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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 NAD(P)H oxidase in [Pt(acac)2(DMS)] inhibition of SH-SY5Y cell migration and invasion.(A) SH-SY5Y cells were treated without or with 0.50 µM [Pt(acac)2(DMS)] for the indicated times. For PKCs translocation studies, cytosol (cyt) and membrane (mem) fractions were analysed by Western blotting with specific antibodies. The purity of fractions was tested with anti β-actin and anti-α subunit of Na+/K+ ATPase monoclonal antibodies. The figures are representative of four independent experiments and results from densitometry are expressed as mean ± SD (n = 4) of sum of the gray level values. (B–E) SH-SY5Y cells were pre incubated or not with different concentration of DPI and then treated with 0.50 µM [Pt(acac)2(DMS)]. (B) Membrane fractions or cell lysates were analysed by Western blotting with specific antibodies. Control loadings are shown by β-actin and representative immunoblots are depicted; results from densitometry are expressed as mean ± SD (n = 4) of sum of the gray level values. (C) NHE1 activities, after acute exposure to an NH4Cl acid load, were measured, (D) cell migration was examined using wound closure assay and (E) cell invasion was measured using the transwell invasion assay system. The data are means ± S.D. obtained from 4 different experiments. (A–E) 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-g006: Role of NAD(P)H oxidase in [Pt(acac)2(DMS)] inhibition of SH-SY5Y cell migration and invasion.(A) SH-SY5Y cells were treated without or with 0.50 µM [Pt(acac)2(DMS)] for the indicated times. For PKCs translocation studies, cytosol (cyt) and membrane (mem) fractions were analysed by Western blotting with specific antibodies. The purity of fractions was tested with anti β-actin and anti-α subunit of Na+/K+ ATPase monoclonal antibodies. The figures are representative of four independent experiments and results from densitometry are expressed as mean ± SD (n = 4) of sum of the gray level values. (B–E) SH-SY5Y cells were pre incubated or not with different concentration of DPI and then treated with 0.50 µM [Pt(acac)2(DMS)]. (B) Membrane fractions or cell lysates were analysed by Western blotting with specific antibodies. Control loadings are shown by β-actin and representative immunoblots are depicted; results from densitometry are expressed as mean ± SD (n = 4) of sum of the gray level values. (C) NHE1 activities, after acute exposure to an NH4Cl acid load, were measured, (D) cell migration was examined using wound closure assay and (E) cell invasion was measured using the transwell invasion assay system. The data are means ± S.D. obtained from 4 different experiments. (A–E) 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: Previous observations indicated that some ROS-mediated events, initiated by [Pt(acac)2(DMS)], led to inhibition of migration of mammary tumour cells [33]. Here, the NADPH oxidase specific ihnibitor DPI was able to inhibit the cytosol-to-membrane translocation of PKC-ε and PKC-δ and the ERK1/2 and p38MAPK phosphorylation (Fig. 6B). DPI also markedly suppressed [Pt(acac)2(DMS)] inhibition of MMP-2 and MMP-9. In addition, the effects of [Pt(acac)2(DMS)] on NHE1 activity (Fig. 6C), wound-healing (Fig. 6D) and transwell invasion (Fig. 6E) were reversed by the pretreatments of cells with DPI.


[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 NAD(P)H oxidase in [Pt(acac)2(DMS)] inhibition of SH-SY5Y cell migration and invasion.(A) SH-SY5Y cells were treated without or with 0.50 µM [Pt(acac)2(DMS)] for the indicated times. For PKCs translocation studies, cytosol (cyt) and membrane (mem) fractions were analysed by Western blotting with specific antibodies. The purity of fractions was tested with anti β-actin and anti-α subunit of Na+/K+ ATPase monoclonal antibodies. The figures are representative of four independent experiments and results from densitometry are expressed as mean ± SD (n = 4) of sum of the gray level values. (B–E) SH-SY5Y cells were pre incubated or not with different concentration of DPI and then treated with 0.50 µM [Pt(acac)2(DMS)]. (B) Membrane fractions or cell lysates were analysed by Western blotting with specific antibodies. Control loadings are shown by β-actin and representative immunoblots are depicted; results from densitometry are expressed as mean ± SD (n = 4) of sum of the gray level values. (C) NHE1 activities, after acute exposure to an NH4Cl acid load, were measured, (D) cell migration was examined using wound closure assay and (E) cell invasion was measured using the transwell invasion assay system. The data are means ± S.D. obtained from 4 different experiments. (A–E) 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-g006: Role of NAD(P)H oxidase in [Pt(acac)2(DMS)] inhibition of SH-SY5Y cell migration and invasion.(A) SH-SY5Y cells were treated without or with 0.50 µM [Pt(acac)2(DMS)] for the indicated times. For PKCs translocation studies, cytosol (cyt) and membrane (mem) fractions were analysed by Western blotting with specific antibodies. The purity of fractions was tested with anti β-actin and anti-α subunit of Na+/K+ ATPase monoclonal antibodies. The figures are representative of four independent experiments and results from densitometry are expressed as mean ± SD (n = 4) of sum of the gray level values. (B–E) SH-SY5Y cells were pre incubated or not with different concentration of DPI and then treated with 0.50 µM [Pt(acac)2(DMS)]. (B) Membrane fractions or cell lysates were analysed by Western blotting with specific antibodies. Control loadings are shown by β-actin and representative immunoblots are depicted; results from densitometry are expressed as mean ± SD (n = 4) of sum of the gray level values. (C) NHE1 activities, after acute exposure to an NH4Cl acid load, were measured, (D) cell migration was examined using wound closure assay and (E) cell invasion was measured using the transwell invasion assay system. The data are means ± S.D. obtained from 4 different experiments. (A–E) 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: Previous observations indicated that some ROS-mediated events, initiated by [Pt(acac)2(DMS)], led to inhibition of migration of mammary tumour cells [33]. Here, the NADPH oxidase specific ihnibitor DPI was able to inhibit the cytosol-to-membrane translocation of PKC-ε and PKC-δ and the ERK1/2 and p38MAPK phosphorylation (Fig. 6B). DPI also markedly suppressed [Pt(acac)2(DMS)] inhibition of MMP-2 and MMP-9. In addition, the effects of [Pt(acac)2(DMS)] on NHE1 activity (Fig. 6C), wound-healing (Fig. 6D) and transwell invasion (Fig. 6E) were reversed by the pretreatments of cells with DPI.

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