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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 Ras/ERK1/2/HIF-1α axis in mesothelioma cellsHMC 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 cells were radiolabeled during the last 24 h with [3H]acetate, then the de novo synthesis of cholesterol (open bars) or FPP (solid bars) was measured as described in Materials and methods. Data are presented as means ± SD (n = 3). Vs untreated (−) cells: *p < 0.01; HMM cells vs HMC: °p < 0.005. (b) The cells were lysed and subjected to the Western blot analysis for Ras-GTP (as index of active Ras), total Ras, phospho(Thr202/Tyr204, Thr185/Tyr187)-ERK1/2, total ERK1/2. The β-tubulin expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results. (c) The ERK activity was measured in cell lysates by a specific ELISA kit. Data are presented as means ± SD (n = 3). Vs untreated (−) cells: *p < 0.01; HMM cells vs HMC: °p < 0.01. (d) Cells were lysed and subjected to the Western blot analysis for phospho(Ser)-HIF-1α and total HIF-1α. The β-tubulin expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results. (e) The amount of HIF-1α was measured in nuclear extracts by Western blotting. The TBP expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results. (f) HIF-1α activity was measured in nuclear extracts by a specific ELISA kit. For each set of experiments, a competition assay (using 20 pmol of the wild type oligonucleotide with nuclear extracts from UPN1 cells grown at 3% O2 for 24 h) was included. In hypoxic conditions, the activity of HIF-1α was 58.32 ± 7.97 U/mg proteins; in the competition assay, the corresponding HIF-1α activity was reduced to 5.09 ± 0.56 U/mg proteins (n = 3; p < 0.001). Data are presented as means ± SD (n = 3). Vs untreated (−) cells: *p < 0.01; HMM cells vs HMC: °p < 0.001.
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Figure 1: Zoledronic acid down-regulates Ras/ERK1/2/HIF-1α axis in mesothelioma cellsHMC 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 cells were radiolabeled during the last 24 h with [3H]acetate, then the de novo synthesis of cholesterol (open bars) or FPP (solid bars) was measured as described in Materials and methods. Data are presented as means ± SD (n = 3). Vs untreated (−) cells: *p < 0.01; HMM cells vs HMC: °p < 0.005. (b) The cells were lysed and subjected to the Western blot analysis for Ras-GTP (as index of active Ras), total Ras, phospho(Thr202/Tyr204, Thr185/Tyr187)-ERK1/2, total ERK1/2. The β-tubulin expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results. (c) The ERK activity was measured in cell lysates by a specific ELISA kit. Data are presented as means ± SD (n = 3). Vs untreated (−) cells: *p < 0.01; HMM cells vs HMC: °p < 0.01. (d) Cells were lysed and subjected to the Western blot analysis for phospho(Ser)-HIF-1α and total HIF-1α. The β-tubulin expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results. (e) The amount of HIF-1α was measured in nuclear extracts by Western blotting. The TBP expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results. (f) HIF-1α activity was measured in nuclear extracts by a specific ELISA kit. For each set of experiments, a competition assay (using 20 pmol of the wild type oligonucleotide with nuclear extracts from UPN1 cells grown at 3% O2 for 24 h) was included. In hypoxic conditions, the activity of HIF-1α was 58.32 ± 7.97 U/mg proteins; in the competition assay, the corresponding HIF-1α activity was reduced to 5.09 ± 0.56 U/mg proteins (n = 3; p < 0.001). Data are presented as means ± SD (n = 3). Vs untreated (−) cells: *p < 0.01; HMM cells vs HMC: °p < 0.001.

Mentions: All HMM samples expressed Pgp, MRP1 and MRP3, and showed variable amounts of MRP4, MRP5, BCRP; such ABC transporters were undetectable in HMC (Supplementary Figure 1). In the subsequent experiments, all the 10 samples (indicated as HMM cells) were investigated, but in blotting experiments, for sake of simplicity, we show four out of the ten samples, including one sarcomatous, two epithelioid (one SV-40-negative and one SV-40-positive) and one biphasic histotypes. However, no significant differences in the parameters examined were observed as a function of the histotype (see below). Compared to the HMC, the HMM cells had higher cholesterol and farnesyl pyrophosphate (FPP) synthesis (Figure 1a), and showed higher levels of GTP-bound Ras (Figure 1b), phosphorylation (Figure 1b) and activity (Figure 1c) of the Ras downstream effectors ERK1/2. In HMM cells, but not in HMC, the transcription factor HIF-1α, a known inducer of Pgp and a target of Ras/ERK1/2 axis [21], was phosphorylated on serine (Figure 1d), translocated in the nucleus (Figure 1e) and bound to the target DNA (Figure 1f). By inhibiting the FPP synthase [26] and reducing the FPP supply (Figure 1a) necessary for Ras, zoledronic acid significantly lowered the activation of Ras, ERK1/2 and HIF-1α (Figures 1b–f).


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 Ras/ERK1/2/HIF-1α axis in mesothelioma cellsHMC 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 cells were radiolabeled during the last 24 h with [3H]acetate, then the de novo synthesis of cholesterol (open bars) or FPP (solid bars) was measured as described in Materials and methods. Data are presented as means ± SD (n = 3). Vs untreated (−) cells: *p < 0.01; HMM cells vs HMC: °p < 0.005. (b) The cells were lysed and subjected to the Western blot analysis for Ras-GTP (as index of active Ras), total Ras, phospho(Thr202/Tyr204, Thr185/Tyr187)-ERK1/2, total ERK1/2. The β-tubulin expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results. (c) The ERK activity was measured in cell lysates by a specific ELISA kit. Data are presented as means ± SD (n = 3). Vs untreated (−) cells: *p < 0.01; HMM cells vs HMC: °p < 0.01. (d) Cells were lysed and subjected to the Western blot analysis for phospho(Ser)-HIF-1α and total HIF-1α. The β-tubulin expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results. (e) The amount of HIF-1α was measured in nuclear extracts by Western blotting. The TBP expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results. (f) HIF-1α activity was measured in nuclear extracts by a specific ELISA kit. For each set of experiments, a competition assay (using 20 pmol of the wild type oligonucleotide with nuclear extracts from UPN1 cells grown at 3% O2 for 24 h) was included. In hypoxic conditions, the activity of HIF-1α was 58.32 ± 7.97 U/mg proteins; in the competition assay, the corresponding HIF-1α activity was reduced to 5.09 ± 0.56 U/mg proteins (n = 3; p < 0.001). Data are presented as means ± SD (n = 3). Vs untreated (−) cells: *p < 0.01; HMM cells vs HMC: °p < 0.001.
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Figure 1: Zoledronic acid down-regulates Ras/ERK1/2/HIF-1α axis in mesothelioma cellsHMC 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 cells were radiolabeled during the last 24 h with [3H]acetate, then the de novo synthesis of cholesterol (open bars) or FPP (solid bars) was measured as described in Materials and methods. Data are presented as means ± SD (n = 3). Vs untreated (−) cells: *p < 0.01; HMM cells vs HMC: °p < 0.005. (b) The cells were lysed and subjected to the Western blot analysis for Ras-GTP (as index of active Ras), total Ras, phospho(Thr202/Tyr204, Thr185/Tyr187)-ERK1/2, total ERK1/2. The β-tubulin expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results. (c) The ERK activity was measured in cell lysates by a specific ELISA kit. Data are presented as means ± SD (n = 3). Vs untreated (−) cells: *p < 0.01; HMM cells vs HMC: °p < 0.01. (d) Cells were lysed and subjected to the Western blot analysis for phospho(Ser)-HIF-1α and total HIF-1α. The β-tubulin expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results. (e) The amount of HIF-1α was measured in nuclear extracts by Western blotting. The TBP expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results. (f) HIF-1α activity was measured in nuclear extracts by a specific ELISA kit. For each set of experiments, a competition assay (using 20 pmol of the wild type oligonucleotide with nuclear extracts from UPN1 cells grown at 3% O2 for 24 h) was included. In hypoxic conditions, the activity of HIF-1α was 58.32 ± 7.97 U/mg proteins; in the competition assay, the corresponding HIF-1α activity was reduced to 5.09 ± 0.56 U/mg proteins (n = 3; p < 0.001). Data are presented as means ± SD (n = 3). Vs untreated (−) cells: *p < 0.01; HMM cells vs HMC: °p < 0.001.
Mentions: All HMM samples expressed Pgp, MRP1 and MRP3, and showed variable amounts of MRP4, MRP5, BCRP; such ABC transporters were undetectable in HMC (Supplementary Figure 1). In the subsequent experiments, all the 10 samples (indicated as HMM cells) were investigated, but in blotting experiments, for sake of simplicity, we show four out of the ten samples, including one sarcomatous, two epithelioid (one SV-40-negative and one SV-40-positive) and one biphasic histotypes. However, no significant differences in the parameters examined were observed as a function of the histotype (see below). Compared to the HMC, the HMM cells had higher cholesterol and farnesyl pyrophosphate (FPP) synthesis (Figure 1a), and showed higher levels of GTP-bound Ras (Figure 1b), phosphorylation (Figure 1b) and activity (Figure 1c) of the Ras downstream effectors ERK1/2. In HMM cells, but not in HMC, the transcription factor HIF-1α, a known inducer of Pgp and a target of Ras/ERK1/2 axis [21], was phosphorylated on serine (Figure 1d), translocated in the nucleus (Figure 1e) and bound to the target DNA (Figure 1f). By inhibiting the FPP synthase [26] and reducing the FPP supply (Figure 1a) necessary for Ras, zoledronic acid significantly lowered the activation of Ras, ERK1/2 and HIF-1α (Figures 1b–f).

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