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Zoledronic acid overcomes chemoresistance and immunosuppression of malignant mesothelioma.

Salaroglio IC, Campia I, Kopecka J, Gazzano E, Orecchia S, Ghigo D, Riganti C - Oncotarget (2015)

Bottom Line: We compared primary HMM samples with non-transformed mesothelial cells.By reducing the activity of the Ras/ERK1/2/STAT3/IDO axis, zoledronic acid lowered the kyurenine synthesis and the expansion of Treg cells, and increased the proliferation of T-lymphocytes.Thanks to its ability to decrease Ras/ERK1/2 activity, which is responsible for both Pgp-mediated chemoresistance and IDO-mediated immunosuppression, zoledronic acid is an effective chemo-immunosensitizing agent in HMM cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, University of Torino, Italy.

ABSTRACT
The human malignant mesothelioma (HMM) is characterized by a chemoresistant and immunosuppressive phenotype. An effective strategy to restore chemosensitivity and immune reactivity against HMM is lacking. We investigated whether the use of zoledronic acid is an effective chemo-immunosensitizing strategy. We compared primary HMM samples with non-transformed mesothelial cells. HMM cells had higher rate of cholesterol and isoprenoid synthesis, constitutive activation of Ras/extracellular signal-regulated kinase1/2 (ERK1/2)/hypoxia inducible factor-1α (HIF-1α) pathway and up-regulation of the drug efflux transporter P-glycoprotein (Pgp). By decreasing the isoprenoid supply, zoledronic acid down-regulated the Ras/ERK1/2/HIF-1α/Pgp axis and chemosensitized the HMM cells to Pgp substrates. The HMM cells also produced higher amounts of kynurenine, decreased the proliferation of T-lymphocytes and expanded the number of T-regulatory (Treg) cells. Kynurenine synthesis was due to the transcription of the indoleamine 1,2 dioxygenase (IDO) enzyme, consequent to the activation of the signal transducer and activator of transcription-3 (STAT3). By reducing the activity of the Ras/ERK1/2/STAT3/IDO axis, zoledronic acid lowered the kyurenine synthesis and the expansion of Treg cells, and increased the proliferation of T-lymphocytes. Thanks to its ability to decrease Ras/ERK1/2 activity, which is responsible for both Pgp-mediated chemoresistance and IDO-mediated immunosuppression, zoledronic acid is an effective chemo-immunosensitizing agent in HMM cells.

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Zoledronic acid down-regulates IDO expression in mesothelioma cells and inhibits the tumor-induced immunosuppressionHMC and HMM cells (UPN, unknown patient number) were incubated in fresh medium (−) or in the presence of 1 μmol/L zoledronic acid (ZA) for 48 h, then subjected to the following investigations. (a) The kynurenine level in cells culture supernatant, taken as an index of IDO enzymatic activity, was measured fluorimetrically (open bars, act); the expression levels of IDO mRNA were measured by qRT-PCR (solid bars, mRNA). Data are presented as means ± SD (n = 4). Vs untreated (−) cells: *p < 0.01; HMM cells vs HMC: °p < 0.002. (b) The cells were lysed and subjected to Western blot analysis for IDO expression. The β-tubulin expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results. (c) The proliferation of activated T-lymphocytes collected from PBMC after a 72 h co-incubation with HMC and HMM cells, grown in fresh medium (−) or in medium containing 1 μmol/L ZA, was measured with the [3H]thymidine assay. As positive control of proliferation, the PBMC were treated with the anti-CD3 and anti-CD28 antibodies, in the absence of cells; as negative control, the PBMC were grown in RPMI-1640 medium, in the absence of anti-CD3 and anti-CD28 antibodies, and of cells. In the presence of the anti-CD3 and anti-CD28 stimulatory antibodies, the [3H]thymidine incorporation was 33,123 ± 3,256 cpm; in the presence of RPMI-1640 medium alone, the [3H]thymidine incorporation was 3,892 ± 297 cpm. The [3H]thymidine incorporation in HMC and HMM cells were: 480 ± 190 cpm (untreated HMC); 482 ± 44 cpm (ZA-treated HMC); 485 ± 277 cpm (untreated HMM cells); 289 ± 28 cpm (ZA-treated HMM cells). Data are presented as means ± SD (n = 3). HMM cells vs HMC: *p < 0.01; ZA-treated HMM cells vs untreated (−) cells: °p < 0.05. (d) The percentage of Tregs (CD4+CD25+CD127low) collected from PBMC, co-incubated as reported in c, was measured by flow cytometry. Data are presented as means ± SD (n = 3). HMM cells vs HMC: *p < 0.02; ZA-treated HMM cells vs untreated (−) cells: °p < 0.05.
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Figure 3: Zoledronic acid down-regulates IDO expression in mesothelioma cells and inhibits the tumor-induced immunosuppressionHMC and HMM cells (UPN, unknown patient number) were incubated in fresh medium (−) or in the presence of 1 μmol/L zoledronic acid (ZA) for 48 h, then subjected to the following investigations. (a) The kynurenine level in cells culture supernatant, taken as an index of IDO enzymatic activity, was measured fluorimetrically (open bars, act); the expression levels of IDO mRNA were measured by qRT-PCR (solid bars, mRNA). Data are presented as means ± SD (n = 4). Vs untreated (−) cells: *p < 0.01; HMM cells vs HMC: °p < 0.002. (b) The cells were lysed and subjected to Western blot analysis for IDO expression. The β-tubulin expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results. (c) The proliferation of activated T-lymphocytes collected from PBMC after a 72 h co-incubation with HMC and HMM cells, grown in fresh medium (−) or in medium containing 1 μmol/L ZA, was measured with the [3H]thymidine assay. As positive control of proliferation, the PBMC were treated with the anti-CD3 and anti-CD28 antibodies, in the absence of cells; as negative control, the PBMC were grown in RPMI-1640 medium, in the absence of anti-CD3 and anti-CD28 antibodies, and of cells. In the presence of the anti-CD3 and anti-CD28 stimulatory antibodies, the [3H]thymidine incorporation was 33,123 ± 3,256 cpm; in the presence of RPMI-1640 medium alone, the [3H]thymidine incorporation was 3,892 ± 297 cpm. The [3H]thymidine incorporation in HMC and HMM cells were: 480 ± 190 cpm (untreated HMC); 482 ± 44 cpm (ZA-treated HMC); 485 ± 277 cpm (untreated HMM cells); 289 ± 28 cpm (ZA-treated HMM cells). Data are presented as means ± SD (n = 3). HMM cells vs HMC: *p < 0.01; ZA-treated HMM cells vs untreated (−) cells: °p < 0.05. (d) The percentage of Tregs (CD4+CD25+CD127low) collected from PBMC, co-incubated as reported in c, was measured by flow cytometry. Data are presented as means ± SD (n = 3). HMM cells vs HMC: *p < 0.02; ZA-treated HMM cells vs untreated (−) cells: °p < 0.05.

Mentions: Primary HMM cells exhibited higher synthesis of the IDO-derived immunosuppressive mediator kynurenine, higher levels of IDO mRNA and protein than HMC, all reduced by zoledronic acid (Figures 3a–b).


Zoledronic acid overcomes chemoresistance and immunosuppression of malignant mesothelioma.

Salaroglio IC, Campia I, Kopecka J, Gazzano E, Orecchia S, Ghigo D, Riganti C - Oncotarget (2015)

Zoledronic acid down-regulates IDO expression in mesothelioma cells and inhibits the tumor-induced immunosuppressionHMC and HMM cells (UPN, unknown patient number) were incubated in fresh medium (−) or in the presence of 1 μmol/L zoledronic acid (ZA) for 48 h, then subjected to the following investigations. (a) The kynurenine level in cells culture supernatant, taken as an index of IDO enzymatic activity, was measured fluorimetrically (open bars, act); the expression levels of IDO mRNA were measured by qRT-PCR (solid bars, mRNA). Data are presented as means ± SD (n = 4). Vs untreated (−) cells: *p < 0.01; HMM cells vs HMC: °p < 0.002. (b) The cells were lysed and subjected to Western blot analysis for IDO expression. The β-tubulin expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results. (c) The proliferation of activated T-lymphocytes collected from PBMC after a 72 h co-incubation with HMC and HMM cells, grown in fresh medium (−) or in medium containing 1 μmol/L ZA, was measured with the [3H]thymidine assay. As positive control of proliferation, the PBMC were treated with the anti-CD3 and anti-CD28 antibodies, in the absence of cells; as negative control, the PBMC were grown in RPMI-1640 medium, in the absence of anti-CD3 and anti-CD28 antibodies, and of cells. In the presence of the anti-CD3 and anti-CD28 stimulatory antibodies, the [3H]thymidine incorporation was 33,123 ± 3,256 cpm; in the presence of RPMI-1640 medium alone, the [3H]thymidine incorporation was 3,892 ± 297 cpm. The [3H]thymidine incorporation in HMC and HMM cells were: 480 ± 190 cpm (untreated HMC); 482 ± 44 cpm (ZA-treated HMC); 485 ± 277 cpm (untreated HMM cells); 289 ± 28 cpm (ZA-treated HMM cells). Data are presented as means ± SD (n = 3). HMM cells vs HMC: *p < 0.01; ZA-treated HMM cells vs untreated (−) cells: °p < 0.05. (d) The percentage of Tregs (CD4+CD25+CD127low) collected from PBMC, co-incubated as reported in c, was measured by flow cytometry. Data are presented as means ± SD (n = 3). HMM cells vs HMC: *p < 0.02; ZA-treated HMM cells vs untreated (−) cells: °p < 0.05.
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Figure 3: Zoledronic acid down-regulates IDO expression in mesothelioma cells and inhibits the tumor-induced immunosuppressionHMC and HMM cells (UPN, unknown patient number) were incubated in fresh medium (−) or in the presence of 1 μmol/L zoledronic acid (ZA) for 48 h, then subjected to the following investigations. (a) The kynurenine level in cells culture supernatant, taken as an index of IDO enzymatic activity, was measured fluorimetrically (open bars, act); the expression levels of IDO mRNA were measured by qRT-PCR (solid bars, mRNA). Data are presented as means ± SD (n = 4). Vs untreated (−) cells: *p < 0.01; HMM cells vs HMC: °p < 0.002. (b) The cells were lysed and subjected to Western blot analysis for IDO expression. The β-tubulin expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results. (c) The proliferation of activated T-lymphocytes collected from PBMC after a 72 h co-incubation with HMC and HMM cells, grown in fresh medium (−) or in medium containing 1 μmol/L ZA, was measured with the [3H]thymidine assay. As positive control of proliferation, the PBMC were treated with the anti-CD3 and anti-CD28 antibodies, in the absence of cells; as negative control, the PBMC were grown in RPMI-1640 medium, in the absence of anti-CD3 and anti-CD28 antibodies, and of cells. In the presence of the anti-CD3 and anti-CD28 stimulatory antibodies, the [3H]thymidine incorporation was 33,123 ± 3,256 cpm; in the presence of RPMI-1640 medium alone, the [3H]thymidine incorporation was 3,892 ± 297 cpm. The [3H]thymidine incorporation in HMC and HMM cells were: 480 ± 190 cpm (untreated HMC); 482 ± 44 cpm (ZA-treated HMC); 485 ± 277 cpm (untreated HMM cells); 289 ± 28 cpm (ZA-treated HMM cells). Data are presented as means ± SD (n = 3). HMM cells vs HMC: *p < 0.01; ZA-treated HMM cells vs untreated (−) cells: °p < 0.05. (d) The percentage of Tregs (CD4+CD25+CD127low) collected from PBMC, co-incubated as reported in c, was measured by flow cytometry. Data are presented as means ± SD (n = 3). HMM cells vs HMC: *p < 0.02; ZA-treated HMM cells vs untreated (−) cells: °p < 0.05.
Mentions: Primary HMM cells exhibited higher synthesis of the IDO-derived immunosuppressive mediator kynurenine, higher levels of IDO mRNA and protein than HMC, all reduced by zoledronic acid (Figures 3a–b).

Bottom Line: We compared primary HMM samples with non-transformed mesothelial cells.By reducing the activity of the Ras/ERK1/2/STAT3/IDO axis, zoledronic acid lowered the kyurenine synthesis and the expansion of Treg cells, and increased the proliferation of T-lymphocytes.Thanks to its ability to decrease Ras/ERK1/2 activity, which is responsible for both Pgp-mediated chemoresistance and IDO-mediated immunosuppression, zoledronic acid is an effective chemo-immunosensitizing agent in HMM cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, University of Torino, Italy.

ABSTRACT
The human malignant mesothelioma (HMM) is characterized by a chemoresistant and immunosuppressive phenotype. An effective strategy to restore chemosensitivity and immune reactivity against HMM is lacking. We investigated whether the use of zoledronic acid is an effective chemo-immunosensitizing strategy. We compared primary HMM samples with non-transformed mesothelial cells. HMM cells had higher rate of cholesterol and isoprenoid synthesis, constitutive activation of Ras/extracellular signal-regulated kinase1/2 (ERK1/2)/hypoxia inducible factor-1α (HIF-1α) pathway and up-regulation of the drug efflux transporter P-glycoprotein (Pgp). By decreasing the isoprenoid supply, zoledronic acid down-regulated the Ras/ERK1/2/HIF-1α/Pgp axis and chemosensitized the HMM cells to Pgp substrates. The HMM cells also produced higher amounts of kynurenine, decreased the proliferation of T-lymphocytes and expanded the number of T-regulatory (Treg) cells. Kynurenine synthesis was due to the transcription of the indoleamine 1,2 dioxygenase (IDO) enzyme, consequent to the activation of the signal transducer and activator of transcription-3 (STAT3). By reducing the activity of the Ras/ERK1/2/STAT3/IDO axis, zoledronic acid lowered the kyurenine synthesis and the expansion of Treg cells, and increased the proliferation of T-lymphocytes. Thanks to its ability to decrease Ras/ERK1/2 activity, which is responsible for both Pgp-mediated chemoresistance and IDO-mediated immunosuppression, zoledronic acid is an effective chemo-immunosensitizing agent in HMM cells.

Show MeSH
Related in: MedlinePlus