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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 cell viability. (A) C6 glioma cells were administrated with mTOR inhibitors. Microglia cells were treated for 48 h with control-conditioned medium (C-CM) (B) or CM from glioma cells activated with lipopolysaccharide (LPS)/IFNγ (LI-CM) (C) and mTOR inhibitors. Effects on cell viability were assessed by the MTS reduction assay. Data were analyzed by one-way analysis of variance followed by Bonferroni post hoc test. ***P <0.001, versus Control, °°°P <0.001 versus C-CM or LI-CM.
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Figure 5: Effects of mammalian target of rapamycin (mTOR) inhibitors on cell viability. (A) C6 glioma cells were administrated with mTOR inhibitors. Microglia cells were treated for 48 h with control-conditioned medium (C-CM) (B) or CM from glioma cells activated with lipopolysaccharide (LPS)/IFNγ (LI-CM) (C) and mTOR inhibitors. Effects on cell viability were assessed by the MTS reduction assay. Data were analyzed by one-way analysis of variance followed by Bonferroni post hoc test. ***P <0.001, versus Control, °°°P <0.001 versus C-CM or LI-CM.

Mentions: A subsequent set of experiments was carried out to assess the effect of mTOR inhibitors on cell viability and proliferation. First, we tested the direct effects of RAPA and RAD on C6 glioma cells and confirmed that both drugs reduce glioma viability (Figure 5A), as reported in the literature. In particular, RAPA and RAD at 1 nM, reduced C6 glioma viability by about 30% after 3 days of treatment. Thereafter, we assessed the effect of mTOR inhibitors on microglia viability in cells exposed to glioma-conditioned media. In this experimental model, RAPA and RAD significantly counteracted the increase in cell viability induced by the exposure to C-CM or LI-CM, although significantly higher concentrations (approximately 1,000-fold higher) were required to achieve a 50% reduction with respect to those found effective on C6 glioma cells (Figure 5B and C) or else on microglial cells not exposed to glioma factors [13]. In untreated microglia, viability was significantly affected by mTOR inhibition even at a low dose (from 10 nM concentration onward) [13]. No significant differences were observed in microglia proliferation in the presence of mTOR inhibitors in the two different experimental paradigms (Figure 6).


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 cell viability. (A) C6 glioma cells were administrated with mTOR inhibitors. Microglia cells were treated for 48 h with control-conditioned medium (C-CM) (B) or CM from glioma cells activated with lipopolysaccharide (LPS)/IFNγ (LI-CM) (C) and mTOR inhibitors. Effects on cell viability were assessed by the MTS reduction assay. Data were analyzed by one-way analysis of variance followed by Bonferroni post hoc test. ***P <0.001, versus Control, °°°P <0.001 versus C-CM or LI-CM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4128534&req=5

Figure 5: Effects of mammalian target of rapamycin (mTOR) inhibitors on cell viability. (A) C6 glioma cells were administrated with mTOR inhibitors. Microglia cells were treated for 48 h with control-conditioned medium (C-CM) (B) or CM from glioma cells activated with lipopolysaccharide (LPS)/IFNγ (LI-CM) (C) and mTOR inhibitors. Effects on cell viability were assessed by the MTS reduction assay. Data were analyzed by one-way analysis of variance followed by Bonferroni post hoc test. ***P <0.001, versus Control, °°°P <0.001 versus C-CM or LI-CM.
Mentions: A subsequent set of experiments was carried out to assess the effect of mTOR inhibitors on cell viability and proliferation. First, we tested the direct effects of RAPA and RAD on C6 glioma cells and confirmed that both drugs reduce glioma viability (Figure 5A), as reported in the literature. In particular, RAPA and RAD at 1 nM, reduced C6 glioma viability by about 30% after 3 days of treatment. Thereafter, we assessed the effect of mTOR inhibitors on microglia viability in cells exposed to glioma-conditioned media. In this experimental model, RAPA and RAD significantly counteracted the increase in cell viability induced by the exposure to C-CM or LI-CM, although significantly higher concentrations (approximately 1,000-fold higher) were required to achieve a 50% reduction with respect to those found effective on C6 glioma cells (Figure 5B and C) or else on microglial cells not exposed to glioma factors [13]. In untreated microglia, viability was significantly affected by mTOR inhibition even at a low dose (from 10 nM concentration onward) [13]. No significant differences were observed in microglia proliferation in the presence of mTOR inhibitors in the two different experimental paradigms (Figure 6).

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