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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 selectively inhibits the release of perforins and granzymes but not of FasL.NK cells were culture for 6d+IL2 with the indicated doses of drugs or in their solvent. Then NK cells were co-cultured with Jurkat or K562 or FO1 (A), (NK/tumor target ratio 2∶1) or incubated with mAbs to CD16 or NKG2D (B) followed by GAM. After 2 h, NK cells were labelled with anti-CD107a mAb and analyzed by flowcytometry. Basal: % of CD107a+ NK cells before interaction with tumor cells (A) or engagement of the indicated receptors (B). Results are expressed as mean±SD of % CD107a+ NK cells from six different donors. (C). BLT esterase activity of SN from NK cells after the engagement of NKG2D. Results are expressed as % of maximal BLT esterase content in NK cells. Statistical significance *p<0.001 vs medium. (D). Evaluation of soluble (s)FasL in SN from cultures of NK cells and Jurkat or K562 or FO1 tumor cell lines (NK/tumor cell ratio 2∶1 at 8 h) or upon the engagement of CD16 or NKG2D (E) receptors with specific mAb and GAM (at 8 h). Data are shown as ng/ml/106 NK cells and are the mean±SD of 6 experiments.
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pone-0062932-g003: Fluvastatin selectively inhibits the release of perforins and granzymes but not of FasL.NK cells were culture for 6d+IL2 with the indicated doses of drugs or in their solvent. Then NK cells were co-cultured with Jurkat or K562 or FO1 (A), (NK/tumor target ratio 2∶1) or incubated with mAbs to CD16 or NKG2D (B) followed by GAM. After 2 h, NK cells were labelled with anti-CD107a mAb and analyzed by flowcytometry. Basal: % of CD107a+ NK cells before interaction with tumor cells (A) or engagement of the indicated receptors (B). Results are expressed as mean±SD of % CD107a+ NK cells from six different donors. (C). BLT esterase activity of SN from NK cells after the engagement of NKG2D. Results are expressed as % of maximal BLT esterase content in NK cells. Statistical significance *p<0.001 vs medium. (D). Evaluation of soluble (s)FasL in SN from cultures of NK cells and Jurkat or K562 or FO1 tumor cell lines (NK/tumor cell ratio 2∶1 at 8 h) or upon the engagement of CD16 or NKG2D (E) receptors with specific mAb and GAM (at 8 h). Data are shown as ng/ml/106 NK cells and are the mean±SD of 6 experiments.

Mentions: Since NK cells can kill tumor targets either through perforin-mediated cytotoxicity or following FasL secretion, we analyzed whether fluvastatin can impair both perforins-granzymes and FasL release. CD107a surface expression on NK cells was used to indirectly evaluate perforin release [32] either after interaction with target cells or upon cross-linking of activating NK receptor with specific mAb and GAM (fig. 3A,B). Fluvastatin impaired CD107a expression at the NK cell surface upon incubation with Jurkat or K562 or FO1 tumor cell lines (fig. 3A). Also, CD107a surface expression induced by engagement of either CD16 or NKG2D was consistently reduced in fluvastatin cultured NK cells (fig. 3B). However, upon CD16 engagement, inhibition was statistically significant when NK cells were cultured with 10 µM but not 1 µM of fluvastatin, while NKG2D-mediated triggering was significantly inhibited at both doses. The low amount of BLT esterase activity elicited upon NKG2D engagement and found in supernatants from fluvastatin-treated NK cells, compared with the high BLT esterase activity found in medium (fig. 3C) or solvent-treated (not shown) NK cells, confirmed that fluvastatin impairs NK cell degranulation. The addition of mevalonate together with fluvastatin completely reverted the effect of fluvastatin (fig. 3A–C). In parallel samples, the release of FasL in culture supernatant was evaluated by ELISA. We found that the interaction of NK cells with Jurkat or K562 or FO1 target cells triggered the release of similar amounts of FasL from NK cells cultured either in medium or with different doses of fluvastatin (fig. 3D). Also, triggering of NK cells by cross-linking of either CD16 or NKG2D could induce the secretion of FasL and, again, fluvastatin-treated NK cells could release FasL in similar amount as untreated (fig. 3E) or solvent treated (not shown) NK cells.


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 selectively inhibits the release of perforins and granzymes but not of FasL.NK cells were culture for 6d+IL2 with the indicated doses of drugs or in their solvent. Then NK cells were co-cultured with Jurkat or K562 or FO1 (A), (NK/tumor target ratio 2∶1) or incubated with mAbs to CD16 or NKG2D (B) followed by GAM. After 2 h, NK cells were labelled with anti-CD107a mAb and analyzed by flowcytometry. Basal: % of CD107a+ NK cells before interaction with tumor cells (A) or engagement of the indicated receptors (B). Results are expressed as mean±SD of % CD107a+ NK cells from six different donors. (C). BLT esterase activity of SN from NK cells after the engagement of NKG2D. Results are expressed as % of maximal BLT esterase content in NK cells. Statistical significance *p<0.001 vs medium. (D). Evaluation of soluble (s)FasL in SN from cultures of NK cells and Jurkat or K562 or FO1 tumor cell lines (NK/tumor cell ratio 2∶1 at 8 h) or upon the engagement of CD16 or NKG2D (E) receptors with specific mAb and GAM (at 8 h). Data are shown as ng/ml/106 NK cells and are the mean±SD of 6 experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3646988&req=5

pone-0062932-g003: Fluvastatin selectively inhibits the release of perforins and granzymes but not of FasL.NK cells were culture for 6d+IL2 with the indicated doses of drugs or in their solvent. Then NK cells were co-cultured with Jurkat or K562 or FO1 (A), (NK/tumor target ratio 2∶1) or incubated with mAbs to CD16 or NKG2D (B) followed by GAM. After 2 h, NK cells were labelled with anti-CD107a mAb and analyzed by flowcytometry. Basal: % of CD107a+ NK cells before interaction with tumor cells (A) or engagement of the indicated receptors (B). Results are expressed as mean±SD of % CD107a+ NK cells from six different donors. (C). BLT esterase activity of SN from NK cells after the engagement of NKG2D. Results are expressed as % of maximal BLT esterase content in NK cells. Statistical significance *p<0.001 vs medium. (D). Evaluation of soluble (s)FasL in SN from cultures of NK cells and Jurkat or K562 or FO1 tumor cell lines (NK/tumor cell ratio 2∶1 at 8 h) or upon the engagement of CD16 or NKG2D (E) receptors with specific mAb and GAM (at 8 h). Data are shown as ng/ml/106 NK cells and are the mean±SD of 6 experiments.
Mentions: Since NK cells can kill tumor targets either through perforin-mediated cytotoxicity or following FasL secretion, we analyzed whether fluvastatin can impair both perforins-granzymes and FasL release. CD107a surface expression on NK cells was used to indirectly evaluate perforin release [32] either after interaction with target cells or upon cross-linking of activating NK receptor with specific mAb and GAM (fig. 3A,B). Fluvastatin impaired CD107a expression at the NK cell surface upon incubation with Jurkat or K562 or FO1 tumor cell lines (fig. 3A). Also, CD107a surface expression induced by engagement of either CD16 or NKG2D was consistently reduced in fluvastatin cultured NK cells (fig. 3B). However, upon CD16 engagement, inhibition was statistically significant when NK cells were cultured with 10 µM but not 1 µM of fluvastatin, while NKG2D-mediated triggering was significantly inhibited at both doses. The low amount of BLT esterase activity elicited upon NKG2D engagement and found in supernatants from fluvastatin-treated NK cells, compared with the high BLT esterase activity found in medium (fig. 3C) or solvent-treated (not shown) NK cells, confirmed that fluvastatin impairs NK cell degranulation. The addition of mevalonate together with fluvastatin completely reverted the effect of fluvastatin (fig. 3A–C). In parallel samples, the release of FasL in culture supernatant was evaluated by ELISA. We found that the interaction of NK cells with Jurkat or K562 or FO1 target cells triggered the release of similar amounts of FasL from NK cells cultured either in medium or with different doses of fluvastatin (fig. 3D). Also, triggering of NK cells by cross-linking of either CD16 or NKG2D could induce the secretion of FasL and, again, fluvastatin-treated NK cells could release FasL in similar amount as untreated (fig. 3E) or solvent treated (not shown) NK cells.

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