Previous studies on the degenerative animal model of multiple sclerosis suggested that the copper-chelator cuprizone might directly suppress T-cell functions. Peripheral T-cell function in the cuprizone model has already been explored; therefore, in the present study, we investigated, for the first time, how cuprizone feeding affects the thymus, the organ of T-cell maturation and selection. We found that even one week of cuprizone treatment induced significant thymic atrophy, affecting the cortex over the medulla. Fluorescent microscopy and flow-cytometric analyses of thymi from cuprizone- and vehicle-treated mice indicated that eradication of the cluster of the differentiation-4 (CD4)-CD8 double-positive T-cell subset was behind the substantial cell loss. This result was confirmed with CD3-CD4-CD8 triple-staining experiments. Ultrastructurally, we observed degraded as well as enlarged mitochondria, myelin-bodies, large lipid droplets, and large lysosomes in the thymi of cuprizone-treated mice. Some of these features were similar to those in physiological and steroid-induced accelerated aging. According to our results, apoptosis was mainly of mitochondrial origin mediated by both caspase-3- and apoptosis inducing factor-mediated mechanisms. Additionally, mitogen activated protein kinase activation and increased pro-apoptotic B cell lymphoma-2 family protein expression were the major underlying processes. Our results do not indicate a functional relationship between cuprizone-induced thymus involution and the absence of inflammatory responses or the selective demyelination observed in the cuprizone model. On the other hand, due to the reversible nature of cuprizone’s deleterious effects, the cuprizone model could be valuable in studying thymus regeneration as well as remyelination processes.

Obesity is a major public health problem worldwide, and it is associated with many diseases and abnormalities, most importantly, type 2 diabetes. The visceral adipose tissue produces an immense variety of adipokines. Leptin is the first identified adipokine which plays a crucial role in the regulation of food intake and metabolism. Sodium glucose co-transport 2 inhibitors are potent antihyperglycemic drugs with various beneficial systemic effects. We aimed to investigate the metabolic state and leptin level among patients with obesity and type 2 diabetes mellitus, and the effect of empagliflozin upon these parameters. We recruited 102 patients into our clinical study, then we performed anthropometric, laboratory, and immunoassay tests. Body mass index, body fat, visceral fat, urea nitrogen, creatinine, and leptin levels were significantly lower in the empagliflozin treated group when compared to obese and diabetic patients receiving conventional antidiabetic treatments. Interestingly, leptin was increased not only among obese patients but in type 2 diabetic patients as well. Body mass index, body fat, and visceral fat percentages were lower, and renal function was preserved in patients receiving empagliflozin treatment. In addition to the known beneficial effects of empagliflozin regarding the cardio-metabolic and renal systems, it may also influence leptin resistance.

Bladder cancer (BC) is a common malignancy of the urinary tract that has a higher frequency in men than in women. Cytostatic resistance and metastasis formation are significant risk factors in BC therapy; therefore, there is great interest in overcoming drug resistance and in initiating research for novel chemotherapeutic approaches. Here, we suggest that desethylamiodarone (DEA)–a metabolite of amiodarone—may have cytostatic potential. DEA activates the collapse of mitochondrial membrane potential (detected by JC-1 fluorescence), and induces cell death in T24 human transitional-cell bladder carcinoma cell line at physiologically achievable concentrations. DEA induces cell cycle arrest in the G0/G1 phase, which may contribute to the inhibition of cell proliferation, and shifts the Bax/Bcl-2 ratio to initiate apoptosis, induce AIF nuclear translocation, and activate PARP-1 cleavage and caspase-3 activation. The major cytoprotective kinases—ERK and Akt—are inhibited by DEA, which may contribute to its cell death-inducing effects. DEA also inhibits the expression of B-cell-specific Moloney murine leukemia virus integration site 1 (BMI1) and reduces colony formation of T24 bladder carcinoma cells, indicating its possible inhibitory effect on metastatic potential. These data show that DEA is a novel anti-cancer candidate of multiple cell death-inducing effects and metastatic potential. Our findings recommend further evaluation of its effects in clinical studies.

Novel compounds significantly interfering with the mitochondrial energy production may have therapeutic value in triple-negative breast cancer (TNBC). This criterion is clearly fulfilled by desethylamiodarone (DEA), which is a major metabolite of amiodarone, a widely used antiarrhythmic drug, since the DEA previously demonstrated anti-neoplastic, anti-metastasizing, and direct mitochondrial effects in B16F10 melanoma cells. Additionally, the more than fifty years of clinical experience with amiodarone should answer most of the safety concerns about DEA. Accordingly, in the present study, we investigated DEA’s potential in TNBC by using a TN and a hormone receptor positive (HR+) BC cell line. DEA reduced the viability, colony formation, and invasive growth of the 4T1 cell line and led to a higher extent of the MCF-7 cell line. It lowered mitochondrial transmembrane potential and induced mitochondrial fragmentation. On the other hand, DEA failed to significantly affect various parameters of the cellular energy metabolism as determined by a Seahorse live cell respirometer. Cyclooxygenase 2 (COX-2), which was upregulated by DEA in the TNBC cell line only, accounted for most of 4T1’s DEA resistance, which was counteracted by the selective COX-2 inhibitor celecoxib. All these data indicate that DEA may have potentiality in the therapy of TNBC.

Previously, we showed that desethylamiodarone (DEA), a major metabolite of the widely used antiarrhythmic drug amiodarone, has direct mitochondrial effects. We hypothesized that these effects account for its observed cytotoxic properties and ability to limit in vivo metastasis. Accordingly, we examined DEA’s rapid (3–12 h) cytotoxicity and its early (3–6 h) effects on various mitochondrial processes in B16F10 melanoma cells. DEA did not affect cellular oxygen radical formation, as determined using two fluorescent dyes. However, it did decrease the mitochondrial transmembrane potential, as assessed by JC-1 dye and fluorescence microscopy. It also induced mitochondrial fragmentation, as visualized by confocal fluorescence microscopy. DEA decreased maximal respiration, ATP production, coupling efficiency, glycolysis, and non-mitochondrial oxygen consumption measured by a Seahorse cellular energy metabolism analyzer. In addition, it induced a cyclosporine A–independent mitochondrial permeability transition, as determined by Co²⁺-mediated calcein fluorescence quenching measured using a high-content imaging system. DEA also caused outer mitochondrial membrane permeabilization, as assessed by the immunoblot analysis of cytochrome C, apoptosis inducing factor, Akt, phospho-Akt, Bad, and phospho-Bad. All of these data supported our initial hypothesis.