Ferroptosis in Different Pathological Contexts Seen through the Eyes of Mitochondria.

Ferroptosis is a recently described form of regulated cell death characterized by intracellular iron accumulation and severe lipid peroxidation due to an impaired cysteine-glutathione-glutathione peroxidase 4 antioxidant defence axis. One of the hallmarks of ferroptosis is a specific morphological phenotype characterized by extensive ultrastructural changes of mitochondria. Increasing evidence suggests that mitochondria play a significant role in the induction and execution of ferroptosis. The present review summarizes existing knowledge about the mitochondrial impact on ferroptosis in different pathological states, primarily cancer, cardiovascular diseases, and neurodegenerative diseases. Additionally, we highlight pathologies in which the ferroptosis/mitochondria relation remains to be investigated, where the process of ferroptosis has been confirmed (such as liver- and kidney-related pathologies) and those in which ferroptosis has not been studied yet, such as diabetes. We will bring attention to avenues that could be followed in future research, based on the use of mitochondria-targeted approaches as anti- and proferroptotic strategies and directed to the improvement of existing and the development of novel therapeutic strategies.

Fractal and stereological analyses of insulin-induced rat exocrine pancreas remodelling.

BACKGROUND: The effect of insulin on the endocrine pancreas has been the subject of extensive study, but quantitative morphometric investigations of the exocrine pancreas are scarce. This study was therefore undertaken to investigate the effect of acute and chronic insulin administration (two doses, 0.4 IU and 4 IU) on the morphology of rat pancreas acini. MATERIALS AND METHODS: Semi-fine sections stained with methylene blue and basic fuchsine or haematoxylin and eosin-stained 5-micrometer thick paraffin sections were used for fractal and stereological analysis of exocrine acini. Acute insulin treatment, independent of applied doses increased fractal dimension in line with decreased lacunarity of pancreas acini. Chronic low dose insulin decreased fractal dimension and increased lacunarity of pancreas acini, but a high dose had the opposite effect. The volume densities (Vv) of cytoplasm, granules and nucleus are affected differently: acute low dose and high chronic dose significantly decreased granules Vv, and in line increased cytoplasmic Vv, whereas other examined structures showed slight changes without statistical significance. RESULTS: The results obtained from this investigation indicate that insulin treatment induced structural remodelling of the exocrine pancreas suggesting a substantial role of insulin in its functioning. CONCLUSIONS: Additionally, we showed that fine architectural changes in acini could be detected by fractal analysis, suggesting this method as an alternative or addition to routine stereology.

GPR55 promotes migration and adhesion of colon cancer cells indicating a role in metastasis.

BACKGROUND AND PURPOSE: Tumour cell migration and adhesion constitute essential features of metastasis. G-protein coupled receptor 55 (GPR55), a lysophospholipid receptor, has been shown to play an important role in carcinogenesis. Here, we investigated the involvement of GPR55 in migration and metastasis of colon cancer cells. EXPERIMENTAL APPROACH: Adhesion and migration assays using the highly metastatic colon cancer cell line HCT116 and an in vivo assay of liver metastasis were performed. The GPR55 antagonist CID16020046, cannabidiol, a putative GPR55 antagonist and GPR55 siRNA were used to block GPR55 activity in HCT116 colon cancer cells. KEY RESULTS: HCT116 cells showed a significant decrease in adhesion to endothelial cells and in migration after blockade with CID16020046 or cannabidiol. The inhibitory effects of CID16020046 or cannabidiol were averted by GPR55 siRNA knock down in cancer cells. The integrity of endothelial cell monolayers was increased after pretreatment of HCT116 cells with the antagonists or after GPR55 siRNA knockdown while pretreatment with lysophosphatidylinositol (LPI), the endogenous ligand of GPR55, decreased integrity of the monolayers. LPI also induced migration in GPR55 overexpressing HCT116 cells that was blocked by GPR55 antagonists. In a mouse model of metastasis, the arrest of HCT116 cancer cells in the liver was reduced after treatment with CID16020046 or cannabidiol. Increased levels of LPI (18:0) were found in colon cancer patients when compared with healthy individuals. CONCLUSIONS AND IMPLICATIONS: GPR55 is involved in the migratory behaviour of colon carcinoma cells and may serve as a pharmacological target for the prevention of metastasis. © 2015 The British Pharmacological Society.

The GPR55 antagonist CID16020046 protects against intestinal inflammation.

BACKGROUND: G protein-coupled receptor 55 (GPR55) is a lysophospholipid receptor responsive to certain cannabinoids. The role of GPR55 in inflammatory processes of the gut is largely unknown. Using the recently characterized GPR55 inhibitor CID16020046, we determined the role of GPR55 in experimental intestinal inflammation and explored possible mechanisms of action. METHODS: Colitis was induced by either 2.5% dextran sulfate sodium (DSS) supplemented in the drinking water of C57BL/6 mice or by a single intrarectal application of trinitrobenzene sulfonic acid (TNBS). KEY RESULTS: Daily application of CID16020046 (20 mg/kg) significantly reduced inflammation scores and myeloperoxidase (MPO) activity. In the DSS colitis model, levels of tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1ß), and the expression of cyclooxygenase (Cox)-2 and signal transducer and activator of transcription 3 (STAT-3) were reduced in colon tissues while in TNBS-induced colitis, levels of Cox-2, IL-1ß and IL-6 were significantly lowered. Evaluation of leukocyte recruitment by flow cytometry indicated reduced presence of lymphocytes and macrophages in the colon following GPR55 inhibition in DSS-induced colitis. In J774A.1 mouse macrophages, inhibition of GPR55 revealed reduced migration of macrophages and decreased CD11b expression, suggesting that direct effects of CID16020046 on macrophages may have contributed to the improvement of colitis. GPR55(-/-) knockout mice showed reduced inflammation scores as compared to wild type mice in the DSS model suggesting a pro-inflammatory role in intestinal inflammation. CONCLUSIONS & INFERENCES: Pharmacological blockade of GPR55 reduces experimental intestinal inflammation by reducing leukocyte migration and activation, in particular that of macrophages. Therefore, CID16020046 represents a possible drug for the treatment of bowel inflammation.

New insights into male (in)fertility: the importance of NO.

Infertility is a global problem that is on the rise, especially during the last decade. Currently, infertility affects approximately 10-15% of the population worldwide. The frequency and origin of different forms of infertility varies. It has been shown that reactive oxygen and nitrogen species (ROS and RNS) are involved in the aetiology of infertility, especially male infertility. Various strategies have been designed to remove or decrease the production of ROS and RNS in spermatozoa, in particular during in vitro fertilization. However, in recent years it has been shown that spermatozoa naturally produce a variety of ROS/RNS, including superoxide anion radical (O2 (⋅-)), hydrogen peroxide and NO. These reactive species, in particular NO, are essential in regulating sperm capacitation and the acrosome reaction, two processes that need to be acquired by sperm in order to achieve fertilization potential. In addition, it has recently been shown that mitochondrial function is positively correlated with human sperm fertilization potential and quality and that NO and NO precursors increase sperm motility by increasing energy production in mitochondria. We will review the new link between sperm NO-driven redox regulation and infertility herein. A special emphasis will be placed on the potential implementation of new redox-active substances that modulate the content of NO in spermatozoa to increase fertility and promote conception.

Calcium-induced alteration of mitochondrial morphology and mitochondrial-endoplasmic reticulum contacts in rat brown adipocytes.

Mitochondria are key organelles maintaining cellular bioenergetics and integrity, and their regulation of [Ca2+]i homeostasis has been investigated in many cell types. We investigated the short-term Ca-SANDOZ® treatment on brown adipocyte mitochondria, using imaging and molecular biology techniques. Two-month-old male Wistar rats were divided into two groups: Ca-SANDOZ® drinking or tap water (control) drinking for three days. Alizarin Red S staining showed increased Ca2+ level in the brown adipocytes of treated rats, and potassium pyroantimonate staining localized electron-dense regions in the cytoplasm, mitochondria and around lipid droplets. Ca-SANDOZ® decreased mitochondrial number, but increased their size and mitochondrial cristae volume. Transmission electron microscopy revealed numerous enlarged and fusioned-like mitochondria in the Ca-SANDOZ® treated group compared to the control, and megamitochondria in some brown adipocytes. The Ca2+ diet affected mitochondrial fusion as mitofusin 1 (MFN1) and mitofusin 2 (MFN2) were increased, and mitochondrial fission as dynamin related protein 1 (DRP1) was decreased. Confocal microscopy showed a higher colocalization rate between functional mitochondria and endoplasmic reticulum (ER). The level of uncoupling protein-1 (UCP1) was elevated, which was confirmed by immunohistochemistry and Western blot analysis. These results suggest that Ca-SANDOZ® stimulates mitochondrial fusion, increases mitochondrial-ER contacts and the thermogenic capacity of brown adipocytes.

Endothelial cell apoptosis in brown adipose tissue of rats induced by hyperinsulinaemia: the possible role of TNF-α.

The aim of the present study was to investigate whether hyperinsulinaemia, which frequently precedes insulin resistance syndrome (obesity, diabetes), induces apoptosis of endothelial cells (ECs) in brown adipose tissue (BAT) and causes BAT atrophy and also, to investigate the possible mechanisms underlying ECs death. In order to induce hyperinsulinaemia, adult male rats of Wistar strain were treated with high dose of insulin (4 U/kg, intraperitonealy) for one or three days. Examinations at ultrastructural level showed apoptotic changes of ECs, allowing us to point out that changes mainly but not exclusively, occur in nuclei. Besides different stages of condensation and alterations of the chromatin, nuclear fragmentation was also observed. Higher number of ECs apoptotic nuclei in the BAT of hyperinsulinaemic rats was also confirmed by propidium iodide staining. Immunohistochemical localization of tumor necrosis factor-alpha (TNF-α) revealed increased expression in ECs of BAT of hyperinsulinaemic animals, indicating its possible role in insulin-induced apoptotic changes. These results suggest that BAT atrophy in hyperinsulinaemia is a result of endothelial and adipocyte apoptosis combined, rather than any of functional components alone.

T-2 toxin affects proliferation of three different neoplastic cell lines.

The antiproliferative effect of T-2 toxin (T-2) towards mouse melanoma B16 cells, human myelogenous leukemia K562 cells, and human cervix carcinoma, HeLa cells, was studied. For the first four days of T-2 presence B16 cell survival was decreased in dose dependent fashion. However, cell survival after eleven days T-2 action may be dual: some stimulation of cell growth that was direct function of the number of seeded cells per well was observed and cell survival (for the highest number of seeded cells) six times greater than control, was noticed at 20 nM T-2 toxin concentration. A smaller cell growth stimulation (cell survival more than 3 times higher than control) was observed with a lower cell number seeded per well. Nevertheless, by eleventh day concentrations of T-2 higher than 35 nM completely inhibited B16 cell proliferation. The same trend was noticed for T-2 action towards K562 cells. Treatment of HeLa cells with various T-2 concentrations led to a marked inhibition of cell survival that was more pronounced at the end of 44th or 72nd hour, than after the 20th hour of agent's action. ICs50 values obtained in the present work, suggest that B16 cells were the most sensitive to T-2 antiproliferative action, while HeLa cells were the most resistant. When PBMC were cultured with HeLa cells the antagonism against various T-2 concentrations was observed; cell survival determined after 44, or 72 hours of cells incubation, was less decreased compared to cultures treated with T-2, or with PBMC only. In addition, it was shown that T-2 and cis-DDP had an antagonist effect on HeLa cells survival.