Mitochondrial division inhibitor (mdivi-1) induces extracellular matrix (ECM)-detachment of viable breast cancer cells by a DRP1-independent mechanism.

Increasing evidence supports the hypothesis that cancer progression is under mitochondrial control. Mitochondrial fission plays a pivotal role in the maintenance of cancer cell homeostasis. The inhibition of DRP1, the main regulator of mitochondrial fission, with the mitochondrial division inhibitor (mdivi-1) had been associated with cancer cell sensitivity to chemotherapeutics and decrease proliferation. Here, using breast cancer cells we find that mdivi-1 induces the detachment of the cells, leading to a bulk of floating cells that conserved their viability. Despite a decrease in their proliferative and clonogenic capabilities, these floating cells maintain the capacity to re-adhere upon re-seeding and retain their migratory and invasive potential. Interestingly, the cell detachment induced by mdivi-1 is independent of DRP1 but relies on inhibition of mitochondrial complex I. Furthermore, mdivi-1 induces cell detachment rely on glucose and the pentose phosphate pathway. Our data evidence a novel DRP1-independent effect of mdivi-1 in the attachment of cancer cells. The generation of floating viable cells restricts the use of mdivi-1 as a therapeutic agent and demonstrates that mdivi-1 effect on cancer cells are more complex than anticipated.

Novel Inositol 1,4,5-Trisphosphate Receptor Inhibitor Antagonizes Hepatic Stellate Cell Activation: A Potential Drug to Treat Liver Fibrosis.

Liver fibrosis, characterized by excessive extracellular matrix (ECM) deposition, can progress to cirrhosis and increases the risk of liver cancer. Hepatic stellate cells (HSCs) play a pivotal role in fibrosis progression, transitioning from a quiescent to activated state upon liver injury, wherein they proliferate, migrate, and produce ECM. Calcium signaling, involving the inositol 1,4,5-trisphosphate receptor (IP3R), regulates HSC activation. This study investigated the efficacy of a novel IP3R inhibitor, desmethylxestospongin B (dmXeB), in preventing HSC activation. Freshly isolated rat HSCs were activated in vitro in the presence of varying dmXeB concentrations. The dmXeB effectively inhibited HSC proliferation, migration, and expression of fibrosis markers without toxicity to the primary rat hepatocytes or human liver organoids. Furthermore, dmXeB preserved the quiescent phenotype of HSCs marked by retained vitamin A storage. Mechanistically, dmXeB suppressed mitochondrial respiration in activated HSCs while enhancing glycolytic activity. Notably, methyl pyruvate, dimethyl α-ketoglutarate, and nucleoside supplementation all individually restored HSC proliferation despite dmXeB treatment. Overall, dmXeB demonstrates promising anti-fibrotic effects by inhibiting HSC activation via IP3R antagonism without adverse effects on other liver cells. These findings highlight dmXeB as a potential therapeutic agent for liver fibrosis treatment, offering a targeted approach to mitigate liver fibrosis progression and its associated complications.

17-ß-estradiol and phytoestrogens elicit NO production and vasodilatation through PI3K, PKA and EGF receptors pathways, evidencing functional selectivity.

Endothelial cells express multiple receptors mediating estrogen responses; including the G protein-coupled estrogen receptor (GPER). Past studies on nitric oxide (NO) production elicited by estrogens raised the question whether 17-ß-estradiol (E2) and natural phytoestrogens activate equivalent mechanisms. We hypothesized that E2 and phytoestrogens elicit NO production via coupling to distinct intracellular pathways signalling. To this aim, perfusion of E2 and phytoestrogens to the precontracted rat mesentery bed examined vasorelaxation, while fluorescence microscopy on primary endothelial cells cultures quantified single cell NO production determined following 4-amino-5-methylamino-2',7'-difluoroescein diacetate (DAF) incubation. Daidzein (DAI) and genistein (GEN) induced rapid vasodilatation associated to NO production. Multiple estrogen receptor activity was inferred based on the reduction of DAF-NO signals; G-36 (GPER antagonist) reduced 75 % of all estrogen responses, while fulvestrant (selective nuclear receptor antagonist) reduced significantly more the phytoestrogens responses than E2. The joint application of both antagonists abolished the E2 response but not the phytoestrogen-induced DAF-NO signals. Wortmannin or LY-294002 (PI3K inhibitors), reduced by 90% the E2-evoked signal while altering significantly less the DAI-induced response. In contrast, H-89 (PKA inhibitor), elicited a 23% reduction of the E2-induced signal while blocking 80% of the DAI-induced response. Desmethylxestospongin-B (IP3 receptor antagonist), decreased to equal extent the E2 or the DAI-induced signal. Epidermal growth factor (EGF) induced NO production, cell treatment with AG-1478, an EGF receptor kinase inhibitor reduced 90% DAI-induced response while only 53% the E2-induced signals; highlighting GPER induced EGF receptor trans-modulation. Receptor functional selectivity may explain distinct signalling pathways mediated by E2 and phytoestrogens.

Impact of platelet-derived mitochondria transfer in the metabolic profiling and progression of metastatic MDA-MB-231 human triple-negative breast cancer cells.

Introduction: An active role of platelets in the progression of triple-negative breast cancer (TNBC) cells has been described. Even the role of platelet-derived extracellular vesicles on the migration of MDA-MB-231 cells has been reported. Interestingly, upon activation, platelets release functional mitochondria into the extracellular environment. However, the impact of these platelet-derived mitochondria on the metabolic properties of MDA-MB-231 cells remains unclear. Methods: MDA-MB-231 and MDA-MB-231-Rho-0 cells were co-cultured with platelets, which were isolated from donor blood. Mitochondrial transfer was assessed through confocal microscopy and flow cytometry, while metabolic analyses were conducted using a Seahorse XF HS Mini Analyzer. The mito-chondrial DNA (mtDNA) copy number was determined via quantitative PCR (qPCR) following platelet co-culture. Finally, cell proliferation and colony formation assay were performed using crystal violet staining. Results and Discussion: We have shown that platelet-derived mitochondria are internalized by MDA-MB-231 cells in co-culture with platelets, increasing ATP production, oxygen (O2) consumption rate (OCR), cell proliferation, and metabolic adaptability. Additionally, we observed that MDA-MB-231 cells depleted from mtDNA restore cell proliferation in uridine/pyruvate-free cell culture medium and mitochondrial O2 consumption after co-culture with platelets, indicating a reconstitution of mtDNA facilitated by platelet-derived mitochondria. In conclusion, our study provides new insights into the role of platelet-derived mitochondria in the metabolic adaptability and progression of metastatic MDA-MB-231 TNBC cells.

Idiopathic inflammatory myopathy human derived cells retain their ability to increase mitochondrial function.

Idiopathic Inflammatory Myopathies (IIMs) have been studied within the framework of autoimmune diseases where skeletal muscle appears to have a passive role in the illness. However, persiting weakness even after resolving inflammation raises questions about the role that skeletal muscle plays by itself in these diseases. "Non-immune mediated" hypotheses have arisen to consider inner skeletal muscle cell processes as trigger factors in the clinical manifestations of IIMs. Alterations in oxidative phosphorylation, ATP production, calcium handling, autophagy, endoplasmic reticulum stress, among others, have been proposed as alternative cellular pathophysiological mechanisms. In this study, we used skeletal muscle-derived cells, from healthy controls and IIM patients to determine mitochondrial function and mitochondrial ability to adapt to a metabolic stress when deprived of glucose. We hypothesized that mitochondria would be dysfunctional in IIM samples, which was partially true in normal glucose rich growing medium as determined by oxygen consumption rate. However, in the glucose-free and galactose supplemented condition, a medium that forced mitochondria to function, IIM cells increased their respiration, reaching values matching normal derived cells. Unexpectedly, cell death significantly increased in IIM cells under this condition. Our findings show that mitochondria in IIM is functional and the decrease respiration observed is part of an adaptative response to improve survival. The increased metabolic function obtained after forcing IIM cells to rely on mitochondrial synthesized ATP is detrimental to the cell's viability. Thus, therapeutic interventions that activate mitochondria, could be detrimental in IIM cell physiology, and must be avoided in patients with IIM.

Antenatal melatonin modulates an enhanced antioxidant/pro-oxidant ratio in pulmonary hypertensive newborn sheep.

Chronic hypobaric hypoxia during fetal and neonatal life induces neonatal pulmonary hypertension. Hypoxia and oxidative stress are driving this condition, which implies an increase generation of reactive oxygen species (ROS) and/or decreased antioxidant capacity. Melatonin has antioxidant properties that decrease oxidative stress and improves pulmonary vascular function when administered postnatally. However, the effects of an antenatal treatment with melatonin in the neonatal pulmonary function and oxidative status are unknown. Therefore, we hypothesized that an antenatal therapy with melatonin improves the pulmonary arterial pressure and antioxidant status in high altitude pulmonary hypertensive neonates. Twelve ewes were bred at high altitude (3600 m); 6 of them were used as a control group (vehicle 1.4% ethanol) and 6 as a melatonin treated group (10 mg d-1 melatonin in vehicle). Treatments were given once daily during the last third of gestation (100-150 days). Lambs were born and raised with their mothers until 12 days old, and neonatal pulmonary arterial pressure and resistance, plasma antioxidant capacity and the lung oxidative status were determined. Furthermore, we measured the pulmonary expression and activity for the antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase, and the oxidative stress markers 8-isoprostanes, 4HNE and nitrotyrosine. Finally, we assessed pulmonary pro-oxidant sources by the expression and function of NADPH oxidase, mitochondria and xanthine oxidase. Melatonin decreased the birth weight. However, melatonin enhanced the plasma antioxidant capacity and decreased the pulmonary antioxidant activity, associated with a diminished oxidative stress during postnatal life. Interestingly, melatonin also decreased ROS generation at the main pro-oxidant sources. Our findings suggest that antenatal administration of melatonin programs an enhanced antioxidant/pro-oxidant status, modulating ROS sources in the postnatal lung.

In Vitro-Generated Tc17 Cells Present a Memory Phenotype and Serve As a Reservoir of Tc1 Cells In Vivo.

Memory CD8+ T cells are ideal candidates for cancer immunotherapy because they can mediate long-term protection against tumors. However, the therapeutic potential of different in vitro-generated CD8+ T cell effector subsets to persist and become memory cells has not been fully characterized. Type 1 CD8+ T (Tc1) cells produce interferon-γ and are endowed with high cytotoxic capacity, whereas IL-17-producing CD8+ T (Tc17) cells are less cytotoxic but display enhanced self-renewal capacity. We sought to evaluate the functional properties of in vitro-generated Tc17 cells and elucidate their potential to become long lasting memory cells. Our results show that in vitro-generated Tc17 cells display a greater in vivo persistence and expansion in response to secondary antigen stimulation compared to Tc1 cells. When transferred into recipient mice, Tc17 cells persist in secondary lymphoid organs, present a recirculation behavior consistent with central memory T cells, and can shift to a Tc1 phenotype. Accordingly, Tc17 cells are endowed with a higher mitochondrial spare respiratory capacity than Tc1 cells and express higher levels of memory-related molecules than Tc1 cells. Together, these results demonstrate that in vitro-generated Tc17 cells acquire a central memory program and provide a lasting reservoir of Tc1 cells in vivo, thus supporting the use of Tc17 lymphocytes in the design of novel and more effective therapies.