Cytostatic Effect of the Hypothalamic Cytokine PRP-1 is Mediated by mTOR and cMyc Inhibition in High Grade Chondrosarcoma

Abstract

This study aimed to further elucidate the molecular mechanisms of antiproliferative action of proline rich polypeptide 1 (PRP-1) cytokine, produced by neurosecretory cells of the hypothalamus to be considered as alternative adjuvant therapy for metastatic chondrosarcoma, which does not respond to chemotherapy or radiation and currently without any effective treatment. Rapid cell proliferation assay of human primary cultures from high grade chondrosarcoma patients biopsies and human chondrosarcoma JJ012 cell line indicated 50 and 80% inhibition in PRP-1 treated samples correspondingly. Videomicroscopy detected that despite the treatment there are still dividing cells, meaning that cells are not in the state of dormancy, rather PRP-1 repressed the cell cycle progression, exhibited cytostatic effect. The mammalian target of rapamycin (mTOR) is an intracellular serine/threonine protein kinase that has a crucial role in a nutrient sensitive signaling pathway that regulates cell growth. Experiments with mTOR pathway after PRP-1 (10 μg/ml) treatment indicated statistically significant 30% inhibition of mTOR activity and its 56% inhibition in immunoprecipitates with PRP-1 concentrations effective for cell proliferation inhibition. Treatment with PRP- caused inhibition of mTOR and downstream target cMyc oncogenic transcription factor sufficient to trigger the cytostatic effect in high grade, but not in low grade chondrosarcomas. The fact that lower concentrations than 10 μg/ml peptide with cytostatic effect did not inhibit mTOR, but inhibited cMyc prompted us to assume that PRP-1 binds to two different receptors facilitating the antiproliferative effect.

Restoration of desmosomal junction protein expression and inhibition of H3K9-specific histone demethylase activity by cytostatic proline-rich polypeptide-1 leads to suppression of tumorigenic potential in human chondrosarcoma cells

“…We previously described the antitumorigenic effect of cytostatic PRP-1 in the JJ012 human chondrosarcoma cell line (13)(14)(15)(16). In our attempt to further elucidate the antiproliferative effect of this peptide, we conducted experiments to determine its possible role in the regulation of intercellular junctions.…”

“…JJ012 chondrosarcoma cells were cultured in a monolayer, as previously described (13)(14)(15)(16), in a medium consisting of Dulbecco's modified Eagle's medium, supplemented with F12, 10% fetal bovine serum, 25 µg/ml ascorbic acid, 100 ng/ml insulin, 100 nM hydrocortisone and 1% penicillin/streptomycin. Upon confluency, the cells were trypsinized and seeded in 6-well cluster dishes at a concentration of 1x10 6 cells/ml and incubated for 24 h in a 5% CO 2 incubator.…”

Epigenetic control of cancer by neuropeptides

“…The antimicrobial, antitumorigenic and antineurodegenerative properties for this immunomodulatory cytokine were previously reported (22) and the quantification of hypothalamic PRP-1 in the blood was recently documented (23). Furthermore, our prevous studies described that PRP-1, mammalian target of rapamycin complex 1 inhibitor, exerts antiproliferative cytostatic effects in chondrosarcoma (7)(8)(9)24) and breast cancer cells (10).…”

“…The stemness markers, targets of the miR302C polycomb protein, BMI-1, NANOG (28) and c-Myc (8,24,28), were markedly downregulated by PRP-1 (28). Interestingly, PRP-1 demonstrated the inhibition of giant cell tumors of the bone and, conversely, supported the growth of human normal bone marrow stromal cells (31).…”

Effect of cytostatic proline rich polypeptide-1 on tumor suppressors of inflammation pathway signaling in chondrosarcoma

“…Understanding the differences in their expression patterns between the normal and malignant states, and the signaling pathways that lead to these differences, require further investigation as important targets for future therapeutic interventions. Metastatic chondrosarcoma does not respond to conventional therapies and the search for new therapeutic approaches is urgent (2)(3)(4)(5)(6)(7)(8)(9)(10)36,46). The ability of PRP-1 as an antiproliferative agent to restore the expression of anti-inflammatory cytokines with tumor suppressor function proves the importance of this neuropeptide for future clinical consideration.…”