Impact of endogenous and exogenous nitrogen species on macrophage extracellular trap (MET) formation by bone marrow-derived macrophages.

Macrophage extracellular traps (METs) represent a novel defense mechanism in the antimicrobial arsenal of macrophages. However, mechanisms of MET formation are still poorly understood and this is at least partially due to the lack of reliable and reproducible models. Thus, we aimed at establishing a protocol of MET induction by bone marrow-derived macrophages (BMDMs) obtained from cryopreserved and then thawed bone marrow (BM) mouse cells. We report that BMDMs obtained in this way were morphologically (F4/80+) and functionally (expression of inducible nitric oxide (NO) synthase and NO production) differentiated and responded to various stimuli of bacterial (lipopolysaccharide, LPS), fungal (zymosan) and chemical (PMA) origin. Importantly, BMDMs were successfully casting METs composed of extracellular DNA (extDNA) serving as their backbone to which proteins such as H2A.X histones and matrix metalloproteinase 9 (MMP-9) were attached. In rendered 3D structure of METs, extDNA and protein components were embedded in each other. Since studies had shown the involvement of oxygen species in MET release, we aimed at studying if reactive nitrogen species (RNS) such as NO are also involved in MET formation. By application of NOS inhibitor - L-NAME or nitric oxide donor (SNAP), we studied the involvement of endogenous and exogenous RNS in traps release. We demonstrated that L-NAME halted MET formation upon stimulation with LPS while SNAP alone induced it. The latter phenomenon was further enhanced in the presence of LPS. Taken together, our findings demonstrate that BMDMs obtained from cryopreserved BM cells are capable of forming METs in an RNS-dependent manner.

Synergistic Action of MCL-1 Inhibitor with BCL-2/BCL-XL or MAPK Pathway Inhibitors Enhances Acute Myeloid Leukemia Cell Apoptosis and Differentiation.

Acute myeloid leukemia (AML) is a hematological malignancy characterized by excessive proliferation of abnormal myeloid precursors accompanied by a differentiation block and inhibition of apoptosis. Increased expression of an anti-apoptotic MCL-1 protein was shown to be critical for the sustained survival and expansion of AML cells. Therefore, herein, we examined the pro-apoptotic and pro-differentiating effects of S63845, a specific inhibitor of MCL-1, in a single-agent treatment and in combination with BCL-2/BCL-XL inhibitor, ABT-737, in two AML cell lines: HL-60 and ML-1. Additionally, we determined whether inhibition of the MAPK pathway had an impact on the sensitivity of AML cells to S63845. To assess AML cells' apoptosis and differentiation, in vitro studies were performed using PrestoBlue assay, Coulter electrical impedance method, flow cytometry, light microscopy and Western blot techniques. S63845 caused a concentration-dependent decrease in the viability of HL-60 and ML-1 cells and increased the percentage of apoptotic cells. Combined treatment with S63845 and ABT-737 or MAPK pathway inhibitor enhanced apoptosis but also induced differentiation of tested cells, as well as altering the expression of the MCL-1 protein. Taken together, our data provide the rationale for further studies regarding the use of MCL-1 inhibitor in combination with other pro-survival protein inhibitors.

Cytotoxic and Antioxidant Activity of Hypericum perforatum L. Extracts against Human Melanoma Cells from Different Stages of Cancer Progression, Cultured under Normoxia and Hypoxia.

Oxidative stress and the hypoxic microenvironment play a key role in the progression of human melanoma, one of the most aggressive skin cancers. The aim of our study was to evaluate the effect of Hypericum perforatum extracts of different origins (both commercially available (HpEx2) and laboratory-prepared from wild grown (HpEx12) and in vitro cultured (HpEx13) plants) and hyperforin salt on WM115 primary and WM266-4 lymph node metastatic human melanoma cells cultured under normoxic and hypoxic conditions. The polyphenol content, radical scavenging activity, and hyperforin concentration were determined in the extracts, while cell viability, apoptosis, ROS production, and expression of NRF2 and HO-1, important oxidative stress-related factors, were analyzed after 24 h of cell stimulation with HpExs and hyperforin salt. We found that cytotoxic, pro-apoptotic and antioxidant effects depend on the extract composition, the stage of melanoma progression, and the oxygen level. Hyperforin salt showed lower activity than H. perforatum extracts. Our study for the first time showed that the anticancer activity of H. perforatum extracts differs in normoxia and hypoxia. Importantly, the composition of extracts of various origins, including in vitro cultured, resulting in their unique properties, may be important in the selection of plants for therapeutic application.

Expression and in vitro effect of phoenixin-14 on the porcine ovarian granulosa cells.

Phoenixin-14 (PNX-14) regulates energy metabolism via the G protein-coupled receptor 173 (GPR173); elevated plasma levels have been described in patients with polycystic ovary syndrome. The aims were to investigate the ovarian expression of PNX-14/GPR173 and the in vitro effect of PNX-14 on granulosa cells (Gc) function. Transcript and protein levels of PNX-14/GRP173 were analysed by real-time PCR, western blot and immunohistochemistry in the porcine ovarian follicles at days 2-3, 10-12 and 16-18 of the oestrous. For in vitro experiments, Gc were isolated from follicles at days 10-12 of the oestrous (4-6 mm) and PNX-14 at doses 1-1000 nM was added for 24-72 h to determine Gc proliferation. Cell cycle progression, E2 secretion, expression of proliferating cells nuclear antigen, cyclins, mitogen-activated kinase (MAP3/1; ERK1/2), protein kinase B (AKT) and signal transducer and activator of transcription 3 (STAT3) were studied. The involvement of these kinases in PNX-14 action on Gc proliferation was analysed using pharmacological inhibitors. Levels of GPR173 were increased in the ovarian follicles with oestrous progression, while only PNX-14 protein was the highest at days 10-12 of the oestrous. Immuno-signal of PNX-14 was detected in Gc and theca cells and oocyte, while GPR173 was mostly in theca. Interestingly, PNX-14 stimulated Gc proliferation, E2 secretion, cell cycle progression and cyclins expression and had a modulatory effect on MAP3/1, AKT and STAT3 activation. Our study suggests that PNX-14 could be an important factor for porcine reproduction by influencing ovarian follicle growth through direct action on Gc function.

Bisphenol S and F affect cell cycle distribution and steroidogenic activity of human ovarian granulosa cells, but not primary granulosa tumour cells.

Bisphenol S (BPS) and F (BPF), a new generation of bisphenols (BPs), are the main substitutes for bisphenol A (BPA). Both have been detected in human body fluids. Importantly, bisphenols are structurally similar to oestrogen, the main sex hormone in females. Because bisphenols bind to nuclear oestrogen receptors (ESR1 and ESR2) and to membrane G-coupled receptor 30 (GPR30), they can disrupt ovarian function. Here, we reveal the molecular mechanism underlying the effects of BPS and BPF on the cell cycle and steroidogenesis in the human ovarian granulosa cell (GC) line HGrC1. We show that BPS and BPF arrest GCs at the G0/G1 phase by inducing expression of cyclin D2, an important event that triggers maximal steroid synthesis in response to the BPS and BPF. We used pharmacological inhibitors to show that BPS and BPF, despite acting via already described pathways, also stimulate steroid secretion via IGF1R pathways in HGrC1 cells. Moreover, we identified differences critical to bisphenols response between normal (HGrC1) and primary tumour granulosa (COV434) cells, that enable COV434 cells to be more resistant to bisphenols. Overall, the data suggest that BPS and BPF drive steroidogenesis in human ovarian GCs by affecting the cell cycle. Furthermore, the results indicate that BPS and BPF act not only via the classical and non-classical ESR pathways, but also via the IGF1R pathway.

Biological Properties of Heparins Modified with an Arylazopyrazole-Based Photoswitch.

Unfractionated heparin (UFH) and enoxaparin (Enox) were substituted with a photoswitch (PS) showing quantitative trans-cis and cis-trans photoisomerizations. Long half-life of the cis photoisomer enabled comparison of the properties of heparins substituted with both PS photoisomers. Hydrodynamic diameter, Dh, of UFH-PS decreased upon trans-cis photoisomerization, the change being more pronounced for UFH-PS with a higher degree of substitution (DS), while Dh of Enox-PS did not significantly change. The anticoagulative properties of substituted heparins were significantly attenuated compared to non-substituted compounds. The interaction of UFH-PS with HSA, lysozyme, and protamine was studied with ITC. Under serum-free conditions, UFH-PS-trans with a high DS stimulated proliferation of murine fibroblasts, while UFH-PS-cis decreased the viability of these cells. Under serum conditions, both UFH-PS-cis and UFH-PS-trans decreased cell viability, the reduction for UFH-PS-cis being higher than that for UFH-PS-trans. Neither Enox-PS-trans nor Enox-PS-cis influenced the viability at concentrations prolonging aPTT, while at higher concentrations their cytotoxicity did not differ.