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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

Lexa and CHX combination treatment induces apoptosis in HCC cells.A, Huh7 cells were treated with TNF-α (10 ng/ml) or Lexa (1 µg/ml) for indicated times. Cell lysates were prepared and subjected to Western blotting to detect the expression of IκB-α and phospho-IκB-α. β-actin protein levels were used as an equal protein loading control (Lexa, lexatumumab). B, Huh7 cells were treated with DMSO (Con), CHX (10 µg/ml), Lexa (1 µg/ml), or a combination of Lexa (1 µg/ml) and CHX (10 µg/ml). Apoptosis was measured by nuclear dye Hoechst 33258 staining to label DNA fragmentation (nuclear morphology changes). (Apoptotic cells were marked with arrows). C, Huh7 cells were treated with DMSO (Con), CHX (10 µg/ml), Lexa (1 µg/ml), or combination treatment of Lexa (1 µg/ml) and CHX (10 µg/ml) and apoptosis was evaluated through Annexin V and PI double staining based FACS analysis using the Annexin-V assay kit (see 'materials and methods'). D, The percentage of apoptotic cells were characterized as those that stained with Annexin-V. Data represent the mean values of three independent experiments (*p<0.05). E, LH86 cells were treated with DMSO (Con), CHX (10 µg/ml), Lexa (1 µg/ml), or a combination of Lexa (1 µg/ml) and CHX (10 µg/ml). Apoptosis was measured as in Fig. 1B (Apoptotic cells were marked with arrows). F, LH86 cells were treated with various stimuli as in Fig. 1E. Apoptosis was measured through FACS analysis as in Fig. 1C and 1D and statistical analysis was performed to show percentage of Annexin-V positive cells for apoptosis ratio. Data represent the mean values of three independent experiments (**p<0.05). G, Huh7 cells were treated with Lexa (1 µg/ml) or combination of Lexa (1 µg/ml) and CHX (10 µg/ml) for up to 6 h and harvested. Cell lysates were prepared for Western blotting to detect the cleavages of caspase 8, caspase 10, caspase 3, and Bid. β-actin protein levels were set up as loading control for equal total protein amounts. H, Huh7 cells treated with indicated conditions for 6 h were harvested and cell lysates were prepared and subjected to Western blotting analysis. Caspase 3 activation was evaluated through detecting cleaved bands with specific antibody. β-actin protein levels were set up as loading control for equal total protein amounts. Each experiment was conducted in triplicate or duplicate and repeated twice independently.
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pone-0016966-g001: Lexa and CHX combination treatment induces apoptosis in HCC cells.A, Huh7 cells were treated with TNF-α (10 ng/ml) or Lexa (1 µg/ml) for indicated times. Cell lysates were prepared and subjected to Western blotting to detect the expression of IκB-α and phospho-IκB-α. β-actin protein levels were used as an equal protein loading control (Lexa, lexatumumab). B, Huh7 cells were treated with DMSO (Con), CHX (10 µg/ml), Lexa (1 µg/ml), or a combination of Lexa (1 µg/ml) and CHX (10 µg/ml). Apoptosis was measured by nuclear dye Hoechst 33258 staining to label DNA fragmentation (nuclear morphology changes). (Apoptotic cells were marked with arrows). C, Huh7 cells were treated with DMSO (Con), CHX (10 µg/ml), Lexa (1 µg/ml), or combination treatment of Lexa (1 µg/ml) and CHX (10 µg/ml) and apoptosis was evaluated through Annexin V and PI double staining based FACS analysis using the Annexin-V assay kit (see 'materials and methods'). D, The percentage of apoptotic cells were characterized as those that stained with Annexin-V. Data represent the mean values of three independent experiments (*p<0.05). E, LH86 cells were treated with DMSO (Con), CHX (10 µg/ml), Lexa (1 µg/ml), or a combination of Lexa (1 µg/ml) and CHX (10 µg/ml). Apoptosis was measured as in Fig. 1B (Apoptotic cells were marked with arrows). F, LH86 cells were treated with various stimuli as in Fig. 1E. Apoptosis was measured through FACS analysis as in Fig. 1C and 1D and statistical analysis was performed to show percentage of Annexin-V positive cells for apoptosis ratio. Data represent the mean values of three independent experiments (**p<0.05). G, Huh7 cells were treated with Lexa (1 µg/ml) or combination of Lexa (1 µg/ml) and CHX (10 µg/ml) for up to 6 h and harvested. Cell lysates were prepared for Western blotting to detect the cleavages of caspase 8, caspase 10, caspase 3, and Bid. β-actin protein levels were set up as loading control for equal total protein amounts. H, Huh7 cells treated with indicated conditions for 6 h were harvested and cell lysates were prepared and subjected to Western blotting analysis. Caspase 3 activation was evaluated through detecting cleaved bands with specific antibody. β-actin protein levels were set up as loading control for equal total protein amounts. Each experiment was conducted in triplicate or duplicate and repeated twice independently.

Mentions: Increasing evidence indicated that many cancer types including breast cancer cells, lung cancer cells, and hepatoma cells, could develop resistance to TRAIL-induced apoptosis through DR5-mediated activation of NF-κB signal pathway [24]–[26]. Here, to investigate whether Lexa triggers NF-κB activation in HCC cell lines, Huh7 cells were treated with TNF-α or Lexa for the indicated times. Western blotting results indicated that TNF-α induced Iκ-B-α decrease after treatment for 15 min and Lexa did that with treatment for 60 min. However, compared with TNF-α, Lexa could not promote Iκ-B-α phosphorylation at 60 min (Fig. 1A); thus Lexa, a DR5 specific monoclonal antibody unlike multifunctional TNF-α, did not activate the anti-apoptotic NF-κB signal pathway through binding to DR5. Then, we tested whether HCC cells are sensitive to Lexa-induced cell death. Huh7 and LH86 cells were treated with Lexa (1 µg/ml) for up to 6 h. Apoptosis assays indicated that both HCC cell lines were resistant to Lexa (see Lexa-treated groups in Fig. 1B, 1C, and 1D for Huh7 cells, and Fig. 1E and 1F for LH86 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 apoptosis in HCC cells.A, Huh7 cells were treated with TNF-α (10 ng/ml) or Lexa (1 µg/ml) for indicated times. Cell lysates were prepared and subjected to Western blotting to detect the expression of IκB-α and phospho-IκB-α. β-actin protein levels were used as an equal protein loading control (Lexa, lexatumumab). B, Huh7 cells were treated with DMSO (Con), CHX (10 µg/ml), Lexa (1 µg/ml), or a combination of Lexa (1 µg/ml) and CHX (10 µg/ml). Apoptosis was measured by nuclear dye Hoechst 33258 staining to label DNA fragmentation (nuclear morphology changes). (Apoptotic cells were marked with arrows). C, Huh7 cells were treated with DMSO (Con), CHX (10 µg/ml), Lexa (1 µg/ml), or combination treatment of Lexa (1 µg/ml) and CHX (10 µg/ml) and apoptosis was evaluated through Annexin V and PI double staining based FACS analysis using the Annexin-V assay kit (see 'materials and methods'). D, The percentage of apoptotic cells were characterized as those that stained with Annexin-V. Data represent the mean values of three independent experiments (*p<0.05). E, LH86 cells were treated with DMSO (Con), CHX (10 µg/ml), Lexa (1 µg/ml), or a combination of Lexa (1 µg/ml) and CHX (10 µg/ml). Apoptosis was measured as in Fig. 1B (Apoptotic cells were marked with arrows). F, LH86 cells were treated with various stimuli as in Fig. 1E. Apoptosis was measured through FACS analysis as in Fig. 1C and 1D and statistical analysis was performed to show percentage of Annexin-V positive cells for apoptosis ratio. Data represent the mean values of three independent experiments (**p<0.05). G, Huh7 cells were treated with Lexa (1 µg/ml) or combination of Lexa (1 µg/ml) and CHX (10 µg/ml) for up to 6 h and harvested. Cell lysates were prepared for Western blotting to detect the cleavages of caspase 8, caspase 10, caspase 3, and Bid. β-actin protein levels were set up as loading control for equal total protein amounts. H, Huh7 cells treated with indicated conditions for 6 h were harvested and cell lysates were prepared and subjected to Western blotting analysis. Caspase 3 activation was evaluated through detecting cleaved bands with specific antibody. β-actin protein levels were set up as loading control for equal total protein amounts. Each experiment was conducted in triplicate or duplicate and repeated twice independently.
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Related In: Results  -  Collection

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

pone-0016966-g001: Lexa and CHX combination treatment induces apoptosis in HCC cells.A, Huh7 cells were treated with TNF-α (10 ng/ml) or Lexa (1 µg/ml) for indicated times. Cell lysates were prepared and subjected to Western blotting to detect the expression of IκB-α and phospho-IκB-α. β-actin protein levels were used as an equal protein loading control (Lexa, lexatumumab). B, Huh7 cells were treated with DMSO (Con), CHX (10 µg/ml), Lexa (1 µg/ml), or a combination of Lexa (1 µg/ml) and CHX (10 µg/ml). Apoptosis was measured by nuclear dye Hoechst 33258 staining to label DNA fragmentation (nuclear morphology changes). (Apoptotic cells were marked with arrows). C, Huh7 cells were treated with DMSO (Con), CHX (10 µg/ml), Lexa (1 µg/ml), or combination treatment of Lexa (1 µg/ml) and CHX (10 µg/ml) and apoptosis was evaluated through Annexin V and PI double staining based FACS analysis using the Annexin-V assay kit (see 'materials and methods'). D, The percentage of apoptotic cells were characterized as those that stained with Annexin-V. Data represent the mean values of three independent experiments (*p<0.05). E, LH86 cells were treated with DMSO (Con), CHX (10 µg/ml), Lexa (1 µg/ml), or a combination of Lexa (1 µg/ml) and CHX (10 µg/ml). Apoptosis was measured as in Fig. 1B (Apoptotic cells were marked with arrows). F, LH86 cells were treated with various stimuli as in Fig. 1E. Apoptosis was measured through FACS analysis as in Fig. 1C and 1D and statistical analysis was performed to show percentage of Annexin-V positive cells for apoptosis ratio. Data represent the mean values of three independent experiments (**p<0.05). G, Huh7 cells were treated with Lexa (1 µg/ml) or combination of Lexa (1 µg/ml) and CHX (10 µg/ml) for up to 6 h and harvested. Cell lysates were prepared for Western blotting to detect the cleavages of caspase 8, caspase 10, caspase 3, and Bid. β-actin protein levels were set up as loading control for equal total protein amounts. H, Huh7 cells treated with indicated conditions for 6 h were harvested and cell lysates were prepared and subjected to Western blotting analysis. Caspase 3 activation was evaluated through detecting cleaved bands with specific antibody. β-actin protein levels were set up as loading control for equal total protein amounts. Each experiment was conducted in triplicate or duplicate and repeated twice independently.
Mentions: Increasing evidence indicated that many cancer types including breast cancer cells, lung cancer cells, and hepatoma cells, could develop resistance to TRAIL-induced apoptosis through DR5-mediated activation of NF-κB signal pathway [24]–[26]. Here, to investigate whether Lexa triggers NF-κB activation in HCC cell lines, Huh7 cells were treated with TNF-α or Lexa for the indicated times. Western blotting results indicated that TNF-α induced Iκ-B-α decrease after treatment for 15 min and Lexa did that with treatment for 60 min. However, compared with TNF-α, Lexa could not promote Iκ-B-α phosphorylation at 60 min (Fig. 1A); thus Lexa, a DR5 specific monoclonal antibody unlike multifunctional TNF-α, did not activate the anti-apoptotic NF-κB signal pathway through binding to DR5. Then, we tested whether HCC cells are sensitive to Lexa-induced cell death. Huh7 and LH86 cells were treated with Lexa (1 µg/ml) for up to 6 h. Apoptosis assays indicated that both HCC cell lines were resistant to Lexa (see Lexa-treated groups in Fig. 1B, 1C, and 1D for Huh7 cells, and Fig. 1E and 1F for LH86 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