Guannan Zhu

Impaired Activation of the Nrf2-ARE Signaling Pathway Undermines H2O2-Induced Oxidative Stress Response: A Possible Mechanism for Melanocyte Degeneration in Vitiligo

Vitiligo melanocytes possess higher susceptibility to oxidative insults. Consistent with this, impairment of the antioxidant defense system has been reported to be involved in the onset and progression of vitiligo. Our previous study showed that the nuclear factor E2-related factor 2-antioxidant response element (Nrf2-ARE) pathway and its downstream antioxidant enzyme heme oxygenase-1 (HO-1) are crucial for melanocytes to cope with H2O2-induced oxidative damage. Here, we sought to determine whether the diminished Nrf2-ARE activity that contributes to reduced downstream antioxidant enzymes and increased oxidative stress could be the reason why melanocytes are more vulnerable to vitiligo. We found that vitiligo melanocytes exhibited hypersensitivity to H2O2-induced oxidative injury because of reduced Nrf2 nuclear translocation and transcriptional activity, which led to decreased HO-1 expression and aberrant redox balance. Moreover, we also found that the level of serum HO-1 was significantly decreased and that of IL-2 was markedly increased in 113 vitiligo patients when compared with healthy controls. These data demonstrate that impaired activation of Nrf2 under oxidative stress could result in decreased expression of antioxidant enzymes and increased death of vitiligo melanocytes.

Oxidative stress drives CD8+ T-cell skin trafficking in patients with vitiligo through CXCL16 upregulation by activating the unfolded protein response in keratinocytes.

Background In patients with vitiligo, an increased reactive oxygen species (ROS) level has been proved to be a key player during disease initiation and progression in melanocytes. Nevertheless, little is known about the effects of ROS on other cells involved in the aberrant microenvironment, such as keratinocytes and the following immune events. CXCL16 is constitutively expressed in keratinocytes and was recently found to mediate homing of CD8+ T cells in human skin. Objective We sought to explicate the effect of oxidative stress on human keratinocytes and its capacity to drive CD8+ T‐cell trafficking through CXCL16 regulation. Methods We first detected putative T‐cell skin‐homing chemokines and ROS in serum and lesions of patients with vitiligo. The production of candidate chemokines was detected by using quantitative real‐time PCR and ELISA in keratinocytes exposed to H2O2. Furthermore, the involved mediators were analyzed by using quantitative real‐time PCR, Western blotting, ELISA, and immunofluorescence. Next, we tested the chemotactic migration of CD8+ T cells from patients with vitiligo mediated by the CXCL16‐CXCR6 pair using the transwell assay. Results CXCL16 expression increased and showed a positive correlation with oxidative stress levels in serum and lesions of patients with vitiligo. The H2O2‐induced CXCL16 expression was due to the activation of 2 unfolded protein response pathways: kinase RNA (PKR)–like ER kinase–eukaryotic initiation factor 2&agr; and inositol‐requiring enzyme 1&agr;–X‐box binding protein 1. CXCL16 produced by stressed keratinocytes induced migration of CXCR6+CD8+ T cells derived from patients with vitiligo. CXCR6+CD8+ T‐cell skin infiltration is accompanied by melanocyte loss in lesions of patients with vitiligo. Conclusion Our study demonstrated that CXCL16‐CXCR6 mediates CD8+ T‐cell skin trafficking under oxidative stress in patients with vitiligo. The CXCL16 expression in human keratinocytes induced by ROS is, at least in part, caused by unfolded protein response activation.

Serum and skin levels of miR-369-3p in patients with psoriasis and their correlation with disease severity

BACKGROUND Psoriasis is a common autoimmune skin disease, characterized by intense proliferation and abnormal differentiation of keratinocytes. Recently, some miRNAs have been proven to show aberrant expression in psoriasis and play a role in the pathogenesis of the disease. OBJECTIVES The aim of this study was to detect serum and skin miR-369-3p levels in patients with psoriasis and confirm their correlation with disease severity. The regulatory mechanism between miR-369-3p and TNF-α was also investigated. METHODS Bioinformatics analysis for target genes of miRNAs was performed using multiple prediction software. Serum samples and skin tissues were collected and serum and skin miR-369-3p levels were measured. The Psoriasis Area Severity Index scores of patients and the correlation with serum and skin miR-369-3p levels were evaluated. Transient transfection and Elisa were applied to HaCaT cells to testify the relationship between expression of miR-369-3p and TNF-α. RESULTS Serum and skin miR-369-3p levels were both higher in patients with psoriasis than those in healthy controls (P<0.05; P<0.001, respectively). And miR-369-3p levels in skin had a positive linear relation with PASI scores in psoriasis patients (r = 0.70, P<0.05). Unexpectedly, miR-369-3p failed to show a regulatory effect on TNF-α levels in HaCaT cells. CONCLUSIONS The expression of miR-369-3p is increased in both serum samples and skin tissues from psoriasis patients, and its level in the skin positively correlates with disease severity. Further studies are needed to clarify the role of miR-369-3p in the pathogenesis of psoriasis.

Aspirin induces Nrf2-mediated transcriptional activation of haem oxygenase-1 in protection of human melanocytes from H2O2-induced oxidative stress

The removal of hydrogen peroxide (H2O2) by antioxidants has been proven to be beneficial to patients with vitiligo. Aspirin (acetylsalicylic acid, ASA) has antioxidant activity and has great preventive and therapeutical effect in many oxidative stress‐relevant diseases. Whether ASA can protect human melanocytes against oxidative stress needs to be further studied. Here, we investigated the potential protective effect and mechanisms of ASA against H2O2‐induced oxidative injury in human melanocytes. Human melanocytes were pre‐treated with different concentrations of ASA, followed by exposure to 1.0 mM H2O2. Cell apoptosis, intracellular reactive oxygen species (ROS) levels were evaluated by flow cytometry, and cell viability was determined by an Cell Counting Kit‐8 assay. Total and phosphorylated NRF2 expression, NRF2 nuclear translocation and antioxidant response element (ARE) transcriptional activity were assayed with or without Nrf2‐siRNA transfection to investigate the possible molecular mechanisms. Concomitant with an increase in viability, pre‐treatment of 10‐90 μmol/l ASA resulted in decreased rate of apoptotic cells, lactate dehydrogenase release and intracellular ROS levels in primary human melanocytes. Furthermore, we found ASA dramatically induced NRF2 nuclear translocation, enhanced ARE‐luciferase activity, increased both p‐ NRF2 and total NRF2 levels, and induced the expression of haem oxygenase‐1 (HO‐1) in human melanocytes. In addition, knockdown of Nrf2 expression or pharmacological inhibition of HO‐1 abrogated the protective action of ASA on melanocytes against H2O2‐induced cytotoxicity and apoptosis. These results suggest that ASA protects human melanocytes against H2O2‐induced oxidative stress via Nrf2‐driven transcriptional activation of HO‐1.

A similar local immune and oxidative stress phenotype in vitiligo and halo nevus

BACKGROUND Vitiligo and halo nevus are two common T-cell-mediated skin disorders. Although autoimmunity has been suggested to be involved in both diseases, the relationship between vitiligo and halo nevus is not fully understood. OBJECTIVE The aim of the current study was to investigate whether vitiligo and halo nevus share the same immunological and oxidative stress response. METHODS Infiltrations of T cells, and expressions of chemokine receptors (CXCR3, CCR4, CCR5) and cytotoxic markers (Granzyme B, Perforin) in the lesions of vitiligo and halo nevus were examined by immunohistochemistry. Enzyme-linked immunosorbent assay was performed to analyze the expressions of chemokines in the serum samples and cytotoxic markers secreted by CD8+ T cells which were sorted from the peripheral blood mononuclear cells in healthy donors, vitiligo and halo nevus patients. Tissue levels of chemokine receptors and CXCR3 ligands in healthy controls, vitiligo patients and halo nevus patients were determined by qRT-PCR analysis. The percentages of CXCR3+ CD4+ T and CXCR3+ CD8+ T cells from the peripheral blood samples were examined by flow cytometry. Tissue and serum hydrogen peroxide (H2O2) concentrations were measured using H2O2 assay kit. RESULTS Immunohistochemistry revealed a significant T-cell response, with pronounced dermal infiltrates of CD8+ T cells in vitiligo and halo nevus. The inflammatory cytotoxic markers such as Granzyme B and Perforin were also elevated in vitiligo and halo nevus, suggesting inflammatory responses in situ. By qRT-PCR and ELISA assay, we found significantly increased expressions of the chemokine receptor CXCR3 and its ligands, especially the accumulated CXCL10 in the skin lesions of vitiligo and halo nevus. Moreover, the level of H2O2, a key player involved in regulation of the immune response was significantly upregulated in the skin lesions of vitiligo and halo nevus. In addition, the increased H2O2 concentration correlated positively with CXCL10 level in skin lesions of vitiligo and halo nevus. CONCLUSIONS These results demonstrate a H2O2-involved autoimmune phenotype in vitiligo and halo nevus, characterized by increased level of IFN-γ-inducible chemokine pair CXCL10-CXCR3, as well as a dense CD8+ T infiltration in the skin lesions, thus suggesting a similar pathogenesis of the two diseases.

The association between trauma and melanoma in the Chinese population: a retrospective study

The association between trauma and melanoma has been a controversial issue.

Aberrant SIRT6 expression contributes to melanoma growth: Role of the autophagy paradox and IGF-AKT signaling

ABSTRACT Melanoma is among the most life-threatening cancers. The pathogenesis of melanoma has not been fully elucidated. Recently, dysregulated macroautophagy/autophagy has been found to play a critical but inconsistent role in modulating melanoma growth at different stages, with the regulatory mechanism unclear. The histone deacetylase SIRT6 (sirtuin 6) is a known autophagy regulator, and its involvement in cancer development has been reported. Therefore, we sought to determine the role of SIRT6 in melanoma growth and detect its possible link with autophagy in the current study. We initially observed that the expression of SIRT6 decreased in primary melanoma but increased in metastatic melanoma compared with melanocytic nevus. Notably, the expression of SIRT6 was significantly correlated with the expression of autophagy biomarkers including MAP1LC3/LC3 and SQSTM1/p62. Furthermore, SIRT6 suppressed the growth of primary melanoma but promoted metastatic melanoma development in an autophagy-dependent way in vitro. Moreover, SIRT6 exerted its regulation on melanoma growth via the IGF-AKT signaling pathway, and the intervention of AKT could partly reverse the effects of SIRT6 on melanoma growth by regulating autophagy. At last, we determined the effects of SIRT6 on melanoma development in vivo. Taken together, our findings demonstrate that the bimodal expression of SIRT6 at different melanoma stages plays a critical role in regulating melanoma growth through an autophagy-dependent manner, which indicates the potential of SIRT6 to be a biomarker and a therapeutic target in melanoma.

Multiple pro-tumorigenic functions of the human minor Histocompatibility Antigen-1 (HA-1) in melanoma progression

BACKGROUND Remodeling of cytoskeleton plays an important role in development of multiple cancers, including melanoma. As a group of F-actin regulators, the Ras homology (Rho) GTPase-activating proteins (ARHGAPs) were reported by accumulating studies as a set of significant mediators in cell morphology, proliferation, migration and invasion. OBJECTIVE To investigate the function of HMHA1 and its encode protein HA-1 in melanoma. METHODS The mRNA microarray was performed to screen the expression of ARHGAP family genes between melanoma tissues and nevi tissues. QRT-PCR and Western Blot were used to detect the expression of mRNA of HMHA1 and its relevant protein HA-1 respectively. Small interfering RNA was used to knock down the expression of HMHA1. Cell-count kit 8 assays and colony formation assays were used to evaluate the cell proliferative viability of melanoma cells. Flow cytometry was employed to analyze cell apoptosis. Transwell assay and the observation of cell morphology were used to evaluate the invasive and migrating activity of melanoma cells. RESULTS In previous study, we first found that both the mRNA level of HMHA1and the expression of HA-1 were up-regulated in melanoma tissues and cell lines compared with nevi tissues and normal human melanocytes respectively. Blocking HMHA1 expression in melanoma cell lines WM35 and A375 suppressed their proliferation and function of colony forming. Moreover, silencing HMHA1 not only significantly increased cell apoptosis but also suppressed cell migration and invasion. CONCLUSION Our results demonstrate that HMHA1 significantly promotes melanoma cells proliferation, invasion and migration, and prevents cell apoptosis. Additionally, it can be considered as a new diagnostic marker and drug target of melanoma.

Combination with γ-secretase inhibitor prolongs treatment efficacy of BRAF inhibitor in BRAF-mutated melanoma cells

Oncogenic triggering of the MAPK pathway in melanocytes results in senescence, and senescence escape is considered as one critical step for melanocytic transformation. In melanoma, induction of a senescent-like state by BRAF-inhibitors (BRAFi) in a fraction of treated cells - instead of killing - contributes to the repression of tumor growth, but may also provide a source for relapse. Here, we demonstrate that NOTCH activation in melanocytes is not only growth-promoting but it also protects these cells against oncogene-induced senescence. In turn, treatment of melanoma cells with an inhibitor of the NOTCH-activating enzyme γ-secretase led to induction of a senescent-like status in a fraction of the cells but overall achieved only a moderate inhibition of melanoma cell growth. However, combination of γ-secretase inhibitor (GSI) with BRAFi markedly increased the treatment efficacy particularly in long-term culture. Moreover, even melanoma cells starting to regrow after continuous BRAFi treatment - the major problem of BRAFi therapy in patients - can still be affected by the combination treatment. Thus, combining GSI with BRAFi increases the therapeutic efficacy by, at least partially, prolonging the senescent-like state of treated cells.

Up-regulated deubiquitinase USP4 plays an oncogenic role in melanoma

Melanoma is the most malignant skin cancer with increasing incidence worldwide. Although innovative therapies such as BRAF inhibitor and immune checkpoint inhibitor have gained remarkable advances, metastatic melanoma remains an incurable disease for its notorious aggressiveness. Therefore, further clarification of the underlying mechanism of melanoma pathogenesis is critical for the improvement of melanoma therapy. Ubiquitination is an important regulatory event for cancer hallmarks and melanoma development, and the deubiquitinating enzymes including ubiquitin‐specific peptidase (USP) families are greatly implicated in modulating cancer biology. Herein, we first found that the expression of the deubiquitinase USP4 was significantly up‐regulated in melanoma tissues and cell lines. Furthermore, although USP4 knockdown had little impact on melanoma cell proliferation, it could increase the sensitivity to DNA damage agent cisplatin. We subsequently showed that USP4 regulated cisplatin‐induced cell apoptosis via p53 signalling. More importantly, USP4 could accentuate the invasive and migratory capacity of melanoma cells by promoting epithelial‐mesenchymal transition. Altogether, our results demonstrate that the up‐regulated USP4 plays an oncogenic role in melanoma by simultaneously suppressing stress‐induced cell apoptosis and facilitating tumour metastasis.