 are significantly expressed by transfection of pcDNA 3.1(-)-Cx43, Cx26 in the HeLa cells. LY uptake was observed in Cx43, Cx26-transfected cells, but was absent in Cx-deficient wild type HeLa cells and these uptake was inhibited by both CBX, 18-AGA. Under cisplatin toxicity, hemichannels opened in Cx43, Cx26-transfected cells. Cx43 and Cx26-transfected cells significantly responded to Ca2+ free condition. Also, LY uptake was observed in the presence of H2O2 in Cx26 transfected cells, it is relatively small compared to Cx43-transfected cells. Under the KCl condition, LY uptake was not observed in both Cxtransfected cells. Live/dead cell assay and western blot assay indicate that cisplatin-induced cell death in Cx43, Cx26-HeLa cells increased (89% and 66%) compared to wild type HeLa cells. The hemichannel blockers prevented increase of cell death.
CONCLUSION : The hemichannel Cx 43 and 26 are opened by cisplatin resulting in apoptosis, which may be closely connected with the suppression of Ca2+.
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