Effect of erythropoietin administration on proteins participating in iron homeostasis in Tmprss6-mutated mask mice.

Tmprss6-mutated mask mice display iron deficiency anemia and high expression of hepcidin. The aim of the study was to determine the effect of erythropoietin administration on proteins participating in the control of iron homeostasis in the liver and spleen in C57BL/6 and mask mice. Administration of erythropoietin for four days at 50 IU/mouse/day increased hemoglobin and hematocrit in C57BL/6 mice, no such increase was seen in mask mice. Erythropoietin administration decreased hepcidin expression in C57BL/6 mice, but not in mask mice. Erythropoietin treatment significantly increased the spleen size in both C57BL/6 and mask mice. Furthermore, erythropoietin administration increased splenic Fam132b, Fam132a and Tfr2 mRNA content. At the protein level, erythropoietin increased the amount of splenic erythroferrone and transferrin receptor 2 both in C57BL/6 and mask mice. Splenic ferroportin content was decreased in erythropoietin-treated mask mice in comparison with erythropoietin-treated C57BL/6 mice. In mask mice, the amount of liver hemojuvelin was decreased in comparison with C57BL/6 mice. The pattern of hemojuvelin cleavage was different between C57BL/6 and mask mice: In both groups, a main hemojuvelin band was detected at approximately 52 kDa; in C57BL/6 mice, a minor cleaved band was seen at 47 kDa. In mask mice, the 47 kDa band was absent, but additional minor bands were detected at approximately 45 kDa and 48 kDa. The results provide support for the interaction between TMPRSS6 and hemojuvelin in vivo; they also suggest that hemojuvelin could be cleaved by another as yet unknown protease in the absence of functional TMPRSS6. The lack of effect of erythropoietin on hepcidin expression in mask mice can not be explained by changes in erythroferrone synthesis, as splenic erythroferrone content increased after erythropoietin administration in both C57BL/6 and mask mice.

Erythropoietin administration increases splenic erythroferrone protein content and liver TMPRSS6 protein content in rats.

Erythroferrone (ERFE) and TMPRSS6 are important proteins in the regulation of iron metabolism. The objective of the study was to examine splenic ERFE and liver TMPRSS6 synthesis in rats treated with a combination of iron and erythropoietin (EPO). EPO was administered to female Wistar rats at 600U/day for four days, iron-pretreated rats received 150mg of iron before EPO treatment. Content of ERFE and TMPRSS6 proteins was determined by commercial antibodies. Iron pretreatment prevented the EPO-induced decrease in hepcidin expression. Content of phosphorylated SMAD 1,5,8 proteins was decreased in the liver by both EPO and iron plus EPO treatment. Fam132b expression in the spleen was increased both by EPO and iron plus EPO treatments; these treatments also significantly induced splenic Fam132a expression. ERFE protein content in the spleen was increased both by EPO and iron plus EPO to a similar extent. EPO administration increased TMPRSS6 content in the plasma membrane-enriched fraction of liver homogenate; in iron-pretreated rats, this increase was abolished. The results confirm that iron pretreatment prevents the EPO-induced decrease in liver Hamp expression. This effect probably occurs despite high circulating ERFE levels, since EPO-induced ERFE protein synthesis is not influenced by iron pretreatment.

Horizontal transfer of whole mitochondria restores tumorigenic potential in mitochondrial DNA-deficient cancer cells.

Recently, we showed that generation of tumours in syngeneic mice by cells devoid of mitochondrial (mt) DNA (ρ0 cells) is linked to the acquisition of the host mtDNA. However, the mechanism of mtDNA movement between cells remains unresolved. To determine whether the transfer of mtDNA involves whole mitochondria, we injected B16ρ0 mouse melanoma cells into syngeneic C57BL/6Nsu9-DsRed2 mice that express red fluorescent protein in their mitochondria. We document that mtDNA is acquired by transfer of whole mitochondria from the host animal, leading to normalisation of mitochondrial respiration. Additionally, knockdown of key mitochondrial complex I (NDUFV1) and complex II (SDHC) subunits by shRNA in B16ρ0 cells abolished or significantly retarded their ability to form tumours. Collectively, these results show that intact mitochondria with their mtDNA payload are transferred in the developing tumour, and provide functional evidence for an essential role of oxidative phosphorylation in cancer.

Tumor-initiating cells of breast and prostate origin show alterations in the expression of genes related to iron metabolism.

The importance of iron in the growth and progression of tumors has been widely documented. In this report, we show that tumor-initiating cells (TICs), represented by spheres derived from the MCF7 cell line, exhibit higher intracellular labile iron pool, mitochondrial iron accumulation and are more susceptible to iron chelation. TICs also show activation of the IRP/IRE system, leading to higher iron uptake and decrease in iron storage, suggesting that level of properly assembled cytosolic iron-sulfur clusters (FeS) is reduced. This finding is confirmed by lower enzymatic activity of aconitase and FeS cluster biogenesis enzymes, as well as lower levels of reduced glutathione, implying reduced FeS clusters synthesis/utilization in TICs. Importantly, we have identified specific gene signature related to iron metabolism consisting of genes regulating iron uptake, mitochondrial FeS cluster biogenesis and hypoxic response (ABCB10, ACO1, CYBRD1, EPAS1, GLRX5, HEPH, HFE, IREB2, QSOX1 and TFRC). Principal component analysis based on this signature is able to distinguish TICs from cancer cells in vitro and also Leukemia-initiating cells (LICs) from non-LICs in the mouse model of acute promyelocytic leukemia (APL). Majority of the described changes were also recapitulated in an alternative model represented by MCF7 cells resistant to tamoxifen (TAMR) that exhibit features of TICs. Our findings point to the critical importance of redox balance and iron metabolism-related genes and proteins in the context of cancer and TICs that could be potentially used for cancer diagnostics or therapy.

Mitochondrially targeted vitamin E succinate modulates expression of mitochondrial DNA transcripts and mitochondrial biogenesis.

AIMS: To assess the effect of mitochondrially targeted vitamin E (VE) analogs on mitochondrial function and biogenesis. RESULTS: Mitochondrially targeted vitamin E succinate (MitoVES) is an efficient inducer of apoptosis in cancer cells. Here, we show that unlike its untargeted counterpart α-tocopheryl succinate, MitoVES suppresses proliferation of cancer cells at sub-apoptotic doses by way of affecting the mitochondrial DNA (mtDNA) transcripts. We found that MitoVES strongly suppresses the level of the displacement loop transcript followed by those of mtDNA genes coding for subunits of mitochondrial complexes. This process is coupled to the inhibition of mitochondrial respiration, dissipation of the mitochondrial membrane potential, and generation of reactive oxygen species. In addition, exposure of cancer cells to MitoVES led to decreased expression of TFAM and diminished mitochondrial biogenesis. The inhibition of mitochondrial transcription was replicated in vivo in a mouse model of HER2(high) breast cancer, where MitoVES lowered the level of mtDNA transcripts in cancer cells but not in normal tissue. INNOVATION: Our data show that mitochondrially targeted VE analogs represent a novel class of mitocans that not only induce apoptosis at higher concentrations but also block proliferation and suppress normal mitochondrial function and transcription at low, non-apoptogenic doses. CONCLUSIONS: Our data indicate a novel, selective anti-cancer activity of compounds that act by targeting mitochondria of cancer cells, inducing significant alterations in mitochondrial function associated with transcription of mtDNA-coded genes. These changes subsequently result in the arrest of cell proliferation.

N-substituted 2-isonicotinoylhydrazinecarboxamides--new antimycobacterial active molecules.

This report presents a new modification of the isoniazid (INH) structure linked with different anilines via a carbonyl group obtained by two synthetic procedures and with N-substituted 5-(pyridine-4-yl)-1,3,4-oxadiazole-2-amines prepared by their cyclisation. All synthesised derivatives were characterised by IR, NMR, MS and elemental analyses and were evaluated in vitro for their antimycobacterial activity against Mycobacterium tuberculosis H37Rv, Mycobacterium avium 330/88, Mycobacterium kansasii 235/80 and one clinical isolated strain of M. kansasii 6509/96. 2-Isonicotinoyl-N-(4-octylphenyl)hydrazinecarboxamide displayed an in vitro efficacy comparable to that of INH for M. tuberculosis with minimum inhibitory concentrations (MICs) of 1-2 µM. Among the halogenated derivatives, the best anti-tuberculosis activity was found for 2-isonicotinoyl-N-(2,4,6-trichlorophenyl)hydrazinecarboxamide (MIC=4 µM). In silico modelling on the enoyl-acyl carrier protein reductase InhA confirmed that longer alkyl substituents are advantageous for the interactions and affinity to InhA. Most of the hydrazinecarboxamides, especially those derived from 4-alkylanilines, exhibited significant activity against INH-resistant nontuberculous mycobacteria.