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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 in A431-AD and CasKi-AD cells and effects of D609 on cell proliferation and stemness potential of CaSki cells.A) Western blot analyses of the relative PC-PLC protein expression in A431-AD and CaSki-AD total cell lysates. β-actin was used as quantitative loading control. Densitometric analyses were performed and results are shown as relative fold change of PC-PLC protein levels normalized to the actin level. Data represent the mean (± SD) of three independent experiments, and are presented relative to the A431-AD cell line. B) Proliferation assays performed on CaSki-AD cells seeded 72 hours before adding different doses of D609 at t = 0 (● = untreated cells; □ = 1.5 μg/ml, ▲ = 3 μg/ml, ▼ = 6 μg/ml, ◇ = 12.5 μg/ml, * = 25 μg/ml and ○ = 50 μg/ml) and monitored for 24h and 48h afterwards. Cell counting (mean ± SD, n = 3) of total present cells (left panel) and of live (white columns) and dead (black columns) cells (right panel) measured by Trypan blue exclusion test and by automated cell counter, as described in the Material and Methods section. C) CaSki-SPH cells were seeded 72 hours before adding different doses of D609 at t = 0 (● = untreated cells; □ = 1.5 μg/ml, ▲ = 3 μg/ml, ▼ = 6 μg/ml, ◇ = 12.5 μg/ml, * = 25 μg/ml and ○ = 50 μg/ml) and monitored for 24h and 48h afterwards. Cell counts (mean ± SD, n = 3) of total present cells (left panel) and of alive (white columns) and dead (black columns) cells (right panel) measured by Trypan blue exclusion test. D) Sphere Formation Efficiency (SFE) of CaSki-AD cells untreated (white column) or treated with 50 μg/ml D609 for 24h (black column) or 48h (grey column) (mean ± SD; n = 2). Statistical analyses were performed using one-way ANOVA, P = 0.064. E) Real Time PCR analyses of the stemness markers OCT4, NANOG, NESTIN (left panel) and ALDH1 (right panel) expressed by the CaSki-AD cell line either untreated (white columns) or in the presence of D609 (50 μg/ml, black columns) for 48h. Statistical analyses were performed using t-test. (OCT4, P = 0.301; NANOG, P = 0.413; NESTIN, P = 0.173; ALDH1, P = 0.019).
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pone.0136120.g006: Comparison of PC-PLC protein expression in A431-AD and CasKi-AD cells and effects of D609 on cell proliferation and stemness potential of CaSki cells.A) Western blot analyses of the relative PC-PLC protein expression in A431-AD and CaSki-AD total cell lysates. β-actin was used as quantitative loading control. Densitometric analyses were performed and results are shown as relative fold change of PC-PLC protein levels normalized to the actin level. Data represent the mean (± SD) of three independent experiments, and are presented relative to the A431-AD cell line. B) Proliferation assays performed on CaSki-AD cells seeded 72 hours before adding different doses of D609 at t = 0 (● = untreated cells; □ = 1.5 μg/ml, ▲ = 3 μg/ml, ▼ = 6 μg/ml, ◇ = 12.5 μg/ml, * = 25 μg/ml and ○ = 50 μg/ml) and monitored for 24h and 48h afterwards. Cell counting (mean ± SD, n = 3) of total present cells (left panel) and of live (white columns) and dead (black columns) cells (right panel) measured by Trypan blue exclusion test and by automated cell counter, as described in the Material and Methods section. C) CaSki-SPH cells were seeded 72 hours before adding different doses of D609 at t = 0 (● = untreated cells; □ = 1.5 μg/ml, ▲ = 3 μg/ml, ▼ = 6 μg/ml, ◇ = 12.5 μg/ml, * = 25 μg/ml and ○ = 50 μg/ml) and monitored for 24h and 48h afterwards. Cell counts (mean ± SD, n = 3) of total present cells (left panel) and of alive (white columns) and dead (black columns) cells (right panel) measured by Trypan blue exclusion test. D) Sphere Formation Efficiency (SFE) of CaSki-AD cells untreated (white column) or treated with 50 μg/ml D609 for 24h (black column) or 48h (grey column) (mean ± SD; n = 2). Statistical analyses were performed using one-way ANOVA, P = 0.064. E) Real Time PCR analyses of the stemness markers OCT4, NANOG, NESTIN (left panel) and ALDH1 (right panel) expressed by the CaSki-AD cell line either untreated (white columns) or in the presence of D609 (50 μg/ml, black columns) for 48h. Statistical analyses were performed using t-test. (OCT4, P = 0.301; NANOG, P = 0.413; NESTIN, P = 0.173; ALDH1, P = 0.019).

Mentions: PC-PLC protein expression, detected by Western Blot analyses, was much lower in CaSki-AD cells (fold change 0.5 ± 0.1) than in A431-AD cells (Fig 6A). Even though the proliferation rate of this cell line was lower than that of A431-AD cells, the inhibition of PC-PLC by D609 was able to induce a substantial cell growth arrest (55% of reduction) under appropriate conditions, i.e at the dose of 50 μg/ml and after 48h of treatment (Fig 6B, left panel). This anti-proliferative effect was similar to that observed in the HaCaT cell line exposed to D609. As indicated by the Trypan blue exclusion assay, the inhibition of PC-PLC did not exert any cytotoxic effect on CaSki-AD cells (Fig 6B, right panel). As previously shown by our group, CaSki cells were also able to grow in suspension thus forming spheres but, in comparison with the A431, the CaSki cell line tumor-initiating potential was much lower [5]. CaSki-SPH were exposed to D609 under the same conditions used for the previous experiments on A431-SPH. Fig 6C (left panel) shows that D609 at the dose of 3 μg/ml, the same dose effective in A431-SPH, was once again extremely potent in inhibiting the proliferation of CaSki-SPH cells (IC50 corresponding to 1.2 μg/ml), also inducing a strong cytotoxic effect (Fig 6C, right panel). As for A431-AD cells, we evaluated the stemness potential of CaSki-AD cells after exposure to D609 (50 μg/ml). After 24h of treatment CaSki-AD cells showed a small decrease in the sphere forming efficiency (SFE = 32.6 ± 7.2% compared with 43.4 ± 7.2% measured in untreated cells). A clear, although only border-line significant effect was detected in cells exposed to D609 for 48h, in which SFE (19.2 ± 13.3%) was 2.3-fold lower than that of untreated cells, and 1.7-fold lower than that observed after 24h of treatment (P = 0.064) (Fig 6D). Real Time PCR performed on untreated CaSki-AD mRNA revealed low expression levels of stem-related markers (Fig 6E). Real Time PCR carried out 48h after D609 treatment showed that the drug did not significantly change the expression level of OCT4 and NANOG stem genes, but it was associated with a significant reduction in ALDH1 expression (P = 0.019) and also with a trend towards a decrease in NESTIN.


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 in A431-AD and CasKi-AD cells and effects of D609 on cell proliferation and stemness potential of CaSki cells.A) Western blot analyses of the relative PC-PLC protein expression in A431-AD and CaSki-AD total cell lysates. β-actin was used as quantitative loading control. Densitometric analyses were performed and results are shown as relative fold change of PC-PLC protein levels normalized to the actin level. Data represent the mean (± SD) of three independent experiments, and are presented relative to the A431-AD cell line. B) Proliferation assays performed on CaSki-AD cells seeded 72 hours before adding different doses of D609 at t = 0 (● = untreated cells; □ = 1.5 μg/ml, ▲ = 3 μg/ml, ▼ = 6 μg/ml, ◇ = 12.5 μg/ml, * = 25 μg/ml and ○ = 50 μg/ml) and monitored for 24h and 48h afterwards. Cell counting (mean ± SD, n = 3) of total present cells (left panel) and of live (white columns) and dead (black columns) cells (right panel) measured by Trypan blue exclusion test and by automated cell counter, as described in the Material and Methods section. C) CaSki-SPH cells were seeded 72 hours before adding different doses of D609 at t = 0 (● = untreated cells; □ = 1.5 μg/ml, ▲ = 3 μg/ml, ▼ = 6 μg/ml, ◇ = 12.5 μg/ml, * = 25 μg/ml and ○ = 50 μg/ml) and monitored for 24h and 48h afterwards. Cell counts (mean ± SD, n = 3) of total present cells (left panel) and of alive (white columns) and dead (black columns) cells (right panel) measured by Trypan blue exclusion test. D) Sphere Formation Efficiency (SFE) of CaSki-AD cells untreated (white column) or treated with 50 μg/ml D609 for 24h (black column) or 48h (grey column) (mean ± SD; n = 2). Statistical analyses were performed using one-way ANOVA, P = 0.064. E) Real Time PCR analyses of the stemness markers OCT4, NANOG, NESTIN (left panel) and ALDH1 (right panel) expressed by the CaSki-AD cell line either untreated (white columns) or in the presence of D609 (50 μg/ml, black columns) for 48h. Statistical analyses were performed using t-test. (OCT4, P = 0.301; NANOG, P = 0.413; NESTIN, P = 0.173; ALDH1, P = 0.019).
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pone.0136120.g006: Comparison of PC-PLC protein expression in A431-AD and CasKi-AD cells and effects of D609 on cell proliferation and stemness potential of CaSki cells.A) Western blot analyses of the relative PC-PLC protein expression in A431-AD and CaSki-AD total cell lysates. β-actin was used as quantitative loading control. Densitometric analyses were performed and results are shown as relative fold change of PC-PLC protein levels normalized to the actin level. Data represent the mean (± SD) of three independent experiments, and are presented relative to the A431-AD cell line. B) Proliferation assays performed on CaSki-AD cells seeded 72 hours before adding different doses of D609 at t = 0 (● = untreated cells; □ = 1.5 μg/ml, ▲ = 3 μg/ml, ▼ = 6 μg/ml, ◇ = 12.5 μg/ml, * = 25 μg/ml and ○ = 50 μg/ml) and monitored for 24h and 48h afterwards. Cell counting (mean ± SD, n = 3) of total present cells (left panel) and of live (white columns) and dead (black columns) cells (right panel) measured by Trypan blue exclusion test and by automated cell counter, as described in the Material and Methods section. C) CaSki-SPH cells were seeded 72 hours before adding different doses of D609 at t = 0 (● = untreated cells; □ = 1.5 μg/ml, ▲ = 3 μg/ml, ▼ = 6 μg/ml, ◇ = 12.5 μg/ml, * = 25 μg/ml and ○ = 50 μg/ml) and monitored for 24h and 48h afterwards. Cell counts (mean ± SD, n = 3) of total present cells (left panel) and of alive (white columns) and dead (black columns) cells (right panel) measured by Trypan blue exclusion test. D) Sphere Formation Efficiency (SFE) of CaSki-AD cells untreated (white column) or treated with 50 μg/ml D609 for 24h (black column) or 48h (grey column) (mean ± SD; n = 2). Statistical analyses were performed using one-way ANOVA, P = 0.064. E) Real Time PCR analyses of the stemness markers OCT4, NANOG, NESTIN (left panel) and ALDH1 (right panel) expressed by the CaSki-AD cell line either untreated (white columns) or in the presence of D609 (50 μg/ml, black columns) for 48h. Statistical analyses were performed using t-test. (OCT4, P = 0.301; NANOG, P = 0.413; NESTIN, P = 0.173; ALDH1, P = 0.019).
Mentions: PC-PLC protein expression, detected by Western Blot analyses, was much lower in CaSki-AD cells (fold change 0.5 ± 0.1) than in A431-AD cells (Fig 6A). Even though the proliferation rate of this cell line was lower than that of A431-AD cells, the inhibition of PC-PLC by D609 was able to induce a substantial cell growth arrest (55% of reduction) under appropriate conditions, i.e at the dose of 50 μg/ml and after 48h of treatment (Fig 6B, left panel). This anti-proliferative effect was similar to that observed in the HaCaT cell line exposed to D609. As indicated by the Trypan blue exclusion assay, the inhibition of PC-PLC did not exert any cytotoxic effect on CaSki-AD cells (Fig 6B, right panel). As previously shown by our group, CaSki cells were also able to grow in suspension thus forming spheres but, in comparison with the A431, the CaSki cell line tumor-initiating potential was much lower [5]. CaSki-SPH were exposed to D609 under the same conditions used for the previous experiments on A431-SPH. Fig 6C (left panel) shows that D609 at the dose of 3 μg/ml, the same dose effective in A431-SPH, was once again extremely potent in inhibiting the proliferation of CaSki-SPH cells (IC50 corresponding to 1.2 μg/ml), also inducing a strong cytotoxic effect (Fig 6C, right panel). As for A431-AD cells, we evaluated the stemness potential of CaSki-AD cells after exposure to D609 (50 μg/ml). After 24h of treatment CaSki-AD cells showed a small decrease in the sphere forming efficiency (SFE = 32.6 ± 7.2% compared with 43.4 ± 7.2% measured in untreated cells). A clear, although only border-line significant effect was detected in cells exposed to D609 for 48h, in which SFE (19.2 ± 13.3%) was 2.3-fold lower than that of untreated cells, and 1.7-fold lower than that observed after 24h of treatment (P = 0.064) (Fig 6D). Real Time PCR performed on untreated CaSki-AD mRNA revealed low expression levels of stem-related markers (Fig 6E). Real Time PCR carried out 48h after D609 treatment showed that the drug did not significantly change the expression level of OCT4 and NANOG stem genes, but it was associated with a significant reduction in ALDH1 expression (P = 0.019) and also with a trend towards a decrease in NESTIN.

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