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Selective role of mevalonate pathway in regulating perforin but not FasL and TNFalpha release in human Natural Killer cells.

Poggi A, Boero S, Musso A, Zocchi MR - PLoS ONE (2013)

Bottom Line: We have analyzed the effects of fluvastatin, an inhibitor of the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase involved in mevalonate synthesis, on human NK cell-mediated anti-tumor cytolysis.Remarkably, fluvastatin did not affect the expression of the inhibiting receptors CD94, KIR2D and LAIR1.Altogether these findings suggest that interference with mevalonate synthesis impairs activation and assembly of cytoskeleton, degranulation and cytotoxic effect of perforins and granzyme but not FasL- and TNFα-mediated cytotoxicity.

View Article: PubMed Central - PubMed

Affiliation: Molecular Oncology and Angiogenesis Unit, IRCCS AOU San Martino - IST National Institute for Cancer Research, Genoa, Italy. alessandro.poggi@istge.it

ABSTRACT
We have analyzed the effects of fluvastatin, an inhibitor of the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase involved in mevalonate synthesis, on human NK cell-mediated anti-tumor cytolysis. Fluvastatin inhibited the activation of the small guanosin triphosphate binding protein (GTP) RhoA and the consequent actin redistribution induced by ligation of LFA1 involved in NK-tumor target cell adhesion. Also, fluvastatin reduced ganglioside M1 rafts formation triggered through the engagement of NK cell activating receptors as FcγRIIIA (CD16), NKG2D and DNAM1. Cytolysis of tumor targets was inhibited up to 90% when NK cells were cultured with fluvastatin by affecting i) receptor-mediated increase of the intracellular free calcium concentration, ii) activation of akt1/PKB and iii) perforin and granzyme release. Fluvastatin displayed a stronger inhibiting effect on NKG2D, DNAM1, 2B4, NKp30, NKp44 and NKp46 than on CD16-mediated NK cell triggering. This was in line with the impairment of surface expression of all these receptors but not of CD16. Remarkably, fluvastatin did not affect the expression of the inhibiting receptors CD94, KIR2D and LAIR1. FasL release elicited by either NK-tumor cell interaction or CD16 or NKG2D engagement, as well as FasL-mediated killing, were not sensitive to fluvastatin. Moreover, TNFα secretion triggered in NK cells upon incubation with tumor target cells or engagement of NKG2D receptor was not impaired in fluvastatin-treated NK cells. Likewise, antibody dependent cellular cytotoxicity (ADCC) triggered through FcγRIIIA engagement with the humanized monoclonal antibody rituximab or trastuzumab was only marginally affected in fluvastatin-treated NK cells. Altogether these findings suggest that interference with mevalonate synthesis impairs activation and assembly of cytoskeleton, degranulation and cytotoxic effect of perforins and granzyme but not FasL- and TNFα-mediated cytotoxicity.

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Fluvastatin does not affect TNFα release in NK cells.(A). TNFα, measured by ELISA, present in supernatant of NK cells cultured for 6d+IL2 in medium alone (medium) or solvent of fluvastatin (solvent) or with fluvastatin (10-1.0-0.1 µM) and incubated with K562 HLAI− target cells, at the NK:target cell ratio of 2∶1. (B). TNFα in SN of NK cells, cultured in A, incubated with HLAI+ Jurkat target cells (2∶1 ratio). (C). TNFα in SN of NK cells, cultured as in A, incubated with P815 target cells in the presence of anti-NKG2D mAb to evaluate receptor-mediated TNFα release. In the different panels: K562 (A), Jurkat (B), P815 (C): basal level of cytokine production of tumor target cells. Left column in panel C indicate the basal level of cytokine production of NK cells. Results are expressed as pg/ml and are representative of three independent experiments. In some experiments, the effect of chelation of intracellular calcium with BAPTA-AM on TNFα release was analyzed (B).
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pone-0062932-g005: Fluvastatin does not affect TNFα release in NK cells.(A). TNFα, measured by ELISA, present in supernatant of NK cells cultured for 6d+IL2 in medium alone (medium) or solvent of fluvastatin (solvent) or with fluvastatin (10-1.0-0.1 µM) and incubated with K562 HLAI− target cells, at the NK:target cell ratio of 2∶1. (B). TNFα in SN of NK cells, cultured in A, incubated with HLAI+ Jurkat target cells (2∶1 ratio). (C). TNFα in SN of NK cells, cultured as in A, incubated with P815 target cells in the presence of anti-NKG2D mAb to evaluate receptor-mediated TNFα release. In the different panels: K562 (A), Jurkat (B), P815 (C): basal level of cytokine production of tumor target cells. Left column in panel C indicate the basal level of cytokine production of NK cells. Results are expressed as pg/ml and are representative of three independent experiments. In some experiments, the effect of chelation of intracellular calcium with BAPTA-AM on TNFα release was analyzed (B).

Mentions: To determine whether secretion of TNFα, another mechanism of NK cell-mediated tumor cell killing (1–5), can be regulated by fluvastatin, we analyzed the content of TNFα in the supernatants of NK cells cultured with two targets expressing or not HLAI: a) HLAI− K562 cells (fig. 5A); b) HLAI+ Jurkat target cell (fig. 5B). Indeed, activation of NK cells can be negatively regulated by interaction with HLAI and some tumor can downregulate HLAI to escape immune system, thus it is relevant to determine whether fluvastatin can have different effects. Ex-vivo isolated NK cells were cultured for 6d in IL2-containing medium in the absence or presence of solvent or fluvastatin before co-incubation with K562 cells. Interaction of NK and K562 cells triggered the release of TNFα; more importantly, TNFα was found in culture supernatants also when NK cells were pretreated with fluvastatin (fig. 5A). The amount of TNFα found in NK-K562 co-cultures from NK cells incubated with 1.0 µM fluvastatin was about two-fold than that detected using untreated NK cells (p<0.01). When Jurkat cells were used as targets, TNFα release was lower than that elicited upon incubation with K562 cells (compare fig. 5A and fig. 5B) and fluvastatin did not affect TNFα production. These findings suggest that HLAI can indeed deliver a negative signal in NK cells leading to low secretion of TNFα but again fluvastatin is not active. Furthermore, to define the role of intracellular calcium in the production of TNFα NK cells were incubated with BAPTA-AM calcium chelator, washed and then co-cultured with Jurkat cells. Of note, TNFα secretion was not altered after chelation of intracellular calcium either in the presence or absence of fluvastatin (fig. 5B) indicating that calcium is not mainly involved in the secretion of this cytokine.


Selective role of mevalonate pathway in regulating perforin but not FasL and TNFalpha release in human Natural Killer cells.

Poggi A, Boero S, Musso A, Zocchi MR - PLoS ONE (2013)

Fluvastatin does not affect TNFα release in NK cells.(A). TNFα, measured by ELISA, present in supernatant of NK cells cultured for 6d+IL2 in medium alone (medium) or solvent of fluvastatin (solvent) or with fluvastatin (10-1.0-0.1 µM) and incubated with K562 HLAI− target cells, at the NK:target cell ratio of 2∶1. (B). TNFα in SN of NK cells, cultured in A, incubated with HLAI+ Jurkat target cells (2∶1 ratio). (C). TNFα in SN of NK cells, cultured as in A, incubated with P815 target cells in the presence of anti-NKG2D mAb to evaluate receptor-mediated TNFα release. In the different panels: K562 (A), Jurkat (B), P815 (C): basal level of cytokine production of tumor target cells. Left column in panel C indicate the basal level of cytokine production of NK cells. Results are expressed as pg/ml and are representative of three independent experiments. In some experiments, the effect of chelation of intracellular calcium with BAPTA-AM on TNFα release was analyzed (B).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3646988&req=5

pone-0062932-g005: Fluvastatin does not affect TNFα release in NK cells.(A). TNFα, measured by ELISA, present in supernatant of NK cells cultured for 6d+IL2 in medium alone (medium) or solvent of fluvastatin (solvent) or with fluvastatin (10-1.0-0.1 µM) and incubated with K562 HLAI− target cells, at the NK:target cell ratio of 2∶1. (B). TNFα in SN of NK cells, cultured in A, incubated with HLAI+ Jurkat target cells (2∶1 ratio). (C). TNFα in SN of NK cells, cultured as in A, incubated with P815 target cells in the presence of anti-NKG2D mAb to evaluate receptor-mediated TNFα release. In the different panels: K562 (A), Jurkat (B), P815 (C): basal level of cytokine production of tumor target cells. Left column in panel C indicate the basal level of cytokine production of NK cells. Results are expressed as pg/ml and are representative of three independent experiments. In some experiments, the effect of chelation of intracellular calcium with BAPTA-AM on TNFα release was analyzed (B).
Mentions: To determine whether secretion of TNFα, another mechanism of NK cell-mediated tumor cell killing (1–5), can be regulated by fluvastatin, we analyzed the content of TNFα in the supernatants of NK cells cultured with two targets expressing or not HLAI: a) HLAI− K562 cells (fig. 5A); b) HLAI+ Jurkat target cell (fig. 5B). Indeed, activation of NK cells can be negatively regulated by interaction with HLAI and some tumor can downregulate HLAI to escape immune system, thus it is relevant to determine whether fluvastatin can have different effects. Ex-vivo isolated NK cells were cultured for 6d in IL2-containing medium in the absence or presence of solvent or fluvastatin before co-incubation with K562 cells. Interaction of NK and K562 cells triggered the release of TNFα; more importantly, TNFα was found in culture supernatants also when NK cells were pretreated with fluvastatin (fig. 5A). The amount of TNFα found in NK-K562 co-cultures from NK cells incubated with 1.0 µM fluvastatin was about two-fold than that detected using untreated NK cells (p<0.01). When Jurkat cells were used as targets, TNFα release was lower than that elicited upon incubation with K562 cells (compare fig. 5A and fig. 5B) and fluvastatin did not affect TNFα production. These findings suggest that HLAI can indeed deliver a negative signal in NK cells leading to low secretion of TNFα but again fluvastatin is not active. Furthermore, to define the role of intracellular calcium in the production of TNFα NK cells were incubated with BAPTA-AM calcium chelator, washed and then co-cultured with Jurkat cells. Of note, TNFα secretion was not altered after chelation of intracellular calcium either in the presence or absence of fluvastatin (fig. 5B) indicating that calcium is not mainly involved in the secretion of this cytokine.

Bottom Line: We have analyzed the effects of fluvastatin, an inhibitor of the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase involved in mevalonate synthesis, on human NK cell-mediated anti-tumor cytolysis.Remarkably, fluvastatin did not affect the expression of the inhibiting receptors CD94, KIR2D and LAIR1.Altogether these findings suggest that interference with mevalonate synthesis impairs activation and assembly of cytoskeleton, degranulation and cytotoxic effect of perforins and granzyme but not FasL- and TNFα-mediated cytotoxicity.

View Article: PubMed Central - PubMed

Affiliation: Molecular Oncology and Angiogenesis Unit, IRCCS AOU San Martino - IST National Institute for Cancer Research, Genoa, Italy. alessandro.poggi@istge.it

ABSTRACT
We have analyzed the effects of fluvastatin, an inhibitor of the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase involved in mevalonate synthesis, on human NK cell-mediated anti-tumor cytolysis. Fluvastatin inhibited the activation of the small guanosin triphosphate binding protein (GTP) RhoA and the consequent actin redistribution induced by ligation of LFA1 involved in NK-tumor target cell adhesion. Also, fluvastatin reduced ganglioside M1 rafts formation triggered through the engagement of NK cell activating receptors as FcγRIIIA (CD16), NKG2D and DNAM1. Cytolysis of tumor targets was inhibited up to 90% when NK cells were cultured with fluvastatin by affecting i) receptor-mediated increase of the intracellular free calcium concentration, ii) activation of akt1/PKB and iii) perforin and granzyme release. Fluvastatin displayed a stronger inhibiting effect on NKG2D, DNAM1, 2B4, NKp30, NKp44 and NKp46 than on CD16-mediated NK cell triggering. This was in line with the impairment of surface expression of all these receptors but not of CD16. Remarkably, fluvastatin did not affect the expression of the inhibiting receptors CD94, KIR2D and LAIR1. FasL release elicited by either NK-tumor cell interaction or CD16 or NKG2D engagement, as well as FasL-mediated killing, were not sensitive to fluvastatin. Moreover, TNFα secretion triggered in NK cells upon incubation with tumor target cells or engagement of NKG2D receptor was not impaired in fluvastatin-treated NK cells. Likewise, antibody dependent cellular cytotoxicity (ADCC) triggered through FcγRIIIA engagement with the humanized monoclonal antibody rituximab or trastuzumab was only marginally affected in fluvastatin-treated NK cells. Altogether these findings suggest that interference with mevalonate synthesis impairs activation and assembly of cytoskeleton, degranulation and cytotoxic effect of perforins and granzyme but not FasL- and TNFα-mediated cytotoxicity.

Show MeSH
Related in: MedlinePlus