Arvanil induces ferroptosis of hepatocellular carcinoma by binding to MICU1.

Hepatocellular carcinoma (HCC) is a primary liver cancer with a high mortality rate that requires research and improved treatment strategies. Chemotherapy is still one of the main methods of HCC treatment, but it may lead to drug resistance and damage to normal organs. Capsaicin, a naturally occurring active ingredient in chili peppers, has demonstrated anticancer properties in a variety of malignant tumor cell lines. However, the anti-cancer mechanism of capsaicin needs to be further explored in HCC. In this study, we utilized Arvanil, a non-stimulating synthetic capsaicin analog, in place of capsaicin. We found that Arvanil induced high mitochondrial calcium flow, which contributed to a decrease in mitochondrial membrane permeability transition pore (mPTP) opening and oxidative phosphorylation levels, ultimately triggering cellular ferroptosis by live cells in real time with a high content screening (HCS) platform and confocal microscopy. It was further confirmed by vina molecular docking and point mutation experiments that Arvanil directly binds to two amino acid sites of mitochondrial calcium uptake protein 1 (MICU1), namely Ser47 and Phe128, to trigger this process, which in turn inhibits the growth of HCC cells. In addition, it was confirmed that Arvanil enhances cisplatin chemosensitivity by inducing HCC cellular ferroptosis in vivo. In conclusion, our study suggests that Arvanil induces ferroptosis in HCC cells and is a candidate drug for the treatment of HCC.

Glucose restriction induces ROS-AMPK-mediated CTR1 expression and increases cisplatin efficiency in NSCLC.

Cisplatin is one of the principal platinum-based chemotherapeutic agents for many types of cancer, including non-small-cell lung cancer (NSCLC). Copper transporter 1 (CTR1) plays a significant role in increasing cellular cisplatin uptake and sensitivity. The current study found that glucose restriction upregulated AMPK (AMP-activated protein kinase) through reactive oxygen species (ROS) to induce CTR1 expression in NSCLC cells. Direct upregulation of ROS levels also activated AMPK expression. The changes in CTR1 expression were consistent with glucose concentrations and AMPK expression. Feeding a low-carbohydrate ketogenic diet (a glucose restriction diet) to a severe combined immune deficiency (SCID) mouse xenograft model significantly enhanced the efficacy of cisplatin. The tumor size was significantly smaller in the group treated with cisplatin plus the low-carbohydrate ketogenic diet than in the group treated with cisplatin alone. Survival was longer in mice treated with the low-carbohydrate ketogenic diet than in the controls. Mice fed the low-carbohydrate ketogenic diet showed increased expression of CTR1 and AMPK in tumor tissues. These results suggest a novel mechanism whereby glucose restriction induces ROS-AMPK-mediated CTR1 expression in NSCLC, indicating glucose restriction as an effective adjuvant NSCLC therapy.

Zn-DTSM, A Zinc Ionophore with Therapeutic Potential for Acrodermatitis Enteropathica?

Acrodermatitis enteropathica (AE) is a rare disease characterised by a failure in intestinal zinc absorption, which results in a host of symptoms that can ultimately lead to death if left untreated. Current clinical treatment involves life-long high-dose zinc supplements, which can introduce complications for overall nutrient balance in the body. Previous studies have therefore explored the pharmacological treatment of AE utilising metal ionophore/transport compounds in an animal model of the disease (conditional knockout (KO) of the zinc transporter, Zip4), with the perspective of finding an alternative to zinc supplementation. In this study we have assessed the utility of a different class of zinc ionophore compound (zinc diethyl bis(N4-methylthiosemicarbazone), Zn-DTSM; Collaborative Medicinal Development, Sausalito, CA, USA) to the one we have previously described (clioquinol), to determine whether it is effective at preventing the stereotypical weight loss present in the animal model of disease. We first utilised an in vitro assay to assess the ionophore capacity of the compound, and then assessed the effect of the compound in three in vivo animal studies (in 1.5-month-old mice at 30 mg/kg/day, and in 5-month old mice at 3 mg/kg/day and 30 mg/kg/day). Our data demonstrate that Zn-DTSM has a pronounced effect on preventing weight loss when administered daily at 30 mg/kg/day; this was apparent in the absence of any added exogenous zinc. This compound had little overall effect on zinc content in various tissues that were assessed, although further characterisation is required to more fully explore the cellular changes underlying the physiological benefit of this compound. These data suggest that Zn-DTSM, or similar compounds, should be further explored as potential therapeutic options for the long-term treatment of AE.

Protective Effect of Znt7 on High Glucose-Induced Epithelial-to-Mesenchymal Transition in Renal Tubular Epithelial Cells.

BACKGROUND/AIMS: Evidence from our and other groups has demonstrated that zinc transporter 7 in SLC30 family (ZnT7) inhibited epithelial-to-mesenchymal transition (EMT) and apoptosis in rat peritoneal mesothelial cells (RPMCs) under high glucose (HG) concentration. In the present study, we investigated the effect of ZnT7 on EMT of renal tubular epithelial cells (RTECs) in an in vitro model of diabetic nephropathy (DN). METHODS: A dual-fluorescent staining protocol was used for detection of ZnT7 in a normal rat kidney tubular epithelial cell line (NRK-52E cells). EMT was induced with HG (30 mM). NRK-52E cells were transfected with plasmids codifying for hZnT7-EGFP and interfering RNA for determination of the effect of ZnT7 over-expression and silencing, respectively. Expression of ZnT7, activation of the MAPK/ERK and TGF-ß/Smad pathways were analyzed with by means of Western blot. RESULTS: ZnT7 was localized in the perinuclear region and Golgi apparatus. In HG-induced EMT of NRK-52E cells, ZnT7 was up-regulated. Over-expression of ZnT7 led to inhibition of HG-induced EMT, while knock-down of ZnT7 increased EMT. Furthermore, knock-down of ZnT7 and increased HG-induced EMT was accompanied by activation of the MAPK/ERK and TGF-ß/Smad pathways. CONCLUSION: The present study provides evidence that ZnT7 has a protective effect over EMT of RTECs in DN and suggests that the inhibition of HG-induced EMT may be achieved through the MAPK/ERK and TGF-ß/Smad pathways. Thereby, ZnT7 could be a potential target for translation medicine and prevention program in DN.

Reduced 64Cu uptake and tumor growth inhibition by knockdown of human copper transporter 1 in xenograft mouse model of prostate cancer.

UNLABELLED: Copper is an element required for cell proliferation and angiogenesis. Human prostate cancer xenografts with increased (64)Cu radioactivity were visualized previously by PET using (64)CuCl2 as a radiotracer ((64)CuCl2 PET). This study aimed to determine whether the increased tumor (64)Cu radioactivity was due to increased cellular uptake of (64)Cu mediated by human copper transporter 1 (hCtr1) or simply due to nonspecific binding of ionic (64)CuCl2 to tumor tissue. In addition, the functional role of hCtr1 in proliferation of prostate cancer cells and tumor growth was also assessed. METHODS: A lentiviral vector encoding short-hairpin RNA specific for hCtr1 (Lenti-hCtr1-shRNA) was constructed for RNA interference-mediated knockdown of hCtr1 expression in prostate cancer cells. The degree of hCtr1 knockdown was determined by Western blot, and the effect of hCtr1 knockdown on copper uptake and proliferation were examined in vitro by cellular (64)Cu uptake and cell proliferation assays. The effects of hCtr1 knockdown on tumor uptake of (64)Cu were determined by PET quantification and tissue radioactivity assay. The effects of hCtr1 knockdown on tumor growth were assessed by PET/CT and tumor size measurement with a caliper. RESULTS: RNA interference-mediated knockdown of hCtr1 was associated with the reduced cellular uptake of (64)Cu and the suppression of prostate cancer cell proliferation in vitro. At 24 h after intravenous injection of the tracer (64)CuCl2, the (64)Cu uptake by the tumors with knockdown of hCtr1 (4.02 ± 0.31 percentage injected dose per gram [%ID/g] in Lenti-hCtr1-shRNA-PC-3 and 2.30 ± 0.59 %ID/g in Lenti-hCtr1-shRNA-DU-145) was significantly lower than the (64)Cu uptake by the control tumors without knockdown of hCtr1 (7.21 ± 1.48 %ID/g in Lenti-SCR-shRNA-PC-3 and 5.57 ± 1.20 %ID/g in Lenti-SCR-shRNA-DU-145, P < 0.001) by PET quantification. Moreover, the volumes of prostate cancer xenograft tumors with knockdown of hCtr1 (179 ± 111 mm(3) for Lenti-hCtr1-shRNA-PC-3 or 39 ± 22 mm(3) for Lenti-hCtr1-shRNA-DU-145) were significantly smaller than those without knockdown of hCtr1 (536 ± 191 mm(3) for Lenti- SCR-shRNA-PC-3 or 208 ± 104 mm(3) for Lenti-SCR-shRNA-DU-145, P < 0.01). CONCLUSION: Overall, data indicated that hCtr1 is a promising theranostic target, which can be further developed for metabolic imaging of prostate cancer using (64)CuCl2 PET/CT and personalized cancer therapy targeting copper metabolism.

Out of balance--systemic iron homeostasis in iron-related disorders.

Iron is an essential element in our daily diet. Most iron is required for the de novo synthesis of red blood cells, where it plays a critical role in oxygen binding to hemoglobin. Thus, iron deficiency causes anemia, a major public health burden worldwide. On the other extreme, iron accumulation in critical organs such as liver, heart, and pancreas causes organ dysfunction due to the generation of oxidative stress. Therefore, systemic iron levels must be tightly balanced. Here we focus on the regulatory role of the hepcidin/ferroportin circuitry as the major regulator of systemic iron homeostasis. We discuss how regulatory cues (e.g., iron, inflammation, or hypoxia) affect the hepcidin response and how impairment of the hepcidin/ferroportin regulatory system causes disorders of iron metabolism.

Rescue of ATP7B function in hepatocyte-like cells from Wilson's disease induced pluripotent stem cells using gene therapy or the chaperone drug curcumin.

Directed hepatocyte differentiation from human induced pluripotent stem cells (iPSCs) potentially provides a unique platform for modeling liver genetic diseases and performing drug-toxicity screening in vitro. Wilson's disease is a genetic disease caused by mutations in the ATP7B gene, whose product is a liver transporter protein responsible for coordinated copper export into bile and blood. Interestingly, the spectrum of ATP7B mutations is vast and can influence clinical presentation (a variable spectrum of hepatic and neural manifestations), though the reason is not well understood. We describe the generation of iPSCs from a Chinese patient with Wilson's disease that bears the R778L Chinese hotspot mutation in the ATP7B gene. These iPSCs were pluripotent and could be readily differentiated into hepatocyte-like cells that displayed abnormal cytoplasmic localization of mutated ATP7B and defective copper transport. Moreover, gene correction using a self-inactivating lentiviral vector that expresses codon optimized-ATP7B or treatment with the chaperone drug curcumin could reverse the functional defect in vitro. Hence, our work describes an attractive model for studying the pathogenesis of Wilson's disease that is valuable for screening compounds or gene therapy approaches aimed to correct the abnormality. In the future, once relevant safety concerns (including the stability of the mature liver-like phenotype) and technical issues for the transplantation procedure are solved, hepatocyte-like cells from similarly genetically corrected iPSCs could be an option for autologous transplantation in Wilson's disease.

Ferroportin is a manganese-responsive protein that decreases manganese cytotoxicity and accumulation.

Although manganese (Mn) is an essential trace element for human development and growth, chronic exposure to excessive Mn levels can result in psychiatric and motor disturbances, referred to as manganism. However, there are no known mechanism(s) for efflux of excess Mn from mammalian cells. Here, we test the hypothesis that the cytoplasmic iron (Fe) exporter ferroportin (Fpn) may also function as a Mn exporter to attenuate Mn toxicity. Using an inducible human embryonic kidney (HEK293T) cell model, we examined the influence of Fpn expression on Mn-induced cytotoxicity and intracellular Mn concentrations. We found that induction of an Fpn-green fluorescent protein fusion protein in HEK293T cells was cytoprotective against several measures of Mn toxicity, including Mn-induced cell membrane leakage and Mn-induced reductions in glutamate uptake. Fpn-green fluorescent protein mediated cytoprotection correlated with decreased Mn accumulation following Mn exposure. Thus, Fpn expression reduces Mn toxicity concomitant with reduced Mn accumulation. To determine if mammalian cells may utilize Fpn in response to increased intracellular Mn concentrations and toxicity, we assessed endogenous Fpn levels in Mn-exposed HEK293T cells and in mouse brain in vivo. We find that 6 h of Mn exposure in HEK293T cells is associated with a significant increase in Fpn levels. Furthermore, mice exposed to Mn showed an increase in Fpn levels in both the cerebellum and cortex. Collectively, these results indicate that (i) Mn exposure promotes Fpn protein expression, (ii) Fpn expression reduces net Mn accumulation, and (iii) reduces cytotoxicity associated with exposure to this metal.