Role Of Cytoglobin Research Articles

Cytoglobin augments ferroptosis through autophagic degradation of ferritin in colorectal cancer cells.

Autophagy has gained importance in the context of ferroptosis. Nevertheless, a deeper understanding of the regulatory mechanism governing autophagy-dependent ferroptosis is necessary. Cytoglobin (CYGB), a member of the globin family, exhibits antifibrotic effects, regulates cellular reactive oxygen species, and stimulates tumor inhibition. Herein, we present further insights into the role of CYGB in ferroptosis regulation. Our investigation confirmed that CYGB impedes cell proliferation and migration. Furthermore, a significant association between CYGB and the lysosomal pathway was suggested based on the RNA sequencing data analysis. Elevated lysosomal signal and colocalization of CYGB with lysosome-associated membrane glycoprotein 1 (LAMP1) were observed. Moreover, upregulated autophagy and augmented ferroptosis induced by RSL3 were confirmed in CYGB-overexpression cells with an obviously increased colocalization of nuclear receptor coactivator 4 (NCOA4) and LC3B. The autophagy inhibitor bafilomycin or chloroquine alleviated autophagy-dependent degradation of ferritin protein under RSL3 treated condition. Additionally, a colocalization of CYGB with the transferrin receptor (TFR) was confirmed. Our results demonstrate an important functional pathway by which CYGB regulates ferroptosis through TFR-binding and autophagic degradation of ferritin, and provide a potential pathway for the treatment of colorectal cancer.

The Role of Cytoglobin in Cancer

Cytoglobin (Cygb) is a new member of the globin protein family, following the discovery of other globin proteins such as hemoglobin (Hb), myoglobin (Mb), and neuroglobin (Ngb). In 2001, Kawada et al. identified Cygb in hepatic stellate cells (HSCs) that play an important role in the repair of damaged liver cell tissue, hence the name stellate cell activation-associated protein (STAP). Cygb has a more universal role than Hb, Mb, and Ngb, and is expressed throughout mammalian tissues. Cygb is a globin with 6 coordination bonds (hexacoordinate globin). Cygb has been shown to play an important role in the normal cell respiratory chain, including oxygen storage, destruction of reactive oxygen species (ROS), terminal oxidase activity, regulation of fibrogenesis, and regulation of apoptosis. The role of Cygb in the respiratory process has been studied because it is associated with the globin family, and because of the upregulation of Cygb during hypoxia, but its specific role has not been elaborated. Recent research reports that Cygb has several implications for cancer. In most cancer cells, Cygb expression is upregulated by hypermethylation, suggesting epigenetic control. In cancer cells, downregulation of Cygb occurs which indicates a possible role as a tumor suppressor gene. In some malignancies, on the contrary, Cygb upregulation may be associated with resistance to hypoxia which indicates Cygb has two sides or Janus faces related to its role in cancer cells.

Cytoglobin attenuates pancreatic cancer growth via scavenging reactive oxygen species

Pancreatic cancer is a highly challenging malignancy with extremely poor prognosis. Cytoglobin (CYGB), a hemeprotein involved in liver fibrosis and cancer development, is expressed in pericytes of all organs. Here, we examined the role of CYGB in the development of pancreatic cancer. CYGB expression appeared predominately in the area surrounding adenocarcinoma and negatively correlated with tumor size in patients with pancreatic cancer. Directly injecting 7, 12-dimethylbenz[a]anthracene into the pancreatic tail in wild-type mice resulted in time-dependent induction of severe pancreatitis, fibrosis, and oxidative damage, which was rescued by Cygb overexpression in transgenic mice. Pancreatic cancer incidence was 93% in wild-type mice but only 55% in transgenic mice. Enhanced CYGB expression in human pancreatic stellate cells in vitro reduced cellular collagen synthesis, inhibited cell activation, increased expression of antioxidant-related genes, and increased CYGB secretion into the medium. Cygb-overexpressing or recombinant human CYGB (rhCYGB) -treated MIA PaCa-2 cancer cells exhibited dose-dependent cell cycle arrest at the G1 phase, diminished cell migration, and reduction in colony formation. RNA sequencing in rhCYGB-treated MIA PaCa-2 cells revealed downregulation of cell cycle and oxidative phosphorylation pathways. An increase in MIA PaCa-2 cell proliferation and reactive oxygen species production by H2O2 challenge was blocked by rhCYGB treatment or Cygb overexpression. PANC-1, OCUP-A2, and BxPC-3 cancer cells showed similar responses to rhCYGB. Known antioxidants N-acetyl cysteine and glutathione also inhibited cancer cell growth. These results demonstrate that CYGB suppresses pancreatic stellate cell activation, pancreatic fibrosis, and tumor growth, suggesting its potential therapeutic application against pancreatic cancer.

Role of cytoglobin in cigarette smoke constituent-induced loss of nitric oxide bioavailability in vascular smooth muscle cells

Cytoglobin (Cygb) has been identified as the major nitric oxide (NO) metabolizing protein in vascular smooth muscle cells (VSMCs) and is crucial for the regulation of vascular tone. In the presence of its requisite cytochrome B5a (B5)/B5 reductase-isoform-3 (B5R) reducing system, Cygb controls NO metabolism through the oxygen-dependent process of NO dioxygenation. Tobacco cigarette smoking (TCS) induces vascular dysfunction; however, the role of Cygb in the pathophysiology of TCS-induced cardiovascular disease has not been previously investigated. While TCS impairs NO biosynthesis, its effect on NO metabolism remains unclear. Therefore, we performed studies in aortic VSMCs with tobacco smoke extract (TSE) exposure to investigate the effects of cigarette smoke constituents on the rates of NO decay, with focus on the alterations that occur in the process of Cygb-mediated NO metabolism. TSE greatly enhanced the rates of NO metabolism by VSMCs. An initial increase in superoxide-mediated NO degradation was seen at 4 h of exposure. This was followed by much larger progressive increases at 24 and 48 h, accompanied by parallel increases in the expression of Cygb and B5/B5R. siRNA-mediated Cygb knockdown greatly decreased these TSE-induced elevations in NO decay rates. Therefore, upregulation of the levels of Cygb and its reducing system accounted for the large increase in NO metabolism rate seen after 24 h of TSE exposure. Thus, increased Cygb-mediated NO degradation would contribute to TCS-induced vascular dysfunction and partial inhibition of Cygb expression or its NO dioxygenase function could be a promising therapeutic target to prevent secondary cardiovascular disease.

Mechanism of NO Decay in Vascular Smooth Muscle Cells: Role of Cytoglobin and Identification of its Cellular Reducing System

Background In smooth muscle, cytoglobin (Cygb) functions as a potent nitric oxide (NO) dioxygenase and regulates NO metabolism and vascular tone. However, major questions remain regarding which cellular reducing systems regulate Cygb-mediated NO metabolism. Therefore, we performed studies to assess the intracellular concentrations of Cygb and its reducing systems in vascular smooth muscle cells (SMCs), and their role in the process of NO decay. Methods Cygb and its reducing systems, cytochrome B5 reductase (B5R), cytochrome B5 (B5) and cytochrome P450 reductase (CPR) were measured in aortic SMCs. Murine aortic smooth muscle cells (SMCs) were purchased from American Type Culture Collection (ATCC; CRL-2797™) and passaged. For depleting Cygb, B5R or B5, siRNAs were used. SMCs were transfected with Cygb siRNA, B5R siRNA, B5 siRNA or CPR siRNA using Lipofectamine RNAiMAX (Invitrogen). The rate of NO metabolism by normal SMCs of different cell numbers was assessed using a Clark-type NO electrode (NOCHM-4, WPI) utilizing two ports on the side wall of a four-port water-jacketed electrochemical chamber (NOCHM-4, WPI) and additionally another oxygen electrode was placed through second port on the side wall of the chamber. For protein assays, Cygb, B5R and B5 were expressed and purified using reported procedures. Quantitative immunoblotting was used to estimate the cellular concentrations of Cygb and each reducing system protein from comparison to standards of known concentration for each purified protein. Results Intracellular Cygb concentration was estimated as 3.5 µM, while B5R, B5, and CPR were 0.88, 0.38 and 0.15 µM, respectively. NO decay in SMCs was measured following bolus addition of 1 µM NO to air-equilibrated cells. The NO dioxygenation rate (VNO) with 3.5 x 106 cells•ml-1 was 12.7 ± 0.3 nM•s-1. siRNA-mediated knockdown experiments indicated that ~78% of NO metabolism in SMCs was Cygb-dependent. Of this, ~87 % was B5R- and B5-dependent. CPR knockdown resulted in a small decrease in VNO, while depletion of ascorbate had no effect. Kinetic analysis of VNO for the B5R/B5/Cygb system with variation of B5 or B5R concentrations from their SMC levels showed that VNO exhibits apparent Michaelis-Menten behavior for B5 and B5R. In contrast, linear variation was seen with change in Cygb concentration. Conclusions B5R/B5 was demonstrated to be the major reducing system supporting Cygb-mediated NO metabolism in SMCs and studies were performed to experimentally model and predict the alterations of VNO with changes in cellular B5R/B5/Cygb levels.

Role of cytoglobin, a novel radical scavenger, in stellate cell activation and hepatic fibrosis.

Cytoglobin (Cygb), a stellate cell-specific globin, has recently drawn attention due to its association with liver fibrosis. In the livers of both humans and rodents, Cygb is expressed only in stellate cells and can be utilized as a marker to distinguish stellate cells from hepatic fibroblast-derived myofibroblasts. Loss of Cygb accelerates liver fibrosis and cancer development in mouse models of chronic liver injury including diethylnitrosamine-induced hepatocellular carcinoma, bile duct ligation-induced cholestasis, thioacetamide-induced hepatic fibrosis, and choline-deficient L-amino acid-defined diet-induced non-alcoholic steatohepatitis. This review focuses on the history of research into the role of reactive oxygen species and nitrogen species in liver fibrosis and discusses the current perception of Cygb as a novel radical scavenger with an emphasis on its role in hepatic stellate cell activation and fibrosis.

Cytoglobin inhibits migration through PI3K/AKT/mTOR pathway in fibroblast cells.

Cell proliferation and migration are crucial in many physiological processes including development, cancer, tissue repair, and wound healing. Cell migration is regulated by several signaling molecules. Identification of genes related to cell migration is required to understand molecular mechanism of non-healing chronic wounds which is a major concern in clinics. In the current study, the role of cytoglobin (CYGB) gene in fıbroblast cell migration and proliferation was described. L929 mouse fibroblast cells were transduced with lentiviral particles for CYGB and GFP, and analyzed for cell proliferation and migration ability. Fibroblast cells overexpressing CYGB displayed decreased cell proliferation, colony formation capacity, and cell migration. Phosphorylation levels of mTOR and two downstream effectors S6 and 4E-BP1 which take part in PI3K/AKT/mTOR signaling declined in CYGB-overexpressing cells. Microarray analysis indicated that CYGB overexpression leads to downregulation of cell proliferation, migration, and tumor growth associated genes in L929 cell line. This study demonstrated the role of CYGB in fibroblast cell motility and proliferation. CYGB could be a promising candidate for further studies as a potential target for diseases related to cell migration such as cancer and chronic wound treatment.

Pathophysiological role of cytoglobin, the fourth globin in mammals, in liver diseases.

Cytoglobin (Cygb), a stellate cell-specific globin, has recently drawn attention due to its association with liver fibrosis and cancer. In human and rodent livers, Cygb is expressed only in stellate cells and can be utilized as a marker to distinguish stellate cells from hepatic fibroblast-derived myofibroblasts. Loss of Cygb accelerates liver fibrosis and cancer development despite its etiology in mouse models of chronic liver injury. This review discusses the current perception of the distribution, regulation and function of Cygb with regard to liver diseases, with an emphasis on its role in tumorigenesis. Further investigation of Cygb may shed new light on the biology of organ carcinogenesis.

ßNp63 controls cellular redox status.

A widely accepted hypothesis indicates that the accumulation of oxidative damage arising from endogenously produced reactive oxygen species (ROS) is one of the main triggers of cellular senescence, age-related disorders and cancer development. High ROS levels are responsible for lipid peroxidation, protein, and DNA oxidation, thus seriously threatening cellular fate and enhancing mutations risks [1]. The regulation of redox homeostasis is crucial to maintain a normal cellular function, ensuring cell proliferation and survival. An important protein, which is able to sustain epithelial proliferation/survival to counteract cellular senescence and organismal aging, is the transcription factor p63 [2]. TP63 belongs to the p53 gene family; it is expressed as multiple isoforms for the presence of two N-terminus promoters and C-terminus alternative splicing [2]. The sNp63 isoforms lack the N-terminal transactivation domain but they are still able to activate transcription of selected genes, using a second transactivation domain. sNp63 is responsible for activation of different cellular programs controlling cell proliferation potential and stemness, epithelial stratification, cell–cell, and cell-matrix adhesion [2, 3]. Interestingly, depletion of sNp63 in primary human keratinocytes and in cancer cell lines (lung, breast) increases endogenous ROS levels [4] suggesting that p63-dependent mechanisms are required in epithelial cells to control redox status. A recent study identified cytoglobin (CYGB) as a novel sNp63 target gene [4]. The role of CYGB in cellular metabolism is partially unknown, its expression increases under oxygen deficiency and oxidative stress. It binds oxygen, therefore it may facilitate oxygen diffusion to mitochondria. It detects O2 concentration in cells and protects them from ROS [4]. CYBG is co-expressed with sNp63 in the basal layer of the epidermis, the proliferative compartment, and in vitro, in proliferating keratinocytes. Down-modulation of CYGB in keratinocytes increases endogenous ROS levels, with consequent p53-mediated induction of apoptosis. The CYGB protecting role is also more evident upon oxidative stress, as CYGB-silenced keratinocytes undergoing to oxidative stress results in elevated level of ROS accumulation that parallel high level of apoptotic events. These results indicate that in physiological conditions, sNp63, via CYGB, plays an important role in maintaining physiological redox status. Thus, this could be one of the mechanisms engaged by sNp63 [4, 5], to counteract senescence/ageing and maintain high proliferative potential in epithelial stem cells. The redox status of cancer cells differs from that of normal cells and the modulation of oxidative stress is very relevant in both tumor formation and response to anti-cancer therapy. Indeed, high ROS levels in cancer cells are consequence of alteration in several signalling pathways that affect cellular metabolism. These ROS levels are balanced to elevated anti-oxidant defence pathways in cancer cells. Therefore, the role of ROS in tumorigenesis is still under debate: the increased anti-oxidant capacity suggests that high ROS level could contribute to a barrier against tumorigenesis, on the other hand, ROS may promote tumorigenesis by inducing DNA mutations and pro-oncogenic signalling [6–8]. The sNp63-CYGB axis could be part of this scenario, controlling cellular redox state in cancer cells. Indeed, experimental evidences show that CYGB-silenced lung SCC cells exhibit elevated ROS levels leading to apoptosis as well as an increased sensitivity to doxorubicin treatments [4]. Furthermore, computational analysis of lung cancer data sets, indicates that sNp63 and CYGB co-expression is a negative prognostic marker for patients, showing significant survival reduction. Glutathione peroxidase (GPX2) is another sNp63 direct target, which inhibits the activation of p53 by reducing the extent of oxidative stresses and oxidative stress-induced apoptosis in cancer cells [4]. In summary, sNp63 maintains redox cellular status in physiological condition, by direct transcriptional control of CYGB and GPX2, possibly preventing senescence/ageing and maintaining epithelial stem cells. Given that sNp63 is frequently amplified in tumors and acts in a dominant-negative manner over p53, sNp63 action as a pro-survival factor is, at least in part, mediated by the inhibition of the p53-dependent oxidative stress-induced apoptotic response. Furthermore, the sNp63-antioxidant properties may modulate therapeutic efficiency of anticancer treatments that act directly and/or indirectly regulating ROS levels.

Effect of permanent middle cerebral artery occlusion on Cytoglobin expression in the mouse brain

Cytoglobin, a new member of the mammalian heme-globin family has been shown to bind oxygen and to have cell protective properties in vitro. Cytoglobin is specifically expressed in a subpopulation of brain neurons. Based on hypoxia-induced up regulation and proposed scavenging of reactive oxygen species Cytoglobin was suggested as a candidate for pharmaceutical stroke treatment. Since production of reactive oxygen species is a hallmark of ischemia, we hypothesized that Cytoglobin expression would be increased and that Cytoglobin expressing neurons would be spared after ischemic injury. Twenty male C57BL/6J mice were used in the experimental design. Ten were sham operated and ten were given permanent middle cerebral artery occlusion (pMCAo). All animals were euthanized after 24h. From each group, three animals were used for histology and seven for QRT-PCR and western blotting. Immunohistochemical examination of the ischemic penumbra revealed neither changes in Cytoglobin immunoreactivity nor any changes in expression in the necrotic infarct area. The lack of expression change was confirmed by western blotting and QRT-PCR showing no significant difference between sham and pMCAo operated mice. This suggests that Cytoglobin is likely not important for global neuronal protection following ischemia and the role of Cytoglobin in relation to endogenous neuroprotection remains unresolved.