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The mTOR kinase inhibitors polarize glioma-activated microglia to express a M1 phenotype.

Lisi L, Laudati E, Navarra P, Dello Russo C - J Neuroinflammation (2014)

Bottom Line: In fact RAPA and RAD significantly increased iNOS expression and activity, while on the same time significantly reducing IL-10 gene expression induced by C-CM, thus suggesting that the drugs prevent the acquisition of a M2 phenotype in response to glioma factors promoting a classic M1 activation.Similar results were obtained using the conditioned media obtained after glioma stimulation with LPS-IFNγ (LI-CM), which was found to induce a mixture of M1 and M2a/b polarization phenotypes.In these conditions, the inhibition of mTOR led to a significant up-regulation of iNOS, and in parallel to the down-regulation of both ARG and IL-10 gene expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Pharmacology, Catholic University Medical School, L,go F Vito 1, 00168 Rome, Italy. pnavarra@rm.unicatt.it.

ABSTRACT

Background: Increased activation of mammalian target of rapamycin (mTOR) is observed in numerous human cancers. Recent studies on the glioma kinome have identified several deregulated pathways that converge and activate mTOR. The available evidence on the role of microglia in CNS cancers would suggest a dual role, a tumoricidal role and -on the contrary- a role favoring tumor growth.

Methods: In the present paper, we have compared the effects of μM concentrations of rapamycin (RAPA) and its analog, RAD001 (RAD), on activated microglia; the latter was obtained by exposing cells to conditioned medium harvested either from inflammatory activated glioma cells (LI-CM) or from glioma cells kept under basal conditions (C-CM).

Results: Here we show that the inhibition of mTOR polarizes glioma-activated microglial cells towards the M1 phenotype, with cytotoxic activities, preventing the induction of the M2 status that promotes tumor growth. In fact RAPA and RAD significantly increased iNOS expression and activity, while on the same time significantly reducing IL-10 gene expression induced by C-CM, thus suggesting that the drugs prevent the acquisition of a M2 phenotype in response to glioma factors promoting a classic M1 activation. Similar results were obtained using the conditioned media obtained after glioma stimulation with LPS-IFNγ (LI-CM), which was found to induce a mixture of M1 and M2a/b polarization phenotypes. In these conditions, the inhibition of mTOR led to a significant up-regulation of iNOS, and in parallel to the down-regulation of both ARG and IL-10 gene expression.

Conclusions: These data suggest that mTOR inhibition may prevent glioma induced M2 polarization of microglial cells and increase their cytotoxic potential, possibly resulting in antitumor actions.

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Effects of mammalian target of rapamycin (mTOR) inhibitors on nitric oxide synthase (NOS)2 in microglia stimulated by both control-conditioned medium (C-CM) and CM from glioma cells activated with LPS/IFNγ (LI-CM). Cells were treated with C-CM alone or in combination with mTOR inhibitors. (A-B) and with LI-CM alone or in combination with mTOR inhibitors (C-D). Total cytosolic RNA was prepared from control, or microglial cells treated with the drugs for 4 h, and used for real time (Q)-PCR analysis of NOS2 expression. Data are expressed as fold change versus control, taken as calibrator for comparative quantitation analysis of mRNA levels. Each sample was measured in triplicate and the experiment was repeated three times with similar results. Data are means ± standard error of the mean (SEM), and were analyzed by one-way analysis of variance followed by Bonferroni post hoc test. ***P <0.001 versus control; °P <0.05 °°°P <0.001 versus C-CM and °P <0.05 °°P <0.01 versus LI-CM. (B-D) After 48-h incubation, the medium was used for nitrite assessment, whereas cells were lysed in 200 mM NaOH and protein content was evaluated by the Bradford method. Results are expressed as μM of nitrites/μg of proteins; data are means ± SEM (n = 6). *P <0.05, ***P <0.001 versus C-CM; *P <0.05 versus control and °P <0.001 °°°P <0.001 versus LI-CM.
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Figure 3: Effects of mammalian target of rapamycin (mTOR) inhibitors on nitric oxide synthase (NOS)2 in microglia stimulated by both control-conditioned medium (C-CM) and CM from glioma cells activated with LPS/IFNγ (LI-CM). Cells were treated with C-CM alone or in combination with mTOR inhibitors. (A-B) and with LI-CM alone or in combination with mTOR inhibitors (C-D). Total cytosolic RNA was prepared from control, or microglial cells treated with the drugs for 4 h, and used for real time (Q)-PCR analysis of NOS2 expression. Data are expressed as fold change versus control, taken as calibrator for comparative quantitation analysis of mRNA levels. Each sample was measured in triplicate and the experiment was repeated three times with similar results. Data are means ± standard error of the mean (SEM), and were analyzed by one-way analysis of variance followed by Bonferroni post hoc test. ***P <0.001 versus control; °P <0.05 °°°P <0.001 versus C-CM and °P <0.05 °°P <0.01 versus LI-CM. (B-D) After 48-h incubation, the medium was used for nitrite assessment, whereas cells were lysed in 200 mM NaOH and protein content was evaluated by the Bradford method. Results are expressed as μM of nitrites/μg of proteins; data are means ± SEM (n = 6). *P <0.05, ***P <0.001 versus C-CM; *P <0.05 versus control and °P <0.001 °°°P <0.001 versus LI-CM.

Mentions: We have previously shown that CM harvested from glioma cells under basal conditions (C-CM), as well as CM from glioma cells activated with LPS/IFNγ (LI-CM), increased the phosphorylation of mTOR at ser 2448, an index of mTORC activation, in microglial cells in 2-h experiments [21]. Here we confirmed this finding, and also observed that mTOR activation is rapid and time-persistent: after 30 minutes of incubation with both conditioned media we detected an increase in phospho-mTOR levels that persisted until 8 h (Figure 1). However, no significant upregulation was found in the mRNA levels of specific interactors of mTORC1 (raptor, Figure 2A) and mTORC2 (rictor, Figure 2B) in 4- and 24-h experiments.The administration of mTOR inhibitors (RAPA and RAD) to microglial cells exposed to C-CM significantly increased iNOS gene expression after 4 h (Figure 3A), followed by an increase in the levels of NO after 48 h (Figure 3B). Furthermore, RAPA and RAD were also able to increase iNOS mRNA levels after 4 h in microglial cells exposed to LI-CM (Figure 3C); such upregulation was followed by a significant augmentation of NO production after 48 h (Figure 3D).In addition, both drugs reduced the upregulation of IL-10 gene expression elicited by C-CM, with significant inhibition from 4 h onward (Figure 4A). In parallel, the drugs were able to reduce the upregulation of IL-10 (Figure 4B) and arginase (ARG) (Figure 4C) elicited by LI-CM. Taken together, these data show that mTOR blockade promotes M1 microglial polarization in both experimental paradigms (that is, conditions resembling the early stage of pathology and conditions mimicking the late stage of pathology).


The mTOR kinase inhibitors polarize glioma-activated microglia to express a M1 phenotype.

Lisi L, Laudati E, Navarra P, Dello Russo C - J Neuroinflammation (2014)

Effects of mammalian target of rapamycin (mTOR) inhibitors on nitric oxide synthase (NOS)2 in microglia stimulated by both control-conditioned medium (C-CM) and CM from glioma cells activated with LPS/IFNγ (LI-CM). Cells were treated with C-CM alone or in combination with mTOR inhibitors. (A-B) and with LI-CM alone or in combination with mTOR inhibitors (C-D). Total cytosolic RNA was prepared from control, or microglial cells treated with the drugs for 4 h, and used for real time (Q)-PCR analysis of NOS2 expression. Data are expressed as fold change versus control, taken as calibrator for comparative quantitation analysis of mRNA levels. Each sample was measured in triplicate and the experiment was repeated three times with similar results. Data are means ± standard error of the mean (SEM), and were analyzed by one-way analysis of variance followed by Bonferroni post hoc test. ***P <0.001 versus control; °P <0.05 °°°P <0.001 versus C-CM and °P <0.05 °°P <0.01 versus LI-CM. (B-D) After 48-h incubation, the medium was used for nitrite assessment, whereas cells were lysed in 200 mM NaOH and protein content was evaluated by the Bradford method. Results are expressed as μM of nitrites/μg of proteins; data are means ± SEM (n = 6). *P <0.05, ***P <0.001 versus C-CM; *P <0.05 versus control and °P <0.001 °°°P <0.001 versus LI-CM.
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Figure 3: Effects of mammalian target of rapamycin (mTOR) inhibitors on nitric oxide synthase (NOS)2 in microglia stimulated by both control-conditioned medium (C-CM) and CM from glioma cells activated with LPS/IFNγ (LI-CM). Cells were treated with C-CM alone or in combination with mTOR inhibitors. (A-B) and with LI-CM alone or in combination with mTOR inhibitors (C-D). Total cytosolic RNA was prepared from control, or microglial cells treated with the drugs for 4 h, and used for real time (Q)-PCR analysis of NOS2 expression. Data are expressed as fold change versus control, taken as calibrator for comparative quantitation analysis of mRNA levels. Each sample was measured in triplicate and the experiment was repeated three times with similar results. Data are means ± standard error of the mean (SEM), and were analyzed by one-way analysis of variance followed by Bonferroni post hoc test. ***P <0.001 versus control; °P <0.05 °°°P <0.001 versus C-CM and °P <0.05 °°P <0.01 versus LI-CM. (B-D) After 48-h incubation, the medium was used for nitrite assessment, whereas cells were lysed in 200 mM NaOH and protein content was evaluated by the Bradford method. Results are expressed as μM of nitrites/μg of proteins; data are means ± SEM (n = 6). *P <0.05, ***P <0.001 versus C-CM; *P <0.05 versus control and °P <0.001 °°°P <0.001 versus LI-CM.
Mentions: We have previously shown that CM harvested from glioma cells under basal conditions (C-CM), as well as CM from glioma cells activated with LPS/IFNγ (LI-CM), increased the phosphorylation of mTOR at ser 2448, an index of mTORC activation, in microglial cells in 2-h experiments [21]. Here we confirmed this finding, and also observed that mTOR activation is rapid and time-persistent: after 30 minutes of incubation with both conditioned media we detected an increase in phospho-mTOR levels that persisted until 8 h (Figure 1). However, no significant upregulation was found in the mRNA levels of specific interactors of mTORC1 (raptor, Figure 2A) and mTORC2 (rictor, Figure 2B) in 4- and 24-h experiments.The administration of mTOR inhibitors (RAPA and RAD) to microglial cells exposed to C-CM significantly increased iNOS gene expression after 4 h (Figure 3A), followed by an increase in the levels of NO after 48 h (Figure 3B). Furthermore, RAPA and RAD were also able to increase iNOS mRNA levels after 4 h in microglial cells exposed to LI-CM (Figure 3C); such upregulation was followed by a significant augmentation of NO production after 48 h (Figure 3D).In addition, both drugs reduced the upregulation of IL-10 gene expression elicited by C-CM, with significant inhibition from 4 h onward (Figure 4A). In parallel, the drugs were able to reduce the upregulation of IL-10 (Figure 4B) and arginase (ARG) (Figure 4C) elicited by LI-CM. Taken together, these data show that mTOR blockade promotes M1 microglial polarization in both experimental paradigms (that is, conditions resembling the early stage of pathology and conditions mimicking the late stage of pathology).

Bottom Line: In fact RAPA and RAD significantly increased iNOS expression and activity, while on the same time significantly reducing IL-10 gene expression induced by C-CM, thus suggesting that the drugs prevent the acquisition of a M2 phenotype in response to glioma factors promoting a classic M1 activation.Similar results were obtained using the conditioned media obtained after glioma stimulation with LPS-IFNγ (LI-CM), which was found to induce a mixture of M1 and M2a/b polarization phenotypes.In these conditions, the inhibition of mTOR led to a significant up-regulation of iNOS, and in parallel to the down-regulation of both ARG and IL-10 gene expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Pharmacology, Catholic University Medical School, L,go F Vito 1, 00168 Rome, Italy. pnavarra@rm.unicatt.it.

ABSTRACT

Background: Increased activation of mammalian target of rapamycin (mTOR) is observed in numerous human cancers. Recent studies on the glioma kinome have identified several deregulated pathways that converge and activate mTOR. The available evidence on the role of microglia in CNS cancers would suggest a dual role, a tumoricidal role and -on the contrary- a role favoring tumor growth.

Methods: In the present paper, we have compared the effects of μM concentrations of rapamycin (RAPA) and its analog, RAD001 (RAD), on activated microglia; the latter was obtained by exposing cells to conditioned medium harvested either from inflammatory activated glioma cells (LI-CM) or from glioma cells kept under basal conditions (C-CM).

Results: Here we show that the inhibition of mTOR polarizes glioma-activated microglial cells towards the M1 phenotype, with cytotoxic activities, preventing the induction of the M2 status that promotes tumor growth. In fact RAPA and RAD significantly increased iNOS expression and activity, while on the same time significantly reducing IL-10 gene expression induced by C-CM, thus suggesting that the drugs prevent the acquisition of a M2 phenotype in response to glioma factors promoting a classic M1 activation. Similar results were obtained using the conditioned media obtained after glioma stimulation with LPS-IFNγ (LI-CM), which was found to induce a mixture of M1 and M2a/b polarization phenotypes. In these conditions, the inhibition of mTOR led to a significant up-regulation of iNOS, and in parallel to the down-regulation of both ARG and IL-10 gene expression.

Conclusions: These data suggest that mTOR inhibition may prevent glioma induced M2 polarization of microglial cells and increase their cytotoxic potential, possibly resulting in antitumor actions.

Show MeSH
Related in: MedlinePlus