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[Pt(O,O'-acac)(γ-acac)(DMS)] alters SH-SY5Y cell migration and invasion by the inhibition of Na+/H+ exchanger isoform 1 occurring through a PKC-ε/ERK/mTOR Pathway.

Muscella A, Vetrugno C, Calabriso N, Cossa LG, De Pascali SA, Fanizzi FP, Marsigliante S - PLoS ONE (2014)

Bottom Line: We previously showed that [Pt(O,O'-acac)(γ-acac)(DMS)] ([Pt(acac)2(DMS)]) exerted substantial cytotoxic effects in SH-SY5Y neuroblastoma cells, and decreased metalloproteases (MMPs) production and cells migration in MCF-7 breast cancer cells.The present study focuses on the effects of [Pt(acac)2(DMS)] in SH-SY5Y cell migration and also on the possibility that NHE1 may be involved in such effect.In conclusion, we have shown a drastic impairment in tumour cell metastatization in response to inhibition of NHE1 and MMPs activities by [Pt(acac)2(DMS)] occurring through a novel mechanism mediated by PKC-δ/-ε activation.

View Article: PubMed Central - PubMed

Affiliation: Cell Pathology Lab, Dipartimento di Scienze e Tecnologie Biologiche e Ambientali (Di.S.Te.B.A.), Salento University, Lecce, Italy.

ABSTRACT
We previously showed that [Pt(O,O'-acac)(γ-acac)(DMS)] ([Pt(acac)2(DMS)]) exerted substantial cytotoxic effects in SH-SY5Y neuroblastoma cells, and decreased metalloproteases (MMPs) production and cells migration in MCF-7 breast cancer cells. The ubiquitously distributed sodium-hydrogen antiporter 1 (NHE1) is involved in motility and invasion of many solid tumours. The present study focuses on the effects of [Pt(acac)2(DMS)] in SH-SY5Y cell migration and also on the possibility that NHE1 may be involved in such effect. After sublethal [Pt(acac)2(DMS)] treatment cell migration was examined by wounding assay and cell invasion by transwell assay. NHE1 activity was measured in BCECF-loaded SH-SY5Y as the rate of Na+-dependent intracellular pH recovery in response to an acute acid pulse. Gelatin zymography for MMP-2/9 activities, Western blottings of MMPs, MAPKs, mTOR, S6 and PKCs and small interfering RNAs to PKC-ε/-δ mRNA were performed. Sublethal concentrations of [Pt(acac)2(DMS)] decreases NHE1 activity, inhibits cell migration and invasion and decreases expression and activity of MMP-2 and -9. [Pt(acac)2(DMS)] administered to SH-SY5Y cells provokes the increment of ROS, generated by NADPH oxidase, responsible for the PKC-ε and PKC-δ activation. Whilst PKC-δ activates p38/MAPK, responsible for the inhibition of MMP-2 and -9 secretion, PKC-ε activates a pathway made of ERK1/2, mTOR and S6K responsible for the inhibition of NHE1 activity and cell migration. In conclusion, we have shown a drastic impairment in tumour cell metastatization in response to inhibition of NHE1 and MMPs activities by [Pt(acac)2(DMS)] occurring through a novel mechanism mediated by PKC-δ/-ε activation.

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Role of p70 S6 Kinase in [Pt(acac)2(DMS)] inhibition of SH-SY5Y cell migration.(A) SH-SY5Y cells were incubated or not with 0.50 µM [Pt(acac)2(DMS)] for the indicated times. Cell lysates were analysed by western blotting with anti- unphosphorylated (6S and mTOR) and phosphorylated (p-6S and p-mTOR) 6S and mTOR antibodies. (B, C and D) SH-SY5Y cells were preincubated with 0.50 µM of [Pt(acac)2(DMS)] in absence or in presence of PD98056, rapamycin or LY294002. (B) Cell lysates were analysed by western blotting with anti-phosphorylated 6S, mTOR and ERK1/2 antibodies. Control loadings are shown by β-actin, representative immunoblots of four experiments are depicted and results from densitometry are expressed as mean ± SD (n = 4) of sum of the gray level values. (C) NHE activities, after acute exposure to an NH4Cl acid load, were measured and cell migration was examined using wound closure assay. Migration rate of wound closure were assessed by measuring the distance between wound edges in at least eight randomly chosen regions of three different experiments (average ± SD) normalized to 100% wound closure for control cells. Representative immunoblots of four experiments are depicted and results from densitometry are expressed as mean ± SD (n = 4) of sum of the gray level values. The data are means ± S.D. obtained from 4 different experiments. (A–D) P<0.0001 by one-way ANOVA (n = 4); values with shared letters are not significantly different according to Bonferroni/Dunn post hoc tests.
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pone-0112186-g003: Role of p70 S6 Kinase in [Pt(acac)2(DMS)] inhibition of SH-SY5Y cell migration.(A) SH-SY5Y cells were incubated or not with 0.50 µM [Pt(acac)2(DMS)] for the indicated times. Cell lysates were analysed by western blotting with anti- unphosphorylated (6S and mTOR) and phosphorylated (p-6S and p-mTOR) 6S and mTOR antibodies. (B, C and D) SH-SY5Y cells were preincubated with 0.50 µM of [Pt(acac)2(DMS)] in absence or in presence of PD98056, rapamycin or LY294002. (B) Cell lysates were analysed by western blotting with anti-phosphorylated 6S, mTOR and ERK1/2 antibodies. Control loadings are shown by β-actin, representative immunoblots of four experiments are depicted and results from densitometry are expressed as mean ± SD (n = 4) of sum of the gray level values. (C) NHE activities, after acute exposure to an NH4Cl acid load, were measured and cell migration was examined using wound closure assay. Migration rate of wound closure were assessed by measuring the distance between wound edges in at least eight randomly chosen regions of three different experiments (average ± SD) normalized to 100% wound closure for control cells. Representative immunoblots of four experiments are depicted and results from densitometry are expressed as mean ± SD (n = 4) of sum of the gray level values. The data are means ± S.D. obtained from 4 different experiments. (A–D) P<0.0001 by one-way ANOVA (n = 4); values with shared letters are not significantly different according to Bonferroni/Dunn post hoc tests.

Mentions: The role of p70S6K in cell migration [41] and in the regulation of NHE activity [42] has been established. We studied the phosphorylation of S6, a downstream component in mTOR signaling pathway, and used it as a marker for p70S6K activation. Phosphorylation of S6 was observed 1 min after [Pt(acac)2(DMS)] treatment reaching a maximum at 10 min (Fig. 3A). [Pt(acac)2(DMS)] had effects also on additional signaling components in the p70S6K pathways, i.e. increased mTOR phosphorylation, with the same kinetic of S6 phosphorylation, without changing the total mTOR level (Fig. 3A). PD98059 prevented the activations of both S6 and mTOR, suggesting that MEK/ERK pathway plays a role in the regulation of both proteins in cells challenged with [Pt(acac)2(DMS)] (Fig. 3B).


[Pt(O,O'-acac)(γ-acac)(DMS)] alters SH-SY5Y cell migration and invasion by the inhibition of Na+/H+ exchanger isoform 1 occurring through a PKC-ε/ERK/mTOR Pathway.

Muscella A, Vetrugno C, Calabriso N, Cossa LG, De Pascali SA, Fanizzi FP, Marsigliante S - PLoS ONE (2014)

Role of p70 S6 Kinase in [Pt(acac)2(DMS)] inhibition of SH-SY5Y cell migration.(A) SH-SY5Y cells were incubated or not with 0.50 µM [Pt(acac)2(DMS)] for the indicated times. Cell lysates were analysed by western blotting with anti- unphosphorylated (6S and mTOR) and phosphorylated (p-6S and p-mTOR) 6S and mTOR antibodies. (B, C and D) SH-SY5Y cells were preincubated with 0.50 µM of [Pt(acac)2(DMS)] in absence or in presence of PD98056, rapamycin or LY294002. (B) Cell lysates were analysed by western blotting with anti-phosphorylated 6S, mTOR and ERK1/2 antibodies. Control loadings are shown by β-actin, representative immunoblots of four experiments are depicted and results from densitometry are expressed as mean ± SD (n = 4) of sum of the gray level values. (C) NHE activities, after acute exposure to an NH4Cl acid load, were measured and cell migration was examined using wound closure assay. Migration rate of wound closure were assessed by measuring the distance between wound edges in at least eight randomly chosen regions of three different experiments (average ± SD) normalized to 100% wound closure for control cells. Representative immunoblots of four experiments are depicted and results from densitometry are expressed as mean ± SD (n = 4) of sum of the gray level values. The data are means ± S.D. obtained from 4 different experiments. (A–D) P<0.0001 by one-way ANOVA (n = 4); values with shared letters are not significantly different according to Bonferroni/Dunn post hoc tests.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4221608&req=5

pone-0112186-g003: Role of p70 S6 Kinase in [Pt(acac)2(DMS)] inhibition of SH-SY5Y cell migration.(A) SH-SY5Y cells were incubated or not with 0.50 µM [Pt(acac)2(DMS)] for the indicated times. Cell lysates were analysed by western blotting with anti- unphosphorylated (6S and mTOR) and phosphorylated (p-6S and p-mTOR) 6S and mTOR antibodies. (B, C and D) SH-SY5Y cells were preincubated with 0.50 µM of [Pt(acac)2(DMS)] in absence or in presence of PD98056, rapamycin or LY294002. (B) Cell lysates were analysed by western blotting with anti-phosphorylated 6S, mTOR and ERK1/2 antibodies. Control loadings are shown by β-actin, representative immunoblots of four experiments are depicted and results from densitometry are expressed as mean ± SD (n = 4) of sum of the gray level values. (C) NHE activities, after acute exposure to an NH4Cl acid load, were measured and cell migration was examined using wound closure assay. Migration rate of wound closure were assessed by measuring the distance between wound edges in at least eight randomly chosen regions of three different experiments (average ± SD) normalized to 100% wound closure for control cells. Representative immunoblots of four experiments are depicted and results from densitometry are expressed as mean ± SD (n = 4) of sum of the gray level values. The data are means ± S.D. obtained from 4 different experiments. (A–D) P<0.0001 by one-way ANOVA (n = 4); values with shared letters are not significantly different according to Bonferroni/Dunn post hoc tests.
Mentions: The role of p70S6K in cell migration [41] and in the regulation of NHE activity [42] has been established. We studied the phosphorylation of S6, a downstream component in mTOR signaling pathway, and used it as a marker for p70S6K activation. Phosphorylation of S6 was observed 1 min after [Pt(acac)2(DMS)] treatment reaching a maximum at 10 min (Fig. 3A). [Pt(acac)2(DMS)] had effects also on additional signaling components in the p70S6K pathways, i.e. increased mTOR phosphorylation, with the same kinetic of S6 phosphorylation, without changing the total mTOR level (Fig. 3A). PD98059 prevented the activations of both S6 and mTOR, suggesting that MEK/ERK pathway plays a role in the regulation of both proteins in cells challenged with [Pt(acac)2(DMS)] (Fig. 3B).

Bottom Line: We previously showed that [Pt(O,O'-acac)(γ-acac)(DMS)] ([Pt(acac)2(DMS)]) exerted substantial cytotoxic effects in SH-SY5Y neuroblastoma cells, and decreased metalloproteases (MMPs) production and cells migration in MCF-7 breast cancer cells.The present study focuses on the effects of [Pt(acac)2(DMS)] in SH-SY5Y cell migration and also on the possibility that NHE1 may be involved in such effect.In conclusion, we have shown a drastic impairment in tumour cell metastatization in response to inhibition of NHE1 and MMPs activities by [Pt(acac)2(DMS)] occurring through a novel mechanism mediated by PKC-δ/-ε activation.

View Article: PubMed Central - PubMed

Affiliation: Cell Pathology Lab, Dipartimento di Scienze e Tecnologie Biologiche e Ambientali (Di.S.Te.B.A.), Salento University, Lecce, Italy.

ABSTRACT
We previously showed that [Pt(O,O'-acac)(γ-acac)(DMS)] ([Pt(acac)2(DMS)]) exerted substantial cytotoxic effects in SH-SY5Y neuroblastoma cells, and decreased metalloproteases (MMPs) production and cells migration in MCF-7 breast cancer cells. The ubiquitously distributed sodium-hydrogen antiporter 1 (NHE1) is involved in motility and invasion of many solid tumours. The present study focuses on the effects of [Pt(acac)2(DMS)] in SH-SY5Y cell migration and also on the possibility that NHE1 may be involved in such effect. After sublethal [Pt(acac)2(DMS)] treatment cell migration was examined by wounding assay and cell invasion by transwell assay. NHE1 activity was measured in BCECF-loaded SH-SY5Y as the rate of Na+-dependent intracellular pH recovery in response to an acute acid pulse. Gelatin zymography for MMP-2/9 activities, Western blottings of MMPs, MAPKs, mTOR, S6 and PKCs and small interfering RNAs to PKC-ε/-δ mRNA were performed. Sublethal concentrations of [Pt(acac)2(DMS)] decreases NHE1 activity, inhibits cell migration and invasion and decreases expression and activity of MMP-2 and -9. [Pt(acac)2(DMS)] administered to SH-SY5Y cells provokes the increment of ROS, generated by NADPH oxidase, responsible for the PKC-ε and PKC-δ activation. Whilst PKC-δ activates p38/MAPK, responsible for the inhibition of MMP-2 and -9 secretion, PKC-ε activates a pathway made of ERK1/2, mTOR and S6K responsible for the inhibition of NHE1 activity and cell migration. In conclusion, we have shown a drastic impairment in tumour cell metastatization in response to inhibition of NHE1 and MMPs activities by [Pt(acac)2(DMS)] occurring through a novel mechanism mediated by PKC-δ/-ε activation.

Show MeSH
Related in: MedlinePlus