Proline rich polypeptide (PRP-1) produced by NPV and NSO cells is released into the general circulation and exerts its effect on the activity of immunocompetent and neuronal cells. PRP-1 is a unique regulator of hematopoiesis, stimulator of bone-marrow hematogenesis. Taking into consideration our preliminary data on antitumor and unique diverse biological properties of PRP-1 previously described by Galoyan et al., we proceeded with investigation of the PRP-1 effect on chondrosarcoma, the second most common malignancy in bone, which tends to be locally invasive and then metastatic. Currently it does not have any effective treatment and does not respond either to radiation or chemotherapy, leaving surgical resection as the only option. Our experimental results of PRP-1 action on human chondrosarcoma JJ012 cells demonstrated inactivation, abolishment of Myc oncogene activity usually upregulated in chondrosarcoma cells and other malignancies. The fact that addition of PRP-1 caused drastic inactivation of Myc-luc response element to the control level in human chondrosarcoma JJ012 cell line prompts to investigate further this neuropeptides powerful antioncogenic potential, opening up possibilities to consider PRP-1 as a potential therapeutic tool for chondrosarcoma treatment.

Studying the influence of brain cathepsin D (EC 3.4.23.5) and high molecular weight (HMW) aspartic endopeptidase (EC 3.4.23.-) on the processing of hypothalamic calmodulin-binding coronaro-constrictory peptide factors from the p-chain of globin it was found that only HMW aspartic endopeptidase generates the fragment 3140 of the p-chain of bovine hemoglobin (Hb) by cleavage of the Leuso-Leu" and Phe40-Phe41 bonds. Digestion of the B-chain of globin was performed at 37°C at an enzyme/substrate ratio of 1:80 at pH 3.5 using different times of incubation (from 4 h to 10 h). The resulting peptides were separated by reversed-phase high-performance liquid chromatography (HPLC) and then identified by amino acid analysis and Edman degradation. The differences in specificity and activity of these two brain aspartic proteinases could be explained by their different structural features. Our finding provides evidence for a different biological function of these two enzymes. Data obtained give us reason to suppose that HMW aspartic proteinase probably can participate in the processing of the coronaro-constrictory peptide in vivo by limited proteolysis of Hb or Hb-like protein.

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.

The following peptide structure in 3 of 5 coronaro-constrictory peptide factors isolated from bovine hypothalamus was determined by amino acid analysis and Edman degradation: 1) (P1)--Val-Val-Tyr-Pro-Trp; 2) (P2)--Val-Val-Tyr-Pro-Trp-Thr; 3) (P3)--Leu-Val-Val-Tyr-Pro-Trp-Thr. A computer search for these amino acid sequences revealed that these peptides represent fragments 33-37; 33-38; 32-38 of the beta-chain of bovine hemoglobin. Solid phase peptide synthesis of 2 peptides (P2 and P3) was carried out. It was established that synthetic peptides had the properties of coronaro-constrictory peptides. The possibility of the formation of hypothalamic coronaro-constrictory peptides in vivo is discussed.

Proline rich polypeptide (PRP-1) produced by neurosecretory cells of the hypothalamus is one of the fragments of neurophysin-vasopressin-associated glycoprotein. The primary structure of the neuropeptide PRP-1 isolated from neurosecretory granules of bovine neurohypophysis. We investigated PRP-1 action on chondrosarcoma, the second most common malignancy in bone, which primarily affects the cartilage cells. This deadly disease does not have any effective treatment. Earlier we demonstrated MYC oncogene inactivating effect by 1 lg/ml concentration brain PRP-1 In the present study we observed reduced viable sarcoma JJ012 cell numbers in comparison with control (89% growth inhibition) when treated with low concentrations of PRP-1 (0.5–1 lg/ml). Higher concentrations did not exhibit inhibitory effect. We assume that PRP-1 in low concentration impedes cell cycle progression. The fact that low concentrations of PRP-1 abolished Myc activity prompts to think that the antitumorigenic effect of PRP-1 in low concentrations is mediated through oncogene inactivation.

Activation of the PI3K-Akt-mTOR pathway is implicated both in the establishment of tumors and as well as a target for therapy in many types of solid malignancy, its blockade represents an opportunity to improve outcomes in patients with tumors that are associated with poor prognosis. Our experimental data indicates that proline-rich polypeptide-1 (PRP-1, galarmin) is immunomodulator cytokine, produced by hypothalamic neurosecretory cells and exerts its antiproliferative effect on the tumor cells of mesenchymal origin via inhibiting mTOR kinase activity and repressing cell cycle progression. The goal of these investigations was to elucidate the antiproliferative action of PRP-1 on the breast carcinoma cell line MDA 231 (ER-) and to compare PRP-1 action previously reported on other mesenchymal tumors. These experiments confirmed maximum inhibition of cell growth at 0.5 and 1 μg/ml PRP-1 (71% and 63%, respectively) and inhibition at 10 μg/ml of 44%. There was no inhibitory effect observed on luminal T47-D (ER+) cells. Videomicroscopy results demonstrated dividing cells in the cytokine-treated MDA 231 (ER-), suggesting that the cells were not in the state of dormancy. The flow cytometry experiments confirmed that PRP-1-treated cells were accumulated in S phase. No apoptosis, caspase activation, or senescence was detected after treatment with this cytokine. Experiments with mTOR with PRP-1 (10 μg/ml) indicated statistically significant 40% inhibition of mTOR kinase activity in immunoprecipitates of the MDA 231 (ER-) cell line. PRP-1 is a novel mTOR inhibitor with strong antiproliferative action in mesenchymal tumors mostly resistant to radiation and chemotherapy.

Objective: The effects of proline-rich polypeptide (PRP) isolated from neurosecretory granules of bovine neurohypophysis produced by nuclei supraopticus and paraventricularis on phagocytosis, bacterial intracellular killing and oxidative burst induction in normal human cells and inflammatory cells from patients with Behçet’s disease (BD), i.e. peripheral blood neutrophils and monocytes, were investigated. Methods: Intracellular killing of Staphylococcus aureus by neutrophils and monocytes of normal controls and BD patients, phagocytic activity as well as spontaneous and N-formyl-Met-Leu-Phe (fMLP)- or phorbol 12-myristate 13-acetate (PMA)-induced activation of their respiratory burst were determined by quantitative flow cytometry using highly specific fluorescence probes. Results: PRP does not affect human peripheral blood neutrophil and monocyte phagocytosis but dramatically enhances spontaneous or fMLP- and PMA-induced oxidative burst as well as the intracellular killing of S. aureus. PRP induced the upregulation of the spontaneous or fMLP- and PMA-induced oxidative burst in normal PMNs and monocytes; the number of inflammatory BD cells did neither increase further nor undergo spontaneous or PMA-stimulated oxidative burst. In BD patients, increased spontaneous production of reactive oxygen intermediates (ROIs) by neutrophils and monocytes is characterized by impaired intracellular protein-kinase-C (PKC)-dependent oxidative burst regulation as well as overregulation of chemotaxis/inflammation-mediated respiratory burst induction. PRP restores rather the impaired intracellular PKC-dependent regulation of ROI production in inflammatory diseased cells than the chemotaxis/induction of the inflammation-mediated respiratory burst. Conclusion: We demonstrated the regulatory role for PRP on oxidative burst in neutrophils and monocytes from normal controls and BD patients. Our results suggest that PRP differentially affects both chemotaxis- and PKC-dependent oxidative burst in normal and inflammatory cells from patients.

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