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AimTo use the antioxidant compounds (sodium selenite, selenomethionine, D-pantethine) for modulation of cytotoxic effect of doxorubicin and cisplatin toward wild type and drug-resistant mutants of several human tumor cells. Similar treatments were applied in vivo toward adult male Wistar rats.MethodsHuman tumor cells of different lines (HCT-116, Jurkat and HL-60) with various mechanisms of drug-resistance were treated with doxorubicin or cisplatin, alone or in combination with sodium selenite, selenomethionine, or D-pantethine. Cell viability, induction of apoptosis, and production of O2- radicals were measured. Activity of redox potential modulating enzymes was measured in the liver and blood plasma of adult male Wistar rats subjected to similar treatments.ResultsAll antioxidants used in physiologically harmless concentration inhibited cytotoxic action of doxorubicin toward tumor cells sensitive to chemotherapy treatment by 15%-30%, and slightly enhanced cytotoxic effect of this medicine toward drug-resistant malignant cells. At the same time, there was no significant effect of these antioxidants on cisplatin action. Such effects were accompanied by a complete inhibition of production of superoxide radicals induced by doxorubicin. The results of in vivo study in adult male Wistar rats were in agreement with the results of in vitro study of human tumor cells.ConclusionProtective effect of specific antioxidant agents during cytotoxic action of doxorubicin was demonstrated in vitro in drug-sensitive human tumor cells and in adult male Wistar rats, while there was no protective effect in drug-resistant sub-lines of these tumor cells during action of doxorubicin and cisplatin.

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Heat shock proteins (HSPs) constitute a major part of the molecular chaperone system and play a fundamental role in cell proteostasis. The HSPA (HSP70) family groups twelve highly homologous HSPA proteins. Certain HSPAs are regarded as important cancer-related proteins, prospective therapeutic targets for cancer treatment, and also as potential cancer biomarkers. Heat Shock Protein A2 (HSPA2), a testis-enriched chaperone and one of the least characterized members of the HSPA family, has recently emerged as an important cancer-relevant protein with potential biomarker significance. Nevertheless, conflicting conclusions have been recently drawn both according to HSPA2 role in cancer cells, as well as to its prognostic value. In this work we have shown that one of the serious limitations in HSPA2 protein research is cross-reactivity of antibodies marketed as specific for HSPA2 with one or more other HSPA(s). Among non-specific antibodies were also those recently used for HSPA2 detection in functional and biomarker studies. We showed how using non-specific antibodies can generate misleading conclusions on HSPA2 expression in non-stressed cancer cells and tumors, as well as in cancer cells exposed to proteotoxic stress. Our findings addressed concerns on some published studies dealing with HSPA2 as a cancer-related protein.

AimTo investigate the potential tissue-protective effects of antioxidants selenomethionine and D-pantethine applied together with doxorubicin (Dx) on NK/Ly lymphoma-bearing mice. The impact of this chemotherapy scheme on animal survival, blood cell profile, hepatotoxicity, glutathione level, and activity of glutathione-converting enzymes in the liver was compared with the action of Dx applied alone.MethodsThe hematological profile of animals was studied by the analysis of blood smears under light microscopy. Hepatotoxicity of studied drugs was evaluated measuring the activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzymes, De Ritis ratio, and coenzyme A fractions by McDougal assay. Glutathione level in animal tissues was measured with Ellman reagent, and the activity of glutathione reductase, transferase, and peroxidase was measured using standard biochemical assays.ResultsD-pantethine (500 mg/kg) and, to a lower extent, selenomethionine (600 µg/kg) partially reduced the negative side effects (leukocytopenia and erythropenia) of Dx (5 mg/kg) in NK/Ly lymphoma bearing animals on the 14th day of their treatment. This increased animal survival time from 47-48 to 60+ days and improved the quality of their life. This ability of D-pantethine and selenomethionine was realized via hepatoprotective and immunomodulating activities. D-pantethine also restored the levels of acid-soluble and free CoA in the liver of tumor-bearing animals, while selenomethionine caused the recovery of glutathione peroxidase levels in the liver, which was significantly diminished under Dx treatment. Both compounds decreased glutathione level in the liver, which was considerably induced by Dx.ConclusionsAntioxidants selenomethionine and D-pantethine partially reversed the negative side effects of Dx in NK/Ly lymphoma-bearing mice and significantly increased the therapeutic efficiency of this drug in tumor treatment.

DOCK7 (dedicator of cytokinesis 7) is a guanidine nucleotide exchange factor (GEF) for Rac1 GTPase that is involved in neuronal polarity and axon generation as well in Schwann cell differentiation and myelination. Recently, we identified DOCK7 as the binding partner of unconventional myosin VI (MVI) in neuronal-lineage PC12 cells and postulated that this interaction could be important in vivo [Majewski et al. (2012) Biochem Cell Biol., 90:565-574]. Herein, we found that MVI-DOCK7 interaction takes also place in other cell lines and demonstrated that MVI cargo domain via its RRL motif binds to DOCK7 C-terminal M2 and DHR2 domains. In MVI knockdown cells, lower Rac1 activity and a decrease of DOCK7 phosphorylation on Tyr1118 were observed, indicating that MVI could contribute to DOCK7 activity. MVI and DOCK7 co-localization was maintained during NGF-stimulated PC12 cell differentiation and observed also in the outgrowths. Also, during differentiation an increase in phosphorylation of DOCK7 as well as of its downstream effector JNK kinase was detected. Interestingly, overexpression of GFP-tagged MVI cargo domain (GFP-GT) impaired protrusion formation indicating that full length protein is important for this process. Moreover, a transient increase in Rac activity observed at 5min of NGF-stimulated differentiation of PC12 cells (overexpressing either GFP or GFP-MVI) was not detected in cells overexpressing the cargo domain. These data indicate that MVI-DOCK7 interaction could have functional implications in the protrusion outgrowth, and full length MVI seems to be important for delivery and maintenance of DOCK7 along the protrusions, and exerting its GEF activity.

We postulated that mobilization, homing, and engraftment of hematopoietic stem/progenitor cells (HSCPs) is facilitated by a state of sterile inflammation induced in bone marrow (BM) after administration of pro-mobilizing drugs or in response to pre-transplant myeloablative conditioning. An important role in this phenomenon plays purinergic signaling that by the release of extracellular adenosine triphosphate (eATP) activates in HSPCs and in cells in the hematopoietic microenvironment an intracellular pattern recognition receptor (PPR) known as Nlrp3 inflammasome. We reported recently that its deficiency results in defective trafficking of HSPCs. Moreover, it is known that eATP after release into extracellular space is processed by cell surface expressed ectonucleotidases CD39 and CD73 to extracellular adenosine (eAdo) that in contrast to eATP shows an anti-inflammatory effect. Based on data that the state of sterile inflammation promotes trafficking of HSPCs, and since eAdo is endowed with anti-inflammatory properties we become interested in how eAdo will affect the mobilization, homing, and engraftment of HSPCs and which of eAdo receptors are involved in these processes. As expected, eAdo impaired HSPCs trafficking and this occurred in autocrine- and paracrine-dependent manner by direct stimulation of these cells or by affecting cells in the BM microenvironment. We report herein for the first time that this defect is mediated by activation of the A2B receptor and a specific inhibitor of this receptor improves eAdo-aggravated trafficking of HSPCs. To explain this at the molecular level eAdo-A2B receptor interaction upregulates in HSPCs in NF-kB-, NRF2- and cAMP-dependent manner heme oxygenase-1 (HO-1), that is Nlrp3 inflammasome inhibitor. This corroborated with our analysis of proteomics signature in murine HSPCs exposed to eAdo that revealed that A2B inhibition promotes cell migration and proliferation. Based on this we postulate that blockage of A2B receptor may accelerate the mobilization of HSPCs as well as their hematopoietic reconstitution and this approach could be potentially considered in the future to be tested in the clinic. Graphical Abstract