Enhancement of depsipeptide-mediated apoptosis of lung or esophageal cancer cells by flavopiridol: Activation of the mitochondria-dependent death-signaling.

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Enhancement of depsipeptide-mediated apoptosis of lung or esophageal cancer cells by flavopiridol: Activation of the mitochondria-dependent death-signaling.

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Enhancement of depsipeptide-mediated apoptosis of lung or esophageal cancer cells by flavopiridol: Activation of the mitochondria-dependent death-signaling pathway Dao M. Nguyen, MD, FRCSC, FACS, William D. Schrump, *, Wilson S. Tsai, MD, Aaron Chen, MS, John H. Stewart, MD, Federico Steiner, MD, David S. Schrump, MD, FACS The Journal of Thoracic and Cardiovascular Surgery Volume 125, Issue 5, Pages 1132-1142 (May 2003) DOI: 10.1067/mtc.2003.180 Copyright © 2003 American Association for Thoracic Surgery Terms and Conditions

Fig. 1 Abrogation of depsipeptide (DP)-mediated upregulation of p21 protein expression in NSCLC or EsC cells by flavopiridol (FLA). Values are means ± SD of 4 independent experiments. The Journal of Thoracic and Cardiovascular Surgery 2003 125, 1132-1142DOI: (10.1067/mtc.2003.180) Copyright © 2003 American Association for Thoracic Surgery Terms and Conditions

Fig. 2A Dose-dependent enhancement of depsipeptide (DP)-mediated cytotoxicity of the NSCLC cells H460 and H322 and the EsC cells TE-12 and TE-13 by flavopiridol (FLA; 50-100 nmol/L). The sensitivity of tumor cells to depsipeptide is indicated by depsipeptide doses that induced 50% growth inhibition (IC50; in nanograms per milliliter). Values shown are means ± SD of 4 independent experiments. The Journal of Thoracic and Cardiovascular Surgery 2003 125, 1132-1142DOI: (10.1067/mtc.2003.180) Copyright © 2003 American Association for Thoracic Surgery Terms and Conditions

Fig. 2B Time course of the synergistic cytocidal effect of the depsipeptide (DP) plus flavopiridol (FLA) drug combination in H460, H322, TE-12, and TE-13 cells. Although individual depsipeptide or flavopiridol treatment resulted in retardation of cell proliferation, significant cell death was observed in cells treated with the drug combination. Cell viability was quantitated by means of MTT assay and expressed as an optical density (OD) at 570 nm. Values shown are means ± SD of 3 independent experiments. The Journal of Thoracic and Cardiovascular Surgery 2003 125, 1132-1142DOI: (10.1067/mtc.2003.180) Copyright © 2003 American Association for Thoracic Surgery Terms and Conditions

Fig. 2C Lack of significant cytotoxic effect of depsipeptide, flavopiridol (FLA), or the depsipeptide plus flavopiridol combination in normal cells. Values shown are means ± SD of 3 independent experiments. The Journal of Thoracic and Cardiovascular Surgery 2003 125, 1132-1142DOI: (10.1067/mtc.2003.180) Copyright © 2003 American Association for Thoracic Surgery Terms and Conditions

Fig. 3 Profound and synergistic induction of apoptosis in NSCLC or EsC cells by the depsipeptide (DP) plus flavopiridol (FLA) drug combination. Values shown are means ± SD of 3 independent experiments. The Journal of Thoracic and Cardiovascular Surgery 2003 125, 1132-1142DOI: (10.1067/mtc.2003.180) Copyright © 2003 American Association for Thoracic Surgery Terms and Conditions

Fig. 4 Western blot analysis of drug-induced alteration of Bax and Bcl2 levels in H322 or TE-12 cells at 18 hours after the onset of drug exposure. The relative levels of Bax and Bcl2 were quantitated by densitometry. This finding was reproduced in another independent experiment. DP, Depsipeptide; FLA, flavopiridol. The Journal of Thoracic and Cardiovascular Surgery 2003 125, 1132-1142DOI: (10.1067/mtc.2003.180) Copyright © 2003 American Association for Thoracic Surgery Terms and Conditions

Fig. 5 Significant accumulation of cytochrome c in the cytosolic fraction of the cell lysates was noted in NSCLC H460 and H322 cells or EsC TE-12 and TE-13 cells after treatment with the depsipeptide plus flavopiridol drug combination. Cells were harvested at 20 hours (H322, TE-12, and TE-13 cells) or 40 hours (H460) after the onset of drug exposure; cytosolic cytochrome c levels were measured by ELISA. Representative data from a series of 3 independent experiments with similar results are shown. The Journal of Thoracic and Cardiovascular Surgery 2003 125, 1132-1142DOI: (10.1067/mtc.2003.180) Copyright © 2003 American Association for Thoracic Surgery Terms and Conditions

Fig. 6 The time course of caspase 9 activation in cancer cell lines after depsipeptide (DP), flavopiridol (FLA), or depsipeptide plus flavopiridol treatments. Relative levels of caspase 9 activities are shown as folds of levels in untreated control cells. The Journal of Thoracic and Cardiovascular Surgery 2003 125, 1132-1142DOI: (10.1067/mtc.2003.180) Copyright © 2003 American Association for Thoracic Surgery Terms and Conditions

Fig. 7 Perturbation of mitochondrial function after drug exposure indicated by the loss of mitochondrial inner membrane potential, as assessed by means of JC-1 staining and flow cytometry. Cells with loss of mitochondrial inner membrane potential are sorted to the right lower quadrant of the dot plots. Representative data from a series of 3 independent experiments with similar results are shown. The Journal of Thoracic and Cardiovascular Surgery 2003 125, 1132-1142DOI: (10.1067/mtc.2003.180) Copyright © 2003 American Association for Thoracic Surgery Terms and Conditions

Fig. 8 Inhibition of depsipeptide (DP) plus flavopiridol (FLA)-mediated induction of apoptosis in NSCLC cells or EsC cells by the selective caspase 9 inhibitor (20 μmol/L added to the culture media just before flavopiridol exposure). Representative data from a series of 3 independent experiments with similar results are shown. The Journal of Thoracic and Cardiovascular Surgery 2003 125, 1132-1142DOI: (10.1067/mtc.2003.180) Copyright © 2003 American Association for Thoracic Surgery Terms and Conditions

Fig. 9 The central role of mitochondria in mediating and amplifying apoptosis initiated by extracellular or intracellular death signals. TNF, Tumor necrosis factor; TRAIL, tumor necrosis factor receptor apoptosis-inducing ligand; DISC, Death-inducing signaling complex; ATP, adenosine triphosphate; Smac/Diablo, second mitochondria-derived activation of caspases; AIF, apoptosis-inducing factor; ROS, reactive oxygen species. The Journal of Thoracic and Cardiovascular Surgery 2003 125, 1132-1142DOI: (10.1067/mtc.2003.180) Copyright © 2003 American Association for Thoracic Surgery Terms and Conditions