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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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Effect of fluvastatin on ADCC and TNFα release triggered with rituximab or trastuzumab.(A). Effect of fluvastatin on ADCC triggered with humanized antibodies. Cytolytic activity of NK cell clones was analyzed upon treatment with fluvastatin in ADCC assay with rituximab (rit) or trastuzumab (tra) using the C1R CD20+ or SKBR3 HER2+ tumor cell lines at 2∶1 NK/tumor cell ratio respectively. Nil: basal cytolysis in absence of humanized antibody. Results are expressed as %51Cr specific release and are the mean±SD of 30 NK cell clones tested from six donors. (B). TNFα released in the NK-tumor cell co-culture after 24 h incubation was assessed by ELISA. Nil: basal level of TNFα produced by unstimulated NK cell clones. Results are expressed as pg/ml and are the mean±SD of TNFα present in SN from 15 NK cell clones. (C). Cytotoxic effect of TNFα released by NK cells in co-cultures with CIR and rituximab or with SKBR3 and trastuzumab antibody. Cytotoxicity was assessed on WEHI164 cell line sensitive to TNFα-mediated killing. In some experiments to block the cytotoxic effect of TNFα a saturating amount of anti-TNFα polyclonal antibody (5 µg/ml) was added (medium plus anti-TNFαAb). *p<0.0001. (D). Expression of CD20 on the cell surface of C1R, Raji and CLL cells (one patients out of ten analyzed) or HER2 on SKBR3 and BT474 breast adenocarcinomas cell lines upon incubation for three days with solvent (upper panels) or 10 µM of fluvastatin (lower panels). Cells were labelled with either anti-CD20 mAb or anti-HER2 mAb followed by alexafluor647 conjugated isotype specific GAM (thick lines). Thin line: cells labelled with an unrelated mAb matched for isotype. Results are expressed as Log far red fluorescence intensity (a.u.) vs number of cells and are representative of three independent experiments for CIR, Raji, SKBR3 and BT474 cell lines or 10 CLL patients. (E). ADCC mediated by ex-vivo NK cells incubated for 36 h with solvent or 10 µM fluvastatin, as indicated, of CIR or SKBR3 cell lines untreated (white columns) or treated with fluvastatin (for three day at 10 µM, black columns). Results are expressed as %51Cr specific release and are the mean±SD of 6 donors of NK cells.
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pone-0062932-g006: Effect of fluvastatin on ADCC and TNFα release triggered with rituximab or trastuzumab.(A). Effect of fluvastatin on ADCC triggered with humanized antibodies. Cytolytic activity of NK cell clones was analyzed upon treatment with fluvastatin in ADCC assay with rituximab (rit) or trastuzumab (tra) using the C1R CD20+ or SKBR3 HER2+ tumor cell lines at 2∶1 NK/tumor cell ratio respectively. Nil: basal cytolysis in absence of humanized antibody. Results are expressed as %51Cr specific release and are the mean±SD of 30 NK cell clones tested from six donors. (B). TNFα released in the NK-tumor cell co-culture after 24 h incubation was assessed by ELISA. Nil: basal level of TNFα produced by unstimulated NK cell clones. Results are expressed as pg/ml and are the mean±SD of TNFα present in SN from 15 NK cell clones. (C). Cytotoxic effect of TNFα released by NK cells in co-cultures with CIR and rituximab or with SKBR3 and trastuzumab antibody. Cytotoxicity was assessed on WEHI164 cell line sensitive to TNFα-mediated killing. In some experiments to block the cytotoxic effect of TNFα a saturating amount of anti-TNFα polyclonal antibody (5 µg/ml) was added (medium plus anti-TNFαAb). *p<0.0001. (D). Expression of CD20 on the cell surface of C1R, Raji and CLL cells (one patients out of ten analyzed) or HER2 on SKBR3 and BT474 breast adenocarcinomas cell lines upon incubation for three days with solvent (upper panels) or 10 µM of fluvastatin (lower panels). Cells were labelled with either anti-CD20 mAb or anti-HER2 mAb followed by alexafluor647 conjugated isotype specific GAM (thick lines). Thin line: cells labelled with an unrelated mAb matched for isotype. Results are expressed as Log far red fluorescence intensity (a.u.) vs number of cells and are representative of three independent experiments for CIR, Raji, SKBR3 and BT474 cell lines or 10 CLL patients. (E). ADCC mediated by ex-vivo NK cells incubated for 36 h with solvent or 10 µM fluvastatin, as indicated, of CIR or SKBR3 cell lines untreated (white columns) or treated with fluvastatin (for three day at 10 µM, black columns). Results are expressed as %51Cr specific release and are the mean±SD of 6 donors of NK cells.

Mentions: It is still debated whether patients treated with statins are more or less prone to occurrence of neoplasia and whether statin treatment can affect the therapy outcome of these patients [15], [16]; thus, we analyzed whether fluvastatin can influence ADCC elicited through the engagement of human FcγRIIIA (namely CD16) on NK cells in two different experimental settings: a) lysis of CD20+ C1R lymphoma target cells with the anti-CD20 rituximab; b) killing of HER2+ SKBR3 breast adenocarcinoma cell line using the anti-HER2 antibody trastuzumab. In these experiments, we used CD16+ NK cell clones expanded with IL2 as more homogeneous effector cells than primary or short-term IL2 activated NK cells. Representative NK cell clones derived from different healthy donors (results from 30 clones from 6 donors) displayed a strong ADCC of either C1R or SKBR3 tumor cell lines using anti-CD20 or anti-HER2 antibody respectively (fig. 6A). Incubation of NK cell clones for 72 h with 10 µM of fluvastatin inhibited by 50% the ADCC triggered through anti-CD20 or anti-HER2 humanized antibodies. No effect was found using 1 µM fluvastatin.


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)

Effect of fluvastatin on ADCC and TNFα release triggered with rituximab or trastuzumab.(A). Effect of fluvastatin on ADCC triggered with humanized antibodies. Cytolytic activity of NK cell clones was analyzed upon treatment with fluvastatin in ADCC assay with rituximab (rit) or trastuzumab (tra) using the C1R CD20+ or SKBR3 HER2+ tumor cell lines at 2∶1 NK/tumor cell ratio respectively. Nil: basal cytolysis in absence of humanized antibody. Results are expressed as %51Cr specific release and are the mean±SD of 30 NK cell clones tested from six donors. (B). TNFα released in the NK-tumor cell co-culture after 24 h incubation was assessed by ELISA. Nil: basal level of TNFα produced by unstimulated NK cell clones. Results are expressed as pg/ml and are the mean±SD of TNFα present in SN from 15 NK cell clones. (C). Cytotoxic effect of TNFα released by NK cells in co-cultures with CIR and rituximab or with SKBR3 and trastuzumab antibody. Cytotoxicity was assessed on WEHI164 cell line sensitive to TNFα-mediated killing. In some experiments to block the cytotoxic effect of TNFα a saturating amount of anti-TNFα polyclonal antibody (5 µg/ml) was added (medium plus anti-TNFαAb). *p<0.0001. (D). Expression of CD20 on the cell surface of C1R, Raji and CLL cells (one patients out of ten analyzed) or HER2 on SKBR3 and BT474 breast adenocarcinomas cell lines upon incubation for three days with solvent (upper panels) or 10 µM of fluvastatin (lower panels). Cells were labelled with either anti-CD20 mAb or anti-HER2 mAb followed by alexafluor647 conjugated isotype specific GAM (thick lines). Thin line: cells labelled with an unrelated mAb matched for isotype. Results are expressed as Log far red fluorescence intensity (a.u.) vs number of cells and are representative of three independent experiments for CIR, Raji, SKBR3 and BT474 cell lines or 10 CLL patients. (E). ADCC mediated by ex-vivo NK cells incubated for 36 h with solvent or 10 µM fluvastatin, as indicated, of CIR or SKBR3 cell lines untreated (white columns) or treated with fluvastatin (for three day at 10 µM, black columns). Results are expressed as %51Cr specific release and are the mean±SD of 6 donors of NK cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3646988&req=5

pone-0062932-g006: Effect of fluvastatin on ADCC and TNFα release triggered with rituximab or trastuzumab.(A). Effect of fluvastatin on ADCC triggered with humanized antibodies. Cytolytic activity of NK cell clones was analyzed upon treatment with fluvastatin in ADCC assay with rituximab (rit) or trastuzumab (tra) using the C1R CD20+ or SKBR3 HER2+ tumor cell lines at 2∶1 NK/tumor cell ratio respectively. Nil: basal cytolysis in absence of humanized antibody. Results are expressed as %51Cr specific release and are the mean±SD of 30 NK cell clones tested from six donors. (B). TNFα released in the NK-tumor cell co-culture after 24 h incubation was assessed by ELISA. Nil: basal level of TNFα produced by unstimulated NK cell clones. Results are expressed as pg/ml and are the mean±SD of TNFα present in SN from 15 NK cell clones. (C). Cytotoxic effect of TNFα released by NK cells in co-cultures with CIR and rituximab or with SKBR3 and trastuzumab antibody. Cytotoxicity was assessed on WEHI164 cell line sensitive to TNFα-mediated killing. In some experiments to block the cytotoxic effect of TNFα a saturating amount of anti-TNFα polyclonal antibody (5 µg/ml) was added (medium plus anti-TNFαAb). *p<0.0001. (D). Expression of CD20 on the cell surface of C1R, Raji and CLL cells (one patients out of ten analyzed) or HER2 on SKBR3 and BT474 breast adenocarcinomas cell lines upon incubation for three days with solvent (upper panels) or 10 µM of fluvastatin (lower panels). Cells were labelled with either anti-CD20 mAb or anti-HER2 mAb followed by alexafluor647 conjugated isotype specific GAM (thick lines). Thin line: cells labelled with an unrelated mAb matched for isotype. Results are expressed as Log far red fluorescence intensity (a.u.) vs number of cells and are representative of three independent experiments for CIR, Raji, SKBR3 and BT474 cell lines or 10 CLL patients. (E). ADCC mediated by ex-vivo NK cells incubated for 36 h with solvent or 10 µM fluvastatin, as indicated, of CIR or SKBR3 cell lines untreated (white columns) or treated with fluvastatin (for three day at 10 µM, black columns). Results are expressed as %51Cr specific release and are the mean±SD of 6 donors of NK cells.
Mentions: It is still debated whether patients treated with statins are more or less prone to occurrence of neoplasia and whether statin treatment can affect the therapy outcome of these patients [15], [16]; thus, we analyzed whether fluvastatin can influence ADCC elicited through the engagement of human FcγRIIIA (namely CD16) on NK cells in two different experimental settings: a) lysis of CD20+ C1R lymphoma target cells with the anti-CD20 rituximab; b) killing of HER2+ SKBR3 breast adenocarcinoma cell line using the anti-HER2 antibody trastuzumab. In these experiments, we used CD16+ NK cell clones expanded with IL2 as more homogeneous effector cells than primary or short-term IL2 activated NK cells. Representative NK cell clones derived from different healthy donors (results from 30 clones from 6 donors) displayed a strong ADCC of either C1R or SKBR3 tumor cell lines using anti-CD20 or anti-HER2 antibody respectively (fig. 6A). Incubation of NK cell clones for 72 h with 10 µM of fluvastatin inhibited by 50% the ADCC triggered through anti-CD20 or anti-HER2 humanized antibodies. No effect was found using 1 µM fluvastatin.

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