Limits...
Role of oxidative stress and intracellular glutathione in the sensitivity to apoptosis induced by proteasome inhibitor in thyroid cancer cells.

Du ZX, Zhang HY, Meng X, Guan Y, Wang HQ - BMC Cancer (2009)

Bottom Line: At concentrations that effectively inhibited proteasome activity, bortezomib induced apoptosis in FRO cells, but not in ARO cells.Furthermore, depletion of GSH increases apoptosis induced by bortezomib, in contrast, repletion of GSH decreases bortezomib-mediated cell death.GSH protects cells from proteasome inhibition-induced oxidative stress and glutathione-dependent redox system might play an important role in the sensitivity to proteasome inhibition-induced apoptosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, China Medical University, Shenyang, PR China. dzx_doctor@hotmail.com

ABSTRACT

Background: The proteasome inhibitor bortezomib has shown impressive clinical activity alone and in combination with conventional and other novel agents for the treatment of multiple myeloma (MM) and some solid cancers. Although bortezomib is known to be a selective proteasome inhibitor, the downstream mechanisms of cytotoxicity and drug resistance are poorly understood.

Methods: Proteasome activity, intracellular glutathione (GSH) and ROS levels, as well as activities of GSH synthesis enzymes were measured using spectrophotometric methods. Cell death was analyzed using flow cytometry and caspase activity assay. The expression level of GSH synthesis enzymes were measured using real-time RT-PCR.

Results: At concentrations that effectively inhibited proteasome activity, bortezomib induced apoptosis in FRO cells, but not in ARO cells. Bortezomib elevated the amount of glutathione (GSH) and the treatment with bortezomib increased the level of mRNA for GCL, a rate-limiting enzyme in glutathione synthesis. Furthermore, depletion of GSH increases apoptosis induced by bortezomib, in contrast, repletion of GSH decreases bortezomib-mediated cell death.

Conclusion: GSH protects cells from proteasome inhibition-induced oxidative stress and glutathione-dependent redox system might play an important role in the sensitivity to proteasome inhibition-induced apoptosis.

Show MeSH

Related in: MedlinePlus

Effects of antioxidants on the bortezomib-induced cell death. Cells were treated with 50 nM bortezomib together with or without tiron for 24 hours, and production of ROS (A) and apoptotic cell (B) were analyzed. C, cells were pretreated with vehicle, GSH or NAC, sequentially treated with 50 nM bortezomib for additional 24 hours, and then intracellular GSH levels were measured. D, cells were treated as in C, and production of ROS was investigated. E, cells were treated as in C, and apoptotic cells were analyzed. *, P< 0.05; ** P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2666756&req=5

Figure 4: Effects of antioxidants on the bortezomib-induced cell death. Cells were treated with 50 nM bortezomib together with or without tiron for 24 hours, and production of ROS (A) and apoptotic cell (B) were analyzed. C, cells were pretreated with vehicle, GSH or NAC, sequentially treated with 50 nM bortezomib for additional 24 hours, and then intracellular GSH levels were measured. D, cells were treated as in C, and production of ROS was investigated. E, cells were treated as in C, and apoptotic cells were analyzed. *, P< 0.05; ** P < 0.001.

Mentions: Because bortezomib treatment led to the enhancement of ROS generation prior to obvious apoptotic effects, it is possible that alterations in the cellular superoxidant state could play a role in the sensitivity to bortezomib-induced apoptosis. To examine whether elevated ROS is crucial for bortezomib-induced apoptosis in thyroid cancer cells, the cell-permeable superoxide scavenger tiron was utilized to perturb the bortezomib-induced ROS generation and then to examine the consequent events. Cotreatment with 1 mM tiron for 24 hours resulted in the complete abrogation of bortezomib-induced generation of ROS (Figure 4A). Abolishment of intracellular superoxide by tiron provided significant protection against apoptosis caused by bortezomib (Figure 4B). To clarify the contribution of intracellular glutathione to ROS suppression, we examined the effect of pretreatment with GSH, or N-acetyl-L-cysteine (NAC), which is a precursor of glutathione and elevates glutathione contents. Glutathione content was significantly elevated by treatment with 10 mM GSH for 4 hours or 10 mM NAC for 24 hours (Figure 4C). Pretreatment with GSH or NAC abrogated the decrease in intracellular GSH level mediated by bortezomib in FRO and KTC2 cells (Figure 4C). Bortezomib alone significantly increased the intracellular GSH in ARO and 8305C cells, whereas GSH or NAC pretreatment had little effect (Figure 4C). Pretreatment with GSH or NAC significantly suppressed bortezomib-induced ROS generation (Figure 4D) and provided significant protection against apoptosis caused by bortezomib in FRO and KTC2 cells (Figure 4E). Minor apoptotic cells were observed in ARO and 8305C cells, and GSH or NAC pretreatment did not provide further protection (Figure 4E).


Role of oxidative stress and intracellular glutathione in the sensitivity to apoptosis induced by proteasome inhibitor in thyroid cancer cells.

Du ZX, Zhang HY, Meng X, Guan Y, Wang HQ - BMC Cancer (2009)

Effects of antioxidants on the bortezomib-induced cell death. Cells were treated with 50 nM bortezomib together with or without tiron for 24 hours, and production of ROS (A) and apoptotic cell (B) were analyzed. C, cells were pretreated with vehicle, GSH or NAC, sequentially treated with 50 nM bortezomib for additional 24 hours, and then intracellular GSH levels were measured. D, cells were treated as in C, and production of ROS was investigated. E, cells were treated as in C, and apoptotic cells were analyzed. *, P< 0.05; ** P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2666756&req=5

Figure 4: Effects of antioxidants on the bortezomib-induced cell death. Cells were treated with 50 nM bortezomib together with or without tiron for 24 hours, and production of ROS (A) and apoptotic cell (B) were analyzed. C, cells were pretreated with vehicle, GSH or NAC, sequentially treated with 50 nM bortezomib for additional 24 hours, and then intracellular GSH levels were measured. D, cells were treated as in C, and production of ROS was investigated. E, cells were treated as in C, and apoptotic cells were analyzed. *, P< 0.05; ** P < 0.001.
Mentions: Because bortezomib treatment led to the enhancement of ROS generation prior to obvious apoptotic effects, it is possible that alterations in the cellular superoxidant state could play a role in the sensitivity to bortezomib-induced apoptosis. To examine whether elevated ROS is crucial for bortezomib-induced apoptosis in thyroid cancer cells, the cell-permeable superoxide scavenger tiron was utilized to perturb the bortezomib-induced ROS generation and then to examine the consequent events. Cotreatment with 1 mM tiron for 24 hours resulted in the complete abrogation of bortezomib-induced generation of ROS (Figure 4A). Abolishment of intracellular superoxide by tiron provided significant protection against apoptosis caused by bortezomib (Figure 4B). To clarify the contribution of intracellular glutathione to ROS suppression, we examined the effect of pretreatment with GSH, or N-acetyl-L-cysteine (NAC), which is a precursor of glutathione and elevates glutathione contents. Glutathione content was significantly elevated by treatment with 10 mM GSH for 4 hours or 10 mM NAC for 24 hours (Figure 4C). Pretreatment with GSH or NAC abrogated the decrease in intracellular GSH level mediated by bortezomib in FRO and KTC2 cells (Figure 4C). Bortezomib alone significantly increased the intracellular GSH in ARO and 8305C cells, whereas GSH or NAC pretreatment had little effect (Figure 4C). Pretreatment with GSH or NAC significantly suppressed bortezomib-induced ROS generation (Figure 4D) and provided significant protection against apoptosis caused by bortezomib in FRO and KTC2 cells (Figure 4E). Minor apoptotic cells were observed in ARO and 8305C cells, and GSH or NAC pretreatment did not provide further protection (Figure 4E).

Bottom Line: At concentrations that effectively inhibited proteasome activity, bortezomib induced apoptosis in FRO cells, but not in ARO cells.Furthermore, depletion of GSH increases apoptosis induced by bortezomib, in contrast, repletion of GSH decreases bortezomib-mediated cell death.GSH protects cells from proteasome inhibition-induced oxidative stress and glutathione-dependent redox system might play an important role in the sensitivity to proteasome inhibition-induced apoptosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, China Medical University, Shenyang, PR China. dzx_doctor@hotmail.com

ABSTRACT

Background: The proteasome inhibitor bortezomib has shown impressive clinical activity alone and in combination with conventional and other novel agents for the treatment of multiple myeloma (MM) and some solid cancers. Although bortezomib is known to be a selective proteasome inhibitor, the downstream mechanisms of cytotoxicity and drug resistance are poorly understood.

Methods: Proteasome activity, intracellular glutathione (GSH) and ROS levels, as well as activities of GSH synthesis enzymes were measured using spectrophotometric methods. Cell death was analyzed using flow cytometry and caspase activity assay. The expression level of GSH synthesis enzymes were measured using real-time RT-PCR.

Results: At concentrations that effectively inhibited proteasome activity, bortezomib induced apoptosis in FRO cells, but not in ARO cells. Bortezomib elevated the amount of glutathione (GSH) and the treatment with bortezomib increased the level of mRNA for GCL, a rate-limiting enzyme in glutathione synthesis. Furthermore, depletion of GSH increases apoptosis induced by bortezomib, in contrast, repletion of GSH decreases bortezomib-mediated cell death.

Conclusion: GSH protects cells from proteasome inhibition-induced oxidative stress and glutathione-dependent redox system might play an important role in the sensitivity to proteasome inhibition-induced apoptosis.

Show MeSH
Related in: MedlinePlus