NAD(P)H:Quinone Oxidoreductase-1 Expression Sensitizes Malignant Melanoma Cells to the HSP90 Inhibitor 17-AAG.
ABSTRACT: The KEAP1-NRF2 pathway regulates cellular redox homeostasis by transcriptional induction of genes associated with antioxidant synthesis and detoxification in response to oxidative stress. Previously, we reported that KEAP1 mutation elicits constitutive NRF2 activation and resistance to cisplatin (CDDP) and dacarbazine (DTIC) in human melanomas. The present study was conducted to clarify whether an HSP90 inhibitor, 17-AAG, efficiently eliminates melanoma with KEAP1 mutation, as the NRF2 target gene, NQO1, is a key enzyme in 17-AAG bioactivation. In melanoma and non-small cell lung carcinoma cell lines with or without KEAP1 mutations, NQO1 expression and 17-AAG sensitivity are inversely correlated. NQO1 is highly expressed in normal melanocytes and in several melanoma cell lines despite the presence of wild-type KEAP1, and the NQO1 expression is dependent on NRF2 activation. Because either CDDP or DTIC produces reactive oxygen species that activate NRF2, we determined whether these agents would sensitize NQO1-low melanoma cells to 17-AAG. Synergistic cytotoxicity of the 17-AAG and CDDP combination was detected in four out of five NQO1-low cell lines, but not in the cell line with KEAP1 mutation. These data indicate that 17-AAG could be a potential chemotherapeutic agent for melanoma with KEAP1 mutation or NQO1 expression.
Project description:Heat shock protein 90 (HSP90) inhibitors, such as 17-allylamino-17-demethoxygeldanamycin (17-AAG, tanespimycin), which is currently in phase II/phase III clinical trials, are promising new anticancer agents. Here, we explored acquired resistance to HSP90 inhibitors in glioblastoma (GB), a primary brain tumor with poor prognosis. GB cells were exposed continuously to increased 17-AAG concentrations. Four 17-AAG-resistant GB cell lines were generated. High-resistance levels with resistance indices (RI = resistant line IC(50)/parental line IC(50)) of 20 to 137 were obtained rapidly (2-8 weeks). After cessation of 17-AAG exposure, RI decreased and then stabilized. Cross-resistance was found with other ansamycin benzoquinones but not with the structurally unrelated HSP90 inhibitors, radicicol, the purine BIIB021, and the resorcinylic pyrazole/isoxazole amide compounds VER-49009, VER-50589, and NVP-AUY922. An inverse correlation between NAD(P)H/quinone oxidoreductase 1 (NQO1) expression/activity and 17-AAG IC(50) was observed in the resistant lines. The NQO1 inhibitor ES936 abrogated the differential effects of 17-AAG sensitivity between the parental and resistant lines. NQO1 mRNA levels and NQO1 DNA polymorphism analysis indicated different underlying mechanisms: reduced expression and selection of the inactive NQO1*2 polymorphism. Decreased NQO1 expression was also observed in a melanoma line with acquired resistance to 17-AAG. No resistance was generated with VER-50589 and NVP-AUY922. In conclusion, low NQO1 activity is a likely mechanism of acquired resistance to 17-AAG in GB, melanoma, and, possibly, other tumor types. Such resistance can be overcome with novel HSP90 inhibitors.
Project description:Alterations in the NRF2/KEAP1 pathway result in the constitutive activation of NRF2, leading to the aberrant induction of antioxidant and detoxification enzymes, including NQO1. The NQO1 bioactivatable agent ?-lapachone can target cells with high NQO1 expression but relies in the generation of reactive oxygen species (ROS), which are actively scavenged in cells with NRF2/KEAP1 mutations. However, whether NRF2/KEAP1 mutations influence the response to ?-lapachone treatment remains unknown. To address this question, we assessed the cytotoxicity of ?-lapachone in a panel of NSCLC cell lines bearing either wild-type or mutant KEAP1. We found that, despite overexpression of NQO1, KEAP1 mutant cells were resistant to ?-lapachone due to enhanced detoxification of ROS, which prevented DNA damage and cell death. To evaluate whether specific inhibition of the NRF2-regulated antioxidant enzymes could abrogate resistance to ?-lapachone, we systematically inhibited the four major antioxidant cellular systems using genetic and/or pharmacologic approaches. We demonstrated that inhibition of the thioredoxin-dependent system or copper-zinc superoxide dismutase (SOD1) could abrogate NRF2-mediated resistance to ?-lapachone, while depletion of catalase or glutathione was ineffective. Interestingly, inhibition of SOD1 selectively sensitized KEAP1 mutant cells to ?-lapachone exposure. Our results suggest that NRF2/KEAP1 mutational status might serve as a predictive biomarker for response to NQO1-bioactivatable quinones in patients. Further, our results suggest SOD1 inhibition may have potential utility in combination with other ROS inducers in patients with KEAP1/NRF2 mutations.
Project description:BACKGROUND: Oesophageal squamous cell carcinoma (OSCC) is a major health burden in Sub-Saharan Africa, and novel chemotherapies are urgently required to combat this disease. The heat shock protein 90 (HSP90) inhibitor 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) has previously been proposed as a possible candidate drug. NADPH quinone oxidoreductase 1 (NQO1) is known to increase the potency of 17-AAG, therefore we investigated the effects of 17-AAG in OSCC cell lines in the context of their NQO1 status. METHODS: We used MTT assays to compare the sensitivity of a panel of OSCC cell lines to 17-AAG. Western blotting, and RT-PCR were used to investigate NQO1 protein and mRNA levels, while an RFLP approach was used to investigate the NQO1 C609T SNP. RESULTS: Expression of NQO1 markedly increased sensitivity to 17-AAG in the OSCC cell lines, while normal fibroblasts, which expressed HSP90 at much lower levels, were more resistant to 17-AAG. In isolation, neither the C609T SNP, nor NQO1 mRNA levels was an accurate predictor of NQO1 protein levels. CONCLUSIONS: Since NQO1 greatly enhances the anti-cancer effects of 17-AAG, this could be used as a selective marker for patients that would benefit most from 17-AAG chemotherapy at low doses. Testing for the presence of the C609T SNP in both alleles could be used as a screen to exclude potentially poor responders to 17-AAG treatment at low dosages.
Project description:Diallyl sulfide, diallyl disulfide, and daillyl trisulfide (DATS) are major volatile components of garlic oil. In this study, we assessed their relative potency in inducing antioxidant enzyme expression. Among the three organosulfur compounds, DATS was found to be most potent in inducing heme oxygenase-1 (HO-1) andquinone oxidoreductase-1 (NQO1) in human gastric epithelial (AGS) cells. Furthermore, DATS administration by gavage increased the expression of HO-1 and NQO1 in C57BL/6 mouse stomach. Treatment with DATS increased the accumulation of nuclear factor-erythroid-2-related factor-2 (Nrf2) in the nucleus of cultured AGS cells and in mouse stomach in vivo. The DATS-induced expression of HO-1 and NQO1 was abrogated in the cells transiently transfected with Nrf2-siRNA or in the embryonic fibroblasts from Nrf2-null mice, indicating that Nrf2 is a key mediator of the cytoprotective effects of DATS. Pretreatment of AGS cells with N-acetylcysteine or dithiothreitol attenuated DATS-induced nuclear localization of Nrf2 and the expression of HO-1 and NQO1. Cysteine-151, -273 and -288 of Kelch-like ECH-associated protein-1 (Keap1), a cytosolic repressor of Nrf2, have been considered to act as a redox sensor and play a role in Nrf2 activation. To determine whether DATS could inactivate Keap1 through thiol modification, we established cell lines constitutively expressing wild type-Keap1 or three different mutant constructs in which cysteine-151, -273, or -288 of Keap1 was replaced with serine by retroviral gene transfer. DATS failed to activate Nrf2, and to induce expression of HO-1 and NQO1 only in Keap1-C288S mutant cells. LC-ESI-MS/MS analysis of recombinant Keap1 treated with DATS revealed that the peptide fragment containing Cys288 gained a molecular mass of 72.1 Da equivalent to the molecular weight of mono-allyl mono-sulfide. Taken together, these findings suggest that DATS may directly interact with the Cys288 residue of Keap1, which partly accounts for its ability to induce Nrf2 activation and upregulate defensive gene expression.
Project description:MIND4-17 is a recently developed NF-E2-related factor 2 (Nrf2) activator, which uniquely causes Nrf2 disassociation from Keap1. Here, we showed that pretreatment with MIND4-17 significantly inhibited hydrogen peroxide (H2O2)-induced viability reduction of primary osteoblasts and OB-6 osteoblastic cells. Meanwhile, MIND4-17 inhibited both apoptotic and non-apoptotic osteoblast cell death by H2O2. MIND4-17 treatment induced Keap1-Nrf2 disassociation, causing Nrf2 stabilization, accumulation and nuclear translocation in osteoblasts, leading to transcription of several Nrf2-dependent genes, including heme oxygenase 1 (HO-1), NAD(P)H quinone oxidoreductase 1 (NQO1), ?-glutamylcysteine synthetase modifier subunit (GCLM) and catalytic subunit (GCLC). Additionally, MIND4-17 largely attenuated H2O2-reactive oxygen species (ROS) production, lipid peroxidation and DNA damages. Nrf2 knockdown by targeted short hairpin RNA (shRNA) exacerbated H2O2-induced cytotoxicity in OB-6 osteoblastic cells, and nullified MIND4-17-mediated cytoprotection against H2O2. Meanwhile, Keap1 shRNA took over MIND4-17's actions and protected OB-6 cells from H2O2. Together, MIND4-17 activates Nrf2 signaling and protects osteoblasts from H2O2.
Project description:At present there are no studies investigating the effects of the kelch?like ECH?associated protein 1 (KEAP1)?nuclear factor erythroid 2?related factor 2 (NRF2)?antioxidant response element (ARE) signaling pathway on Hep2 cell line. The present study aimed to investigate this topic through knockdown of the KEAP1 gene. A stable Hep2 cell line specifically silencing the human KEAP1 gene was initially constructed. Hydrogen peroxide (H2O2) was added to the culture medium at various concentrations for various durations to interact with the short hairpin (sh)KEAP1?transfected Hep2 cells. Subsequently, the gene and protein expression levels of KEAP1, NRF2, NAD(P)H quinone oxidoreductase1 (NQO1) and heme oxygenase 1 (HO1) in experimental and control cells were measured by reverse transcription?quantitative polymerase chain reaction and western blotting, respectively. Furthermore, the viability and apoptotic rate of the shKEAP1?transfected Hep2 cells were detected by a Cell Counting?Kit 8 assay and flow cytometry, respectively. In the shKEAP1 Hep2 cell line, the mRNA and protein expression levels of NRF2, NQO1 and HO1 were markedly higher compared with the scramble control?transfected Hep2 and parent Hep2 cell lines. Immunofluorescence staining indicated that NRF2 was primarily located in the cytoplasm of scHep2 and parent Hep2 cell lines, but was present in the nuclei and cytoplasm of the shKEAP1 Hep2 cell line, where it translocates into the nuclei in response to H2O2. Following knockdown of the KEAP1 gene Hep2 cells, the apoptosis rates were 31.8 and 45.3% in scHep2 cells at 0.1 and 0.25 mmol/l H2O2 respectively and 14.1 and 27.9% in shKEAP1 cells. The present study indicated that the KEAP1?NRF2?ARE signaling pathway may exhibit an antioxidative effect within Hep2 cells and may be used for clinical treatment of cancer.
Project description:Oxidative stress is a major risk factor for acute pancreatitis. Reactive oxygen species (ROS) mediate expression of inflammatory cytokines such as interleukin-6 (IL-6) which reflects the severity of acute pancreatitis. The nuclear factor erythroid-2-related factor 2 (Nrf2) pathway is activated to induce the expression of antioxidant enzymes such as NAD(P)H: quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) as a cytoprotective response to oxidative stress. In addition, binding of Kelch-like ECH-associated protein 1 (Keap1) to Nrf2 promotes degradation of Nrf2. Docosahexaenoic acid (DHA)-an omega-3 fatty acid-exerts anti-inflammatory and antioxidant effects. Oxidized omega-3 fatty acids react with Keap1 to induce Nrf2-regulated gene expression. In this study, we investigated whether DHA reduces ROS levels and inhibits IL-6 expression via Nrf2 signaling in pancreatic acinar (AR42J) cells stimulated with cerulein, as an in vitro model of acute pancreatitis. The cells were pretreated with or without DHA for 1 h and treated with cerulein (10-8 M) for 1 (ROS levels, protein levels of NQO1, HO-1, pNrf2, Nrf2, and Keap1), 6 (IL-6 mRNA expression), and 24 h (IL-6 protein level in the medium). Our results showed that DHA upregulates the expression of NQO1 and HO-1 in cerulein-stimulated AR42J cells by promoting phosphorylation and nuclear translocation of Nrf2. DHA increased interaction between Keap1 and Nrf2 in AR42J cells, which may increase Nrf2 activity by inhibiting Keap1-mediated sequestration of Nrf2. In addition, DHA-induced expression of NQO1 and HO-1 is related to reduction of ROS and IL-6 levels in cerulein-stimulated AR42J cells. In conclusion, DHA inhibits ROS-mediated IL-6 expression by upregulating Nrf2-mediated expression of NQO1 and HO-1 in cerulein-stimulated pancreatic acinar cells. DHA may exert positive modulatory effects on acute pancreatitis by inhibiting oxidative stress and inflammatory cytokine production by activating Nrf2 signaling in pancreatic acinar cells.
Project description:The Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response elements pathway enables cells to survive oxidative stress conditions through regulating the expression of cytoprotective enzymes such asquinone oxidoreductase 1 (NQO1). This work presents the design and synthesis of novel anilinoquinazoline derivatives (2-16a) and evaluation of their NQO1 inducer activity in murine cells. Molecular docking of the new compounds was performed to assess their ability to inhibit Keap1-Nrf2 protein-protein interaction through occupying the Keap1-Nrf2-binding domain, which leads to Nrf2 accumulation and enhanced gene expression of NQO1. Docking results showed that all compounds can potentially interact with Keap1; however, 1,5-dimethyl-2-phenyl-4-(2-phenylquinazolin-4-ylamino)-1,2-dihydropyrazol-3-one (9), the most potent inducer, showed the largest number of interactions with key amino acids in the binding pocket (Arg483, Tyr525, and Phe478) compared to the native ligand or any other compound in this series.
Project description:Heat shock protein 90 (HSP90) is a molecular chaperone which stabilizes client proteins with important roles in tumor growth. 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of HSP90 ATPase activity, occupies the ATP binding site of HSP90 causing a conformational change which destabilizes client proteins and directs them towards proteosomal degradation. Malignant melanomas have active RAF-MEK-ERK signaling which can occur either through an activating mutation in BRAF (BRAFV600E) or through activation of signal transduction upstream of BRAF. Prior work showed that 17-AAG inhibits cell growth in BRAFV600E and BRAF wildtype (BRAFWT) melanomas, although there were conflicting reports about the dependence of BRAFV600E and BRAFWT upon HSP90 activity for stability. Here, we demonstrate that BRAFWT and CRAF are bound by HSP90 in BRAFWT, NRAS mutant melanoma cells. HSP90 inhibition by 17-AAG inhibits ERK signaling and cell growth by destabilizing CRAF but not BRAFWT in the majority of NRAS mutant melanoma cells. The highly-selective BRAFV600E inhibitor, PLX4032 (vemurafenib), inhibits ERK signaling and cell growth in mutant BRAF melanoma cells, but paradoxically enhances signaling in cells with wild-type BRAF. In our study, we examined whether 17-AAG could inhibit PLX4032-enhanced ERK signaling in BRAFWT melanoma cells. As expected, PLX4032 alone enhanced ERK signaling in the BRAFWT melanoma cell lines Mel-Juso, SK-Mel-2, and SK-Mel-30, and inhibited signaling and cell growth in BRAFV600E A375 cells. However, HSP90 inhibition by 17-AAG inhibited PLX4032-enhanced ERK signaling and inhibited cell growth by destabilizing CRAF. Surprisingly, 17-AAG also stimulated melanin production in SK-Mel-30 cells and enhanced TYRP1 and DCT expression without stimulating TYR production in all three BRAFWT cell lines studied as well as in B16F10 mouse melanoma cells. In vivo, the combination of 17-AAG and cellular immunotherapy directed against Tyrp1 enhanced the inhibition of tumor growth compared to either therapy alone. Our studies support a role for 17-AAG and HSP90 inhibition in enhancing cellular immunotherapy for melanoma.
Project description:BACKGROUND: Carbon monoxide (CO) is a cytoprotective and homeostatic molecule with important signaling capabilities in physiological and pathophysiological situations. CO protects cells/tissues from damage by free radicals or oxidative stress. NAD(P)H:quinone oxidoreductase (NQO1) is a highly inducible enzyme that is regulated by the Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway, which is central to efficient detoxification of reactive metabolites and reactive oxygen species (ROS). METHODS: We generated NQO1 promoter construct. HepG2 cells were treated with CO Releasing Molecules-2 (CORM-2) or CO gas and the gene expressions were measured by RT-PCR, immunoblot, and luciferase assays. RESULTS: CO induced expression of NQO1 in human hepatocarcinoma cell lines by activation of Nrf2. Exposure of HepG2 cells to CO resulted in significant induction of NQO1 in dose- and time-dependent manners. Analysis of the NQO1 promoter indicated that an antioxidant responsible element (ARE)-containing region was critical for the CO-induced Nrf2-dependent increase of NQO1 gene expression in HepG2 cells. CONCLUSION: Our results suggest that CO-induced Nrf2 increases the expression of NQO1 which is well known to detoxify reactive metabolites and ROS.