NQO1 Protein Research Articles

Imatinib Induces Apoptosis of NK/T Cell Neoplasms by Inhibiting Hematopoietic Cell Kinase and NQO2

▪[Background] Natural killer and T (NK/T)-cell malignancies are uncommon in Western countries but relatively frequent in Asia. Although regimens that include L-asparaginase have improved outcomes, these disorders, particularly aggressive NK-cell leukemias/lymphomas, have poor prognoses. No molecular-targeting drugs have been developed so far. Current reports have provided evidence that a subset of NK cell malignancies is of myeloid origin. Some tyrosine kinase inhibitors (TKIs) that target multiple kinases, including LcK involved in the growth of myeloid malignancies, may therefore show inhibitory effects against NK/T cell neoplasms where LcK members are highly expressed. We investigated the potential anti-tumor activities of multitarget TKIs against malignant NK/T cell neoplasms. [Methods and Results] Three TKIs (imatinib, nilotinib, and dasatinib) were tested for their apoptosis-inducing activities against various NK cell lines including KHYG1, NK92, and NKL after 24-h incubation. Therapeutic doses of imatinib (7.5-27 µM) induced variable annexinV(+) cells in 5 out of 7 NK lines, ranging from 27.1%±6.9% to 87.8%±3.3% (Figure 1), while the apoptosis-inducing effects of therapeutic doses of nilobinib and dasatinib were marginal. Membrane array screenings, which can quantitate 43 phosphorylated proteins, showed that hematopoietic cell kinase (HcK) was constitutively active in KHYG1 cells, and treatment with imatinib inhibited pHcK by 80%. Other known imatinib-targets, including PDGFR, LcK, c-kit, and BCR/ABL, were undetectable in the NK cell lines tested. Further immunoblot analyses using anti-pHcK antibodies confirmed pHcK inhibition in imatinib-treated KHYG1 and NKL (Figure 2). Immunohistochemistry studies with anti-pHcK antibodies revealed two of eight extranodal NK/T-cell lymphomas, nasal type, to be strongly positive for pHcK (Figure 3). In addition, immunohistochemistry studies revealed that NAD(P)H dehydrogenase, quinone 2 (NQO2), a lesser-known imatinib target, was highly expressed in tumor tissues of all seven NK/T-cell lymphoma patients examined, including two pHcK(+) patients, and in vitro studies showed 50% NQO2 protein downregulation and 80% inhibition of its enzymatic activity in imatinib-treated cells. Notably, imatinib enhanced the pro-apoptotic and anti-proliferative activities of L-asparaginase against malignant NK and T cells (CI 0.77 and 0.86 respectively). [Conclusions] Imatinib induces the apoptosis of a subset of NK/T cell malignancies by inhibiting pHcK and NQO2. Imatinib and HcK inhibitors may have therapeutic potential against NK/T cell malignancies either alone or in combination with L-asparaginase. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Quinone oxidoreductase 1 is overexpressed in gastric cancer and associated with outcome of adjuvant chemotherapy and survival.

BACKGROUNDQuinine oxidoreductase 1 (NQO1) plays a vital role in protecting normal cells against oxidative damage and electrophilic attack. It is highly expressed in many solid tumors, suggesting a role in cancer development and progression. However, the role of NQO1 in gastric cancer and its effect on cancer development and prognosis have not been fully investigated.AIMTo investigate the clinical relevance of NQO1 protein expression in gastric cancer and to explore the potential of NQO1 to serve as a prognostic biomarker and therapeutic target.METHODSIn this retrospective study, gastric cancer specimens of 175 patients who were treated between 1995 and 2011 were subjected to immunohistochemistry analyses for NQO1. The correlation of NQO1 expression with gastric cancer prognosis and clinical and pathological parameters was investigated.RESULTSNQO1 protein was overexpressed in 59.43% (104/175) of the analyzed samples. Overexpression of NQO1 was associated with a significantly inferior prognosis. In addition, multivariate analysis suggested that NQO1 overexpression, along with tumor stage and patient age, are prominent prognostic biomarkers for gastric cancer. Moreover, NQO1 overexpression was correlated to a better response to 5-fluorouracil (5-FU)-based adjuvant chemotherapy.CONCLUSIONNQO1 overexpression is associated with a significantly poor prognosis and better response to 5-FU in patients with gastric cancer. These findings are relevant for improving therapeutic approaches for gastric cancer patients.

Bisavenathramide Analogues as Nrf2 Inductors and Neuroprotectors in In Vitro Models of Oxidative Stress and Hyperphosphorylation.

Oxidative stress is crucial to the outbreak and advancement of neurodegenerative diseases and is a common factor to many of them. We describe the synthesis of a library of derivatives of the 4-arylmethylen-2-pyrrolin-5-one framework by sequential application of a three-component reaction of primary amines, β-dicarbonyl compounds, and α-haloketones and a Knoevenagel condensation. These compounds can be viewed as cyclic amides of caffeic and ferulic acids, and are also structurally related to the bisavenanthramide family of natural antioxidants. Most members of the library showed low cytotoxicity and good activity as inductors of Nrf2, a transcription factor that acts as the master regulator of the antioxidant response associated with activation of the antioxidant response element (ARE). Nrf2-dependent protein expression was also proved by the significant increase in the levels of the HMOX1 and NQO1 proteins. Some compounds exerted neuroprotective properties in oxidative stress situations, such as rotenone/oligomycin-induced toxicity, and also against protein hyperphosphorylation induced by the phosphatase inhibitor okadaic acid. Compound 3i, which can be considered a good candidate for further hit-to-lead development against neurodegenerative diseases due to its well-balanced multitarget profile, was further characterized by proving its ability to reduce phosphorylated Tau levels.

High intensity muscle stimulation activates a systemic Nrf2-mediated redox stress response

High intensity exercise is a popular mode of exercise to elicit similar or greater adaptive responses compared to traditional moderate intensity continuous exercise. However, the molecular mechanisms underlying these adaptive responses are still unclear. The purpose of this pilot study was to compare high and low intensity contractile stimulus on the Nrf2-mediated redox stress response in mouse skeletal muscle. An intra-animal design was used to control for variations in individual responses to muscle stimulation by comparing a stimulated limb (STIM) to the contralateral unstimulated control limb (CON). High Intensity (HI – 100Hz), Low Intensity (LI – 50Hz), and Naïve Control (NC – Mock stimulation vs CON) groups were used to compare these effects on Nrf2-ARE binding, Keap1 protein, and downstream gene and protein expression of Nrf2 target genes. Muscle stimulation significantly increased Nrf2-ARE binding in LI-STIM compared to LI-CON (p = 0.0098), while Nrf2-ARE binding was elevated in both HI-CON and HI-STIM compared to NC (p = 0.0007). The Nrf2-ARE results were mirrored in the downregulation of Keap1, where Keap1 expression in HI-CON and HI-STIM were both significantly lower than NC (p = 0.008) and decreased in LI-STIM compared to LI-CON (p = 0.015). In addition, stimulation increased NQO1 protein compared to contralateral control regardless of stimulation intensity (p = 0.019), and HO1 protein was significantly higher in high intensity compared to the Naïve control group (p = 0.002). Taken together, these data suggest a systemic redox signaling exerkine is activating Nrf2-ARE binding and is intensity gated, where Nrf2-ARE activation in contralateral control limbs were only seen in the HI group. Other research in exercise induced Nrf2 signaling support the general finding that Nrf2 is activated in peripheral tissues in response to exercise, however the specific exerkine responsible for the systemic signaling effects is not known. Future work should aim to delineate these redox sensitive systemic signaling mechanisms.

Porous SiO2 -coated ultrasmall selenium particles nanospheres attenuate cerulein-induce acute pancreatitis in mice by downregulating oxidative stress.

To investigate the potential therapeutic role of porous SiO2 -coated ultrasmall selenium particles nanospheres (Se@SiO2 nanospheres) pretreatment in acute pancreatitis (AP) and to investigate the related mechanism. C57BL/6 mice were randomized to the normal control (CON) group, the AP (induced by cerulein injection) (CAE) group, and AP pretreated with Se@SiO2 nanocomposites at 1 and 2mg/kg (CAE + 1 or 2 mg/kg Se@SiO2 ) groups, respectively. Serum levels of amylase and lipase, inflammatory cytokines (interleukin [IL]-6, IL-1β and tumor necrosis factor [TNF]-α), alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (Cr) were measured, and histopathology was performed to examine the tissue samples of the pancreas, lungs, kidneys and liver. Immunofluorescence assay of reactive oxygen species (ROS), myeloperoxidase (MPO) and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling were conducted, and levels of MPO, malondialdehyde, superoxide dismutase and glutathione were evaluated. Finally, Western blot analysis was used to evaluate protein expressions of Nrf2, HO-1, NQO1, TLR4, MyD88 and p-p65 in pancreatic tissue. Se@SiO2 nanospheres alleviated pathological damage to the pancreas, and reduced pancreatic enzymes and inflammatory cytokines. Injury to other organs such as the liver, lungs and kidneys was also alleviated, as indicated by decreased ALT, AST, BUN, and Cr levels as well as improved histopathology. Moreover, Se@SiO2 nanospheres reduced oxidative stress, and ultimately inhibited TLR4/ MyD88/p-p65 pathway and increased the protein expressions of NQO1, Nrf2, and HO-1. Se@SiO2 nanospheres may alleviate AP by relieving oxidative stress and targeting the TLR4/Myd88/p-p65 and NQO1/Nrf2/HO-1 pathways.

Asperuloside suppressing oxidative stress and inflammation in DSS-induced chronic colitis and RAW 264.7 macrophages via Nrf2/HO-1 and NF-κB pathways

BackgroundInflammatory bowel diseases (IBDs), which mainly include Crohn's disease (CD) and ulcerative colitis (UC), are chronic idiopathic inflammatory disease of the gastrointestinal tract for which effective pharmacological treatments are lacking or options are very limited. PurposeHere, we aim to investigate the therapeutic effects of an iridoid glycoside, asperuloside (ASP) on mice experimental chronic colitis induced by dextran sulfate sodium (DSS) and further explore underlying mechanisms in vitro and in vivo. MethodsLPS-treated RAW 264.7 cells showed inflammation and were assessed for various physiological, morphological and biochemical parameters in the absence or presence of ASP. Chronic colitis was induced by 2% DSS in mice, which were used as an animal model to explore the pharmacodynamics of ASP. We detected p65 and Nrf2 pathway proteins via Western blot and RT-PCR analysis, assessed the cytokines TNF-α and IL-6 via ELISA, tested p65 and Nrf2 nuclear translocation via fluorescence. In addition, the docking affinity of ASP and p65 or Nrf2 proteins in the MOE 2015 software. ResultsWe found that ASP attenuated weight loss, disease activity index (DAI) and colonic pathological damage in colitis mice and restored the expressions of inflammatory cytokines in the colon. In addition, ASP restored antioxidant capacity in DSS-induced chronic colitis mice and lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Furthermore, ASP suppressed oxidative stress through increasing Nrf2, HO-1 and NQO-1 proteins expressions, and down-regulated nuclear levels of p65 to inhibit DSS-induced colonic oxidative stress and inflammation. Validation of the molecular docking results also indicated that ASP interacts with Nrf2 or p65 proteins. In summary, ASP improved DSS-induced chronic colitis by alleviating inflammation and oxidative stress, activating Nrf2/HO-1 signaling and limiting NF-κB signaling pathway, which may be an effective candidate for the treatment of IBD.

Hepatic oxidative damage and Nrf2 pathway protein changes in rats following long-term manganese exposure.

This study investigated hepatic oxidative damage in rats following long-term manganese (Mn) exposure and clarified the underlying mechanisms. Forty-eight rats (SPF, male) were randomly assigned to receive low (10 mg/kg, n = 16) or high doses of Mn (50 mg/kg, n = 16) or sterilized distilled water (control group, n = 16). Rats were euthanized after 12 months, and liver Mn levels and histopathological changes were determined. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels and liver malondialdehyde (MDA), glutathione peroxidase (GSH-PX), nuclear factor E2-related factor-2 (Nrf2), heme oxygenase-1 (HO-1), and NAD(P)H quinine oxidoreductase-1 (NQO1) levels were also determined. The Mn concentration and relative liver weights were significantly higher in the high-dose Mn group than in the control and low-dose Mn exposure groups. Low-dose Mn exposure resulted in mild expansion of hepatic sinuses and intact nuclei, whereas high-dose exposure led to pathological alterations in hepatocytes. High-dose Mn treatment significantly increased AST, ALT, and MDA activities and decreased GSH-PX activity. Additionally, liver Nrf2, HO-1, and NQO1 protein expression were markedly reduced by Mn exposure. Under the study conditions, long-term low-dose Mn exposure resulted in slight pathological changes in liver structure, but high-dose Mn exposure affected both liver structure and function, which might be related to the inhibition of Nrf2 expression, suppression of the transcription of its underlying antioxidant genes, and down regulation of the corresponding proteins. Consequently, the antioxidant capacity in the rat liver was weakened.

Aucubin ameliorates the LPS‐induced inflammatory response in bovine endometrial epithelial cells by inhibiting NF‐κB and activating the Keap1/Nrf2 signalling pathway

Cows are susceptible to pathogenic bacterial infection after pregnancy, leading to inflammation of the endometrium. Aucubin (AU) has been proven to exhibit highly effective anti-inflammatory activity, but its ability to protect against endometritis in dairy cows remains unclear. Therefore, the goal of the present study was to evaluate the protective effect of AU on the LPS-induced inflammatory response of bovine endometrial epithelial cells (BEECs). After pre-treating BEECs with AU (10, 20 and 50μM) for 6hr, the cells were stimulated with LPS for 3hr. Subsequently, BEECs apoptosis was analysed by flow cytometry, the expression of pro-inflammatory cytokine mRNA was detected by qRT-PCR, and changes in NF-κB and Keap1/Nrf2 signalling were analysed by western blotting and immunofluorescence analyses. The results showed that AU can reduce TNF-α, IL-1β, IL-6, COX-2 and iNOS mRNA expression in BEECs and reduce cell apoptosis. Furthermore, AU significantly reduced the level of NF-κB p65 and IκB phosphorylation and inhibited the nuclear translocation of NF-κB p65. AU also activated the Keap1/Nrf2 pathway, promoting the nuclear transfer of Nrf2 and increasing Keap1, Nrf2, HO-1 and NQO1 mRNA and protein levels. Taken together, these results indicate that AU ameliorates the LPS-induced inflammatory response by inhibiting NF-κB and activating the Keap1/Nrf2 signalling pathway, which has a protective effect on BEECs.

Upregulation of p53 by tannic acid treatment suppresses the proliferation of human colorectal carcinoma.

The present study's objective is to clarify the molecular mechanisms of tannic acid effects on the viability of human colorectal carcinoma (CRC). Tannic acid is stable for up to 48 h and is localized in both cytoplasm and nucleus. It dose-dependently inhibited the viability of CRC cell lines; SW-620 and HT-29 with IC 50 values of 7.2 ± 0.8 and 37.6 ± 1.4 µmol L-1. Besides, metastatic, invasive, and colony formation properties of CRC cells were significantly inhibited following the tannic acid treatment (p < 0.001). Tannic acid has been found to modulate enzyme, protein, and gene expressions of NQO1 in different levels and the upregulation of protein/gene expressions of p53 (p < 0.001), which leads the cells to trigger apoptosis. In conclusion, the present in vitro study may supply a significant background for in vivo studies in which the molecular mechanisms of antioxidant and chemopreventive activities of tannic acid will completely clarify.

Chronic treatment with acetaminophen protects against liver aging by targeting inflammation and oxidative stress.

The liver exhibits a variety of functions that are well-preserved during aging. However, the cellular hallmarks of aging increase the risk of hepatic alterations and development of chronic liver diseases. Acetaminophen (APAP) is a first choice for relieving mild-to-moderate pain. Most of the knowledge about APAP-mediated hepatotoxicity arises from acute overdose studies due to massive oxidative stress and inflammation, but little is known about its effect in age-related liver inflammation after chronic exposure. Our results show that chronic treatment of wild-type mice on the B6D2JRcc/Hsd genetic background with APAP at an infratherapeutic dose reduces liver alterations during aging without affecting body weight. This intervention attenuates age-induced mild oxidative stress by increasing HO-1, MnSOD and NQO1 protein levels and reducing ERK1/2 and p38 MAPK phosphorylation. More importantly, APAP treatment counteracts the increase in Cd8+ and the reduction in Cd4+ T lymphocytes observed in the liver with age. This response was also found in peripheral blood mononuclear cells. In conclusion, chronic infratherapeutic APAP treatment protects mice from age-related liver alterations by attenuating oxidative stress and inflammation.

New insights into the role of the Nrf2 signaling pathway in green tea catechin applications

Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a transcriptional signaling pathway that plays a crucial role in numerous clinical complications. Pivotal roles of Nrf2 have been proved in cancer, autoimmune diseases, neurodegeneration, cardiovascular diseases, diabetes mellitus, renal injuries, respiratory conditions, gastrointestinal disturbances, and general disorders related to oxidative stress, inflammation, apoptosis, gelatinolysis, autophagy, and fibrogenesis processes. Green tea catechins as a rich source of phenolic compounds can deal with various clinical problems and manifestations. In this review, we attempted to focus on intervention between green tea catechins and Nrf2. Green tea catechins especially epigallocatechin gallate (EGCG) elucidated the protective role of Nrf2 and its downstream molecules in various disorders through Keap-1, HO-1, NQO-1, GPx, GCLc, GCLm, NF-kB cross-link, kinases, and apoptotic proteins. Subsequently, we compiled an updated expansions of the Nrf2 role as a gate to manage and protect different disorders and feasible indications of green tea catechins through this signaling pathway. The present review highlighted recent evidence-based data in silico, in vitro, and in vivo studies on an outline for future clinical trials.

The anti-melanogenic effects of ellagic acid through induction of autophagy in melanocytes and suppression of UVA-activated α-MSH pathways via Nrf2 activation in keratinocytes

Ellagic acid (EA) is a natural phenol antioxidant in different fruits, vegetables, and nuts. As a copper iron chelator from the tyrosinase enzyme's active site, EA was reported to inhibit melanogenesis in melanocytes. Here, we demonstrated the anti-melanogenic mechanisms of EA through autophagy induction in melanoma B16F10 cells and the role of Nrf2 and UVA (3 J/cm2)-activated α-melanocyte stimulating hormone (α-MSH) pathways in keratinocyte HaCaT cells. In vitro data showed that EA suppressed the tyrosinase activity and melanogenesis by suppressing cAMP-mediated CREB and MITF signaling mechanisms in α-MSH-stimulated B16F10 cells. ERK, JNK, and AKT pathways were involved in this EA-regulated MITF downregulation. Notably, EA induced autophagy in B16F10 cells was evidenced from increased LC3-II accumulation, p62/SQSTM1 activation, ATG4B downregulation, acidic vesicular organelle (AVO) formation, PI3K/AKT/mTOR inhibition, and Beclin-1/Bcl-2 dysregulation. Interestingly, 3-MA (an autophagy inhibitor) pretreatment or LC3 silencing (siRNA transfection) of B16F10 cells significantly reduced EA-induced anti-melanogenic activity. Besides this, in UVA-irradiated keratinocyte HaCaT cells, EA suppressed ROS production and α-MSH generation. Moreover, EA mediated the activation and nuclear translocation of Nrf2, leading to antioxidant γ-GCLC, HO-1, and NQO-1 protein expression in HaCaT cells. However, Nrf2 knockdown has significantly impaired this effect, and there was an uncontrolled ROS generation following UVA irradiation. JNK, PKC, and ROS pathways were involved in the activation of Nrf2 in HaCaT cells. In vivo experiments using the zebrafish model confirmed that EA inhibited tyrosinase activity and endogenous pigmentation. In conclusion, ellagic acid is an effective skin-whitening agent and might be used as a topical applicant.

Mechanism of circadian regulation of the NRF2/ARE pathway in renal ischemia-reperfusion.

The nuclear erythroid 2-related factor 2 (NRF2)/antioxidant response element (ARE) pathway has been shown to provide strong protection against oxidative stress injury induced by renal ischemia-reperfusion (IR). However, the endogenous regulatory mechanism of the NRF2/ARE pathway in renal IR injury is incompletely understood. A rat model of renal IR was established by occlusion of the bilateral renal pedicle for 45 min, followed by reperfusion for 24 h. Renal injury was assessed by light microscopy and levels of serum creatinine, blood urea nitrogen and neutrophil gelatinase-associated lipocalin was measured using enzyme-linked immunosorbent assay. Renal oxidative stress was also evaluated by measuring superoxide dismutase and malondialdehyde in renal tissues. Protein expression levels of brain and muscle ARNT-like 1 (BMAL1), nuclear factor erythroid 2-related factor 2 (NRF2), NAD(P)H dehydrogenase [quinone] 1 (NQO1), glutamate-cysteine ligase modifier (GCLM) and heme oxygenase 1 (HO1) in the kidney were determined by western blotting and immunohistochemistry. Reverse transcription-quantitative PCR was used to evaluate rhythmic transcription of the core clock genes (CLOCK and BMAL1) and the NRF2 gene. The nature of the binding of BMAL1 to the promoter regions in the NRF2 gene was assessed by chromatin immunoprecipitation assays in rat kidneys. BMAL1 was found to bind to the promoter of the NRF2 gene through an E-BOX element associated with strongly rhythmic activation of NRF2 in both the normal kidney and those exposed to IR. The ARE-regulated anti-oxidative stress protein was affected by the circadian rhythm of the NRF2 gene. As the NRF2 level was at a circadian nadir, the expression of the proteins NQO1, GCLM and HO1 was weakened, resulting in more serious renal oxidative stress injury and pathological and functional impairment induced by IR. It can be concluded that the circadian rhythm of the NRF2/ARE pathway controlled by the circadian clock is essential for regulating antioxidant stress in renal IR injury, which might prompt new therapeutic strategies associated with the diurnal variability of human kidney disease, including renal transplantation.

Hydrogen-Rich Water Ameliorates Murine Chronic Graft-versus-Host Disease through Antioxidation.

Background Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an important treatment option for various hematopoietic diseases and certain hereditary diseases. Chronic graft-versus-host disease (cGVHD) has become the main life-threatening complication and cause of death in later stage postallo-HSCT. Current treatment options for cGVHD are limited. Hydrogen gas (H2) has been demonstrated that has antioxidative, anti-inflammatory, and antifibrosis effects. The aim of this study was to confirm whether oral administration hydrogen-rich water exerted therapeutic effects on a scleroderma cGVHD mouse model and tried to explain the mechanism underly it. Methods A mouse cGVHD model was established by haploidentical bone marrow transplantation. To evaluate therapeutic effects of H2 on cGVHD, survival rate, changes in clinical scores, and skin pathologic characteristics of cGVHD mice were observed. To evaluate its therapeutic mechanism, we detected the expression levels of antioxidative enzymes heme oxygenase-1(HO-1) and NAD (P)H: quinone acceptor oxidoreductase 1(NQO1) in skin homogenates. We also detected the expression level of the apoptotic protein caspase-3 in skin homogenates. Results 1-month survival rate of cGVHD mice in the hydrogen group reached 93.3%, significantly higher than 66.7% in the nonhydrogen group (p < 0.05). Clinical score of cGVHD mice was improved by hydrogen-rich water at 96 days posttransplantation (2.2 versus 4.5, p < 0.05). The skin pathological condition of cGVHD mice was significantly improved by hydrogen-rich water. At 96 days posttransplantation, average skin pathological hematoxylin and eosin (HE) staining score in the hydrogen group was 1.05, which was significantly lower than 3.2 in the nonhydrogen group (p < 0.01). Average Masson staining score was 0.6 point in the hydrogen group, lower than 0.9 point in the nonhydrogen group (p < 0.05). Both the relative expression levels of HO-1 and NQO1 proteins in skin specimens of cGVHD mice in the hydrogen group were lower than that in the nonhydrogen group (2.47 versus 6.21 and 1.83 versus 3.59, p < 0.05). The relative expression level of caspase-3 protein in skin specimens of cGVHD mice increased to 7.17 on the 96th day after transplantation, significantly higher than 4.36 in the hydrogen group. Conclusion In this study, we found that oral hydrogen-rich water improved the survival rate and clinical symptoms of cGVHD mice by antioxidant and antiapoptosis. This study would pave the way for further clinical study, which may provide a new treatment option for cGVHD.

Puerarin induces apoptosis in prostate cancer cells via inactivation of the Keap1/Nrf2/ARE signaling pathway

ABSTRACT In this study, we examined the antitumor effects of Puerarin (PEU) on androgen-independent (DU145 and PC-3) and androgen-dependent (LNCaP) prostate cancer cells, and explored its potential mechanisms. Supplement with PEU (2.5 μM, 5 μM, and 10 μM) exhibited a marked inhibitory effect against the growth of DU145 and PC-3 cells, especially beyond 24 h, whereas there is only slight growth inhibitory effect on LNCaP cells at the high concentration of 10 μM at 72 h. This loss of cell viability in DU145 and PC-3 cells by PEU was mediated by the induction of apoptosis via up-regulation of Bax and cleaved-caspase-3, but downregulation of Bcl-2. Moreover, more intracellular ROS and LDH production were observed in DU145 and PC-3 cells upon PEU treatment. Meanwhile, the amount of pro-inflammatory cytokines (IL-1β and IL-6) was increased, but the content of anti-inflammatory cytokines IL-10 was attenuated. Additionally, PEU pretreatment resulted in an increase of Keap1 protein expression, and a decline of Nrf2, HO-1 and NQO1 protein expression in DU145 and PC3 cells. The present findings indicated that PEU exerted its antitumor activities toward androgen-independent prostate cancer cells via inactivation of Keap1/NrF2/ARE signaling pathway.

Design, synthesis and biological activity of 1,4-quinone moiety attached to betulin derivatives as potent DT-diaphorase substrate

In this research, betulin derivatives were bonded to the 1,4-quinone fragment by triazole linker. Furthermore, the enzymatic assay used has shown that these compounds are a good DT-diaphorase (NQO1) substrates as evidenced by increasing enzymatic conversion rates relative to that of streptonigrin. The anticancer activities of the hybrids were tested against a panel of human cell lines, like: melanoma, ovarian, breast, colon, and lung cancers. The structure–activity relationship showed that the activity depends on the type of 1,4-quinone moiety and the tumor cell lines used. It was also found that the anticancer effects were increasing against the cell line with higher NQO1 protein level, like: breast (T47D, MCF-7), colon (Caco-2), and lung (A549) cancers. The transcriptional activity of the gene encoding a proliferation marker (H3 histone), cell cycle regulators (p53 and p21) and apoptosis pathway (BCL-2 and BAX) for selected compounds were determined. The molecular docking study was carried out to examine the interaction between the hybrids and NQO1 enzyme. The computational simulation showed that the type of the 1,4-quinone moiety influences location of the compound in the active site of the enzyme. It is worth noting that the study of new hybrids of betulin as substrate for NQO1 protein may lead to new medical therapeutic applications in the future.

Naturally-Occurring Rare Mutations Cause Mild to Catastrophic Effects in the Multifunctional and Cancer-Associated NQO1 Protein.

The functional and pathological implications of the enormous genetic diversity of the human genome are mostly unknown, primarily due to our unability to predict pathogenicity in a high-throughput manner. In this work, we characterized the phenotypic consequences of eight naturally-occurring missense variants on the multifunctional and disease-associated NQO1 protein using biophysical and structural analyses on several protein traits. Mutations found in both exome-sequencing initiatives and in cancer cell lines cause mild to catastrophic effects on NQO1 stability and function. Importantly, some mutations perturb functional features located structurally far from the mutated site. These effects are well rationalized by considering the nature of the mutation, its location in protein structure and the local stability of its environment. Using a set of 22 experimentally characterized mutations in NQO1, we generated experimental scores for pathogenicity that correlate reasonably well with bioinformatic scores derived from a set of commonly used algorithms, although the latter fail to semiquantitatively predict the phenotypic alterations caused by a significant fraction of mutations individually. These results provide insight into the propagation of mutational effects on multifunctional proteins, the implementation of in silico approaches for establishing genotype-phenotype correlations and the molecular determinants underlying loss-of-function in genetic diseases.

Indole-3-carbinol regulates microglia homeostasis and protects the retina from degeneration

BackgroundRetinal degenerative diseases significantly contribute to visual impairment and blindness. Microglia reactivity is a hallmark of neurodegenerative diseases including retinal cell death and immunomodulation emerges as a therapeutic option. Indole-3-carbinol (I3C) is a natural ligand of aryl hydrocarbon receptor (AhR), with potent immunomodulatory properties. Here, we hypothesized that I3C may inhibit microglia reactivity and exert neuroprotective effects in the light-damaged murine retina mimicking important immunological aspects of retinal degeneration.MethodsBV-2 microglia were treated in vitro with I3C followed by lipopolysaccharide (LPS) stimulation to analyze pro-inflammatory and anti-oxidant responses by quantitative real-time PCR (qRT-PCR) and Western blots. Nitric oxide (NO) secretion, caspase 3/7 levels, phagocytosis rates, migration, and morphology were analyzed in control and AhR knockdown cells. I3C or vehicle was systemically applied to light-treated BALB/cJ mice as an experimental model of retinal degeneration. Pro-inflammatory and anti-oxidant responses in the retina were examined by qRT-PCR, ELISA, and Western blots. Immunohistochemical staining of retinal flat mounts and cryosections were performed. The retinal thickness and structure were evaluated by in vivo imaging using spectral domain-optical coherence tomography (SD-OCT).ResultsThe in vitro data showed that I3C potently diminished LPS-induced pro-inflammatory gene expression of I-NOS, IL-1ß, NLRP3, IL-6, and CCL2 and induced anti-oxidants gene levels of NQO1, HMOX1, and CAT1 in BV-2 cells. I3C also reduced LPS-induced NO secretion, phagocytosis, and migration as important functional microglia parameters. siRNA-mediated knockdown of AhR partially prevented the previously observed gene regulatory events. The in vivo experiments revealed that I3C treatment diminished light-damage induced I-NOS, IL-1ß, NLRP3, IL-6, and CCL2 transcripts and also reduced CCL2, I-NOS, IL-1ß, p-NFkBp65 protein levels in mice. Moreover, I3C increased anti-oxidant NQO1 and HMOX1 protein levels in light-exposed retinas. Finally, I3C therapy prevented the accumulation of amoeboid microglia in the subretinal space and protected from retinal degeneration.ConclusionsThe AhR ligand I3C potently counter-acts microgliosis and light-induced retinal damage, highlighting a potential treatment concept for retinal degeneration.

Resveratrol Regulates Nrf2-Mediated Expression of Antioxidant and Xenobiotic Metabolizing Enzymes in Pesticides-Induced Parkinsonism.

Combined maneb (MB) and paraquat (PQ), two widely used pesticides, increases oxidative stress leading to Parkinsonism. Xenobiotic metabolizing enzymes, cytochrome P450 (CYP) 2D6 and its mouse ortholog Cyp2d22 protect against Parkinsonism. Resveratrol, an antioxidant, restores antioxidant defense system through the activation of nuclear factor erythroid 2- related factor 2 (Nrf2). However, a crosstalk between Cyp2d22/CYP2D6-mediated protection and resveratrol-induced Nrf2 activation leading to neuroprotection is not yet elucidated. The study aimed to decipher the effect of resveratrol on Nrf2 activation and expression of its downstream mediators, nicotinamide adenine dinucleotide phosphate quinone oxidoreductase 1 (NQO1) and thioredoxin 1 (Trx1) along with Cyp2d22/CYP2D6 activity in combined MB and PQ mouse model of Parkinsonism and differentiated neuroblastoma cells. MB and PQ reduced the dopamine content (mouse) and Cyp2d22/CYP2D6 activity (mouse/neuroblastoma cells) and increased the nuclear translocation of Nrf2 and expression of NQO1 and Trx1 (both). Resveratrol ameliorated pesticides-induced changes in dopamine content and Cyp2d22/CYP2D6 activity. It was found to promote nuclear translocation of Nrf2 and expression of NQO1 and Trx1 proteins. Since Cyp2d22/CYP2D6 inhibitor (ketoconazole/quinidine) per se reduced Cyp2d22/CYP2D6 activity and dopamine content, it was found to substantially increase the pesticides-induced reduction in Cyp2d22/CYP2D6 activity and dopamine content. Inhibitors normalized the pesticides induced changes in Nrf2 translocation and NQO1 and Trx1 levels in pesticides treated groups. The results suggest that resveratrol promotes the catalytic activity of xenobiotic metabolizing enzyme, Cyp2d22/CYP2D6, which partially contributes to Nrf2 activation in pesticides- induced Parkinsonism.

Inducible expression of heat shock protein 20 protects airway epithelial cells against oxidative injury involving the Nrf2-NQO-1 pathway

BackgroundHeat shock protein (HSP) 20 is a molecular chaperone that exerts multiple protective functions in various kinds of tissues. However, the expression of HSP20 and its specific functions in airway epithelial cells (AECs) remain elusive.ResultsIn current study, we first confirmed the inducible expression of HSP20 in mouse AECs and in a human bronchial epithelial cell line BEAS-2B cells, under different oxidant stressors. Then by establishing a HSP20-abundant mouse model with repeated low-level-ozone exposures and stimulating this model with a single high-level ozone exposure, we found that the HSP20 abundance along with its enhanced phosphorylation potentially contributed to the alleviation of oxidative injuries, evidenced by the decreases in the bodyweight reduction, the BAL neutrophil accumulation, the AECs shedding, and the BAL concentrations of albumin and E-cadherin. The biological function of HSP20 and its molecular mechanisms were further investigated in BEAS-2B cells that were transfected with HSP20-, unphosphorylatable HSP20(Ala) or empty vector plasmids prior to the stimulation of H2O2, of which its oxidant capacity has been proved to be similar with those of ozone in an air–liquid culture system. We found that the H2O2-induced intracellular ROS level and the early cell apoptosis were attenuated in the HSP20- but not HSP20(Ala)- transfected cells. The intracellular expression of NQO-1 (mRNA and protein) and the intranuclear content of Nrf2 were significantly increased in the HSP20- transfected cells but not in the HSP20(Ala)- and empty vector-transfected cells after the stimulation of H2O2.ConclusionsThe inducible expression of HSP20 in AECs by oxidative stress exerts protective roles against oxidative damages, which may involve the activation of the Nrf2-NQO-1 pathway.