Immunohistochemical analysis of ALCAM.

<p>Membranous positive staining in surrounding non-tumor tissue (A and C) and in medullary thyroid carcinomas (MTCs) (B). Cytoplasmic and membranous positive staining in papillary thyroid carcinomas (PTCs) (D). 1, low-power field; 2, high-power field; N (normal tissue), T (tumor tissue).</p

PC-PLC protein expression and activity in the non-tumoral keratinocyte HaCaT and in the A431-AD squamous carcinoma cell lines.

<p>Subcellular localization of PC-PLC (grey) on the plasma membrane of unfixed cells (<b>A</b>) and in different cellular compartments of fixed and permeabilized cells (<b>B)</b> detected by CLSM analyses. Single central optical sections are shown. Nuclei were stained with DAPI (blue). Scale bars, 20 μm. <b>C</b>) 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. <b>D)</b> 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.</p

Release of ALCAM is sensitive to ADAM17/TACE silencing.

<p>(A) Analysis of lysates from TPC-1 and A2774 cells, resolved on 4–12% SDS-PAGE and immunoblotted with anti-ADAM17/TACE antibodies. Arrows indicate inactive (130 kDa) and active (80 kDa) forms of ADAM17/TACE enzyme. Normalization of results was obtained with immunoblotting analysis of beta-actin. (B) Expression of ADAM17/TACE protein by TPC-1 cells transfected with an ADAM17/TACE-specific small interfering RNA (siRNA) (OTP17), or with non-targeting siRNA (NT) as detected by western blot. The amount of protein was calculated by comparative densitometric scanning with beta-actin. (C) ELISA detection of ALCAM release by TPC-1 cells after transfection with siRNA specifically inhibiting ADAM-17/TACE (OTP17, black column) or with non-targeting siRNA (NT, white column). (D) Conditioned medium (CM) from TPC-1 cells, cultured with pervanadate (PV) (60 min), epidermal growth factor (EGF) (24 h), or medium alone (ctr, 24 h) in the presence (black columns) or in absence (white columns) of CGS27023A (CGS) was assessed by ELISA for ALCAM. Columns, means of three experiments (cells cultured in presence of 10, 1, or 0.1 µM CGS); bars, SD. *, P<0.05. Grey bar: in absence of CGS, but in presence of orthovanadate (OV).</p

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

<div><p>Purpose</p><p>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.</p><p>Results</p><p>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).</p><p>Conclusions</p><p>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.</p></div

ALCAM expression in thyroid cell lines.

<p>Analysis of TT and MZ-CRC1 cell lysates showing ALCAM protein expression in these thyroid cell lines. SKOV-3 and TPC-1 cell lysates were included as controls. Total lysates from each cell line were resolved by 4–12% SDS-PAGE and immunoblotted with anti-ALCAM antibodies. Arrow indicates the fully glycosylated ALCAM isoform. Beta-actin was used as a loading control.</p

ALCAM expression in normal and tumor human thyroid tissue samples.

<p>(A) Total protein extracts (30 µg) from 11 papillary thyroid carcinomas (Ca Pap), 5 medullary thyroid carcinomas (Ca Mid), and 5 normal thyroid tissues (Thyroid) were resolved by 4–12% SDS-PAGE, transferred onto a nitrocellulose membrane, and immunoblotted with anti-ALCAM antibody. (*) indicates a non-specific background band. Beta-actin was used as a loading control. (B) Western blot analysis of ALCAM expression of total protein extracts (30 µg) from three PTCs, two MTCs, and three CTRLs, treated (+) or not (−) with N-glycosidase F. Beta-actin was used as a loading control.</p

Western blot analysis of ALCAM expression in lysate and conditioned medium of TPC-1 cell line.

<p>(A) Analysis of TPC-1 cell lysate (Lys) and conditioned medium (CM) using anti-ALCAM antibody. Arrow indicates membrane-localized ALCAM isoform in lysate, and arrowheads indicate shed-ALCAM isoforms in CM, respectively. The SKOV-3 cell line was used as positive control. (B) TPC-1 cell lysate and CM were treated or not with a mixture of O-glycosidase (O) and sialidase (S) and analyzed by western blot using anti-ALCAM antibody. Continuous arrows indicate membrane and shed ALCAM forms before digestion, and dashed arrows indicate digestion products. (*) indicates less evident bands of ALCAM in CM. (C) TPC-1 cell lysate and CM were treated or not with O-glycosidase (O), sialidase (S), or PNGaseF (F) and analyzed by western blot using anti-ALCAM antibodies. Continuous arrows indicate membrane and shed ALCAM forms before digestion, and dashed arrows indicate digestion products.</p

Effects of PC-PLC inhibition on EGFR, ERK and AKT phosphorylation in the HaCat and A431-AD cells.

<p>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.</p

Changes in ALCAM expression induced by ionomycin or phorbol myristate acetate (PMA).

<p>Total lysates (Lys) and conditioned medium (CM) from TPC-1 cells, treated or not with 1 µM ionomycin (iono) or 50 ng/mL PMA for 2 h, were resolved by 3–8% SDS-PAGE and immunoblotted with ALCAM antibody. Both treatments led to an increase of secreted ALCAM isoforms; especially PMA induced a gain of 60-kDa isoform expression. Arrow indicates the membrane-localized ALCAM isoform in lysate, and arrowheads indicate shed-ALCAM isoforms in CM, respectively. Beta-actin was used as a loading control.</p

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.

<p><b>A</b>) 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>B</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. <b>C</b>) 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.</p