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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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Related in: MedlinePlus

Protein expression in HCC cells treated with Lexa or the combination of Lexa and CHX.LH86 cells were treated with Lexa (1 µg/ml) or pre-treated with CHX (10 µg/ml) followed by Lexa (1 µg/ml) for up to 6 h. Cells were harvested and cell lysates were prepared for Western blotting. Bcl-xL, survivin, Bim, Bad, Bak, Bax, DR4, and DR5 were detected with specific antibodies respectively. β-actin was detected with anti-β-actin mouse antibody for an equal protein loading control.
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pone-0016966-g004: Protein expression in HCC cells treated with Lexa or the combination of Lexa and CHX.LH86 cells were treated with Lexa (1 µg/ml) or pre-treated with CHX (10 µg/ml) followed by Lexa (1 µg/ml) for up to 6 h. Cells were harvested and cell lysates were prepared for Western blotting. Bcl-xL, survivin, Bim, Bad, Bak, Bax, DR4, and DR5 were detected with specific antibodies respectively. β-actin was detected with anti-β-actin mouse antibody for an equal protein loading control.

Mentions: Since CHX is a protein synthesis inhibitor, it may sensitize Lexa to induce apoptosis through down-regulating anti-apoptotic proteins. We analyzed the apoptosis associated protein expression changes in HCC cells treated with Lexa alone or combination-treated with Lexa and CHX for up to 6 h. Western blotting results showed that there were no decreases in anti-apoptotic molecules Bcl-xL or survivin expression; of the pro-apoptotic proteins, both Bad and Bim were down-regulated, while Bak was down-regulated with Lexa single treatment and up-regulated with combination treatment. Additionally, protein levels of pro-apoptotic Bax were not affected, and the expression levels of death receptors DR4 and DR5 showed no significant changes (Fig. 4). These results suggest that the inhibitor CHX could not completely block protein synthesis stimulated by Lexa in HCC cells and that apoptosis induced by combination treatment of Lexa and CHX may be mediated by Bax/Bak activation.


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)

Protein expression in HCC cells treated with Lexa or the combination of Lexa and CHX.LH86 cells were treated with Lexa (1 µg/ml) or pre-treated with CHX (10 µg/ml) followed by Lexa (1 µg/ml) for up to 6 h. Cells were harvested and cell lysates were prepared for Western blotting. Bcl-xL, survivin, Bim, Bad, Bak, Bax, DR4, and DR5 were detected with specific antibodies respectively. β-actin was detected with anti-β-actin mouse antibody for an equal protein loading control.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0016966-g004: Protein expression in HCC cells treated with Lexa or the combination of Lexa and CHX.LH86 cells were treated with Lexa (1 µg/ml) or pre-treated with CHX (10 µg/ml) followed by Lexa (1 µg/ml) for up to 6 h. Cells were harvested and cell lysates were prepared for Western blotting. Bcl-xL, survivin, Bim, Bad, Bak, Bax, DR4, and DR5 were detected with specific antibodies respectively. β-actin was detected with anti-β-actin mouse antibody for an equal protein loading control.
Mentions: Since CHX is a protein synthesis inhibitor, it may sensitize Lexa to induce apoptosis through down-regulating anti-apoptotic proteins. We analyzed the apoptosis associated protein expression changes in HCC cells treated with Lexa alone or combination-treated with Lexa and CHX for up to 6 h. Western blotting results showed that there were no decreases in anti-apoptotic molecules Bcl-xL or survivin expression; of the pro-apoptotic proteins, both Bad and Bim were down-regulated, while Bak was down-regulated with Lexa single treatment and up-regulated with combination treatment. Additionally, protein levels of pro-apoptotic Bax were not affected, and the expression levels of death receptors DR4 and DR5 showed no significant changes (Fig. 4). These results suggest that the inhibitor CHX could not completely block protein synthesis stimulated by Lexa in HCC cells and that apoptosis induced by combination treatment of Lexa and CHX may be mediated by Bax/Bak activation.

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