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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 chemosensitizes mesothelioma cells to Pgp substratesHMC 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) ChIP of HIF-1α on MDR1 promoter (pro MDR1). wo Ab: samples precipitated without anti-HIF-1α antibody; bl: blank; input: genomic DNA. The figure is representative of 3 experiments with similar results. (b) Cells were lysed and subjected to the Western blot analysis for Pgp expression. The β-tubulin expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results. (c) IC50 for doxorubicin, vinblastine, etoposide, cisplatin, gemcitabine and pemetrexed in HMM cells. The cells were incubated with increasing concentrations of each drug (1-10-100 pmol/L, 1-10-100 nmol/L, 1-10-100 μmol/L, 1 mmol/L) for 48 h, in the absence (CTRL) or in the presence of 1 μmol/L ZA. Cell viability was assessed with neutral red staining, as detailed under Materials and methods. Data are presented as means ± SD (n = 4). Doxorubicin-, vinblastine-, etoposide-treated cells vs CTRL cells: *p < 0.02.
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Figure 2: Zoledronic acid chemosensitizes mesothelioma cells to Pgp substratesHMC 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) ChIP of HIF-1α on MDR1 promoter (pro MDR1). wo Ab: samples precipitated without anti-HIF-1α antibody; bl: blank; input: genomic DNA. The figure is representative of 3 experiments with similar results. (b) Cells were lysed and subjected to the Western blot analysis for Pgp expression. The β-tubulin expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results. (c) IC50 for doxorubicin, vinblastine, etoposide, cisplatin, gemcitabine and pemetrexed in HMM cells. The cells were incubated with increasing concentrations of each drug (1-10-100 pmol/L, 1-10-100 nmol/L, 1-10-100 μmol/L, 1 mmol/L) for 48 h, in the absence (CTRL) or in the presence of 1 μmol/L ZA. Cell viability was assessed with neutral red staining, as detailed under Materials and methods. Data are presented as means ± SD (n = 4). Doxorubicin-, vinblastine-, etoposide-treated cells vs CTRL cells: *p < 0.02.

Mentions: HIF-1α was constitutively bound to the promoter of MDR1 gene, which encodes for Pgp, in HMM cells (Figure 2a) that – differently from HMC – were characterized by constitutively detectable levels of Pgp protein (Figure 2b). Zoledronic acid reduced the binding of HIF-1α to the MDR1 promoter (Figure 2a) and the expression of Pgp (Figure 2b). Consequently, it lowered the IC50 of chemotherapeutic drugs that are substrates of Pgp (Supplementary Table 1), such as doxorubicin, vinblastine and etoposide (Figure 2c). By contrast, zoledronic acid did not affect the IC50 of cisplatin, gemcitabine and pemetrexed (Figure 2c) that are not effluxed by Pgp (Supplementary Table 1).


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 chemosensitizes mesothelioma cells to Pgp substratesHMC 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) ChIP of HIF-1α on MDR1 promoter (pro MDR1). wo Ab: samples precipitated without anti-HIF-1α antibody; bl: blank; input: genomic DNA. The figure is representative of 3 experiments with similar results. (b) Cells were lysed and subjected to the Western blot analysis for Pgp expression. The β-tubulin expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results. (c) IC50 for doxorubicin, vinblastine, etoposide, cisplatin, gemcitabine and pemetrexed in HMM cells. The cells were incubated with increasing concentrations of each drug (1-10-100 pmol/L, 1-10-100 nmol/L, 1-10-100 μmol/L, 1 mmol/L) for 48 h, in the absence (CTRL) or in the presence of 1 μmol/L ZA. Cell viability was assessed with neutral red staining, as detailed under Materials and methods. Data are presented as means ± SD (n = 4). Doxorubicin-, vinblastine-, etoposide-treated cells vs CTRL cells: *p < 0.02.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4359222&req=5

Figure 2: Zoledronic acid chemosensitizes mesothelioma cells to Pgp substratesHMC 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) ChIP of HIF-1α on MDR1 promoter (pro MDR1). wo Ab: samples precipitated without anti-HIF-1α antibody; bl: blank; input: genomic DNA. The figure is representative of 3 experiments with similar results. (b) Cells were lysed and subjected to the Western blot analysis for Pgp expression. The β-tubulin expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results. (c) IC50 for doxorubicin, vinblastine, etoposide, cisplatin, gemcitabine and pemetrexed in HMM cells. The cells were incubated with increasing concentrations of each drug (1-10-100 pmol/L, 1-10-100 nmol/L, 1-10-100 μmol/L, 1 mmol/L) for 48 h, in the absence (CTRL) or in the presence of 1 μmol/L ZA. Cell viability was assessed with neutral red staining, as detailed under Materials and methods. Data are presented as means ± SD (n = 4). Doxorubicin-, vinblastine-, etoposide-treated cells vs CTRL cells: *p < 0.02.
Mentions: HIF-1α was constitutively bound to the promoter of MDR1 gene, which encodes for Pgp, in HMM cells (Figure 2a) that – differently from HMC – were characterized by constitutively detectable levels of Pgp protein (Figure 2b). Zoledronic acid reduced the binding of HIF-1α to the MDR1 promoter (Figure 2a) and the expression of Pgp (Figure 2b). Consequently, it lowered the IC50 of chemotherapeutic drugs that are substrates of Pgp (Supplementary Table 1), such as doxorubicin, vinblastine and etoposide (Figure 2c). By contrast, zoledronic acid did not affect the IC50 of cisplatin, gemcitabine and pemetrexed (Figure 2c) that are not effluxed by Pgp (Supplementary Table 1).

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