Effects of statins and controls on the glycosylation status of CD147.

<p>Total cell lysates were subjected to immunoblotting to assess the glycosylation status of CD147. The effects of fluvastatin, rescue compounds and inhibitors of crucial steps in the cholesterol biosynthesis pathway are shown. Alpha tubulin served as a loading control for the immunoblots. Experiments were conducted in duplicate; one representative immunoblot is shown (A). B: Quantitative analysis of LG CD147 bands shown in (A). The value of the band intensity of PMA-differentiated cells was set as 1.0. Diamonds and thirteenth column: non-glycosylated core protein (27 kDa) after tunicamycin treatment. Basal, untreated cells; PMA, PMA-differentiated cells; FS, cells treated with fluvastatin (1 μM or 10 μM). Rescue compounds: MEV, mevalonate; SQU, squalene; CHOL, cholesterol. Cholesterol pathway inhibitors: FTI-277; GGTI-298; FPT-1; AP-9, antagonistic peptide 9; TUN, tunicamycin. * on top of columns: p < 0.05, compared with PMA-differentiated cells whose value was set as 1.0. * on horizontal lines: p < 0.05, fluvastatin plus rescue compounds compared with fluvastatin only.</p

Alterations in CD147 expression on PMA-differentiated THP-1 cells upon statin and control treatments.

<p>The cell surface expression of CD147 on THP-1 cells treated with statins, rescue compounds, and cholesterol pathway inhibitors was analyzed by flow cytometry. THP-1 cells were left untreated or treated with 200 nM PMA for 24 h in the presence or absence of statins or controls. Changes in CD147 expression are shown in overlaid histograms. A: PMA-differentiated cells versus untreated cells (basal). B: PMA-differentiated cells versus cells treated with PMA in the presence of atorvastatin (AS), pravastatin (PS), or fluvastatin (FS). C-D: Rescue experiments: C: PMA-differentiated cells versus cells treated with fluvastatin (FS) and fluvastatin-treated cells rescued with dolichol (DOL). D: PMA-differentiated cells versus cells treated with fluvastatin (FS) and fluvastatin-treated cells rescued with farnesylpyrophosphate (FPP). E: PMA-differentiated cells versus cells treated with fluvastatin (FS) and fluvastatin-treated cells rescued with geranylgeranylpyrophosphate (GGPP). F: PMA-differentiated cells versus cells treated with the cholesterol pathway inhibitors FTI-277, GGTI-298 or tunicamycin (TUN). Gray histograms represent isotype controls. Experiments were conducted in triplicate; one representative result is shown.</p

Alteration of the morphology of PMA-differentiated THP-1 cells by treatment with statins and various controls.

<p>THP-1 cells were (A) left untreated or (B-L) treated with 200 nM PMA for 24 h (B) without statins or in the presence of (C) 10 μM pravastatin (PS); (D) 10 μM atorvastatin (AS); (E) 10 μM fluvastatin (FS); (F) 10 μM fluvastatin plus 100 μM mevalonate (MEV); (G) 10 μM fluvastatin plus 10 μM farnesylpyrophosphate (FPP); (H) 10 μM fluvastatin plus 10 μM geranylgeranylpyrophosphate (GGPP); (I) 10 μM fluvastatin plus 10 μM dolichol (DOL); (J) 10 μg/ml tunicamycin (TUN); (K) 10 μM of the farnesyl transferase inhibitor (FTI)-277; or (L) 10 μM of the geranylgeranyl transferase inhibitor (GGTI)-298. Cellular morphology was assessed by light microscopy at 50x magnification. Numbers represent percentage of THP-1 cells with adherent, amoeboid morphology indicating differentiation (examples are marked by arrows). Experiments were conducted in triplicate; one representative result is shown.</p

Effects of statins and controls on surface versus total CD147 expression.

<p>Cell permeabilization studies were conducted to confirm cellular retention of CD147. Results are shown as overlaid histograms. Light lines: surface-expression of CD147 in unpermeabilized THP-1 cells; bold lines: total cellular expression of CD147 in permeabilized cells. A: PMA-differentiated cells. B: PMA-differentiated cells treated with fluvastatin (FS). C: fluvastatin-treated, PMA-differentiated cells rescued with farnesylpyrophosphate (FPP). D: PMA-differentiated cells treated with tunicamycin (TUN). Gray histograms, isotype controls. Experiments were conducted in triplicate; one representative result is shown.</p

Downregulation of <i>de novo</i> synthesized CD147 glycoprotein at the cell surface after statin treatment.

<p>Levels of biotinylated cell surface proteins were compared with cellular total protein levels (in whole cell lysates) by immunoblot. A: biotinylated cell surface proteins; B: whole cell lysates. Note that the lowly glycosylated (LG) form of CD147 was not present on the cell surface and that the expression of the highly glycosylated (HG) form of CD147 was reduced predominantly on the cell surface. Experiments were conducted in duplicate; one representative immunoblot is shown. C: Quantitative analysis of bands shown in (A) and (B). The value of the band intensity of PMA-differentiated cells was set as 1.0, respectively. Black bars: HG CD147 on the cell surface. Grey bars: HG CD147 in whole cell lysate. White bars: LG CD147 in whole cell lysate. Basal, untreated cells; PMA, PMA-differentiated cells; PS, pravastatin; AS, atorvastatin; FS, fluvastatin; TUN, tunicamycin. * p < 0.05, compared with PMA-differentiated cells whose value was set as 1.0.</p

Statins and the cholesterol biosynthesis pathway.

<p>Statins inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase-mediated formation of mevalonate, which is the rate-limiting step of the cholesterol biosynthesis pathway. Farnesylpyrophosphate (-PP) and its derivative, geranylgeranyl-PP, are lipids that posttranslationally modify immunologically important guanosine triphosphate (GTP)-binding proteins, such as Ras and Rho. This isoprenylation permits the subsequent activation and membrane translocation of these proteins, which is necessary for a number of their cellular functions. Dolichol production is dependent on the presence of farnesyl-PP, and dolichol is a necessary substrate for the <i>N</i>-glycosylation of immunologically important transmembrane glycoproteins such as CD147. Inhibitors of the production of specific intermediates downstream of mevalonate include the farnesyl transferase inhibitor (FTI)-277, the geranylgeranyl transferase inhibitor (GGTI)-298, and tunicamycin, a selective inhibitor of <i>N</i>-glycosylation.</p

Alterations in CD14 expression in PMA-differentiated THP-1 cells induced by statin and control treatment.

<p>The cell surface expression of CD14 in THP-1 cells treated with statins, rescue compounds, and cholesterol pathway inhibitors was analyzed by flow cytometry. THP-1 cells were left untreated or treated with PMA in the presence or absence of statins or controls. The changes in CD14 expression are shown in overlaid histograms. A: PMA-differentiated cells versus untreated cells (basal). B: PMA-differentiated cells versus cells treated with PMA in the presence of atorvastatin (AS), pravastatin (PS), and fluvastatin (FS). C-E: PMA-differentiated cells versus cells treated with FS: FS-treated cells rescued with (C) dolichol (DOL), (D) farnesylpyrophosphate (FPP), or (E) geranylgeranylpyrophosphate (GGPP). F: PMA-differentiated cells versus cells treated with the cholesterol pathway inhibitors FTI-277, GGTI-298 or tunicamycin (TUN). Gray histograms, isotype controls. Experiments were conducted in triplicate; one representative result is shown.</p

Shh pathway affects lung fibroblast migration, invasion and collagen synthesis.

<p>(<b>A</b>) Accumulated distance of migration of primary human lung fibroblasts treated with Shh (500 ng/ml) or cyclopamine (10 µM) and monitored by live cell microscopy for 48 hours. The accumulated distance of migration of each cell was determined using ImageJ. ***p<0,01. (<b>B</b>) Scratch wound assay was performed in lung fibroblasts CCL206 treated or not with Shh at the doses indicated or with 10 µM cyclopamine (cyclop) for up to 48 hours. Migration of the cells was recorded using live cell microscopy and representative pictures at 1,5, 12,5 and 26 hours are shown. The colored lines indicate the border of cell migration in each case. (<b>C</b>) The area of the wound was quantified after 26 hours and the percentage of wound closure, relative to the initial area of scratch for each case, was determined. (<b>D</b>) Transmembrane invasion assay was performed in lung fibroblasts treated with Shh (500 ng/ml) or with cyclopamine (10 µM). (<b>E</b>) RT-qPCR was performed to assess MMP2 and MMP9 expression in fibroblasts treated or not with Shh (500 ng/ml) for 48hr. Results are presented as fold of mRNA levels in treated cells compared with non-treated cells. *p<0,1. (<b>F</b>) The total collagen content of lung fibroblasts treated with Shh (500 ng/ml) or TGF-ß1 (5 ng/ml) for the indicated times was quantified using the Sircol collagen assay. *p<0,1, **p<0,05.</p

Shh mediates NSCLC/lung fibroblast reciprocal crosstalk.

<p>CCL206 lung fibroblasts were cultured or not with the supernatant of H520 transfected with a NC siRNA(Sup) or with Shh siRNA (Sup siShh) for 48 h. (A) Levels of secreted Leukemia Inhibitory Factor (LIF) were evaluated in CCL206 supernatant by multiplex biometric ELISA-based immunoassay (Bioplex system). Results are presented in percentage as relative secretion compared with cells cultured without H520 supernatant. *p<0,1; **p<0,05 (B) Levels of secreted Vascular Endothelial Growth Factor (VEGF were assessed in CCL206 supernatant by multiplex biometric ELISA-based immunoassay (Bioplex system). Results are presented in percentage as relative secretion compared with cells cultured without H520 supernatant. **p<0,05 (C) A549 and H520 cells were pre-stained with Hoechst (1 µg/ml) and then cultured for 48 h with CCL206 lung fibroblasts pre-treated with Shh (500 ng/ml) or with SAG (3 nM). The number of Hoechst positive cells was evaluated by fluorescent microscopy and is presented in percentage as relative number of cells compared with the number of cancer cells cultured alone. (D) NSCLC cells A549 and H520 were pre-stained with Hoechst (1 µg/ml) and then co-cultured for 72 h either alone or with CCL206 pre-treated with Shh (500 ng/ml) or with 3 nM SAG. The number of NSCLC cells transmigrating is expressed in percentage as the relative number of cells migrating compared with the total number of cancer cells per transwell. *p<0,1; ***p<0,01.</p

Inhibition of Hedgehog signaling reduces the proliferation of NSCLC cells.

<p>Lung adenocarcinoma A549 cells (<b>A</b>) and lung squamous carcinoma H520 cells (<b>B)</b> were cultured in presence or absence of 10 µM cyclopamine for 5 days. Proliferation was assessed by cell counting and cell survival by MTT assay. *p<0,1; **p<0,05. (<b>C</b>) The Hedgehog-responsive transcription factors Gli1, Gli2 or Gli3 were knocked down with siRNA in A549 cells. RT-qPCR was performed to confirm the specific silencing of each Gli and to assess the expression of the Hedgehog receptor Ptch1. Results are presented as fold differences of mRNA levels (2^∧∧Ct) compared with cells transfected with a negative control siRNA (NC siRNA) having no homology in vertebrate transcriptome. **p<0,05, ***p<0,01. The impact of silencing Gli1, Gli2 or Gli3 in A549 cell proliferation was assessed by cell counting (<b>D</b>) and in cell survival by MTT assay (<b>E</b>)<b>.</b> Results are presented in percentage as relative proliferation and relative survival compared with cells transfected with the negative control siRNA (NC). *p<0,1<b>.</b> (<b>F</b>) Representative phase-contrast microscopic pictures after 72 hours of siRNA are presented<b>.</b> (<b>G</b>) RT-qPCR was performed to evaluate the effect of the siRNA of Gli1, Gli2 and Gli3 in the expression of the G1/S phase cyclins D (Cyc D1, Cyc D2, Cyc D3) and cyclin E (Cyc E1)<b>.</b> Results are presented as fold of mRNA levels (2^∧∧Ct) compared with cells transfected with a negative control siRNA (NC siRNA) having no homology in vertebrate transcriptome. *p<0,1; **p<0,05. (<b>H</b>) Western blot of cyclin D2 in A549 cells transfected with Gli siRNA or with a negative control siRNA (NC). Blotting of ß-actin was used as loading control.</p