NRF2 regulates viability, proliferation, resistance to oxidative stress, and differentiation of murine myoblasts and muscle satellite cells

Increased oxidative stress can slow down the regeneration of skeletal muscle and affect the activity of muscle satellite cells (mSCs). Therefore, we evaluated the role of the NRF2 transcription factor (encoded by the Nfe2l2 gene), the main regulator of the antioxidant response, in muscle cell biology. We used (i) an immortalized murine myoblast cell line (C2C12) with stable overexpression of NRF2 and (ii) primary mSCs isolated from wild-type and Nfe2l2 (transcriptionally)-deficient mice (Nfe2l2$^{tKO}$). NRF2 promoted myoblast proliferation and viability under oxidative stress conditions and decreased the production of reactive oxygen species. Furthermore, NRF2 overexpression inhibited C2C12 cell differentiation by down-regulating the expression of myogenic regulatory factors (MRFs) and muscle-specific microRNAs. We also showed that NRF2 is indispensable for the viability of mSCs since the lack of its transcriptional activity caused high mortality of cells cultured in vitro under normoxic conditions. Concomitantly, Nfe2l2$^{tKO}$ mSCs grown and differentiated under hypoxic conditions were viable and much more differentiated compared to cells isolated from wild-type mice. Taken together, NRF2 significantly influences the properties of myoblasts and muscle satellite cells. This effect might be modulated by the muscle microenvironment

Preliminary analysis of the expression of selected proangiogenic and antioxidant genes and MicroRNAs in patients with non-muscle-invasive bladder cancer

Heme oxygenase-1 (HO-1) is an enzyme contributing to the development and progression of different cancer types. HO-1 plays a role in pathological angiogenesis in bladder cancer and contributes to the resistance of this cancer to therapy. It also regulates the expression of microRNAs in rhabdomyosarcoma and non-small cell lung cancer. The expression of HO-1 may be regulated by hypoxia inducible factors (HIFs) and Nrf2 transcription factor. The expression of HO-1 has not so far been examined in relation to Nrf2, HIF-1α, and potential mediators of angiogenesis in human bladder cancer. We measured the concentration of proinflammatory and proangiogenic cytokines and the expression of cytoprotective and proangiogenic mRNAs and miRNAs in healthy subjects and patients with bladder cancer. HO-1 expression was upregulated together with HIF-1α, HIF-2α, and Nrf2 in bladder cancer in comparison to healthy tissue. VEGF was elevated both at mRNA and protein level in the tumor and in sera, respectively. Additionally, IL-6 and IL-8 were increased in sera of patients affected with urothelial bladder cancer. Moreover, miR-155 was downregulated whereas miR-200c was elevated in cancer biopsies in comparison to healthy tissue. The results indicate that the increased expression of HO-1 in bladder cancer is paralleled by changes in the expression of other potentially interacting genes, like Nrf2, HIF-1α, HIF-2α, IL-6, IL-8, and VEGF. Further studies are necessary to also elucidate the potential links with miR-155 and miR-200c

Myoblast-conditioned media improve regeneration and revascularization of ischemic muscles in diabetic mice

INTRODUCTION: Diabetes is associated with reduced expression of heme oxygenase-1 (HO-1), a heme-degrading enzyme with cytoprotective and proangiogenic properties. In myoblasts and muscle satellite cells HO-1 improves survival, proliferation and production of proangiogenic growth factors. Induction of HO-1 in injured tissues facilitates neovascularization, the process impaired in diabetes. We aimed to examine whether conditioned media from the HO-1 overexpressing myoblast cell line can improve a blood-flow recovery in ischemic muscles of diabetic mice. METHODS: Analysis of myogenic markers was performed at the mRNA level in primary muscle satellite cells, isolated by a pre-plate technique from diabetic db/db and normoglycemic wild-type mice, and then cultured under growth or differentiation conditions. Hind limb ischemia was performed by femoral artery ligation in db/db mice and blood recovery was monitored by laser Doppler measurements. Mice were treated with a single intramuscular injection of conditioned media harvested from wild-type C2C12 myoblast cell line, C2C12 cells stably transduced with HO-1 cDNA, or with unconditioned media. RESULTS: Expression of HO-1 was lower in muscle satellite cells isolated from muscles of diabetic db/db mice when compared to their wild-type counterparts, what was accompanied by increased levels of Myf5 or CXCR4, and decreased Mef2 or Pax7. Such cells also displayed diminished differentiation potential when cultured in vitro, as shown by less effective formation of myotubes and reduced expression of myogenic markers (myogenic differentiation antigen - myoD, myogenin and myosin). Blood flow recovery after induction of severe hind limb ischemia was delayed in db/db mice compared to that in normoglycemic individuals. To improve muscle regeneration after ischemia, conditioned media collected from differentiating C2C12 cells (control and HO-1 overexpressing) were injected into hind limbs of diabetic mice. Analysis of blood flow revealed that media from HO-1 overexpressing cells accelerated blood-flow recovery, while immunohistochemical staining assessment of vessel density in injected muscle confirmed increased angiogenesis. The effect might be mediated by stromal-cell derived factor-1α proangiogenic factor, as its secretion is elevated in HO-1 overexpressing cells. CONCLUSIONS: In conclusion, paracrine stimulation of angiogenesis in ischemic skeletal muscle using conditioned media may be a safe approach exploiting protective and proangiogenic properties of HO-1 in diabetes

Effect of heme oxygenase-1 on the differentiation of human myoblasts and the regeneration of murine skeletal muscles after acute and chronic injury

Background Impaired muscle regeneration is a hallmark of Duchenne muscular dystrophy (DMD), a neuromuscular disorder caused by mutations in the DMD gene encoding dystrophin. The lack of heme oxygenase-1 (HO-1, Hmox1), a known anti-inflammatory and cytoprotective enzyme, was shown to aggravate DMD pathology. Methods We evaluated the role of HO-1 overexpression in human induced pluripotent stem cell (hiPSC)-derived skeletal muscle cells (hiPSC-SkM) in vitro and in the regeneration process in vivo in wild-type mice. Furthermore, the effect of cobalt protoporphyrin IX (CoPP), a pharmacological inducer of HO-1 expression, on regeneration markers during myogenic hiPSC differentiation and progression of the dystrophic phenotype was analysed in the mdx mouse DMD model. Results HO-1 has an impact on hiPSC-SkM generation by decreasing cell fusion capacity and the expression of myogenic regulatory factors and muscle-specific microRNAs (myomiRs). Also, strong induction of HO-1 by CoPP totally abolished hiPSC-SkM differentiation. Injection of HO-1-overexpressing hiPSC-SkM into the cardiotoxin (CTX)-injured muscle of immunodeficient wild-type mice was associated with decreased expression of miR-206 and Myh3 and lower number of regenerating fibers, suggesting some advanced regeneration. However, the very potent induction of HO-1 by CoPP did not exert any protective effect on necrosis, leukocyte infiltration, fibrosis, myofiber regeneration biomarkers, and exercise capacity of mdx mice. Conclusions In summary, HO-1 inhibits the expression of differentiation markers in human iPSC-derived myoblasts. Although moderate overexpression of HO-1 in the injected myoblast was associated with partially advanced muscle regeneration, the high systemic induction of HO-1 did not improve muscle regeneration. The appropriate threshold of HO-1 expression must be established for the therapeutic effect of HO-1 on muscle regeneration

Curcumin enhances the cytogenotoxic effect of etoposide in leukemia cells through induction of reactive oxygen species

Curcumin may exert a more selective cytotoxic effect in tumor cells with elevated levels of free radicals. Here, we investigated whether curcumin can modulate etoposide action in myeloid leukemia cells and in normal cells of hematopoietic origin. HL-60 cell line, normal myeloid progenitor cluster of differentiation (CD)-34+ cells, and granulocytes were incubated for 4 or 24 hours at different concentrations of curcumin and/or etoposide. Brown Norway rats with acute myeloid leukemia (BNML) were used to prove the influence of curcumin on etoposide action in vivo. Rats were treated with curcumin for 23 days and etoposide was administered for the final 3 days of the experiment. Curcumin synergistically potentiated the cytotoxic effect of etoposide, and it intensified apoptosis and phosphorylation of the histone H2AX induced by this cytostatic drug in leukemic HL-60 cells. In contrast, curcumin did not significantly modify etoposide-induced cytotoxicity and H2AX phosphorylation in normal CD34+ cells and granulocytes. Curcumin modified the cytotoxic action of etoposide in HL-60 cells through intensification of free radical production because preincubation with N-acetyl-l-cysteine (NAC) significantly reduced the cytotoxic effect of curcumin itself and a combination of two compounds. In contrast, NAC did not decrease the cytotoxic effect of etoposide. Thus, oxidative stress plays a greater role in the cytotoxic effect of curcumin than that of etoposide in HL-60 cells. In vitro results were confirmed in a BNML model. Pretreatment with curcumin enhanced the antileukemic activity of etoposide in BNML rats (1.57-fold tumor reduction versus etoposide alone; P<0.05) and induced apoptosis of BNML cells more efficiently than etoposide alone (1.54-fold change versus etoposide alone; P<0.05), but this treatment protected nonleukemic B-cells from apoptosis. Thus, curcumin can increase the antileukemic effect of etoposide through reactive oxygen species in sensitive myeloid leukemia cells, and it is harmless to normal human cells

Role of HMOX1 promoter genetic variants in chemoresistance and chemotherapy induced neutropenia in children with acute lymphoblastic leukemia

Whilst the survival rates of childhood acute lymphoblastic leukemia (ALL) have increased remarkably over the last decades, the therapy resistance and toxicity are still the major causes of treatment failure. It was shown that overexpression of heme oxygenase-1 (HO-1) promotes proliferation and chemoresistance of cancer cells. In humans, the HO-1 gene (HMOX1) expression is modulated by two polymorphisms in the promoter region: (GT)n-length polymorphism and single-nucleotide polymorphism (SNP) A(−413)T, with short GT repeat sequences and 413-A variants linked to an increased HO-1 inducibility. We found that the short alleles are significantly more frequent in ALL patients in comparison to the control group, and that their presence may be associated with a higher risk of treatment failure, reflecting the role of HO-1 in chemoresistance. We also observed that the presence of short alleles may predispose to develop chemotherapy-induced neutropenia. In case of SNP, the 413-T variant co-segregated with short or long alleles, while 413-A almost selectively co-segregated with long alleles, hence it is not possible to determine if SNPs are actually of phenotypic significance. Our results suggest that HO-1 can be a potential target to overcome the treatment failure in ALL patients

Heme oxygenase-1 is required for angiogenic function of bone marrow-derived progenitor cells : role in therapeutic revascularization

Aims: Heme oxygenase-1 (HO-1) is a cytoprotective enzyme that can be down-regulated in diabetes. Its importance for mature endothelium has been described, but its role in proangiogenic progenitors is not well known. We investigated the effect of HO-1 on the angiogenic potential of bone marrow-derived cells (BMDCs) and on blood flow recovery in ischemic muscle of diabetic mice. Results: Lack of HO-1 decreased the number of endothelial progenitor cells (Lin−CD45−cKit-Sca-1+VEGFR-2+) in murine bone marrow, and inhibited the angiogenic potential of cultured BMDCs, affecting their survival under oxidative stress, proliferation, migration, formation of capillaries, and paracrine proangiogenic potential. Transcriptome analysis of HO-1−/− BMDCs revealed the attenuated up-regulation of proangiogenic genes in response to hypoxia. Heterozygous HO-1+/− diabetic mice subjected to hind limb ischemia exhibited reduced local expression of vascular endothelial growth factor (VEGF), placental growth factor (PlGF), stromal cell-derived factor 1 (SDF-1), VEGFR-1, VEGFR-2, and CXCR-4. This was accompanied by impaired revascularization of ischemic muscle, despite a strong mobilization of bone marrow-derived proangiogenic progenitors (Sca-1+CXCR-4+) into peripheral blood. Blood flow recovery could be rescued by local injections of conditioned media harvested from BMDCs, but not by an injection of cultured BMDCs. Innovation: This is the first report showing that HO-1 haploinsufficiency impairs tissue revascularization in diabetes and that proangiogenic in situ response, not progenitor cell mobilization, is important for blood flow recovery. Conclusions: HO-1 is necessary for a proper proangiogenic function of BMDCs. A low level of HO-1 in hyperglycemic mice decreases restoration of perfusion in ischemic muscle, which can be rescued by a local injection of conditioned media from cultured BMDCs