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Reactive oxygen species is essential for cycloheximide to sensitize lexatumumab-induced apoptosis in hepatocellular carcinoma cells.

Zhao X, Cao M, Liu JJ, Zhu H, Nelson DR, Liu C - PLoS ONE (2011)

Bottom Line: ROS generation induced by combination treatment of Lexa and CHX triggered pro-apoptotic protein Bax oligomerization, conformation change, and translocation to mitochondria, which resulted in the release of cytochrome c and subsequent cell death.More importantly, we observed that combination treatment of Lexa and CHX did not cause apoptotic toxicity in normal human primary hepatocytes.These results suggest that Lexa and CHX combination treatment merits investigation for the development of therapies for patients with HCC.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Florida College of Medicine, Gainesville, Florida, United States of America.

ABSTRACT
This study aims to investigate apoptosis induced by lexatumumab (Lexa) in hepatocellular carcinoma (HCC) cells. We assessed the sensitivity of HCC cell lines and normal human hepatocytes to Lexa and explored the sensitization of HCC cells to Lexa-induced apoptosis by cycloheximide (CHX). Our data indicated that CHX sensitized HCC cell lines to Lexa-induced apoptosis, whereas treatment using solely CHX or Lexa was ineffective. The sequential treatment of CHX followed by Lexa dramatically induced caspase-dependent apoptosis in HCC cells and had synergistically increased intracellular rates of reactive oxygen species (ROS). Additionally, when ROS production was blocked by N-acetyl-L-cysteine (NAC), HCC cells were protected against Lexa and CHX combination treatment-induced apoptosis. ROS generation induced by combination treatment of Lexa and CHX triggered pro-apoptotic protein Bax oligomerization, conformation change, and translocation to mitochondria, which resulted in the release of cytochrome c and subsequent cell death. Furthermore, HSP90 was involved in mediating Lexa and CHX combination treatment-induced ROS increase and apoptotic death. More importantly, we observed that combination treatment of Lexa and CHX did not cause apoptotic toxicity in normal human primary hepatocytes. These results suggest that Lexa and CHX combination treatment merits investigation for the development of therapies for patients with HCC.

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Lexa and CHX combination treatment induces caspase-dependent apoptosis.A, Huh7 cells were treated with different conditions as indicated. Apoptosis was tested by DNA ladder assays as described in ‘Materials and Methods’. B, Huh7 cells were treated with various stimuli as indicated (Lexa, (1 µg/ml), CHX, (10 µg/ml), or caspase 8 inhibitor IETD (50 µM)). Apoptotic cells were examined as described in Fig. 1B (representative apoptotic cells were marked with arrows). C, Huh7 cells were treated with conditions as indicated and apoptotic ratio was determined by counting cells with apoptotic nuclei as described in ‘Materials and Methods’. Data represent the mean values of three independent experiments (*p<0.05). D, Huh7 cells were treated with various conditions as indicated (Lexa, (1 µg/ml), CHX, (10 µg/ml), IETD (50 µM), z-VAD (50 µM)). Apoptosis was evaluated as in Fig. 1C. E, Huh7 cells were treated with different conditions as indicated and apoptosis percentage was determined as in Fig. 1D. Data represent the mean values of three independent experiments (**p<0.05).
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pone-0016966-g002: Lexa and CHX combination treatment induces caspase-dependent apoptosis.A, Huh7 cells were treated with different conditions as indicated. Apoptosis was tested by DNA ladder assays as described in ‘Materials and Methods’. B, Huh7 cells were treated with various stimuli as indicated (Lexa, (1 µg/ml), CHX, (10 µg/ml), or caspase 8 inhibitor IETD (50 µM)). Apoptotic cells were examined as described in Fig. 1B (representative apoptotic cells were marked with arrows). C, Huh7 cells were treated with conditions as indicated and apoptotic ratio was determined by counting cells with apoptotic nuclei as described in ‘Materials and Methods’. Data represent the mean values of three independent experiments (*p<0.05). D, Huh7 cells were treated with various conditions as indicated (Lexa, (1 µg/ml), CHX, (10 µg/ml), IETD (50 µM), z-VAD (50 µM)). Apoptosis was evaluated as in Fig. 1C. E, Huh7 cells were treated with different conditions as indicated and apoptosis percentage was determined as in Fig. 1D. Data represent the mean values of three independent experiments (**p<0.05).

Mentions: Death receptor-mediated apoptosis can be classified as either caspase dependent or caspase independent [30], [31]. To examine whether Lexa and CHX combination-induced apoptosis is controlled by caspase activation, Huh7 cells were pre-incubated with pan-caspase inhibitor z-VAD-FMK (50 µM) for 1 h, followed by various stimuli as indicated. DNA ladder assays (Fig. 2A), DNA fragmentation analysis (Fig. 2B and 2C) and FACS analysis (Fig. 2D and 2E) demonstrated that apoptosis induced by combination treatment with Lexa and CHX were totally blocked by pan-caspase inhibitor z-VAD-FMK, suggesting that CHX-sensitized and Lexa-induced apoptosis is caspase dependent. Meanwhile, we measured the effects of caspase 8 specific inhibitor z-IETD-FMK on apoptosis induced by Lexa and CHX combination treatment. As shown in Fig. 2A, 2B, 2C, 2D, and 2E, blockade of caspase 8 activation completely abolished Lexa and CHX combination-induced apoptosis in Huh7 cells. Taken together, these results suggest that caspase activation plays a key role in Lexa-mediated apoptosis induction in HCC cells.


Reactive oxygen species is essential for cycloheximide to sensitize lexatumumab-induced apoptosis in hepatocellular carcinoma cells.

Zhao X, Cao M, Liu JJ, Zhu H, Nelson DR, Liu C - PLoS ONE (2011)

Lexa and CHX combination treatment induces caspase-dependent apoptosis.A, Huh7 cells were treated with different conditions as indicated. Apoptosis was tested by DNA ladder assays as described in ‘Materials and Methods’. B, Huh7 cells were treated with various stimuli as indicated (Lexa, (1 µg/ml), CHX, (10 µg/ml), or caspase 8 inhibitor IETD (50 µM)). Apoptotic cells were examined as described in Fig. 1B (representative apoptotic cells were marked with arrows). C, Huh7 cells were treated with conditions as indicated and apoptotic ratio was determined by counting cells with apoptotic nuclei as described in ‘Materials and Methods’. Data represent the mean values of three independent experiments (*p<0.05). D, Huh7 cells were treated with various conditions as indicated (Lexa, (1 µg/ml), CHX, (10 µg/ml), IETD (50 µM), z-VAD (50 µM)). Apoptosis was evaluated as in Fig. 1C. E, Huh7 cells were treated with different conditions as indicated and apoptosis percentage was determined as in Fig. 1D. Data represent the mean values of three independent experiments (**p<0.05).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3037406&req=5

pone-0016966-g002: Lexa and CHX combination treatment induces caspase-dependent apoptosis.A, Huh7 cells were treated with different conditions as indicated. Apoptosis was tested by DNA ladder assays as described in ‘Materials and Methods’. B, Huh7 cells were treated with various stimuli as indicated (Lexa, (1 µg/ml), CHX, (10 µg/ml), or caspase 8 inhibitor IETD (50 µM)). Apoptotic cells were examined as described in Fig. 1B (representative apoptotic cells were marked with arrows). C, Huh7 cells were treated with conditions as indicated and apoptotic ratio was determined by counting cells with apoptotic nuclei as described in ‘Materials and Methods’. Data represent the mean values of three independent experiments (*p<0.05). D, Huh7 cells were treated with various conditions as indicated (Lexa, (1 µg/ml), CHX, (10 µg/ml), IETD (50 µM), z-VAD (50 µM)). Apoptosis was evaluated as in Fig. 1C. E, Huh7 cells were treated with different conditions as indicated and apoptosis percentage was determined as in Fig. 1D. Data represent the mean values of three independent experiments (**p<0.05).
Mentions: Death receptor-mediated apoptosis can be classified as either caspase dependent or caspase independent [30], [31]. To examine whether Lexa and CHX combination-induced apoptosis is controlled by caspase activation, Huh7 cells were pre-incubated with pan-caspase inhibitor z-VAD-FMK (50 µM) for 1 h, followed by various stimuli as indicated. DNA ladder assays (Fig. 2A), DNA fragmentation analysis (Fig. 2B and 2C) and FACS analysis (Fig. 2D and 2E) demonstrated that apoptosis induced by combination treatment with Lexa and CHX were totally blocked by pan-caspase inhibitor z-VAD-FMK, suggesting that CHX-sensitized and Lexa-induced apoptosis is caspase dependent. Meanwhile, we measured the effects of caspase 8 specific inhibitor z-IETD-FMK on apoptosis induced by Lexa and CHX combination treatment. As shown in Fig. 2A, 2B, 2C, 2D, and 2E, blockade of caspase 8 activation completely abolished Lexa and CHX combination-induced apoptosis in Huh7 cells. Taken together, these results suggest that caspase activation plays a key role in Lexa-mediated apoptosis induction in HCC cells.

Bottom Line: ROS generation induced by combination treatment of Lexa and CHX triggered pro-apoptotic protein Bax oligomerization, conformation change, and translocation to mitochondria, which resulted in the release of cytochrome c and subsequent cell death.More importantly, we observed that combination treatment of Lexa and CHX did not cause apoptotic toxicity in normal human primary hepatocytes.These results suggest that Lexa and CHX combination treatment merits investigation for the development of therapies for patients with HCC.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Florida College of Medicine, Gainesville, Florida, United States of America.

ABSTRACT
This study aims to investigate apoptosis induced by lexatumumab (Lexa) in hepatocellular carcinoma (HCC) cells. We assessed the sensitivity of HCC cell lines and normal human hepatocytes to Lexa and explored the sensitization of HCC cells to Lexa-induced apoptosis by cycloheximide (CHX). Our data indicated that CHX sensitized HCC cell lines to Lexa-induced apoptosis, whereas treatment using solely CHX or Lexa was ineffective. The sequential treatment of CHX followed by Lexa dramatically induced caspase-dependent apoptosis in HCC cells and had synergistically increased intracellular rates of reactive oxygen species (ROS). Additionally, when ROS production was blocked by N-acetyl-L-cysteine (NAC), HCC cells were protected against Lexa and CHX combination treatment-induced apoptosis. ROS generation induced by combination treatment of Lexa and CHX triggered pro-apoptotic protein Bax oligomerization, conformation change, and translocation to mitochondria, which resulted in the release of cytochrome c and subsequent cell death. Furthermore, HSP90 was involved in mediating Lexa and CHX combination treatment-induced ROS increase and apoptotic death. More importantly, we observed that combination treatment of Lexa and CHX did not cause apoptotic toxicity in normal human primary hepatocytes. These results suggest that Lexa and CHX combination treatment merits investigation for the development of therapies for patients with HCC.

Show MeSH
Related in: MedlinePlus