Expression of the integrin subunits and heterodimers that are targeted by cilengitide.

<p>(A) Relative mRNA levels of cilengitide target integrin subunits were measured by real-time qPCR and normalised to the Glyceraldehyde 3-phosphate dehydrogenase (<i>GAPDH</i>) control gene expression. Each cell line was analysed at least twice with two independent RNA samples. (B) Western blot analysis of αv integrins under non-reducing condition. The β-actin protein serves as loading control. Molecular sizes are indicated on the left of the blots. (C) Immunofluorescence of αvβ3 in MPM cells. The cells were incubated with a rabbit monoclonal antibody against αvβ3 and detected with an Alexa Fluor 488-conjugated secondary antibody.</p

Cilengitide Inhibits Attachment and Invasion of Malignant Pleural Mesothelioma Cells through Antagonism of Integrins αvβ3 and αvβ5

<div><p>Malignant pleural mesothelioma (MPM) is an almost invariably fatal, asbestos-related malignancy arising from the mesothelial membrane lining the thoracic cavities. Despite some improvements in treatment, therapy is not considered curative and median survival following diagnosis is less than 1 year. Although still classed as a rare cancer, the incidence of MPM is increasing, and the limited progress in treating the disease makes the identification of new therapies a priority. As there is evidence for expression of the integrins αvβ3 and αvβ5 in MPM, there is a rationale for investigating the effects on MPM of cilengitide, a synthetic peptide inhibitor of integrin αv heterodimer with high specificity for αvβ3 and αvβ5. In mesothelial cells (MC) and 7 MPM cell lines, growth inhibition by cilengitide was associated with the expression level of its target integrins. Furthermore, cilengitide caused cell detachment and subsequent death of anoikis-sensitive cells. It also suppressed invasion of MPM cells in monolayer and three-dimensional cultures. Gene knockdown experiments indicated that these effects of cilengitide were, at least partly, due to antagonism of αvβ3 and αvβ5.</p></div

Summary of results in MPM: cilengitide treatment and target gene knockdown.

1<p>Cytotoxicity (see ): +++, highly sensitive; ++, intermediate; +, relatively resistant.</p>2<p>Anoikis resistance indicated by relative % viable cells in detached culture to attached culture (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090374#pone.0090374.s003" target="_blank">Figure S3B</a>): +++, high resistance; ++, intermediate; +, low resistance.</p>3<p>Anoikis death in non-adherent culture (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090374#pone.0090374.s003" target="_blank">Figure S3C</a>): +++, high; ++, intermediate; + low.</p

Effect of cilengitide on MPM cell viability and anchorage-independent growth.

<p>(A) Growth inhibition: cells were incubated in a concentration series of cilengitide (CGT) for 3 days. Viability was determined with the alamar blue metabolic assay. Results shown are the mean ± SD from 3 independent experiments. For simplicity, results of the 2 most sensitive and 2 most resistant cell lines were shown. (B) Anoikis resistance: cells were grown for 3 days on both ultra-low attachment plates (non-adherent condition) or on normal tissue culture plates (adherent) and viability was determined with the alamar blue metabolic assay. Anoikis-resistance is expressed as relative percentage of viable cells in non-adherent <i>versus</i> adherent culture. (C) Anoikis sensitivity is expressed as the proportion of dead cells in the non-adherent cultures, detected by ethidium homodimer III staining and calibrated to a 100% cell death control induced by saponin treatment. (D) The effect of cilengitide on proliferation of MPM cells grown on various extracellular matrix coatings. Uncoated plates were compared to plates coated with type I collagen or basal membrane extract (BME). Cells were incubated in a concentration series of cilengitide for 3 days and cell viability determined with the alamar blue assay. Results in B, C and D are means ± SD from 3 replicates.</p

siRNA-mediated knockdown of integrin <i>ITGB3</i> and <i>ITGB5</i> in H28 cells.

<p>(A) Western analysis of αvβ3 expression 3–7 days after siRNA transfection. Three siRNAs <i>ITGB3-1</i>, <i>ITGB3-2</i> and <i>ITGB3</i>-3 were compared. Ten µg of lysates were loaded on gel except lane 2. The β-actin protein serves as loading control. (B) Immunocytometry of αvβ3 and αvβ5 in H28 cells transfected for 3 days with siRNA for <i>ITGB3</i> or <i>ITGB5</i>. Cells were immunostained with antibodies against αvβ3 or αvβ5, detected with an Alexa Fluor 488-conjugated secondary antibody and analysed with the TALI image-based cytometer. Representative fields of images captured on the cytometer are shown. (C) Invasion of collagen matrix by H28 spheroids with <i>ITGB3</i> or <i>ITGB5</i> knockdown. Spheroids were generated one day after siRNA transfection and assayed for invasion into collagen gel; each experiment was performed in triplicate and on at least 3 occasions.</p

Effects of cilengitide on 3D invasion by MPM spheroids.

<p>Spheroids were embedded in collagen gel, overlaid with medium ±1 µM cilengitide and incubated at 37°C for 1–4 days. Spheroids were stained with calcein-AM or SYBR green and live images were captured. Representative images from four cell lines are shown; each experiment was performed in triplicate and on at least 3 occasions.</p

Cilengitide inhibits agarose spot invasion by H28 cells.

<p>(A) Mitotically inactivated cells were plated on wells with an agarose spot in medium ±1 µM cilengitide. After 1–2 days cells were fixed and stained with crystal violet and imaged with an EVOS-FL digital imaging system. Experiments were performed at least 3 times; representative images from three cell lines are shown. The whole agarose spots are shown in the first three columns, with the last column showing images of H28 cells at higher magnification. (B) Dose-dependent inhibition of agarose spot invasion by H28 cells. Cells were plated in medium containing various concentrations of cilengitide and imaged as above. One series is shown from 4 replicates.</p

Effects of siRNA-mediated knockdown of <i>ITGB5</i> in MPM cells.

<p>(A) Morphology of MPM cells with <i>ITGB5</i> down-regulation 3 days post-siRNA transfection (compare with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090374#pone-0090374-g002" target="_blank">Figure 2</a>). (B) 3D invasion by cells from MPM spheroids with <i>ITGB5</i> knockdown. Spheroids were generated starting one day after siRNA transfection and cultured for 3 days. They were subsequently embedded in collagen and incubated at 37°C for 1–4 days, followed by live staining with SYBR green and imaged. Representative images for 4 cell lines are shown; each experiment was performed on two occasions with 3 replicates each.</p

Cilengitide causes detachment of MPM cells in monolayer culture.

<p>Cells were allowed to adhere to tissue culture surface before incubation with 0, 1, or 10 µM cilengitide (CGT). Images were captured with the Zeiss Axiovert inverted microscope 1 day post-cilengitide treatment.</p

Relative expression (expressed in Δ<i>C</i>T) of the HH pathway genes.

<p>For each gene, the relative expression of mRNA was normalized to an endogenous <i>PGK1</i> control. Values represent the mean ± S.E.M. of three independent experiments each performed in duplicates. <b>A</b>, <i>GLI1</i>, <b>B</b>, <i>GLI2</i> and <b>C</b>, <i>PTCH1</i> in 7 MMe cell lines and 2 primary cultures of normal mesothelial cells.</p