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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.

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Related in: MedlinePlus

Alteration of glutathione upon bortezomib exposure. Cells were treated with 50 nM bortezomib for the indicated time, intracellular GSH (A) and GSSG (B) levels were measured. ** P < 0.001 vs 0 hour treatment.
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Figure 2: Alteration of glutathione upon bortezomib exposure. Cells were treated with 50 nM bortezomib for the indicated time, intracellular GSH (A) and GSSG (B) levels were measured. ** P < 0.001 vs 0 hour treatment.

Mentions: Distinct ROS generation patterns prompted us to compare the intracellular levels of the reducing agent GSH, the most abundant intracellular antioxidant, in thyroid cancer cells. Without bortezomib treatment, there were no significant differences in GSH levels among the panel of thyroid cancer cells (30–40 nmol/mg protein). With bortezomib treatment, no obvious alterations in GSH levels were observed in FRO and KTC2 cells at 4–8 hours, a significant fall was demonstrated starting at 12 hours after exposure (Figure 2A). ARO and 8305C cells had levels of GSH that were even higher than vehicle-treated controls at 8 hours after exposure. The amount of reduced GSH was further increased at 12–16 hours, and peaked at about 24 hours after exposure (Figure 2A). Since the increase in reduced GSH could be derived from reduction of oxidized glutathione (GSSG) by glutathione reductase or from de novo synthesis [23], we investigated the amount of GSSG upon exposure to proteasome. No marked alteration in GSSG amount was observed in FRO and KTC2 cells within 24 hours (Figure 2B). In ARO and 8305C cells, the amount of GSSG was even elevated at 16–24 hours of bortezomib exposure and restored to the basal level after 36 hours (Figure 2B). As the amount of GSSG was not decreased, but even elevated at some time points in ARO and 8305C cells, it is suggested that reduction of GSSG was not responsible for GSH elevation in these cells.


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)

Alteration of glutathione upon bortezomib exposure. Cells were treated with 50 nM bortezomib for the indicated time, intracellular GSH (A) and GSSG (B) levels were measured. ** P < 0.001 vs 0 hour treatment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Alteration of glutathione upon bortezomib exposure. Cells were treated with 50 nM bortezomib for the indicated time, intracellular GSH (A) and GSSG (B) levels were measured. ** P < 0.001 vs 0 hour treatment.
Mentions: Distinct ROS generation patterns prompted us to compare the intracellular levels of the reducing agent GSH, the most abundant intracellular antioxidant, in thyroid cancer cells. Without bortezomib treatment, there were no significant differences in GSH levels among the panel of thyroid cancer cells (30–40 nmol/mg protein). With bortezomib treatment, no obvious alterations in GSH levels were observed in FRO and KTC2 cells at 4–8 hours, a significant fall was demonstrated starting at 12 hours after exposure (Figure 2A). ARO and 8305C cells had levels of GSH that were even higher than vehicle-treated controls at 8 hours after exposure. The amount of reduced GSH was further increased at 12–16 hours, and peaked at about 24 hours after exposure (Figure 2A). Since the increase in reduced GSH could be derived from reduction of oxidized glutathione (GSSG) by glutathione reductase or from de novo synthesis [23], we investigated the amount of GSSG upon exposure to proteasome. No marked alteration in GSSG amount was observed in FRO and KTC2 cells within 24 hours (Figure 2B). In ARO and 8305C cells, the amount of GSSG was even elevated at 16–24 hours of bortezomib exposure and restored to the basal level after 36 hours (Figure 2B). As the amount of GSSG was not decreased, but even elevated at some time points in ARO and 8305C cells, it is suggested that reduction of GSSG was not responsible for GSH elevation in these cells.

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