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Monika A Papież,1 Wirginia Krzyściak,2 Krzysztof Szade,3 Karolina Bukowska-Straková,3,4 Magdalena Kozakowska,3 Karolina Hajduk,3 Beata Bystrowska,5 Jozef Dulak,3,6 Alicja Jozkowicz31Department of Cytobiology, 2Department of Medical Diagnostic, Faculty of Pharmacy, Jagiellonian University Medical College, 3Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 4Department of Clinical Immunology, Institute of Pediatrics, 5Department of Toxicology, Faculty of Pharmacy, Jagiellonian University Medical College, 6Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, PolandAbstract: Curcumin may exert a more selective cytotoxic effect in tumor cells with elevated levels of free radicals. Here, we investigated whether curcumin can modulate etoposide action in myeloid leukemia cells and in normal cells of hematopoietic origin. HL-60 cell line, normal myeloid progenitor cluster of differentiation (CD)-34+ cells, and granulocytes were incubated for 4 or 24 hours at different concentrations of curcumin and/or etoposide. Brown Norway rats with acute myeloid leukemia (BNML) were used to prove the influence of curcumin on etoposide action in vivo. Rats were treated with curcumin for 23 days and etoposide was administered for the final 3 days of the experiment. Curcumin synergistically potentiated the cytotoxic effect of etoposide, and it intensified apoptosis and phosphorylation of the histone H2AX induced by this cytostatic drug in leukemic HL-60 cells. In contrast, curcumin did not significantly modify etoposide-induced cytotoxicity and H2AX phosphorylation in normal CD34+ cells and granulocytes. Curcumin modified the cytotoxic action of etoposide in HL-60 cells through intensification of free radical production because preincubation with N-acetyl-l-cysteine (NAC) significantly reduced the cytotoxic effect of curcumin itself and a combination of two compounds. In contrast, NAC did not decrease the cytotoxic effect of etoposide. Thus, oxidative stress plays a greater role in the cytotoxic effect of curcumin than that of etoposide in HL-60 cells. In vitro results were confirmed in a BNML model. Pretreatment with curcumin enhanced the antileukemic activity of etoposide in BNML rats (1.57-fold tumor reduction versus etoposide alone; P

Summary: Bone marrow endothelial cells (BM-ECs) are the essential components of the BM niche and support the function of hematopoietic stem cells (HSCs). However, conditioning for HSC transplantation causes damage to the recipients’ BM-ECs and may lead to transplantation-related morbidity. Here, we investigated the cellular and clonal mechanisms of BM-EC regeneration after irradiative conditioning. Using single-cell RNA sequencing, imaging, and flow cytometry, we revealed how the heterogeneous pool of BM-ECs changes during regeneration from irradiation stress. Next, we developed a single-cell in vitro clonogenic assay and demonstrated that all EC fractions hold a high potential to reenter the cell cycle and form vessel-like structures. Finally, we used Rainbow mice and a machine-learning-based model to show that the regeneration of BM-ECs after irradiation is mostly polyclonal and driven by the broad fraction of BM-ECs; however, the cell output among clones varies at later stages of regeneration.

Abstract Background HO-1 participates in the degradation of heme. Its products can exert unique cytoprotective effects. Numerous tumors express high levels of HO-1 indicating that this enzyme might be a potential therapeutic target. In this study we decided to evaluate potential cytostatic/cytotoxic effects of zinc protoporphyrin IX (Zn(II)PPIX), a selective HO-1 inhibitor and to evaluate its antitumor activity in combination with chemotherapeutics. Methods Cytostatic/cytotoxic effects of Zn(II)PPIX were evaluated with crystal violet staining and clonogenic assay. Western blotting was used for the evaluation of protein expression. Flow cytometry was used to evaluate the influence of Zn(II)PPIX on the induction of apoptosis and generation of reactive oxygen species. Knock-down of HO-1 expression was achieved with siRNA. Antitumor effects of Zn(II)PPIX alone or in combination with chemotherapeutics were measured in transplantation tumor models. Results Zn(II)PPIX induced significant accumulation of reactive oxygen species in tumor cells. This effect was partly reversed by administration of exogenous bilirubin. Moreover, Zn(II)PPIX exerted potent cytostatic/cytotoxic effects against human and murine tumor cell lines. Despite a significant time and dose-dependent decrease in cyclin D expression in Zn(II)PPIX-treated cells no accumulation of tumor cells in G1 phase of the cell cycle was observed. However, incubation of C-26 cells with Zn(II)PPIX increased the percentage of cells in sub-G1 phase of the cells cycle. Flow cytometry studies with propidium iodide and annexin V staining as well as detection of cleaved caspase 3 by Western blotting revealed that Zn(II)PPIX can induce apoptosis of tumor cells. B16F10 melanoma cells overexpressing HO-1 and transplanted into syngeneic mice were resistant to either Zn(II)PPIX or antitumor effects of cisplatin. Zn(II)PPIX was unable to potentiate antitumor effects of 5-fluorouracil, cisplatin or doxorubicin in three different tumor models, but significantly potentiated toxicity of 5-FU and cisplatin. Conclusion Inhibition of HO-1 exerts antitumor effects but should not be used to potentiate antitumor effects of cancer chemotherapeutics unless procedures of selective tumor targeting of HO-1 inhibitors are developed.

Inflammation has a critical role in the development and progression of atherosclerosis. On the molecular level, inflammatory pathways negatively impact endothelial barrier properties and thus, tissue homeostasis. Conformational changes and destruction of the glycocalyx further promote pro-inflammatory pathways also contributing to pro-coagulability and a prothrombotic state. In addition, changes in the extracellular matrix composition lead to (peri-)vascular remodelling and alterations of the vessel wall, e.g., aneurysm formation. Moreover, progressive fibrosis leads to reduced tissue perfusion due to loss of functional capillaries. The present review aims at discussing the molecular and clinical effects of inflammatory processes on the micro- and macrovasculature with a focus on peripheral artery disease.

Toll-like receptors (TLRs) have a critical role in the pathogenesis and disease course of viral infections. The induced pro-inflammatory responses result in the disturbance of the endovascular surface layer and impair vascular homeostasis. The injury of the vessel wall further promotes pro-thrombotic and pro-coagulatory processes, eventually leading to micro-vessel plugging and tissue necrosis. Moreover, TLRs have a direct role in the sensing of viruses and platelet activation. TLR-mediated upregulation of von Willebrand factor release and neutrophil, as well as macrophage extra-cellular trap formation, further contribute to (micro-) thrombotic processes during inflammation. The following review focuses on TLR signaling pathways of TLRs expressed in humans provoking pro-thrombotic responses, which determine patient outcome during viral infections, especially in those with cardiovascular diseases.

Up to date, there is no information on the influence of 2,2,2-tribromoethanol (TBE; Avertin), a commonly used anaesthetic, on mice with impaired antioxidant capacity. We aimed to analyse the effect of a single dose of Avertin on anaesthesia duration time, inflammatory response, oxidative stress and collagen deposition in the large intestine of Nrf2 transcriptional knockout mice (tNrf2−/−). The studies were performed on six-month-old female mice Nrf2+/+ and tNrf2−/− randomly assigned to Avertin (250 mg/kg b.w. single i.p. injection) or vehicle group. We observed a 2-fold increase in anaesthesia time and longer recovery time (p = 0.015) in tNrf2−/− in comparison to Nrf2+/+. However, no hepato- or nephrotoxicity was detected. Interestingly, we found severe changes in colon morphology of untreated tNrf2-/- mice associated with colon shortening (p = 0.02) and thickening (p = 0.015). Avertin treatment caused colon damage manifested with epithelial layer damage and goblet depletion in Nrf2+/+ mice but not in tNrf2−/− individuals. Additionally, Avertin did not induce oxidative stress in colon tissue, but it increased leukocyte infiltration in Nrf2+/+ mice (p = 0.02). Immunofluorescent staining also revealed enhanced deposition of collagen I and collagen III in the colon of untreated tNrf2−/− mice. Avertin contributed to increased deposition of collagen I in Nrf2+/+ mice but reduced deposition of collagen I and III in tNrf2−/− individuals. In conclusion, tNrf2−/− respond to Avertin with prolonged anaesthesia that is not associated with acute toxicity, inflammatory reaction or enhanced oxidative stress. Avertin does not impair intestine morphology in tNrf2−/− mice but can normalise the enhanced fibrosis.

Abstract In the present study, we showed that in the retina of Drosophila, the expression of the ho gene, encoding haeme oxygenase (HO), is regulated by light but only at the beginning of the day. This timing must be set by the circadian clock as light pulses applied at other time points during the day do not increase the ho mRNA level. Moreover, light-induced activation of HO does not depend on the canonical phototransduction pathway but instead involves cryptochrome and is enhanced by ultraviolet (UV) light. Interestingly, the level of DNA damage in the retina after UV exposure was inversely related to the circadian oscillation of the ho mRNA level during the night, being the highest when the HO level was low and reversed during the day. Accordingly, induction of HO by hemin was associated with low DNA damage, while inhibition of HO activity by SnPPIX aggravated the damage. Our data suggest that HO acts in the retina to decrease oxidative DNA damage in photoreceptors caused by UV-rich light in the morning.