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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

PC-PLC protein expression and activity in the non-tumoral keratinocyte HaCaT and in the A431-AD squamous carcinoma cell lines.Subcellular localization of PC-PLC (grey) on the plasma membrane of unfixed cells (A) and in different cellular compartments of fixed and permeabilized cells (B) detected by CLSM analyses. Single central optical sections are shown. Nuclei were stained with DAPI (blue). Scale bars, 20 μm. C) Western Blot analysis of the relative PC-PLC protein expression in HaCaT and A431-AD total cell lysates (left) and in their cytoplasmic and nuclear fractions (right). Nucleoporin and β-actin were used to ensure the quality of fractions’ separation and protein quantitative loading, respectively. D) Absolute PC-PLC activity (pmoles/μg protein x min; mean ± SD, n = 6) measured by Amplex Red assay in total cell lysates. P = 0.0001.
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pone.0136120.g001: PC-PLC protein expression and activity in the non-tumoral keratinocyte HaCaT and in the A431-AD squamous carcinoma cell lines.Subcellular localization of PC-PLC (grey) on the plasma membrane of unfixed cells (A) and in different cellular compartments of fixed and permeabilized cells (B) detected by CLSM analyses. Single central optical sections are shown. Nuclei were stained with DAPI (blue). Scale bars, 20 μm. C) Western Blot analysis of the relative PC-PLC protein expression in HaCaT and A431-AD total cell lysates (left) and in their cytoplasmic and nuclear fractions (right). Nucleoporin and β-actin were used to ensure the quality of fractions’ separation and protein quantitative loading, respectively. D) Absolute PC-PLC activity (pmoles/μg protein x min; mean ± SD, n = 6) measured by Amplex Red assay in total cell lysates. P = 0.0001.

Mentions: In a study aimed at the characterization of genomic and metabolic profiles of normal keratinocytes and SCC cell lines, we found that the immortalized keratinocyte HaCaT cells exhibit a gene profiling similar to normal keratinocytes (5 samples) and distinct from that of three human SCC (A431, CaSki and SiHa) (Bortolomai I, personal communication), as evaluated by unsupervised hierarchical clustering (see representative dendrogram in S1 Fig). Thus, PC-PLC protein expression was measured in A431-AD cells, as a typical model of SCC, and in HaCaT cells, as a representative model of normal epithelial cells. Confocal Laser Scanning Microscopy (CLSM) analyses of either unfixed or fixed and permeabilized cells showed that PC-PLC is similarly distributed on the plasma membrane (Fig 1A) and in cytoplasmic compartments (Fig 1B) of HaCaT and A431-AD cell lines. PC-PLC also localized as large granules inside the nucleus of A431-AD cancer cells, whereas no PC-PLC-positivity was detected in HaCaT nuclei (Fig 1B). Western blot analysis of sub-cellular fractions further demonstrated the nuclear localization of a PC-PLC isoform (66 kDa) in A431-AD cancer cells only (Fig 1C, right panel). The PC-PLC enzymatic activity, measured with Amplex Red assay in total lysates of cells harvested at early confluence, was about 2.5 fold higher in the A431-AD than in HaCaT cells (P = 0.0001; Fig 1D).


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)

PC-PLC protein expression and activity in the non-tumoral keratinocyte HaCaT and in the A431-AD squamous carcinoma cell lines.Subcellular localization of PC-PLC (grey) on the plasma membrane of unfixed cells (A) and in different cellular compartments of fixed and permeabilized cells (B) detected by CLSM analyses. Single central optical sections are shown. Nuclei were stained with DAPI (blue). Scale bars, 20 μm. C) Western Blot analysis of the relative PC-PLC protein expression in HaCaT and A431-AD total cell lysates (left) and in their cytoplasmic and nuclear fractions (right). Nucleoporin and β-actin were used to ensure the quality of fractions’ separation and protein quantitative loading, respectively. D) Absolute PC-PLC activity (pmoles/μg protein x min; mean ± SD, n = 6) measured by Amplex Red assay in total cell lysates. P = 0.0001.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0136120.g001: PC-PLC protein expression and activity in the non-tumoral keratinocyte HaCaT and in the A431-AD squamous carcinoma cell lines.Subcellular localization of PC-PLC (grey) on the plasma membrane of unfixed cells (A) and in different cellular compartments of fixed and permeabilized cells (B) detected by CLSM analyses. Single central optical sections are shown. Nuclei were stained with DAPI (blue). Scale bars, 20 μm. C) Western Blot analysis of the relative PC-PLC protein expression in HaCaT and A431-AD total cell lysates (left) and in their cytoplasmic and nuclear fractions (right). Nucleoporin and β-actin were used to ensure the quality of fractions’ separation and protein quantitative loading, respectively. D) Absolute PC-PLC activity (pmoles/μg protein x min; mean ± SD, n = 6) measured by Amplex Red assay in total cell lysates. P = 0.0001.
Mentions: In a study aimed at the characterization of genomic and metabolic profiles of normal keratinocytes and SCC cell lines, we found that the immortalized keratinocyte HaCaT cells exhibit a gene profiling similar to normal keratinocytes (5 samples) and distinct from that of three human SCC (A431, CaSki and SiHa) (Bortolomai I, personal communication), as evaluated by unsupervised hierarchical clustering (see representative dendrogram in S1 Fig). Thus, PC-PLC protein expression was measured in A431-AD cells, as a typical model of SCC, and in HaCaT cells, as a representative model of normal epithelial cells. Confocal Laser Scanning Microscopy (CLSM) analyses of either unfixed or fixed and permeabilized cells showed that PC-PLC is similarly distributed on the plasma membrane (Fig 1A) and in cytoplasmic compartments (Fig 1B) of HaCaT and A431-AD cell lines. PC-PLC also localized as large granules inside the nucleus of A431-AD cancer cells, whereas no PC-PLC-positivity was detected in HaCaT nuclei (Fig 1B). Western blot analysis of sub-cellular fractions further demonstrated the nuclear localization of a PC-PLC isoform (66 kDa) in A431-AD cancer cells only (Fig 1C, right panel). The PC-PLC enzymatic activity, measured with Amplex Red assay in total lysates of cells harvested at early confluence, was about 2.5 fold higher in the A431-AD than in HaCaT cells (P = 0.0001; Fig 1D).

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