Limits...
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.

Show MeSH

Related in: MedlinePlus

Lexa and CHX combination treatment induces apoptosis via ROS.A, Huh7 cells were treated with the combination of Lexa (1 µg/ml) and CHX (10 µg/ml). Nuclei were stained by Hoechst 33258 (right panel-blue, thin arrow marks the nucleus in non-apoptotic cells; thick arrow marks apoptotic condensed nuclei). Intracellular ROS level was assessed by DHED-based fluorescent staining (middle panel-red, thin arrow marks ROS in non-apoptotic cells, thick arrow marks ROS in apoptotic cells), and merged figure (left) to show that ROS levels in non-apoptotic cells are lower than that in apoptotic cells (white boxes marked areas were enlarged (Zoom in)). B, Huh7 cells were treated with DMSO (Con), Lexa (1 µg/ml), CHX (10 µg/ml), NAC (10 mM), a combination of Lexa (1 µg/ml) and CHX (10 µg/ml) in the absence or presence of NAC (10 mM). Intracellular ROS level was measured with DHED dye (red fluorescence). C, Huh7 cells were treated with various stimuli as indicated and apoptosis was assessed by DNA ladder. D, Huh7 cells treated with DMSO (Con), Lexa (1 µg/ml), CHX (10 µg/ml), NAC (10 mM), the combination of Lexa (1 µg/ml) and CHX (10 µg/ml) in the absence or the presence of NAC (10 µM) for 6 h. Then cells were stained with Hoechst 33258 to test apoptotic cell death (representative apoptotic cells are marked with arrows). E and F, Huh7 cells were treated with conditions as indicated. Apoptosis was evaluated as in Fig. 1C and 1D. Data represent the mean values of three independent experiments (**p<0.05).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3037406&req=5

pone-0016966-g003: Lexa and CHX combination treatment induces apoptosis via ROS.A, Huh7 cells were treated with the combination of Lexa (1 µg/ml) and CHX (10 µg/ml). Nuclei were stained by Hoechst 33258 (right panel-blue, thin arrow marks the nucleus in non-apoptotic cells; thick arrow marks apoptotic condensed nuclei). Intracellular ROS level was assessed by DHED-based fluorescent staining (middle panel-red, thin arrow marks ROS in non-apoptotic cells, thick arrow marks ROS in apoptotic cells), and merged figure (left) to show that ROS levels in non-apoptotic cells are lower than that in apoptotic cells (white boxes marked areas were enlarged (Zoom in)). B, Huh7 cells were treated with DMSO (Con), Lexa (1 µg/ml), CHX (10 µg/ml), NAC (10 mM), a combination of Lexa (1 µg/ml) and CHX (10 µg/ml) in the absence or presence of NAC (10 mM). Intracellular ROS level was measured with DHED dye (red fluorescence). C, Huh7 cells were treated with various stimuli as indicated and apoptosis was assessed by DNA ladder. D, Huh7 cells treated with DMSO (Con), Lexa (1 µg/ml), CHX (10 µg/ml), NAC (10 mM), the combination of Lexa (1 µg/ml) and CHX (10 µg/ml) in the absence or the presence of NAC (10 µM) for 6 h. Then cells were stained with Hoechst 33258 to test apoptotic cell death (representative apoptotic cells are marked with arrows). E and F, Huh7 cells were treated with conditions as indicated. Apoptosis was evaluated as in Fig. 1C and 1D. Data represent the mean values of three independent experiments (**p<0.05).

Mentions: To assess whether ROS plays a major role in regulating Lexa and CHX combination-induced apoptosis, HCC cells were treated with Lexa and CHX for 6 h. Intracellular ROS levels were determined by a DHED-based fluorescence assay. As shown in Fig. 3A, Lexa and CHX combination treatment induced ROS dramatic increase in apoptotic cells, but not in non-apoptotic cells, suggesting ROS may contribute to CHX-and Lexa-induced apoptosis. To further determine whether generation of ROS can regulate apoptosis, N-acetyl-L-cysteine (NAC), a precursor of reduced glutathione (GSH) widely used as a thiol-containing antioxidant to scavenge intracellular ROS, was added before administration of Lexa and CHX. As shown in Fig. 3B, the ROS increase induced by combined treatment of Lexa and CHX was completely negated. More importantly, apoptosis assays indicated that the blockade of ROS by NAC can also protect cells against Lexa-and CHX-induced apoptosis (Fig. 3C, 3D, 3E, and 3F). These results suggest that Lexa and CHX combination treatment-induced ROS increase can trigger apoptosis 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 apoptosis via ROS.A, Huh7 cells were treated with the combination of Lexa (1 µg/ml) and CHX (10 µg/ml). Nuclei were stained by Hoechst 33258 (right panel-blue, thin arrow marks the nucleus in non-apoptotic cells; thick arrow marks apoptotic condensed nuclei). Intracellular ROS level was assessed by DHED-based fluorescent staining (middle panel-red, thin arrow marks ROS in non-apoptotic cells, thick arrow marks ROS in apoptotic cells), and merged figure (left) to show that ROS levels in non-apoptotic cells are lower than that in apoptotic cells (white boxes marked areas were enlarged (Zoom in)). B, Huh7 cells were treated with DMSO (Con), Lexa (1 µg/ml), CHX (10 µg/ml), NAC (10 mM), a combination of Lexa (1 µg/ml) and CHX (10 µg/ml) in the absence or presence of NAC (10 mM). Intracellular ROS level was measured with DHED dye (red fluorescence). C, Huh7 cells were treated with various stimuli as indicated and apoptosis was assessed by DNA ladder. D, Huh7 cells treated with DMSO (Con), Lexa (1 µg/ml), CHX (10 µg/ml), NAC (10 mM), the combination of Lexa (1 µg/ml) and CHX (10 µg/ml) in the absence or the presence of NAC (10 µM) for 6 h. Then cells were stained with Hoechst 33258 to test apoptotic cell death (representative apoptotic cells are marked with arrows). E and F, Huh7 cells were treated with conditions as indicated. Apoptosis was evaluated as in Fig. 1C and 1D. Data represent the mean values of three independent experiments (**p<0.05).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0016966-g003: Lexa and CHX combination treatment induces apoptosis via ROS.A, Huh7 cells were treated with the combination of Lexa (1 µg/ml) and CHX (10 µg/ml). Nuclei were stained by Hoechst 33258 (right panel-blue, thin arrow marks the nucleus in non-apoptotic cells; thick arrow marks apoptotic condensed nuclei). Intracellular ROS level was assessed by DHED-based fluorescent staining (middle panel-red, thin arrow marks ROS in non-apoptotic cells, thick arrow marks ROS in apoptotic cells), and merged figure (left) to show that ROS levels in non-apoptotic cells are lower than that in apoptotic cells (white boxes marked areas were enlarged (Zoom in)). B, Huh7 cells were treated with DMSO (Con), Lexa (1 µg/ml), CHX (10 µg/ml), NAC (10 mM), a combination of Lexa (1 µg/ml) and CHX (10 µg/ml) in the absence or presence of NAC (10 mM). Intracellular ROS level was measured with DHED dye (red fluorescence). C, Huh7 cells were treated with various stimuli as indicated and apoptosis was assessed by DNA ladder. D, Huh7 cells treated with DMSO (Con), Lexa (1 µg/ml), CHX (10 µg/ml), NAC (10 mM), the combination of Lexa (1 µg/ml) and CHX (10 µg/ml) in the absence or the presence of NAC (10 µM) for 6 h. Then cells were stained with Hoechst 33258 to test apoptotic cell death (representative apoptotic cells are marked with arrows). E and F, Huh7 cells were treated with conditions as indicated. Apoptosis was evaluated as in Fig. 1C and 1D. Data represent the mean values of three independent experiments (**p<0.05).
Mentions: To assess whether ROS plays a major role in regulating Lexa and CHX combination-induced apoptosis, HCC cells were treated with Lexa and CHX for 6 h. Intracellular ROS levels were determined by a DHED-based fluorescence assay. As shown in Fig. 3A, Lexa and CHX combination treatment induced ROS dramatic increase in apoptotic cells, but not in non-apoptotic cells, suggesting ROS may contribute to CHX-and Lexa-induced apoptosis. To further determine whether generation of ROS can regulate apoptosis, N-acetyl-L-cysteine (NAC), a precursor of reduced glutathione (GSH) widely used as a thiol-containing antioxidant to scavenge intracellular ROS, was added before administration of Lexa and CHX. As shown in Fig. 3B, the ROS increase induced by combined treatment of Lexa and CHX was completely negated. More importantly, apoptosis assays indicated that the blockade of ROS by NAC can also protect cells against Lexa-and CHX-induced apoptosis (Fig. 3C, 3D, 3E, and 3F). These results suggest that Lexa and CHX combination treatment-induced ROS increase can trigger apoptosis 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