Amputation-induced reactive oxygen species are required for successful Xenopus tadpole tail regeneration

Mitochondria Transfer from Hematopoietic Stem and Progenitor Cells to Pdgfrα+/Sca-1-/CD48dim BM Stromal Cells Via CX43 Gap Junctions and AMPK Signaling Inversely Regulate ROS Generation in Both Cell Populations

49 - Inhibition of Amputation-Induced Reactive Oxygen Species Blocks Salamander Tail Regeneration

The iron chelator deferasirox (DFX) prevents complications related to transfusional iron overload in several haematological disorders characterized by marrow failure. It is also able to induce haematological responses in a percentage of treated patients, particularly in those affected by myelodysplastic syndromes. The underlying mechanisms responsible for this feature, however, are still poorly understood. In this study, we investigated the effect of DFX-treatment in human haematopoietic/progenitor stem cells, focussing on its impact on the redox balance, which proved to control the interplay between stemness maintenance, self-renewal and differentiation priming. Here we show, for the first time, that DFX treatment induces a significant diphenyleneiodonium-sensitive reactive oxygen species (ROS) production that leads to the activation of POU5F1 (OCT4), SOX2 and SOX17 gene expression, relevant in reprogramming processes, and the reduction of the haematopoietic regulatory proteins CTNNB1 (β-Catenin) and BMI1. These DFX-mediated events were accompanied by decreased CD34 expression, increased mitochondrial mass and up-regulation of the erythropoietic marker CD71 (TFRC) and were compound-specific, dissimilar to deferoxamine. Our findings would suggest a novel mechanism by which DFX, probably independently on its iron-chelating property but through ROS signalling activation, may influence key factors involved in self-renewal/differentiation of haematopoietic stem cells.

IntroductionElevated levels of reactive oxygen species (ROS) frequently observed in tumours are part of the metabolic reprogramming, which is critical for cancer initiation and progression. However, cancer cells remain sensitive to treatments which further increase or decrease ROS. Tailored modulation of ROS levels thus may become a new strategy in cancer therapy. In contrast to other tumours, melanoma have low ROS levels and we have shown previously that RAF kinases are able to prevent excessive mitochondrial ROS production. To understand the underlying reasons for impaired ROS production in BRAFV600E transformed cells, we studied a possible link to the oxidoreductase p66Shc, a protein that is directly involved in the generation of mitochondrial ROS and frequently overexpressed in tumours.Material and methodsMitochondrial ROS production was analysed in fibroblasts singly transformed by mutant BRAFV600E, the melanoma cell lines A375 and M238 carrying the identical BRAF mutation, and the vemurafenib resistant variant M238R. Mitochondrial ROS production was analysed by microscopic imaging or FACS analysis of MitoTracker Red CM-H2XRos stained cells. Intracellular signalling was monitored through the use of phosphorylation-specific and control antibodies.Results and discussionsWe show that following treatment with the cell death inducing agent phenethyl isothiocyanate (PEITC) oncogenic BRAFV600E renders cells refractory to phosphorylation of S36 in p66Shc, which is essential for p66Shc activation, mitochondrial translocation and ROS production. Consistent with this observation, the activation of JNK1/2, the kinases phosphorylating S36, as demonstrated by us previously, was blunted, while other MAPKs were not affected. Use of a JNK1/2 inhibitor most efficiently prevented ROS production while inhibition of BRAF or MEK increased ROS levels, without affecting S36 phosphorylation. Melanoma cells, which had become resistant to the mutant BRAF-specific inhibitor vemurafenib, were impaired in S36 phosphorylation and ROS production after PEITC treatment. Moreover, they failed to increase ROS levels after MEK/BRAF inhibition. Finally, shRNA-mediated knockdown of p66Shc led to increased colony size of BRAFV600E transformed cells in soft agar. Taken together these data suggest that phoshpo-p66Shc functions as a tumour suppressor in melanoma.ConclusionOur findings demonstrate that interference with cytoplasmic signalling pathways regulating p66Shc activation may be therapeutically exploited to alter ROS levels in tumour cells.

Background: Nuclear events play a well-established role in the cytotoxicity of doxorubicin (DOX). Other reported mechanisms include redox cycling for reactive oxygen species (ROS) production, which is getting recognized for a larger role than previously recognized. In addition, ROS production may be implicated in lysosome membrane permeabilization (LMP) and subsequent cell death. We have previously reported DOX-induced LMP in breast cancer cells. The goal of this study is to establish the role of LMP in the cytotoxic process and whether ROS are the initiator of this LMP. Methods: Studies were conducted using DOX concentrations ranging from 0.1 μM to 10 μM for 2, 6, and 24 h exposure periods in MCF-7 and MDA-MB-231 breast cancer cells. Intracellular localization of DOX was examined by fluorescence microscopy using lysosome and nuclear markers. ROS production was determined by the DCFDA assay using a fluorescence plate reader. LMP was demonstrated using fluorescence microscopy as the cytosolic release of a 10 kDa AlexaFluor-488 Dextran conjugate that was preloaded into lysosomes. Mitochondrial membrane potential (MMP) was determined by JC-1 assay. Viability was determined by MTT. Cathepsin B activity will be determined by Z-Arg-Arg-AMC cathepsin B substrate. Experiments will be conducted in the presence of E-64 cathepsin B inhibitor and Tiron superoxide scavenger to establish the importance of LMP and ROS towards DOX cytotoxicity. Results: Nuclear uptake was seen in both cell lines as early as 2 h at a concentration of 10 μM. Furthermore, lysosome accumulation of DOX, although small, was visible by 2 h in MDA cells, increasing in degree with time and concentration. ROS levels were found to be highest at 2 h in MCF-7 cells, increasing by up to 100% of the untreated control, and occurring concurrently with LMP at higher concentrations. Preliminary results suggest much lower ROS levels in MDA-MB-231 cells. In MCF-7 cells, ROS production decreased at 6 h, but increased by 24 h with concurrent increases in LMP. Although less than the MCF-7 cells, MDA cells demonstrated notable LMP for the first time at 5 and 10 μM conditions, occurring concurrently with MMP. By 24 h, viability was substantially reduced for both cell lines, with MCF-7s demonstrating a greater reduction in viability. Significant MMP was not observable until 24 h in MCF-7 cells. Conclusions: The early and strong ROS occurring with LMP suggest a previously unrecognized DOX cytotoxic combination in MCF-7 cells. LMP does not appear to result from lysosomal accumulation of DOX. A reduced role of ROS and LMP may account for the lesser cytotoxicity in MDA-MB-231 cells. Both ROS and LMP appear to be necessary for cytotoxicity in MCF-7 cells. Citation Format: Rachel E. Nicoletto, Clyde M. Ofner. Investigating the roles of reactive oxygen species and lysosomes in the cytotoxicity of doxorubicin in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB103.

Higher levels of reactive oxygen species are associated with anergy in chronic lymphocytic leukemia2][3] As in other cells, mitochondria appear to be the main source of ROS and CLL cells have an increased mitochondrial mass compared to that of normal B cells. 2,4Higher levels of ROS confer increased sensitivity to induction of apoptosis by agents which further enhance ROS and it may be possible to exploit this as the basis for new treatments. 1,2,5]5,6 Analyses have mainly been performed on samples from patients with more advanced disease and, in that setting, prior therapy appeared to be a major determinant of ROS levels. 3,5,6This may reflect a direct effect of drugs on ROS production, perhaps linked to accumulation of mitochondrial DNA damage. 6However, a recent study demonstrated variable ROS levels in cells from untreated patients 2 indicating an influence of other factors.The B-cell receptor (BCR) is now recognized as a key determinant of variable behavior of CLL 7 and is a target for therapeutic attack.Antigen engagement appears to be iterative, with the outcome being either proliferation or anergy, a balance likely to influence disease outcome. 7,8Anergy

TGF-β1 signaling is essential for tissue regeneration in the Xenopus tadpole tail

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease without clearly known disease causes. Recent epidemiological and animal studies suggest that the supplementation of dietary antioxidants (e.g., vitamins C and E) decreases cancer risk, implying that increased reactive oxygen species (ROS) may play a role in pancreatic carcinogenesis. However, oncogenic Kras mutations (e.g., KrasG12D), which are present in more than 90% of PDAC, have been proven to foster low intracellular ROS levels. Here, oncogenic Kras activates expression of a series of anti-oxidant genes via Nrf2 (nuclear factor, erythroid derived 2, like 2) and also mediates an unusual metabolic pathway of glutamine to generate NADPH. This can then be used as the reducing power for ROS detoxification, leading collectively to low ROS levels in pancreatic pre-neoplastic cells and in cancer cells. In adult stem cells and cancer stem cells, low ROS levels have been associated with the formation of a proliferation-permissive intracellular environment and with perseverance of self-renewal capacities. Therefore, it is conceivable that low intracellular ROS levels may contribute significantly to oncogenic Kras-mediated PDAC formation.

UVB Radiation Induces Apoptosis in Keratinocytes by Activating a Pathway Linked to “BLT2-Reactive Oxygen Species”

Research into different species has verified the negative correlation between longevity and the level of reactive oxygen species (ROS) production. ROS creates oxidative damage and, consequently, fuels the aging process. As such, the astonishing longevity of avian species correlates well with their lower levels of ROS production, in comparison to mammals of similar size. Apart from this inter-species difference, caloric restriction (CR) is a widely-documented means of increasing intra-species longevity, and it works by decreasing ROS production. However, little is known about the mechanisms responsible, either for the retardation of aging in CR or for the longevity of long-living species. Recent findings have shown an increase in uncoupling protein (UCP) activity with lower ROS levels, after CR stress. These UCPs are stimulated by fatty acids. Moreover, in numerous studies, fatty acids have been demonstrated to generate a reduction in ROS generation. Thus, the decreased ROS production seen in both CR and longer lifespan may occur via up-regulation of free fatty acid stimulation of UCP activity. Consequently, free fatty acids may play an important regulatory role in longevity, by reducing ROS, via actions on UCPs.

Psoriasis is a chronic inflammatory skin disease resulting from immune dysregulation. Regulatory T cells (Tregs) are important in the prevention of psoriasis. Traditionally, reactive oxygen species (ROS) are known to be implicated in the progression of inflammatory diseases, including psoriasis, but many recent studies suggested the protective role of ROS in immune-mediated diseases. In particular, severe cases of psoriasis vulgaris have been reported to be successfully treated by hyperbaric oxygen therapy (HBOT), which raises tissue level of ROS. Also it was reported that Treg function was closely associated with ROS level. However, it has been only investigated in lowered levels of ROS so far. Thus, in this study, to clarify the relationship between ROS level and Treg function, as well as their role in the pathogenesis of psoriasis, we investigated imiquimod-induced psoriatic dermatitis (PD) in association with Treg function both in elevated and lowered levels of ROS by using knockout mice, such as glutathione peroxidase-1−/− and neutrophil cytosolic factor-1−/− mice, as well as by using HBOT or chemicals, such as 2,3-dimethoxy-1,4-naphthoquinone and N-acetylcysteine. The results consistently showed Tregs were hyperfunctional in elevated levels of ROS, whereas hypofunctional in lowered levels of ROS. In addition, imiquimod-induced PD was attenuated in elevated levels of ROS, whereas aggravated in lowered levels of ROS. For the molecular mechanism that may link ROS level and Treg function, we investigated the expression of an immunoregulatory enzyme, indoleamine 2,3-dioxygenase (IDO) which is induced by ROS, in PD lesions. Taken together, it was implied that appropriately elevated levels of ROS might prevent psoriasis through enhancing IDO expression and Treg function.

Increased production of reactive oxygen species (ROS) in the vascular wall is a characteristic feature of disease states, including atherosclerosis, diabetes and hypertension. ROS, such as superoxide, reduce nitric oxide bioactivity by scavenging and cause oxidation of lipids and target proteins. In addition, recent work has revealed that ROS mediate a wide range of pathological processes in the endothelium, smooth muscle cells, and inflammatory cells.1 ROS are generated by enzyme systems present in cells in the vascular wall, including NAD(P)H oxidase, xanthine oxidase, and nitric oxide synthase. The activities and levels of these enzyme systems are increased in association with vascular disease risk factors2 and in vascular disease states in which oxidative stress is prominent, for example, in diabetes3 and atherosclerosis. See page 1838 The NAD(P)H oxidases appear to be particularly important sources of ROS production in blood vessels,4 where they are constitutively active, producing relatively low levels of ROS under basal conditions, but generating higher levels of oxidants in response to stimuli such as growth factors and cytokines. These factors are consistent with a role for nonphagocytic NAD(P)H oxidases in cellular signaling rather than the high-level burst activity characteristic of the phagocyte NAD(P)H oxidase. The NAD(P)H oxidases are multimeric enzymes composed of plasma membrane associated–proteins as well as cytosolic factors. In the phagocytic-type NAD(P)H oxidase, the plasma membrane–associated proteins gp91phox and p22phox compose the flavocytochrome b558 complex, which forms the catalytic subunit of the oxidase. The cytosolic subunits, including p47phox, p67phox, and the G-protein Rac, provide regulatory function. Azumi and colleagues,5 in this issue of Arteriosclerosis, Thrombosis and Vascular Biology …

Notch-Mediated Suppression of PKCθ Reduces Reactive Oxygen Species and Promotes Leukemic Stem Cell Activity In T-Cell Acute Lymphoblastic Leukemia (T-ALL)

ROS: Redux and paradox in fatty liver disease

Cul5-type Ubiquitin Ligase KLHDC1 Contributes to the Elimination of Truncated SELENOS Produced by Failed UGA/Sec Decoding.