PRP­1 significantly decreases the ALDHhigh cancer stem cell population and regulates the aberrant Wnt/ß­catenin pathway in human chondrosarcoma JJ012 cells.

Chondrosarcomas are malignant bone tumors refractory to chemotherapy and radiation treatment; thus, novel therapeutic strategies are required. Proline­rich polypeptide 1 (PRP­1) has previously demonstrated antitumor properties in chondrosarcoma. To further investigate the role of PRP­1 in chondrosarcoma cells, its effects on cancer stem cell (CSC) populations were determined by analyzing aldehyde dehydrogenase (ALDH) activity, an established marker of CSCs, in association with regulation of the Wnt/ß­catenin signaling. A significant decrease in ALDHhigh CSCs was observed following treatment of chondrosarcoma JJ012 cells with PRP­1. For RT2 profiler PCR array analysis of Wnt/ß­catenin signaling genes, cells were sorted into: i) Bulk JJ012 cells; ii) ALDHhigh cells sorted from untreated JJ012 cells (ALDHhigh­untreated); and iii) ALDHlow cells sorted from PRP­1­treated JJ012 cells (ALDHlow­PRP­1). The expression levels of Wnt/ß­catenin signaling genes were determined to be downregulated in the ALDHhigh­untreated cells and upregulated in ALDHlow­PRP­1 cells when compared to the bulk JJ012 cells. Additionally, two important oncogenes involved in this pathway, MMP7 and CCND2, were found to be downregulated in the ALDHlow­PRP­1 cells. Immunocytochemistry demonstrated the localization of ß­catenin in the nuclei of the PRP­1­treated cells. Western blotting indicated increased ß­catenin expression in the ALDHlow­PRP­1 cells compared with the bulk JJ012 cells. Analysis of the cytoplasmic and nuclear fractions of cells treated with increasing concentrations of PRP­1 and ß­catenin nuclear translocation inhibitor CGP57380, suggested the nuclear translocation of ß­catenin following PRP­1 treatment. In addition, treatment of JJ012 cells with a specific ALDH inhibitor, diethylaminobenzaldehyde, and PRP­1 resulted in a significant decrease in cytoplasmic ß­catenin protein expression. This indicated that ALDH inactivation may be associated with the nuclear translocation of ß­catenin. Derivation of sarcomas from mesenchymal stem cells via inactivation of the Wnt pathway has been previously documented. The findings of the present study support the notion that Wnt/ß­catenin activation may serve a differential role in sarcomas, limiting tumor progression in association with decreased CSC activity.

The protective and immunomodulatory effects of hypothalamic proline-rich polypeptide galarmin against methicillin-resistant Staphylococcus aureus infection in mice.

The present research summarizes the protective and immunomodulatory activity of hypothalamic proline-rich polypeptide galarmin against methicillin-resistant Staphylococcus aureus (MRSA). The protective effect of galarmin was shown on MRSA-infected animals' survival and weight loss recovery. The immunological impact of galarmin was evaluated in terms of immunocompetent cell recruitment, serum immunoglobulins, complement components C3 and C4, and pro- and anti-inflammatory cytokines (IL-6, IL-8, IL-10, IL-1b, TNFa, and KC) secretion. Galarmin efficiently protects mice against lethal MRSA infection (100% of survival vs. 0% in the untreated group) when intramuscularly injected 24 h before infection and during the 1-h post-infection period at a concentration of 1 µg per mouse, while its higher concentrations (5 and 10 µg) were protective when injected in parallel to the infection process. The protective effect of galarmin was not due to a direct effect on MRSA, but should be attributed to an action on the host response to infection. Galarmin significantly increased and modulated the levels of IL-6, IL-8, IL-1b, IL-10, and KC in both peritoneal lavages and blood, leukocyte and platelet counts, lymphocytes percentage, serum IgM and IgG, and complement C3 and C4 components secretion. The experimental results allow concluding that galarmin is a powerful immunomodulatory and protective agent for the in vivo prophylaxis and treatment of MRSA-induced infection.

Proline rich polypeptide (PRP-1) increases the superoxide-producing and ferrihemoglobin reducing activities of cytochrome B(558) isoforms from human lymphosarcoma tissue cells.

The two cytochromes (cyt) b(558) of acidic nature, one-95-100 kDa and another one, 60-70 kDa were isolated for the first time from the human's lymphosarcoma tissue cells using gel filtration and ion exchange chromatography. These hemoproteins possess NADPH dependent O(2)(-)-producing and ferrihemoglobin-reducing activities. The incubation of neuropeptide PRP-1 (5 µg) with cytochrome b(558), caused elevation of these activities. The gel filtration results indicated possible binding of PRP-1 to these cytochromes b(558). PRP-1 activated both NADPH dependent O(2)(-)-producing and ferriHb-reducing activities of the cyt b(1)(558) and cyt b(2)(558), obtained from human lymphosarcoma tissue cells. One can assume that PRP-1 associated with cyt b(558) on the surface of the tumor cells by increasing both NADPH dependent O(2)(-)-producing and ferriHb-reducing activities of cyt b(558), increases the oxidation- reduction status. Changing the oxidation-reduction status and oxygen homeostasis of the tumor cells by PRP-1 can serve as one of the possible explanation of antitumorigenic effect of this cytokine.

Antioxidant and electron donating function of hypothalamic polypeptides: galarmin and Gx-NH2.

Chemical mechanisms of antioxidant and electron donating function of the hypothalamic proline-rich polypeptides have been clarified on the molecular level. The antioxidant-chelating property of Galarmin and Gx-NH(2) was established by their capability to inhibit copper(II) dichloride catalyzed H(2)O(2) decomposition, thus preventing formation of HO(*) and HOO(*) radicals. The antiradical activity of Galarmin and Gx-NH(2) was determined by their ability to react with 2,2-diphenyl-1-picrylhydrazyl radical applying differential pulse voltammetry and UV-Vis spectrophotometry methods. Galarmin manifest antiradical activity towards 2,2-diphenyl-1-picrylhydrazyl radical, depending on the existence of phenolic OH group in tyrosine residue at the end of the molecule. The presence of antiradical activity and reduction properties of Galarmin are confirmed by the existence of an oxidation specific peak in voltammograms made by differential pulse voltammetry at E ( composite function) = 0.795 V vs. Ag/Ag(+) aq.