Reductive stress induced by NRF2/G6PD through glucose metabolic reprogramming promotes malignant transformation in Arsenite-exposed human keratinocytes.

Our previous research found that the nuclear factor-E2-related factor 2 (NRF2) protein was sustained activated in malignant transformation of human keratinocyte (HaCaT cells) caused by NaAsO2, but the role of NRF2 in it remains unknown. In this study, malignant transformation of HaCaT cells and labeled HaCaT cells used to detect mitochondrial glutathione levels (Mito-Grx1-roGFP2 HaCaT cells) were induced by 1.0 µM NaAsO2. Redox levels were measured at passages 0, early stage (passages 1, 7, 14), later stage (passages 21, 28 and 35) of arsenite-treated HaCaT cells. Oxidative stress levels increased at early stage. The NRF2 pathway was sustained activated. Cells and mitochondrial reductive stress levels (GSH/GSSG and NADPH/NADP+) increased. The mitochondrial GSH/GSSG levels of Mito-Grx1-roGFP2 HaCaT cells also increased. The indicators of glucose metabolism glucose-6-phosphate, lactate and the glucose-6-phosphate dehydrogenase (G6PD) levels increased, however Acetyl-CoA level decreased. Expression levels of glucose metabolic enzymes increased. After transfection with NRF2 siRNA, the indicators of glucose metabolism were reversed. After transfection with NRF2 or G6PD siRNA, cells and mitochondrial reductive stress levels decreased and the malignant phenotype was reversed. In conclusion, oxidative stress occurred in the early stage and the NRF2 was sustained high expression. In the later stage, increased NRF2/G6PD through glucose metabolic reprogramming induced reductive stress, thereby leading to malignant transformation.

The Potential Key Role of the NRF2/NQO1 Pathway in the Health Effects of Arsenic Pollution on SCC.

Arsenic is widely present in nature and is a common environmental poison that seriously damages human health. Chronic exposure to arsenic is a major environmental poisoning factor that promotes cell proliferation and leads to malignant transformation. However, its molecular mechanism remains unclear. In this study, we found that arsenite can promote the transformation of immortalized human keratinocyte cells (HaCaT) from the G0/G1 phase to S phase and demonstrated malignant phenotypes. This phenomenon is accompanied by obviously elevated levels of NRF2, NQO1, Cyclin E, and Cyclin-dependent kinase 2 (CDK2). Silencing the NRF2 expression with small interfering RNA (siRNA) in arsenite-transformed (T-HaCaT) cells was shown to reverse the malignant phenotype. Furthermore, the siRNA silencing of NQO1 significantly decreased the levels of the cyclin E-CDK2 complex, inhibiting the G0/G1 to S phase cell cycle progression and transformation to the T-HaCaT phenotypes. Thus, we hypothesized that the NRF2/NQO1 pathway played a key role in the arsenite-induced malignancy of HaCaT cells. By increasing the expression of Cyclin E-CDK2, the NRF2/NQO1 pathway can affect cell cycle progression and cell proliferation. A new common health effect mechanism of arsenic carcinogenesis has been identified; thus, it would contribute to the development of novel treatments to prevent and treat skin cancer caused by arsenic.

Possible differences in the mechanism of malignant transformation of HaCaT cells by arsenite and its dimethyl metabolites, particularly dimethylthioarsenics.

BACKGROUND: As a confirmed human carcinogen, arsenic can cause skin cancer, lung cancer, etc. However, its carcinogenic mechanism is still unclear. In recent years, the oxidative stress hypothesis has become widely accepted. In mammals it has been found that arsenic can be converted to dimethylarsinous acid (DMAIII) and dimethylmonothioarsinic acid (DMMTAV) through a series of methylation and redox reactions. DMAIII and DMMTAV are highly toxic. METHODS: Human keratinocytes (HaCaT) were exposed to different concentrations of NaAsO2 (IAsIII), DMMTAV and DMAIII for 24 h. Reactive oxygen species (hydrogen peroxide and superoxide), oxidative damage markers (8-hydroxydeoxyguanosine and malondialdehyde), and antioxidant markers (glutathione and superoxide dismutase) were measured. In addition, sulfane sulfurs were measured in HaCaT cells and a cell-free system. RESULTS: In the DMMTAV and DMAIII treatment groups, the levels of hydrogen peroxide and superoxide in HaCaT cells were higher than in the IAsIII treatment groups at the same dose. Levels of 8-OHdG and MDA in the DMMTAV and DMAIII treatment groups were also higher than those in the IAsIII treatment groups at the same dose. However, in the DMMTAV and DMAIII treatment groups, the levels of GSH and SOD activity were lower than that in the IAsIII treatment groups. In DMMTAV-treated HaCaT cells, sulfane sulfurs were produced. Further, it was found that DMMTAV could react with DMDTAV to form persulfide in the cell-free system, which may explain the mechanism of the formation of sulfane sulfurs in DMMTAV-treated HaCaT cells. CONCLUSIONS: DMMTAV and DMAIII more readily induce reactive oxygen species (ROS) and cause oxidative damage in HaCaT cells than inorganic arsenic. Further, the persulfide formed by the reaction of DMMTAV and DMDTAV produced from the metabolism of DMMTAV may induce a stronger reductive defense mechanism than GSH against the intracellular oxidative stress of DMMTAV. However, the cells exposed to arsenite are transformed by the continuous nuclear translocation of Nrf2 due to oxidative stress, and the persulfide from dimethylthioarsenics may promote Nrf2 by the combination with thiol groups, especially redox control key protein, Keap1, eventually cause nuclear translocation of sustained Nrf2.

High NF-E2-related factor 2 expression predicts poor prognosis in patients with lung cancer: a meta-analysis of cohort studies.

NF-E2-related factor 2 (Nrf2) plays an important role in tumour proliferation and chemoresistance. Nrf2 expression levels may be associated with prognosis of lung cancer, but previous results have been inconsistent. Pooled hazard ratios (HRs) and odds ratios were calculated to assess the prognostic value of the Nrf2 expression in this meta-analysis. Nine studies with 940 patients were included. A high Nrf2 expression level was significantly related to decreased overall survival (OS) (HR = 1.948, 95% CI = 1.564-2.427), lower response rate (HR = 2.675, 95% CI = 1.553-4.610), and poor progression-free survival (HR = 3.078, 95% CI = 1.791-5.293). Subgroup analysis demonstrated that high-Nrf2-expression was significantly correlated with worse OS of patients possessing epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) or undergoing chemotherapeutic treatments (HR = 2.500, 95% CI = 1.556-4.018). Conversely, this high expression was not significantly related to the OS of patients with surgical resection (HR = 1.750, 95% CI = 0.995-3.080, and p=.052). High Nrf2 expression was significantly correlated with worse OS of patients in advanced stage (HR = 2.500, 95% CI = 1.556-4.018), compared with early cancer stage (HR = 1.609, 95% CI = 0.675-3.835, and p=.283). This meta-analysis suggests that high Nrf2 expression may be a predictive factor of poor outcomes in lung cancer. Therefore, Nrf2 likely plays an important role in prognostic evaluation and may be a therapeutic target for EGFR-TKIs therapy and chemotherapy.

The protective effect of silk fibroin on high glucose induced insulin resistance in HepG2 cells.

The therapeutic use of silk-derived materials such as fibroin in biomedicine is well-established in Southeast Asian countries. Studies indicated that silk fibroin (SF) peptide enhances insulin sensitivity and glucose metabolism phenomena associated with type 2 diabetes mellitus (T2DM) suggesting this peptide may be beneficial to treat this disease. However, the mechanisms underlying protective effect of SF in insulin-mediated hepatic metabolic dysfunction remains unclear. The aim of this study was to investigate the influence of SF on insulin resistant HepG2 cells which were used a model of T2DM. Treatment of cells with 30 mmol/L of glucose and 10-6 mol/L insulin for 48 h significantly reduced glucose consumptions and intracellular glycogen levels but increased triglyceride (TG) levels. SF or metformin alone elevated glucose consumptions and glycogen accumulation accompanied by lower TG content. Greater effects in these metabolic parameters were found when SF and metformin were combined. Treatment of insulin resistant cells with SF or metformin alone decreased levels of reactive oxygen species (ROS), malondialdehyde (MDA), tumor necrosis factor (TNF-α) and interleukin-6 (IL-6); whereas antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) activity, as well as total antioxidant capacity (T-AOC) ability increased. The combination of SF and metformin produced greater changes in these parameters compared to metformin alone. Data indicated that the protective effect of SF or metformin in insulin resistant HepG2 cells involves inhibition of oxidant processes and that the combination of agents may prove more effective therapeutically.