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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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Bortezomib increases paclitaxel sensitivity in chemoresistant ovarian cancer cellsA) A2780cp55 and B) SKOV3TRip2 cell viability following exposure to DMSO or bortezomib combined with increasing concentrations of paclitaxel. C) A2780cp55 and D) SKOV3TRip2 cell viability following treatment with DMSO alone, LDE225 alone, bortezomib alone or LDE225+bortezomib combined with increasing concentrations of paclitaxel. Cell viability was determined using MTT assay. E) Cell cycle analysis was performed on A2780cp55 and SKOV3TRip2 cells treated with DMSO alone, bortezomib alone, paclitaxel alone or combined bortezomib+paclitaxel for 72 hours using propidium iodide (PI) staining. F) Representative histograms of each treatment group in A2780cp55 and SKOV3TRip2 cells are shown. G) Protein expression of PARP, an indcator of apoptosis, was examined in SKOV3TRip2 cells exposed to DMSO alone, bortezomib alone, paclitaxel alone or combined bortezomib+paclitaxel for 72 hours using Western blot analysis. β-actin was used as a loading control. Data are representative of at least 3 independent experiments.
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Figure 4: Bortezomib increases paclitaxel sensitivity in chemoresistant ovarian cancer cellsA) A2780cp55 and B) SKOV3TRip2 cell viability following exposure to DMSO or bortezomib combined with increasing concentrations of paclitaxel. C) A2780cp55 and D) SKOV3TRip2 cell viability following treatment with DMSO alone, LDE225 alone, bortezomib alone or LDE225+bortezomib combined with increasing concentrations of paclitaxel. Cell viability was determined using MTT assay. E) Cell cycle analysis was performed on A2780cp55 and SKOV3TRip2 cells treated with DMSO alone, bortezomib alone, paclitaxel alone or combined bortezomib+paclitaxel for 72 hours using propidium iodide (PI) staining. F) Representative histograms of each treatment group in A2780cp55 and SKOV3TRip2 cells are shown. G) Protein expression of PARP, an indcator of apoptosis, was examined in SKOV3TRip2 cells exposed to DMSO alone, bortezomib alone, paclitaxel alone or combined bortezomib+paclitaxel for 72 hours using Western blot analysis. β-actin was used as a loading control. Data are representative of at least 3 independent experiments.

Mentions: We have previously shown that antagonism of the hedgehog pathway, using LDE225 or siRNAs designed against hedgehog signaling components, can reverse taxane resistance in ovarian cancer cells [16]. The inhibitory effect of bortezomib on hedgehog signaling led us to consider whether this compound could be used to increase paclitaxel sensitivity. The chemoresistant ovarian cancer cell lines A2780cp55 and SKOV3TRip2 were exposed to DMSO or bortezomib combined with increasing concentrations of paclitaxel. Interestingly, we found that bortezomib combined with paclitaxel act in a synergistic manner. Up to a 2.3-fold decrease in paclitaxel IC50 compared to DMSO control was observed in A2780cp55 cells (CI=0.47) (Figure 4A) and up to a 2.6-fold decrease in paclitaxel IC50 compared to DMSO control was observed in SKOV3TRip2 cells (CI=0.41 at 30nM) (Figure 4B). To determine whether LDE225 combined with bortezomib could have a greater effect on paclitaxel sensitization, we exposed A2780cp55 and SKOV3TRip2 cells to DMSO, LDE225, bortezomib or LDE225+bortezomib combined with increasing concentrations of paclitaxel. In agreement with previous findings [16], LDE225 alone increased the sensitivity of A2780cp55 and SKOV3TRip2 cells to paclitaxel (Figure 4C, D); however, combined LDE225+bortezomib did not further enhance paclitaxel sensitization compared to single agents, indicating an additive effect. These results suggest that bortezomib can sensitize chemoresistant ovarian cancer cells to a similar degree as inhibition of hedgehog signaling.


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)

Bortezomib increases paclitaxel sensitivity in chemoresistant ovarian cancer cellsA) A2780cp55 and B) SKOV3TRip2 cell viability following exposure to DMSO or bortezomib combined with increasing concentrations of paclitaxel. C) A2780cp55 and D) SKOV3TRip2 cell viability following treatment with DMSO alone, LDE225 alone, bortezomib alone or LDE225+bortezomib combined with increasing concentrations of paclitaxel. Cell viability was determined using MTT assay. E) Cell cycle analysis was performed on A2780cp55 and SKOV3TRip2 cells treated with DMSO alone, bortezomib alone, paclitaxel alone or combined bortezomib+paclitaxel for 72 hours using propidium iodide (PI) staining. F) Representative histograms of each treatment group in A2780cp55 and SKOV3TRip2 cells are shown. G) Protein expression of PARP, an indcator of apoptosis, was examined in SKOV3TRip2 cells exposed to DMSO alone, bortezomib alone, paclitaxel alone or combined bortezomib+paclitaxel for 72 hours using Western blot analysis. β-actin was used as a loading control. Data are representative of at least 3 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Bortezomib increases paclitaxel sensitivity in chemoresistant ovarian cancer cellsA) A2780cp55 and B) SKOV3TRip2 cell viability following exposure to DMSO or bortezomib combined with increasing concentrations of paclitaxel. C) A2780cp55 and D) SKOV3TRip2 cell viability following treatment with DMSO alone, LDE225 alone, bortezomib alone or LDE225+bortezomib combined with increasing concentrations of paclitaxel. Cell viability was determined using MTT assay. E) Cell cycle analysis was performed on A2780cp55 and SKOV3TRip2 cells treated with DMSO alone, bortezomib alone, paclitaxel alone or combined bortezomib+paclitaxel for 72 hours using propidium iodide (PI) staining. F) Representative histograms of each treatment group in A2780cp55 and SKOV3TRip2 cells are shown. G) Protein expression of PARP, an indcator of apoptosis, was examined in SKOV3TRip2 cells exposed to DMSO alone, bortezomib alone, paclitaxel alone or combined bortezomib+paclitaxel for 72 hours using Western blot analysis. β-actin was used as a loading control. Data are representative of at least 3 independent experiments.
Mentions: We have previously shown that antagonism of the hedgehog pathway, using LDE225 or siRNAs designed against hedgehog signaling components, can reverse taxane resistance in ovarian cancer cells [16]. The inhibitory effect of bortezomib on hedgehog signaling led us to consider whether this compound could be used to increase paclitaxel sensitivity. The chemoresistant ovarian cancer cell lines A2780cp55 and SKOV3TRip2 were exposed to DMSO or bortezomib combined with increasing concentrations of paclitaxel. Interestingly, we found that bortezomib combined with paclitaxel act in a synergistic manner. Up to a 2.3-fold decrease in paclitaxel IC50 compared to DMSO control was observed in A2780cp55 cells (CI=0.47) (Figure 4A) and up to a 2.6-fold decrease in paclitaxel IC50 compared to DMSO control was observed in SKOV3TRip2 cells (CI=0.41 at 30nM) (Figure 4B). To determine whether LDE225 combined with bortezomib could have a greater effect on paclitaxel sensitization, we exposed A2780cp55 and SKOV3TRip2 cells to DMSO, LDE225, bortezomib or LDE225+bortezomib combined with increasing concentrations of paclitaxel. In agreement with previous findings [16], LDE225 alone increased the sensitivity of A2780cp55 and SKOV3TRip2 cells to paclitaxel (Figure 4C, D); however, combined LDE225+bortezomib did not further enhance paclitaxel sensitization compared to single agents, indicating an additive effect. These results suggest that bortezomib can sensitize chemoresistant ovarian cancer cells to a similar degree as inhibition of hedgehog signaling.

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