Supplementary MaterialsAdditional file 1: Figure S1
May 16, 2021
Supplementary MaterialsAdditional file 1: Figure S1. monolayer. Then, A2780 cells suspended in growth medium plus/minus 10?nM RI-3 were seeded onto the mesothelial cell monolayer and invasion of mesothelium by A2780 cells Anavex2-73 HCl was monitored in real-time as changes in Cell Index due to breaking of the monolayer integrity. Data represent mean??SD from a quadruplicate experiment representative of 2replicates. Figure S2. Uncropped images of immunoblots from Fig. ?Fig.55c. 13046_2019_1465_MOESM1_ESM.zip (217K) GUID:?7F968B4B-BD9E-40AD-9679-1C115286EF66 Data Availability StatementAll data generated or analyzed during this study are included in this published article and its supplementary information file. Further details are available Rabbit polyclonal to ANXA13 from the corresponding author on reasonable request. Abstract Background The biological behavior of epithelial ovarian cancer (EOC) is unique since EOC cells metastasize early to the peritoneum. Thereby, new anti-target agents designed to block trans-coelomic dissemination of EOC cells may be useful as anti-metastatic drugs. The Urokinase Plasminogen Activator Receptor (uPAR) is overexpressed in EOC tissues, and its truncated forms released in sera and/or ascitic fluid are associated with poor prognosis and unfavorable clinical outcome. We documented that uPAR triggers intra-abdominal dissemination of EOC cells through the interaction of its 84C95 sequence with the Formyl Peptide Receptor type 1 (FPR1), even as short linear peptide Ser-Arg-Ser-Arg-Tyr (SRSRY). While the pro-metastatic role of uPAR is well documented, Anavex2-73 HCl little information regarding the expression and role of FPR1 in EOC is currently available. Methods Expression levels of uPAR and FPR1 in EOC cells and tissues were assessed by immunofluorescence, Western blot, or immunohystochemistry. Cell adhesion to extra-cellular matrix proteins and mesothelium as Anavex2-73 HCl well as mesothelium invasion kinetics by EOC cells were monitored using the xCELLigence technology or assessed by measuring cell-associated fluorescence. Cell internalization of FPR1 was identified on multiple z-series by confocal microscopy. Data from in vitro assays were analysed by one-way ANOVA and post-hoc Dunnett t-test for multiple comparisons. Tissue microarray data were analyzed with the Pearsons Chi-square (2) test. Results Co-expression of uPAR and FPR1 by SKOV-3 and primary EOC cells confers a marked adhesion to vitronectin. The extent of cell adhesion decreases to basal level by pre-exposure to anti-uPAR84C95 Abs, or to the RI-3 peptide, blocking the uPAR84C95/FPR1 interaction. Furthermore, EOC cells exposed to RI-3 or desensitized with an excess of SRSRY, fail to adhere also to mesothelial cell monolayers, losing the ability to cross them. Finally, primary and metastatic EOC tissues express a high level of FPR1. Conclusions Our findings identify for the first time FPR1 as a potential biomarker of aggressive EOC and suggests that inhibitors of the uPAR84C95/FPR1 crosstalk may be useful for the treatment of metastatic EOC. residue in the Ser88-Arg-Ser-Arg-Tyr92 sequence inhibiting the uPAR/FPR1 interaction, directional cell migration, invasion and angiogenesis [32C35]. Later, to improve their chemical stability and half-life, we developed a new library of retro-inverso peptides . The lead compound Ac-(D)-Tyr-(D)-Arg-Aib-(D)-Arg-NH2 (RI-3) is stable in human serum, adopts the turn structure typical of uPAR/FPR1 antagonists, and competes with fMLF and SRSRY for binding to FPR1, preventing SRSRY-induced FPR1 internalization as well as p38 MAPK and PI3K/AKT signaling cascades , which are documented to mediate FPR1 signal transduction pathways . Interestingly, RI-3 inhibits migration and invasion of sarcoma and melanoma cells in a dose dependent manner, an overall 50% reduction of cell migration and invasion being reached in the picomolar and nanomolar range, respectively [36, 37]. Recently, to understand the structural basis of the RI-3 inhibitory effects, the FPR1/fMLF, FPR1/SRSRY and FPR1/RI-3 complexes were modeled and analyzed, focusing on the binding pocket of FPR1 and the interaction between the amino acids that signal to the FPR1 C-terminal loop. We found that RI-3 Anavex2-73 HCl shares the same binding site of fMLF and SRSRY on FPR1. However, while fMLF and Anavex2-73 HCl SRSRY display the same agonist activation signature, RI-3 does not interact with the activation region of FPR1, keeping receptor anchored on cell membrane and hence unable to internalize and activate signaling, . In this study, we analyzed the expression of FPR1 in tissues from patients affected by EOC. Then, by using primary EOC cells, we analyzed the role of uPAR/FPR1 crosstalk enabling cancer cells to adhere onto matrices and mesothelial cell monolayers. We also show that RI-3 successfully prevents the capability of ovarian cancer cells to adhere onto vitronectin and invade mesothelium. Methods EOC cell line, EOC primary cultures and transfection Human ovarian carcinoma SKOV-3 and A2780 cell lines, obtained from the Cell Factory of the National Cancer Institute of Genova, were cultured in DMEM or RPMI, respectively, supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100?g/mL), streptomycin (100?U/mL) and maintained at 37?C in a humidified atmosphere of 5% CO2. To obtain primary cultures, a representative sample from the EOC excision.