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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

Comparison of PC-PLC protein expression, activity and cellular localization in A431-AD and in a model of cancer-initiating A431-SPH.A) Western blot analysis of PC-PLC relative levels in total cell lysates (left panels) and in the cytoplasmic and nuclear fractions (right panels). Nucleoporin and actin were used to ensure the quality of fractions separation and protein quantitative loading control, respectively. Densitometric analyses were performed and results (mean ± SD of three independent experiments) are shown as relative fold change of PC-PLC protein levels normalized to the actin (for total lysates and cytoplasmic fractions) or nucleoporin (for nuclear fractions) levels. Data are presented relative to the A431-AD cell line. B) PC-PLC activity (mean ± SD, n = 3) measured by Amplex Red assay in total cell lysates. P = 0.002. C, D) CSLM analyses of the single central optical section (top panels) and Z-projection of 25 optical sections taken from the bottom to the edge (3-D reconstruction, bottom panels) of the A431 sphere cells cultured for 72 hours, then fixed, permeabilized and stained for PC-PLC (green), EGFR (red) and nuclei (blue) detection. Scale bar, 40 μm.
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pone.0136120.g004: Comparison of PC-PLC protein expression, activity and cellular localization in A431-AD and in a model of cancer-initiating A431-SPH.A) Western blot analysis of PC-PLC relative levels in total cell lysates (left panels) and in the cytoplasmic and nuclear fractions (right panels). Nucleoporin and actin were used to ensure the quality of fractions separation and protein quantitative loading control, respectively. Densitometric analyses were performed and results (mean ± SD of three independent experiments) are shown as relative fold change of PC-PLC protein levels normalized to the actin (for total lysates and cytoplasmic fractions) or nucleoporin (for nuclear fractions) levels. Data are presented relative to the A431-AD cell line. B) PC-PLC activity (mean ± SD, n = 3) measured by Amplex Red assay in total cell lysates. P = 0.002. C, D) CSLM analyses of the single central optical section (top panels) and Z-projection of 25 optical sections taken from the bottom to the edge (3-D reconstruction, bottom panels) of the A431 sphere cells cultured for 72 hours, then fixed, permeabilized and stained for PC-PLC (green), EGFR (red) and nuclei (blue) detection. Scale bar, 40 μm.

Mentions: The A431-SPH cells were first analyzed for PC-PLC protein expression level, subcellular localization and activity. Comparison of Western blot analyses of total cell lysates and subcellular fractions of A431-AD and A431-SPH cells showed that the overall PC-PLC content was lower in the spheres (fold change 0.6 ± 0.1), and its subcellular distribution indicates that the main differences between A431-AD and A431-SPH rely exclusively on the nuclear fraction (fold change in A431-SPH 0.7 ± 0.1) (Fig 4A). In substantial agreement with the difference in the PC-PLC protein level, Amplex Red assays showed a significant 2.8 ± 0.2 fold lower enzymatic activity in A431-SPH than in A431-AD cells (Fig 4B, P = 0.002). CLSM analyses on fixed and permeabilized A431-SPH cells showed that PC-PLC was expressed to a substantial level only in a subset of cells constituting the sphere (Fig 4C). Since it is well known that the A431-AD cells are characterized by over-expression of EGFR, we also evaluated the expression of this receptor on the A431-SPH cells. Interestingly, the PC-PLC+ subset also showed a remarkable degree of co-localization between the enzyme and the EGFR receptor (Fig 4D), suggesting that PC-PLC might play an important role in regulating the EGFR-driven oncogene signaling in this cellular component.


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)

Comparison of PC-PLC protein expression, activity and cellular localization in A431-AD and in a model of cancer-initiating A431-SPH.A) Western blot analysis of PC-PLC relative levels in total cell lysates (left panels) and in the cytoplasmic and nuclear fractions (right panels). Nucleoporin and actin were used to ensure the quality of fractions separation and protein quantitative loading control, respectively. Densitometric analyses were performed and results (mean ± SD of three independent experiments) are shown as relative fold change of PC-PLC protein levels normalized to the actin (for total lysates and cytoplasmic fractions) or nucleoporin (for nuclear fractions) levels. Data are presented relative to the A431-AD cell line. B) PC-PLC activity (mean ± SD, n = 3) measured by Amplex Red assay in total cell lysates. P = 0.002. C, D) CSLM analyses of the single central optical section (top panels) and Z-projection of 25 optical sections taken from the bottom to the edge (3-D reconstruction, bottom panels) of the A431 sphere cells cultured for 72 hours, then fixed, permeabilized and stained for PC-PLC (green), EGFR (red) and nuclei (blue) detection. Scale bar, 40 μm.
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Related In: Results  -  Collection

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pone.0136120.g004: Comparison of PC-PLC protein expression, activity and cellular localization in A431-AD and in a model of cancer-initiating A431-SPH.A) Western blot analysis of PC-PLC relative levels in total cell lysates (left panels) and in the cytoplasmic and nuclear fractions (right panels). Nucleoporin and actin were used to ensure the quality of fractions separation and protein quantitative loading control, respectively. Densitometric analyses were performed and results (mean ± SD of three independent experiments) are shown as relative fold change of PC-PLC protein levels normalized to the actin (for total lysates and cytoplasmic fractions) or nucleoporin (for nuclear fractions) levels. Data are presented relative to the A431-AD cell line. B) PC-PLC activity (mean ± SD, n = 3) measured by Amplex Red assay in total cell lysates. P = 0.002. C, D) CSLM analyses of the single central optical section (top panels) and Z-projection of 25 optical sections taken from the bottom to the edge (3-D reconstruction, bottom panels) of the A431 sphere cells cultured for 72 hours, then fixed, permeabilized and stained for PC-PLC (green), EGFR (red) and nuclei (blue) detection. Scale bar, 40 μm.
Mentions: The A431-SPH cells were first analyzed for PC-PLC protein expression level, subcellular localization and activity. Comparison of Western blot analyses of total cell lysates and subcellular fractions of A431-AD and A431-SPH cells showed that the overall PC-PLC content was lower in the spheres (fold change 0.6 ± 0.1), and its subcellular distribution indicates that the main differences between A431-AD and A431-SPH rely exclusively on the nuclear fraction (fold change in A431-SPH 0.7 ± 0.1) (Fig 4A). In substantial agreement with the difference in the PC-PLC protein level, Amplex Red assays showed a significant 2.8 ± 0.2 fold lower enzymatic activity in A431-SPH than in A431-AD cells (Fig 4B, P = 0.002). CLSM analyses on fixed and permeabilized A431-SPH cells showed that PC-PLC was expressed to a substantial level only in a subset of cells constituting the sphere (Fig 4C). Since it is well known that the A431-AD cells are characterized by over-expression of EGFR, we also evaluated the expression of this receptor on the A431-SPH cells. Interestingly, the PC-PLC+ subset also showed a remarkable degree of co-localization between the enzyme and the EGFR receptor (Fig 4D), suggesting that PC-PLC might play an important role in regulating the EGFR-driven oncogene signaling in this cellular component.

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