Metastatic chondrosarcoma is a bone malignancy not responsive to conventional therapies; new approaches and therapies are urgently needed. We have previously reported that mTORC1 inhibitor, antitumorigenic cytostatic proline rich polypeptide 1 (PRP-1), galarmin caused a significant upregulation of tumor suppressors including TET1/2 and SOCS3 (known to be involved in inflammatory processes), downregulation of oncoproteins and embryonic stem cell marker miR-302C and its targets Nanog, c-Myc and Bmi-1 in human chondrosarcoma. To understand better the mechanism of PRP-1 action it was very important to identify the receptor it binds to. Nuclear pathway receptor and GPCR assays indicated that PRP-1 receptors are not G protein coupled, neither do they belong to family of nuclear or orphan receptors. In the present study, we have demonstrated that PRP-1 binding interacting partners belong to innate immunity pattern recognition toll like receptors TLR1/2 and TLR6 and gel forming secreted mucin MUC5B. MUC5B was identified as PRP-1 receptor in human chondrosarcoma JJ012 cell line using Ligand-receptor capture technology. Toll like receptors TLR1/2 and TLR6 were identified as binding interaction partners with PRP-1 by western blot analysis in human chondrosarcoma JJ012 cell line lysates. Immunocytochemistry experiments confirmed the finding and indicated the localization of PRP-1 receptors in the tumor nucleus predominantly. TLR1/2, TLR6 and MUC5B were downregulated in human chondrosarcoma and upregulated in dose-response manner upon PRP-1 treatment. Experimental data indicated that in this cellular context the mentioned receptors had tumor suppressive function.

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A new type of bioactive polypeptides of the neurosecretory hypothalamus called proline-rich peptides (PRPs), which are isolated from bovine neurosecretory granules of the neurohypophysis, are synthesized in the form of a common precursor protein (neurophysin vasopressin-associated glycoprotein). Proline-rich polypetide 1 (PRP-1; also known as galarmin) is comprised of 15 amino acids residues, and has been suggested to possess anti-neurodegenerative, immunoregulatory, hematopoietic, antimicrobial and antitumor properties. The cytostatic, antiproliferative effect of PRP-1 was demonstrated in the human chondrosarcoma JJ012 and triple negative breast carcinoma MDA MB 231 cell lines. PRP-1 action is disease and tissue specific. To further explore the antitumorigenic and possible cytotoxic effects of PRP-1, a morpho-functional study on the effect of PRP-1 on a mouse Ehrlich ascites carcinoma (EAC) model was conducted. The PRP-1-induced morphological features of EAC cells confirmed the apoptotic nature of PRP-1, as manifested by cell shrinkage, membrane blebbing, chromosome condensation (pyknosis) and nuclear fragmentation (karyorrhexis). The effect of PRP-1 on the number of tumor cells incubated for 24 h and their viability in trypan blue-stained samples lead to a 44% reduction in the number of viable cells on day 11 post-inoculation vs. 22% inhibition of viable cells after PRP-1 treatment (0.1 µg/ml) on day 7 post-inoculation. Apoptosis experiments using an Annexin V-cyanine 3 apoptosis detection kit indicated that 24 h incubation with 0.1 µg/ml PRP-1 caused a significant increase in the number of apoptotic cells, reaching 50.33%, compared to 8.33% in the sample control on day 7 post-inoculation.