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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 reduces the phosphorylation of STAT3, which is critical to up-regulate IDO in mesothelioma cellsHMM samples (UPN: unknown patient number) were grown for 24 h in fresh medium (−), treated with a non-targeting scrambled siRNA (scr) or with a specific siRNA pool targeting STAT1 or STAT3 (siSTAT1, siSTAT3). (a) The expression of STAT1 and STAT3 was measured in whole cell lysates (left panel) and in nuclear extracts (right panel) by Western blotting. The β-tubulin and TBP expression were used as controls of equal protein loading in whole cell lysates and nuclear extracts, respectively. The figure is representative of 3 experiments with similar results. (b) The expression levels of IDO mRNA were measured by qRT-PCR. Data are presented as means ± SD (n = 4). Vs untreated (−) cells: *p < 0.001. (c) The kynurenine level in the cell culture supernatant, taken as an index of IDO enzymatic activity, was measured fluorimetrically. Data are presented as means ± SD (n = 4). Vs untreated (−) cells: *p < 0.001. (d) The cells were grown in fresh medium (−) or in the presence of 1 μmol/L zoledronic acid (ZA) for 48 h, then lysed and subjected to the Western blot analysis of phospho(Tyr701)-STAT1, phospho(Ser727)-STAT1, total STAT1, phospho(Tyr705)-STAT3, phospho(Ser727)-STAT3, total STAT3. The β-tubulin expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results.
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Figure 4: Zoledronic acid reduces the phosphorylation of STAT3, which is critical to up-regulate IDO in mesothelioma cellsHMM samples (UPN: unknown patient number) were grown for 24 h in fresh medium (−), treated with a non-targeting scrambled siRNA (scr) or with a specific siRNA pool targeting STAT1 or STAT3 (siSTAT1, siSTAT3). (a) The expression of STAT1 and STAT3 was measured in whole cell lysates (left panel) and in nuclear extracts (right panel) by Western blotting. The β-tubulin and TBP expression were used as controls of equal protein loading in whole cell lysates and nuclear extracts, respectively. The figure is representative of 3 experiments with similar results. (b) The expression levels of IDO mRNA were measured by qRT-PCR. Data are presented as means ± SD (n = 4). Vs untreated (−) cells: *p < 0.001. (c) The kynurenine level in the cell culture supernatant, taken as an index of IDO enzymatic activity, was measured fluorimetrically. Data are presented as means ± SD (n = 4). Vs untreated (−) cells: *p < 0.001. (d) The cells were grown in fresh medium (−) or in the presence of 1 μmol/L zoledronic acid (ZA) for 48 h, then lysed and subjected to the Western blot analysis of phospho(Tyr701)-STAT1, phospho(Ser727)-STAT1, total STAT1, phospho(Tyr705)-STAT3, phospho(Ser727)-STAT3, total STAT3. The β-tubulin expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results.

Mentions: The transcriptional activators of the IDO gene STAT1 and STAT3 [28, 29] were present in HMM cells and constitutively translocated in the nucleus (Figure 4a). To investigate their involvement in the transcription of IDO, STAT1 and STAT3 were separately silenced in two primary HMM samples (one epithelioid and one sarcomatous; Figure 4a). STAT3-, but not STAT1-silenced cells showed decreased IDO mRNA (Figure 4b) and activity (Figure 4c).


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 reduces the phosphorylation of STAT3, which is critical to up-regulate IDO in mesothelioma cellsHMM samples (UPN: unknown patient number) were grown for 24 h in fresh medium (−), treated with a non-targeting scrambled siRNA (scr) or with a specific siRNA pool targeting STAT1 or STAT3 (siSTAT1, siSTAT3). (a) The expression of STAT1 and STAT3 was measured in whole cell lysates (left panel) and in nuclear extracts (right panel) by Western blotting. The β-tubulin and TBP expression were used as controls of equal protein loading in whole cell lysates and nuclear extracts, respectively. The figure is representative of 3 experiments with similar results. (b) The expression levels of IDO mRNA were measured by qRT-PCR. Data are presented as means ± SD (n = 4). Vs untreated (−) cells: *p < 0.001. (c) The kynurenine level in the cell culture supernatant, taken as an index of IDO enzymatic activity, was measured fluorimetrically. Data are presented as means ± SD (n = 4). Vs untreated (−) cells: *p < 0.001. (d) The cells were grown in fresh medium (−) or in the presence of 1 μmol/L zoledronic acid (ZA) for 48 h, then lysed and subjected to the Western blot analysis of phospho(Tyr701)-STAT1, phospho(Ser727)-STAT1, total STAT1, phospho(Tyr705)-STAT3, phospho(Ser727)-STAT3, total STAT3. The β-tubulin expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Zoledronic acid reduces the phosphorylation of STAT3, which is critical to up-regulate IDO in mesothelioma cellsHMM samples (UPN: unknown patient number) were grown for 24 h in fresh medium (−), treated with a non-targeting scrambled siRNA (scr) or with a specific siRNA pool targeting STAT1 or STAT3 (siSTAT1, siSTAT3). (a) The expression of STAT1 and STAT3 was measured in whole cell lysates (left panel) and in nuclear extracts (right panel) by Western blotting. The β-tubulin and TBP expression were used as controls of equal protein loading in whole cell lysates and nuclear extracts, respectively. The figure is representative of 3 experiments with similar results. (b) The expression levels of IDO mRNA were measured by qRT-PCR. Data are presented as means ± SD (n = 4). Vs untreated (−) cells: *p < 0.001. (c) The kynurenine level in the cell culture supernatant, taken as an index of IDO enzymatic activity, was measured fluorimetrically. Data are presented as means ± SD (n = 4). Vs untreated (−) cells: *p < 0.001. (d) The cells were grown in fresh medium (−) or in the presence of 1 μmol/L zoledronic acid (ZA) for 48 h, then lysed and subjected to the Western blot analysis of phospho(Tyr701)-STAT1, phospho(Ser727)-STAT1, total STAT1, phospho(Tyr705)-STAT3, phospho(Ser727)-STAT3, total STAT3. The β-tubulin expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results.
Mentions: The transcriptional activators of the IDO gene STAT1 and STAT3 [28, 29] were present in HMM cells and constitutively translocated in the nucleus (Figure 4a). To investigate their involvement in the transcription of IDO, STAT1 and STAT3 were separately silenced in two primary HMM samples (one epithelioid and one sarcomatous; Figure 4a). STAT3-, but not STAT1-silenced cells showed decreased IDO mRNA (Figure 4b) and activity (Figure 4c).

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