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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 inhibit NK cell mediated cytotoxicity of target cells mediated by FasL/Fas receptor ligation.(A). Cytotoxic activity of NK cells cultured with solvent or fluvastatin 10 µM for 6d+IL2 was analyzed after 4 h or 24 h incubation with the indicated target cells (Jurkat, K562 or FO1). Results are expressed as percentage of 51Cr specific release and are the mean±SD of six experiments. (B, first row). Side scatter (SSC) and forward scatter (FSC) of ex-vivo isolated NK cells cultured for 6d+IL2 in solvent (DMSO) or in presence of fluvastatin (10-1.0-0.1 µM). FSC and SSC of Jurkat cell line is also shown. R1 gate: living cells. (B, second and third row) NK cells, in the indicated culture conditions, were co-incubated with Jurkat cells for 4 h (second row) or 24 h (third row) at 2∶1 E:T ratio. R1 gate: living cells; R2 gate: dying cells. Results are representative of three independent experiments and number in panels indicate the % of R2 gated cells. (C–E). NK cells were cultured for 6d+IL2 in medium alone (medium) or solvent of fluvastatin (solvent) or with fluvastatin (10-1-0.1 µM) or with mevalonate 1 mM either alone or with fluvastatin 10 µM as indicated. NK cell-mediated cytotoxicity of Jurkat cells at 2∶1 E:T ratio analyzed after 4 h (C) or 24 h (D) without mAbs added; (E): anti-FasL and anti-Fas mAbs added at the onset of the 24 h cytotoxicity assay.
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pone-0062932-g004: Fluvastatin does not inhibit NK cell mediated cytotoxicity of target cells mediated by FasL/Fas receptor ligation.(A). Cytotoxic activity of NK cells cultured with solvent or fluvastatin 10 µM for 6d+IL2 was analyzed after 4 h or 24 h incubation with the indicated target cells (Jurkat, K562 or FO1). Results are expressed as percentage of 51Cr specific release and are the mean±SD of six experiments. (B, first row). Side scatter (SSC) and forward scatter (FSC) of ex-vivo isolated NK cells cultured for 6d+IL2 in solvent (DMSO) or in presence of fluvastatin (10-1.0-0.1 µM). FSC and SSC of Jurkat cell line is also shown. R1 gate: living cells. (B, second and third row) NK cells, in the indicated culture conditions, were co-incubated with Jurkat cells for 4 h (second row) or 24 h (third row) at 2∶1 E:T ratio. R1 gate: living cells; R2 gate: dying cells. Results are representative of three independent experiments and number in panels indicate the % of R2 gated cells. (C–E). NK cells were cultured for 6d+IL2 in medium alone (medium) or solvent of fluvastatin (solvent) or with fluvastatin (10-1-0.1 µM) or with mevalonate 1 mM either alone or with fluvastatin 10 µM as indicated. NK cell-mediated cytotoxicity of Jurkat cells at 2∶1 E:T ratio analyzed after 4 h (C) or 24 h (D) without mAbs added; (E): anti-FasL and anti-Fas mAbs added at the onset of the 24 h cytotoxicity assay.

Mentions: Cytolysis that occurs upon polarized release of granules containing perforin and granzymes leads to necrosis of target cells in a short time, usually detected after 4 h of incubation, while FasL induces apoptosis of targets after more than 8 h, being detectable at 24 h [1]–[5], [29], [30]. Thus, we analyzed whether fluvastatin can differently affect these two mechanisms of killing. We found that NK cell-mediated cytotoxicity of Jurkat, K562 or FO1 tumor targets strongly increased when analyzed after 24 h compared to the killing detected at 4 h (fig. 4A). At 4 h, NK cells incubated with fluvastatin exerted a lower cytotoxic activity vs Jurkat or K562 or FO1 compared to that of NK cells cultured in medium (not shown) or solvent (fig. 4A). Conversely, at 24 h, we found that the killing of the same targets was not inhibited by fluvastatin (fig. 4A). Focusing on Jurkat cells, which are sensitive to Fas/FasL mediated apoptosis [29], [30], we analyzed more in detail the mechanism of cytotoxicity at 24 h, that was not inhibited by fluvastatin. First, we found that the shape and size of cells analyzed as forward scatter (FSC) and side scatter (SSC) in NK/Jurkat cell co-cultures were not altered at 4 h when NK cells were incubated with fluvastatin (a few cells present in the region R2 of fig. 4B plots at 4 h). On the contrary, FSC and SSC of co-cultures changed at 24 h (large amount of cells in R2 region, fig. 4B). Accordingly, killing of Jurkat cells was inhibited in fluvastatin treated cells at 4 h (fig. 4C) but not at 24 h (fig. 4D). Furthermore, the addition (after 4 h from the onset of the cytotoxicity assay) of anti-Fas and anti-FasL mAb, to block the interaction between FasL secreted by NK cells and Fas molecule on target cells, markedly inhibited the killing detected at 24 h suggesting that it is Fas/FasL-dependent (fig. 4E). It is of note that the degree of killing of Jurkat cells chelating extracellular calcium with EGTA, and evaluated after 24 h, was superimposable using as effector cells either untreated or fluvastatin treated NK cells (not shown). This would suggest that long-term target cell lysis is not dependent on extracellular calcium.


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 inhibit NK cell mediated cytotoxicity of target cells mediated by FasL/Fas receptor ligation.(A). Cytotoxic activity of NK cells cultured with solvent or fluvastatin 10 µM for 6d+IL2 was analyzed after 4 h or 24 h incubation with the indicated target cells (Jurkat, K562 or FO1). Results are expressed as percentage of 51Cr specific release and are the mean±SD of six experiments. (B, first row). Side scatter (SSC) and forward scatter (FSC) of ex-vivo isolated NK cells cultured for 6d+IL2 in solvent (DMSO) or in presence of fluvastatin (10-1.0-0.1 µM). FSC and SSC of Jurkat cell line is also shown. R1 gate: living cells. (B, second and third row) NK cells, in the indicated culture conditions, were co-incubated with Jurkat cells for 4 h (second row) or 24 h (third row) at 2∶1 E:T ratio. R1 gate: living cells; R2 gate: dying cells. Results are representative of three independent experiments and number in panels indicate the % of R2 gated cells. (C–E). NK cells were cultured for 6d+IL2 in medium alone (medium) or solvent of fluvastatin (solvent) or with fluvastatin (10-1-0.1 µM) or with mevalonate 1 mM either alone or with fluvastatin 10 µM as indicated. NK cell-mediated cytotoxicity of Jurkat cells at 2∶1 E:T ratio analyzed after 4 h (C) or 24 h (D) without mAbs added; (E): anti-FasL and anti-Fas mAbs added at the onset of the 24 h cytotoxicity assay.
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Related In: Results  -  Collection

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pone-0062932-g004: Fluvastatin does not inhibit NK cell mediated cytotoxicity of target cells mediated by FasL/Fas receptor ligation.(A). Cytotoxic activity of NK cells cultured with solvent or fluvastatin 10 µM for 6d+IL2 was analyzed after 4 h or 24 h incubation with the indicated target cells (Jurkat, K562 or FO1). Results are expressed as percentage of 51Cr specific release and are the mean±SD of six experiments. (B, first row). Side scatter (SSC) and forward scatter (FSC) of ex-vivo isolated NK cells cultured for 6d+IL2 in solvent (DMSO) or in presence of fluvastatin (10-1.0-0.1 µM). FSC and SSC of Jurkat cell line is also shown. R1 gate: living cells. (B, second and third row) NK cells, in the indicated culture conditions, were co-incubated with Jurkat cells for 4 h (second row) or 24 h (third row) at 2∶1 E:T ratio. R1 gate: living cells; R2 gate: dying cells. Results are representative of three independent experiments and number in panels indicate the % of R2 gated cells. (C–E). NK cells were cultured for 6d+IL2 in medium alone (medium) or solvent of fluvastatin (solvent) or with fluvastatin (10-1-0.1 µM) or with mevalonate 1 mM either alone or with fluvastatin 10 µM as indicated. NK cell-mediated cytotoxicity of Jurkat cells at 2∶1 E:T ratio analyzed after 4 h (C) or 24 h (D) without mAbs added; (E): anti-FasL and anti-Fas mAbs added at the onset of the 24 h cytotoxicity assay.
Mentions: Cytolysis that occurs upon polarized release of granules containing perforin and granzymes leads to necrosis of target cells in a short time, usually detected after 4 h of incubation, while FasL induces apoptosis of targets after more than 8 h, being detectable at 24 h [1]–[5], [29], [30]. Thus, we analyzed whether fluvastatin can differently affect these two mechanisms of killing. We found that NK cell-mediated cytotoxicity of Jurkat, K562 or FO1 tumor targets strongly increased when analyzed after 24 h compared to the killing detected at 4 h (fig. 4A). At 4 h, NK cells incubated with fluvastatin exerted a lower cytotoxic activity vs Jurkat or K562 or FO1 compared to that of NK cells cultured in medium (not shown) or solvent (fig. 4A). Conversely, at 24 h, we found that the killing of the same targets was not inhibited by fluvastatin (fig. 4A). Focusing on Jurkat cells, which are sensitive to Fas/FasL mediated apoptosis [29], [30], we analyzed more in detail the mechanism of cytotoxicity at 24 h, that was not inhibited by fluvastatin. First, we found that the shape and size of cells analyzed as forward scatter (FSC) and side scatter (SSC) in NK/Jurkat cell co-cultures were not altered at 4 h when NK cells were incubated with fluvastatin (a few cells present in the region R2 of fig. 4B plots at 4 h). On the contrary, FSC and SSC of co-cultures changed at 24 h (large amount of cells in R2 region, fig. 4B). Accordingly, killing of Jurkat cells was inhibited in fluvastatin treated cells at 4 h (fig. 4C) but not at 24 h (fig. 4D). Furthermore, the addition (after 4 h from the onset of the cytotoxicity assay) of anti-Fas and anti-FasL mAb, to block the interaction between FasL secreted by NK cells and Fas molecule on target cells, markedly inhibited the killing detected at 24 h suggesting that it is Fas/FasL-dependent (fig. 4E). It is of note that the degree of killing of Jurkat cells chelating extracellular calcium with EGTA, and evaluated after 24 h, was superimposable using as effector cells either untreated or fluvastatin treated NK cells (not shown). This would suggest that long-term target cell lysis is not dependent on extracellular calcium.

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