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Inhibition of Phosphatidylcholine-Specific Phospholipase C Interferes with Proliferation and Survival of Tumor Initiating Cells in Squamous Cell Carcinoma.

Cecchetti S, Bortolomai I, Ferri R, Mercurio L, Canevari S, Podo F, Miotti S, Iorio E - PLoS ONE (2015)

Bottom Line: Compared with A431-AD, A431-SPH cells showed about 2.8-fold lower PC-PLC protein and activity levels, but similar nuclear content.Exposure of adherent cells to the PC-PLC inhibitor D609 (48h) induced a 50% reduction of cell proliferation at doses comprised between 33 and 50 μg/ml, without inducing any relevant cytotoxic effect (cell viability 95±5%).Furthermore, D609 treatment of A431-AD and CaSki-AD cells affected the sphere-forming efficiency, which dropped in both cells, and induced down-modulation of stem-related markers mRNA levels (Oct4, Nestin, Nanog and ALDH1 in A431; Nestin and ALDH1 in CaSki cells).

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Rome, Italy.

ABSTRACT

Purpose: The role of phosphatidylcholine-specific phospholipase C (PC-PLC), the enzyme involved in cell differentiation and proliferation, has not yet been explored in tumor initiating cells (TICs). We investigated PC-PLC expression and effects of PC-PLC inhibition in two adherent (AD) squamous carcinoma cell lines (A431 and CaSki), with different proliferative and stemness potential, and in TIC-enriched floating spheres (SPH) originated from them.

Results: Compared with immortalized non-tumoral keratinocytes (HaCaT) A431-AD cells showed 2.5-fold higher PC-PLC activity, nuclear localization of a 66-kDa PC-PLC isoform, but a similar distribution of the enzyme on plasma membrane and in cytoplasmic compartments. Compared with A431-AD, A431-SPH cells showed about 2.8-fold lower PC-PLC protein and activity levels, but similar nuclear content. Exposure of adherent cells to the PC-PLC inhibitor D609 (48h) induced a 50% reduction of cell proliferation at doses comprised between 33 and 50 μg/ml, without inducing any relevant cytotoxic effect (cell viability 95±5%). In A431-SPH and CaSki-SPH D609 induced both cytostatic and cytotoxic effects at about 20 to 30-fold lower doses (IC50 ranging between 1.2 and 1.6 μg/ml). Furthermore, D609 treatment of A431-AD and CaSki-AD cells affected the sphere-forming efficiency, which dropped in both cells, and induced down-modulation of stem-related markers mRNA levels (Oct4, Nestin, Nanog and ALDH1 in A431; Nestin and ALDH1 in CaSki cells).

Conclusions: These data suggest that the inhibition of PC-PLC activity may represent a new therapeutic approach to selectively target the most aggressive and tumor promoting sub-population of floating spheres originated from squamous cancer cells possessing different proliferative and stemness potential.

No MeSH data available.


Related in: MedlinePlus

Effects of PC-PLC inhibition on EGFR, ERK and AKT phosphorylation in the HaCat and A431-AD cells.Representative Western blot analyses of total cell lysates from HaCaT (left panels) and A431-AD (right panels) cells cultured in the presence or absence of 50 μg/ml of D609. Cell lysates were immunoblotted with the following antibodies: pEGFR (Tyr1068), EGFR, pERK1/2 (Thr202/Tyr204), ERK 1/2, pAKT (Ser473), AKT and β-actin. β-actin was used as a quantitative loading control. Histograms below each panel represent the relative optical densities of phospho-protein levels normalized to the total protein level (mean values ± SD of three independent experiments) and are presented relative to the untreated sample at 24h. Statistical analyses were performed between treated samples (24h and 48 h) and untreated control sample at 24h, using the t-test.
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pone.0136120.g003: Effects of PC-PLC inhibition on EGFR, ERK and AKT phosphorylation in the HaCat and A431-AD cells.Representative Western blot analyses of total cell lysates from HaCaT (left panels) and A431-AD (right panels) cells cultured in the presence or absence of 50 μg/ml of D609. Cell lysates were immunoblotted with the following antibodies: pEGFR (Tyr1068), EGFR, pERK1/2 (Thr202/Tyr204), ERK 1/2, pAKT (Ser473), AKT and β-actin. β-actin was used as a quantitative loading control. Histograms below each panel represent the relative optical densities of phospho-protein levels normalized to the total protein level (mean values ± SD of three independent experiments) and are presented relative to the untreated sample at 24h. Statistical analyses were performed between treated samples (24h and 48 h) and untreated control sample at 24h, using the t-test.

Mentions: As shown in Fig 3, quantitative Western blot analyses performed on total cell lysates showed that the level of EGFR phosphorylation (Y1068 residue) was substantially reduced by D609 in A431-AD cells at 24h (to about 72%, P = 0.039) and 48h (to about 62%, P = 0.002), while no effect was observed on the total level of EGFR protein. On the contrary, in HaCaT cells the phosphorylated Y1068 form of EGFR was not affected by D609, whereas the total level of the EGFR protein decreased (Fig 3, top pair of panels). In both cell lines the relative phosphorylation of ERK1/2 (pERK/total ERK ratio) was decreased to about 50% at 24h and persisted up to 48h of D609 exposure (Fig 3, middle pair of panels, P = 0.002). Compared with HaCaT, A431-AD cells showed a higher AKT content. A similar percent decrease in the relative AKT phosphorylation however occurred in the two cell lines at 48h (pAKT/total AKT ratio reduced to 50% in the HaCat, P = 0.005, and to 60% in the A431-AD cells, P = 0.001) but not at 24h of D609 treatment (Fig 3, bottom pair of panels).


Inhibition of Phosphatidylcholine-Specific Phospholipase C Interferes with Proliferation and Survival of Tumor Initiating Cells in Squamous Cell Carcinoma.

Cecchetti S, Bortolomai I, Ferri R, Mercurio L, Canevari S, Podo F, Miotti S, Iorio E - PLoS ONE (2015)

Effects of PC-PLC inhibition on EGFR, ERK and AKT phosphorylation in the HaCat and A431-AD cells.Representative Western blot analyses of total cell lysates from HaCaT (left panels) and A431-AD (right panels) cells cultured in the presence or absence of 50 μg/ml of D609. Cell lysates were immunoblotted with the following antibodies: pEGFR (Tyr1068), EGFR, pERK1/2 (Thr202/Tyr204), ERK 1/2, pAKT (Ser473), AKT and β-actin. β-actin was used as a quantitative loading control. Histograms below each panel represent the relative optical densities of phospho-protein levels normalized to the total protein level (mean values ± SD of three independent experiments) and are presented relative to the untreated sample at 24h. Statistical analyses were performed between treated samples (24h and 48 h) and untreated control sample at 24h, using the t-test.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581859&req=5

pone.0136120.g003: Effects of PC-PLC inhibition on EGFR, ERK and AKT phosphorylation in the HaCat and A431-AD cells.Representative Western blot analyses of total cell lysates from HaCaT (left panels) and A431-AD (right panels) cells cultured in the presence or absence of 50 μg/ml of D609. Cell lysates were immunoblotted with the following antibodies: pEGFR (Tyr1068), EGFR, pERK1/2 (Thr202/Tyr204), ERK 1/2, pAKT (Ser473), AKT and β-actin. β-actin was used as a quantitative loading control. Histograms below each panel represent the relative optical densities of phospho-protein levels normalized to the total protein level (mean values ± SD of three independent experiments) and are presented relative to the untreated sample at 24h. Statistical analyses were performed between treated samples (24h and 48 h) and untreated control sample at 24h, using the t-test.
Mentions: As shown in Fig 3, quantitative Western blot analyses performed on total cell lysates showed that the level of EGFR phosphorylation (Y1068 residue) was substantially reduced by D609 in A431-AD cells at 24h (to about 72%, P = 0.039) and 48h (to about 62%, P = 0.002), while no effect was observed on the total level of EGFR protein. On the contrary, in HaCaT cells the phosphorylated Y1068 form of EGFR was not affected by D609, whereas the total level of the EGFR protein decreased (Fig 3, top pair of panels). In both cell lines the relative phosphorylation of ERK1/2 (pERK/total ERK ratio) was decreased to about 50% at 24h and persisted up to 48h of D609 exposure (Fig 3, middle pair of panels, P = 0.002). Compared with HaCaT, A431-AD cells showed a higher AKT content. A similar percent decrease in the relative AKT phosphorylation however occurred in the two cell lines at 48h (pAKT/total AKT ratio reduced to 50% in the HaCat, P = 0.005, and to 60% in the A431-AD cells, P = 0.001) but not at 24h of D609 treatment (Fig 3, bottom pair of panels).

Bottom Line: Compared with A431-AD, A431-SPH cells showed about 2.8-fold lower PC-PLC protein and activity levels, but similar nuclear content.Exposure of adherent cells to the PC-PLC inhibitor D609 (48h) induced a 50% reduction of cell proliferation at doses comprised between 33 and 50 μg/ml, without inducing any relevant cytotoxic effect (cell viability 95±5%).Furthermore, D609 treatment of A431-AD and CaSki-AD cells affected the sphere-forming efficiency, which dropped in both cells, and induced down-modulation of stem-related markers mRNA levels (Oct4, Nestin, Nanog and ALDH1 in A431; Nestin and ALDH1 in CaSki cells).

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Rome, Italy.

ABSTRACT

Purpose: The role of phosphatidylcholine-specific phospholipase C (PC-PLC), the enzyme involved in cell differentiation and proliferation, has not yet been explored in tumor initiating cells (TICs). We investigated PC-PLC expression and effects of PC-PLC inhibition in two adherent (AD) squamous carcinoma cell lines (A431 and CaSki), with different proliferative and stemness potential, and in TIC-enriched floating spheres (SPH) originated from them.

Results: Compared with immortalized non-tumoral keratinocytes (HaCaT) A431-AD cells showed 2.5-fold higher PC-PLC activity, nuclear localization of a 66-kDa PC-PLC isoform, but a similar distribution of the enzyme on plasma membrane and in cytoplasmic compartments. Compared with A431-AD, A431-SPH cells showed about 2.8-fold lower PC-PLC protein and activity levels, but similar nuclear content. Exposure of adherent cells to the PC-PLC inhibitor D609 (48h) induced a 50% reduction of cell proliferation at doses comprised between 33 and 50 μg/ml, without inducing any relevant cytotoxic effect (cell viability 95±5%). In A431-SPH and CaSki-SPH D609 induced both cytostatic and cytotoxic effects at about 20 to 30-fold lower doses (IC50 ranging between 1.2 and 1.6 μg/ml). Furthermore, D609 treatment of A431-AD and CaSki-AD cells affected the sphere-forming efficiency, which dropped in both cells, and induced down-modulation of stem-related markers mRNA levels (Oct4, Nestin, Nanog and ALDH1 in A431; Nestin and ALDH1 in CaSki cells).

Conclusions: These data suggest that the inhibition of PC-PLC activity may represent a new therapeutic approach to selectively target the most aggressive and tumor promoting sub-population of floating spheres originated from squamous cancer cells possessing different proliferative and stemness potential.

No MeSH data available.


Related in: MedlinePlus