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Proteasome inhibition reverses hedgehog inhibitor and taxane resistance in ovarian cancer.

Steg AD, Burke MR, Amm HM, Katre AA, Dobbin ZC, Jeong DH, Landen CN - Oncotarget (2014)

Bottom Line: Expression of the multi-drug resistance gene ABCB1/MDR1 was decreased and acetylation of α-tubulin, a marker of microtubule stabilization, was increased following bortezomib treatment.HDAC6 inhibitor tubastatin-a demonstrated that microtubule effects are associated with hedgehog inhibition and sensitization to paclitaxel and LDE225.These results suggest that proteasome inhibition, through alteration of microtubule dynamics and hedgehog signaling, can reverse taxane-mediated chemoresistance.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, University of Alabama at Birmingham, Birmingham, AL. McWhorter School of Pharmacy, Samford University, Birmingham, AL. These authors contributed equally to this work.

ABSTRACT
The goal of this study was to determine whether combined targeted therapies, specifically those against the Notch, hedgehog and ubiquitin-proteasome pathways, could overcome ovarian cancer chemoresistance. Chemoresistant ovarian cancer cells were exposed to gamma-secretase inhibitors (GSI-I, Compound E) or the proteasome inhibitor bortezomib, alone and in combination with the hedgehog antagonist, LDE225. Bortezomib, alone and in combination with LDE225, was evaluated for effects on paclitaxel efficacy. Cell viability and cell cycle analysis were assessed by MTT assay and propidium iodide staining, respectively. Proteasome activity and gene expression were determined by luminescence assay and qPCR, respectively. Studies demonstrated that GSI-I, but not Compound E, inhibited proteasome activity, similar to bortezomib. Proteasome inhibition decreased hedgehog target genes (PTCH1, GLI1 and GLI2) and increased LDE225 sensitivity in vitro. Bortezomib, alone and in combination with LDE225, increased paclitaxel sensitivity through apoptosis and G2/M arrest. Expression of the multi-drug resistance gene ABCB1/MDR1 was decreased and acetylation of α-tubulin, a marker of microtubule stabilization, was increased following bortezomib treatment. HDAC6 inhibitor tubastatin-a demonstrated that microtubule effects are associated with hedgehog inhibition and sensitization to paclitaxel and LDE225. These results suggest that proteasome inhibition, through alteration of microtubule dynamics and hedgehog signaling, can reverse taxane-mediated chemoresistance.

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Modification of microtubule dynamics plays a role in the sensitization of paclitaxel and LDE225 by bortezomibA) Gene expression of ABCB1/MDR1 was examined in SKOV3TRip2 cells treated with DMSO or bortezomib at the indicated doses for 72 hours, using quantitative PCR. *P < 0.05, compared to DMSO vehicle control. B) Protein expression of acetylated α-tubulin was examined in A2780cp55 cells treated with DMSO, bortezomib, paclitaxel or LDE225 for 24 hours using Western blot analysis. -actin was used as a loading control. C) Gene expression of PTCH1, GLI1 and GLI2 was examined in A2780cp55 cells treated with DMSO, paclitaxel or tubastatin-a for 24 hours, using quantitative PCR. *P < 0.05, compared to DMSO vehicle control. D) A2780cp55 and E) SKOV3TRip2 cell viability following exposure to DMSO or tubastatin-a combined with increasing concentrations of paclitaxel. F) SKOV3TRip2 cell viability following treatment with DMSO or tubastatin-a combined with increasing concentrations of LDE225. Cell viability was determined using MTT assay. Data are representative of at least 3 independent experiments.
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Figure 5: Modification of microtubule dynamics plays a role in the sensitization of paclitaxel and LDE225 by bortezomibA) Gene expression of ABCB1/MDR1 was examined in SKOV3TRip2 cells treated with DMSO or bortezomib at the indicated doses for 72 hours, using quantitative PCR. *P < 0.05, compared to DMSO vehicle control. B) Protein expression of acetylated α-tubulin was examined in A2780cp55 cells treated with DMSO, bortezomib, paclitaxel or LDE225 for 24 hours using Western blot analysis. -actin was used as a loading control. C) Gene expression of PTCH1, GLI1 and GLI2 was examined in A2780cp55 cells treated with DMSO, paclitaxel or tubastatin-a for 24 hours, using quantitative PCR. *P < 0.05, compared to DMSO vehicle control. D) A2780cp55 and E) SKOV3TRip2 cell viability following exposure to DMSO or tubastatin-a combined with increasing concentrations of paclitaxel. F) SKOV3TRip2 cell viability following treatment with DMSO or tubastatin-a combined with increasing concentrations of LDE225. Cell viability was determined using MTT assay. Data are representative of at least 3 independent experiments.

Mentions: To determine how inhibition of hedgehog signaling by bortezomib could promote increased paclitaxel sensitivity in vitro, we first examined expression of a primary mediator of taxane resistance ABCB1/MDR1. This gene encodes for P-glycoprotein, a drug efflux pump that has been shown by our laboratory to play a role in the synergy between LDE225 and paclitaxel [16]. Gene expression of ABCB1/MDR1 was measured using qPCR after treatment with increasing concentrations of bortezomib for 72 hours. As shown in Figure 5A, bortezomib (10, 20, 30 nM) significantly (p<0.05) decreased ABCB1/MDR1 expression in a dose-dependent manner (by 25%, 73% and 83%, respectively), indicating that bortezomib increases paclitaxel sensitivity, at least in part, through inhibition of drug efflux.


Proteasome inhibition reverses hedgehog inhibitor and taxane resistance in ovarian cancer.

Steg AD, Burke MR, Amm HM, Katre AA, Dobbin ZC, Jeong DH, Landen CN - Oncotarget (2014)

Modification of microtubule dynamics plays a role in the sensitization of paclitaxel and LDE225 by bortezomibA) Gene expression of ABCB1/MDR1 was examined in SKOV3TRip2 cells treated with DMSO or bortezomib at the indicated doses for 72 hours, using quantitative PCR. *P < 0.05, compared to DMSO vehicle control. B) Protein expression of acetylated α-tubulin was examined in A2780cp55 cells treated with DMSO, bortezomib, paclitaxel or LDE225 for 24 hours using Western blot analysis. -actin was used as a loading control. C) Gene expression of PTCH1, GLI1 and GLI2 was examined in A2780cp55 cells treated with DMSO, paclitaxel or tubastatin-a for 24 hours, using quantitative PCR. *P < 0.05, compared to DMSO vehicle control. D) A2780cp55 and E) SKOV3TRip2 cell viability following exposure to DMSO or tubastatin-a combined with increasing concentrations of paclitaxel. F) SKOV3TRip2 cell viability following treatment with DMSO or tubastatin-a combined with increasing concentrations of LDE225. Cell viability was determined using MTT assay. Data are representative of at least 3 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4196184&req=5

Figure 5: Modification of microtubule dynamics plays a role in the sensitization of paclitaxel and LDE225 by bortezomibA) Gene expression of ABCB1/MDR1 was examined in SKOV3TRip2 cells treated with DMSO or bortezomib at the indicated doses for 72 hours, using quantitative PCR. *P < 0.05, compared to DMSO vehicle control. B) Protein expression of acetylated α-tubulin was examined in A2780cp55 cells treated with DMSO, bortezomib, paclitaxel or LDE225 for 24 hours using Western blot analysis. -actin was used as a loading control. C) Gene expression of PTCH1, GLI1 and GLI2 was examined in A2780cp55 cells treated with DMSO, paclitaxel or tubastatin-a for 24 hours, using quantitative PCR. *P < 0.05, compared to DMSO vehicle control. D) A2780cp55 and E) SKOV3TRip2 cell viability following exposure to DMSO or tubastatin-a combined with increasing concentrations of paclitaxel. F) SKOV3TRip2 cell viability following treatment with DMSO or tubastatin-a combined with increasing concentrations of LDE225. Cell viability was determined using MTT assay. Data are representative of at least 3 independent experiments.
Mentions: To determine how inhibition of hedgehog signaling by bortezomib could promote increased paclitaxel sensitivity in vitro, we first examined expression of a primary mediator of taxane resistance ABCB1/MDR1. This gene encodes for P-glycoprotein, a drug efflux pump that has been shown by our laboratory to play a role in the synergy between LDE225 and paclitaxel [16]. Gene expression of ABCB1/MDR1 was measured using qPCR after treatment with increasing concentrations of bortezomib for 72 hours. As shown in Figure 5A, bortezomib (10, 20, 30 nM) significantly (p<0.05) decreased ABCB1/MDR1 expression in a dose-dependent manner (by 25%, 73% and 83%, respectively), indicating that bortezomib increases paclitaxel sensitivity, at least in part, through inhibition of drug efflux.

Bottom Line: Expression of the multi-drug resistance gene ABCB1/MDR1 was decreased and acetylation of α-tubulin, a marker of microtubule stabilization, was increased following bortezomib treatment.HDAC6 inhibitor tubastatin-a demonstrated that microtubule effects are associated with hedgehog inhibition and sensitization to paclitaxel and LDE225.These results suggest that proteasome inhibition, through alteration of microtubule dynamics and hedgehog signaling, can reverse taxane-mediated chemoresistance.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, University of Alabama at Birmingham, Birmingham, AL. McWhorter School of Pharmacy, Samford University, Birmingham, AL. These authors contributed equally to this work.

ABSTRACT
The goal of this study was to determine whether combined targeted therapies, specifically those against the Notch, hedgehog and ubiquitin-proteasome pathways, could overcome ovarian cancer chemoresistance. Chemoresistant ovarian cancer cells were exposed to gamma-secretase inhibitors (GSI-I, Compound E) or the proteasome inhibitor bortezomib, alone and in combination with the hedgehog antagonist, LDE225. Bortezomib, alone and in combination with LDE225, was evaluated for effects on paclitaxel efficacy. Cell viability and cell cycle analysis were assessed by MTT assay and propidium iodide staining, respectively. Proteasome activity and gene expression were determined by luminescence assay and qPCR, respectively. Studies demonstrated that GSI-I, but not Compound E, inhibited proteasome activity, similar to bortezomib. Proteasome inhibition decreased hedgehog target genes (PTCH1, GLI1 and GLI2) and increased LDE225 sensitivity in vitro. Bortezomib, alone and in combination with LDE225, increased paclitaxel sensitivity through apoptosis and G2/M arrest. Expression of the multi-drug resistance gene ABCB1/MDR1 was decreased and acetylation of α-tubulin, a marker of microtubule stabilization, was increased following bortezomib treatment. HDAC6 inhibitor tubastatin-a demonstrated that microtubule effects are associated with hedgehog inhibition and sensitization to paclitaxel and LDE225. These results suggest that proteasome inhibition, through alteration of microtubule dynamics and hedgehog signaling, can reverse taxane-mediated chemoresistance.

Show MeSH
Related in: MedlinePlus