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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
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. Non-junctional 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 Gene- transfection 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 Cx-transfected 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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