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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 the PC-PLC inhibitor D609 on PC-PLC activity, PC-PLC protein expression and cell proliferation in HaCaT keratinocytes and in the squamous carcinoma cell line A431-AD.A) Proliferation assays performed on 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 count (mean ± SD, n = 3) of live cells was obtained by Trypan blue exclusion assays and by automated cell counter, as described in the Materials and Methods section. B) Cell counting (mean percentage ± SD, n = 3) of either live (white columns) or dead cells (black columns) measured by Trypan blue exclusion test in the cultures used for the proliferation assays shown in panel A. C) Top panel: PC-PLC activity (mean ± SD, n = 3) measured by Amplex Red assay in total lysates of control (untreated = black columns) or 50 μg/ml D609-treated cells (grey columns). Statistical analyses were performed using t-test; HaCaT, P = 0.006 at 24h and P = 0.002 at 48h; A431-AD, P<0.0001 at 24h and at 48h. Bottom panel: Representative Western blot analyses of PC-PLC protein expression performed in total lysates of cells cultured in the presence or absence of 50 μg/ml D609 (n = 3 independent experiments); t0 = untreated cells at 72 hours after seeding; β actin was used as quantitative loading control.
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pone.0136120.g002: Effects of the PC-PLC inhibitor D609 on PC-PLC activity, PC-PLC protein expression and cell proliferation in HaCaT keratinocytes and in the squamous carcinoma cell line A431-AD.A) Proliferation assays performed on 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 count (mean ± SD, n = 3) of live cells was obtained by Trypan blue exclusion assays and by automated cell counter, as described in the Materials and Methods section. B) Cell counting (mean percentage ± SD, n = 3) of either live (white columns) or dead cells (black columns) measured by Trypan blue exclusion test in the cultures used for the proliferation assays shown in panel A. C) Top panel: PC-PLC activity (mean ± SD, n = 3) measured by Amplex Red assay in total lysates of control (untreated = black columns) or 50 μg/ml D609-treated cells (grey columns). Statistical analyses were performed using t-test; HaCaT, P = 0.006 at 24h and P = 0.002 at 48h; A431-AD, P<0.0001 at 24h and at 48h. Bottom panel: Representative Western blot analyses of PC-PLC protein expression performed in total lysates of cells cultured in the presence or absence of 50 μg/ml D609 (n = 3 independent experiments); t0 = untreated cells at 72 hours after seeding; β actin was used as quantitative loading control.

Mentions: To better understand the functional role of PC-PLC in cancer cells, as well as in their non-tumoral counterpart, we investigated the effects of the PC-PLC competitive inhibitor D609 on cell proliferation by exposing HaCaT and A431-AD cell lines to different doses of the drug. Cells were seeded 72h before adding D609 to the culture medium (t = 0) and the cell proliferation rate was determined 24h and 48h after treatment. In both cell lines D609 at doses up to 12.5 μg/ml induced minimal or no changes in cell proliferation (Fig 2A), while 48h after treatment with 25 μg/ml of D609, a 37.5% reduction of cell proliferation was observed in A431-AD cells only. We found that the 50% of cell proliferation inhibition was achieved at about 50 μg/ml for HaCat and 33.4 μg/ml for A431 cell lines. Continuous exposure to 50 μg/ml of D609 progressively induced cell growth arrest in A431-AD cells, whereas D609-treated HaCaT cells underwent a more limited growth inhibition (83.8% and 56.9% reduction after 48h of treatment, respectively). D609 treatment mainly exerted a cytostatic rather than a cytotoxic effect, as indicated by dead cell counting (restricted to a range of 5–15%, independently from dose and time of treatment) (Fig 2B). This anti-proliferative effect was more evident and remarkable in the A431 cancer cells than in the non-tumoral HaCaT cells. In order to compare the results derived from the further experiments, we decided to use the dose of 50 μg/ml for all the adherent cell lines.


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 the PC-PLC inhibitor D609 on PC-PLC activity, PC-PLC protein expression and cell proliferation in HaCaT keratinocytes and in the squamous carcinoma cell line A431-AD.A) Proliferation assays performed on 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 count (mean ± SD, n = 3) of live cells was obtained by Trypan blue exclusion assays and by automated cell counter, as described in the Materials and Methods section. B) Cell counting (mean percentage ± SD, n = 3) of either live (white columns) or dead cells (black columns) measured by Trypan blue exclusion test in the cultures used for the proliferation assays shown in panel A. C) Top panel: PC-PLC activity (mean ± SD, n = 3) measured by Amplex Red assay in total lysates of control (untreated = black columns) or 50 μg/ml D609-treated cells (grey columns). Statistical analyses were performed using t-test; HaCaT, P = 0.006 at 24h and P = 0.002 at 48h; A431-AD, P<0.0001 at 24h and at 48h. Bottom panel: Representative Western blot analyses of PC-PLC protein expression performed in total lysates of cells cultured in the presence or absence of 50 μg/ml D609 (n = 3 independent experiments); t0 = untreated cells at 72 hours after seeding; β actin was used as quantitative loading control.
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Related In: Results  -  Collection

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pone.0136120.g002: Effects of the PC-PLC inhibitor D609 on PC-PLC activity, PC-PLC protein expression and cell proliferation in HaCaT keratinocytes and in the squamous carcinoma cell line A431-AD.A) Proliferation assays performed on 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 count (mean ± SD, n = 3) of live cells was obtained by Trypan blue exclusion assays and by automated cell counter, as described in the Materials and Methods section. B) Cell counting (mean percentage ± SD, n = 3) of either live (white columns) or dead cells (black columns) measured by Trypan blue exclusion test in the cultures used for the proliferation assays shown in panel A. C) Top panel: PC-PLC activity (mean ± SD, n = 3) measured by Amplex Red assay in total lysates of control (untreated = black columns) or 50 μg/ml D609-treated cells (grey columns). Statistical analyses were performed using t-test; HaCaT, P = 0.006 at 24h and P = 0.002 at 48h; A431-AD, P<0.0001 at 24h and at 48h. Bottom panel: Representative Western blot analyses of PC-PLC protein expression performed in total lysates of cells cultured in the presence or absence of 50 μg/ml D609 (n = 3 independent experiments); t0 = untreated cells at 72 hours after seeding; β actin was used as quantitative loading control.
Mentions: To better understand the functional role of PC-PLC in cancer cells, as well as in their non-tumoral counterpart, we investigated the effects of the PC-PLC competitive inhibitor D609 on cell proliferation by exposing HaCaT and A431-AD cell lines to different doses of the drug. Cells were seeded 72h before adding D609 to the culture medium (t = 0) and the cell proliferation rate was determined 24h and 48h after treatment. In both cell lines D609 at doses up to 12.5 μg/ml induced minimal or no changes in cell proliferation (Fig 2A), while 48h after treatment with 25 μg/ml of D609, a 37.5% reduction of cell proliferation was observed in A431-AD cells only. We found that the 50% of cell proliferation inhibition was achieved at about 50 μg/ml for HaCat and 33.4 μg/ml for A431 cell lines. Continuous exposure to 50 μg/ml of D609 progressively induced cell growth arrest in A431-AD cells, whereas D609-treated HaCaT cells underwent a more limited growth inhibition (83.8% and 56.9% reduction after 48h of treatment, respectively). D609 treatment mainly exerted a cytostatic rather than a cytotoxic effect, as indicated by dead cell counting (restricted to a range of 5–15%, independently from dose and time of treatment) (Fig 2B). This anti-proliferative effect was more evident and remarkable in the A431 cancer cells than in the non-tumoral HaCaT cells. In order to compare the results derived from the further experiments, we decided to use the dose of 50 μg/ml for all the adherent cell lines.

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