Apoptotic insults to human HepG2 cells induced by S-(+)-ketamine occurs through activation of a Bax-mitochondria-caspase protease pathway S.-T. Lee,

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Apoptotic insults to human HepG2 cells induced by S-(+)-ketamine occurs through activation of a Bax-mitochondria-caspase protease pathway S.-T. Lee, T.-T. Wu, P.-Y. Yu, R.-M. Chen British Journal of Anaesthesia Volume 102, Issue 1, Pages 80-89 (January 2009) DOI: 10.1093/bja/aen322 Copyright © 2009 British Journal of Anaesthesia Terms and Conditions

Fig 1 Effects of S-(+)-ketamine on the release of GPT and LDH, and cell viability. HepG2 cells were exposed to 10, 50, 100, and 200 μM ketamine for 24 h, or to 200 μM ketamine for 1, 6, and 24 h. The amounts of GPT (a and b) and LDH (c and d) released from human hepatocytes to the culture medium were quantified using an automatic autoanalyzer. Cell viability was determined by a trypan blue exclusion method (e and f). The figures are drawn as box and whisker plots showing median, inter-quartile, and full ranges. *Values significantly differ from the respective control, P<0.05, n=6. British Journal of Anaesthesia 2009 102, 80-89DOI: (10.1093/bja/aen322) Copyright © 2009 British Journal of Anaesthesia Terms and Conditions

Fig 2 Effects of S-(+)-ketamine on cell morphologies, DNA fragmentation, and cell apoptosis. HepG2 cells were exposed to 200 μM ketamine for 1, 6, and 24 h. Cell morphologies were observed and photographed using a reverse-phase microscope (a). Fragmentation of genomic DNA was quantified using a BrdU-labelled histone-associated enzyme-linked immunosorbent assay (b). Apoptotic cells were analysed using flow cytometry (c). The figures are drawn as box and whisker plots showing median, inter-quartile, and full ranges. *Values significantly differ from the respective control, P<0.05, n=6. British Journal of Anaesthesia 2009 102, 80-89DOI: (10.1093/bja/aen322) Copyright © 2009 British Journal of Anaesthesia Terms and Conditions

Fig 3 Effects of S-(+)-ketamine on Bax translocation, the mitochondrial membrane potential, cellular ATP levels, and release of cytochrome c (Cyt c). HepG2 cells were exposed to 200 μM ketamine for 1, 6, and 24 h. The amounts of mitochondrial and cytosolic Bax proteins were immunodetected (a, top and middle panels). β-Actin was immunodetected as the internal control (a, bottom panel). These immunoreactive protein bands were quantified and analysed (b). The mitochondrial membrane potential was detected by staining with DiOC6 and quantified using flow cytometry (c). The levels of cellular ATP were analysed using a bioluminescence assay (d). Cyt c in the cytoplasm was immunodetected (e, top panel). β-Actin was analysed as the internal control (e, bottom panel). These protein bands were quantified and analysed (f). The figures are drawn as box and whisker plots showing median, inter-quartile, and full ranges. *Values significantly differ from the respective control, P<0.05, n=6. British Journal of Anaesthesia 2009 102, 80-89DOI: (10.1093/bja/aen322) Copyright © 2009 British Journal of Anaesthesia Terms and Conditions

Fig 4 Effects of S-(+)-ketamine on the activities of caspases-9, -3, and -6. Human hepatocytes were exposed to 200 μM ketamine for 1, 6, 16, and 24 h. Activities of caspases-9, -3, and -6 were analysed by fluorogenic assays using LEHD, DEVD, and VEID as the respective substrates (a–c). The figures are drawn as box and whisker plots showing median, inter-quartile, and full ranges. *Values significantly differ from the respective control, P<0.05, n=6. British Journal of Anaesthesia 2009 102, 80-89DOI: (10.1093/bja/aen322) Copyright © 2009 British Journal of Anaesthesia Terms and Conditions

Fig 5 Effects of S-(+)-ketamine and Z-VEID-FMK on caspase-6 activity, DNA fragmentation, and cell apoptosis. Human hepatocytes were pretreated with 50 μM Z-VEID-FMK, an inhibitor of caspase-6, for 1 h, and then exposed to 200 μM ketamine for another 16 h. Caspase-6 activity was determined by a fluorogenic assay (a). DNA fragmentation was quantified using a BrdU-labelled histone-associated enzyme-linked immunosorbent assay kit (b). Apoptotic cells were quantified using flow cytometry (c). The figures are drawn as box and whisker plots showing median, inter-quartile, and full ranges. The symbols * and # indicate that a value significantly (P<0.05) differs from the respective control and ketamine-treated groups, respectively (n=6). British Journal of Anaesthesia 2009 102, 80-89DOI: (10.1093/bja/aen322) Copyright © 2009 British Journal of Anaesthesia Terms and Conditions

Fig 6 Signal-transducing mechanism of S-(+)-ketamine-induced apoptotic HepG2 cells. British Journal of Anaesthesia 2009 102, 80-89DOI: (10.1093/bja/aen322) Copyright © 2009 British Journal of Anaesthesia Terms and Conditions