ABSTRACT
Approximately 50%-55% of high-grade serous ovarian carcinoma (HGSOC) patients have MYC oncogenic pathway activation. Because MYC is not directly targetable, we have analyzed molecular pathways enriched in MYC-high HGSOC tumors to identify potential therapeutic targets. Here, we report that MYC-high HGSOC tumors show enrichment in genes controlled by NRF2, an antioxidant signaling pathway, along with increased thioredoxin redox activity. Treatment of MYC-high HGSOC tumors cells with US Food and Drug Administration (FDA)-approved thioredoxin reductase 1 (TrxR1) inhibitor auranofin resulted in significant growth suppression and apoptosis in MYC-high HGSOC cells in vitro and also significantly reduced tumor growth in an MYC-high HGSOC patient-derived tumor xenograft. We found that auranofin treatment inhibited glycolysis in MYC-high cells via oxidation-induced GAPDH inhibition. Interestingly, in response to auranofin-induced glycolysis inhibition, MYC-high HGSOC cells switched to glutamine metabolism for survival. Depletion of glutamine with either glutamine starvation or glutaminase (GLS1) inhibitor CB-839 exerted synergistic anti-tumor activity with auranofin in HGSOC cells and OVCAR-8 cell line xenograft. These findings suggest that applying a combined therapy of GLS1 inhibitor and TrxR1 inhibitor could effectively treat MYC-high HGSOC patients.
Subject(s)
Auranofin , Genes, myc , Glutamine , Ovarian Neoplasms , Thioredoxin-Disulfide Reductase , Female , Humans , Auranofin/pharmacology , Auranofin/therapeutic use , Cell Line, Tumor , Genes, myc/genetics , Glutaminase/genetics , Glutaminase/metabolism , Glutamine/genetics , Glutamine/metabolism , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/genetics , Thioredoxin-Disulfide Reductase/genetics , Thioredoxin-Disulfide Reductase/metabolism , Thioredoxins/antagonists & inhibitors , Thioredoxins/genetics , Thioredoxins/metabolismABSTRACT
BACKGROUND: High-grade serous ovarian carcinomas (HGSCs) are a heterogeneous subtype of epithelial ovarian cancers and include serous cancers arising in the fallopian tube and peritoneum. These cancers are now subdivided into homologous recombination repair (HR)-deficient and proficient subgroups as this classification impacts on management and prognosis. PARP inhibitors (PARPi) have shown significant clinical efficacy, particularly as maintenance therapy following response to platinum-based chemotherapy in BRCA-mutant or homologous recombination (HR)-deficient HGSCs in both the 1st and 2nd line settings. However, PARPi have limited clinical benefit in HR-proficient HGSCs which make up almost 50% of HGSC and improving outcomes in these patients is now a high priority due to the poor prognosis with ineffectiveness of the current standard of care. There are a number of potential lines of investigation including efforts in sensitizing HR-proficient tumors to PARPi. Herein, we aimed to develop a novel combination therapy by targeting SSRP1 using a small molecule inhibitor CBL0137 with PARPi in HR-proficient HGSCs. EXPERIMENTAL DESIGN: We tested anti-cancer activity of CBL0137 monotherapy using a panel of HGSC cell lines and patient-derived tumor cells in vitro. RNA sequencing was used to map global transcriptomic changes in CBL0137-treated patient-derived HR-proficient HGSC cells. We tested efficacy of CBL0137 in combination with PARPi using HGSC cell lines and patient-derived tumor cells in vitro and in vivo. RESULTS: We show that SSRP1 inhibition using a small molecule, CBL0137, that traps SSRP1 onto chromatin, exerts a significant anti-growth activity in vitro against HGSC cell lines and patient-derived tumor cells, and also reduces tumor burden in vivo. CBL0137 induced DNA repair deficiency via inhibition of the HR repair pathway and sensitized SSRP1-high HR-proficient HGSC cell lines and patient-derived tumor cells/xenografts to the PARPi, Olaparib in vitro and in vivo. CBL0137 also enhanced the efficacy of DNA damaging platinum-based chemotherapy in HGSC patient-derived xenografts. CONCLUSION: Our findings strongly suggest that combination of CBL0137 and PARP inhibition represents a novel therapeutic strategy for HR-proficient HGSCs that express high levels of SSRP1 and should be investigated in the clinic.
Subject(s)
Ovarian Neoplasms , Poly(ADP-ribose) Polymerase Inhibitors , Female , Humans , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use , Recombinational DNA Repair , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , Carcinoma, Ovarian Epithelial/drug therapy , Cell Line, Tumor , DNA-Binding Proteins/genetics , High Mobility Group Proteins/metabolism , Transcriptional Elongation Factors/geneticsABSTRACT
[This corrects the article DOI: 10.18632/oncotarget.3795.].
ABSTRACT
Breast cancer (BC) is one of the most common cancers in women. TNBC (Triple-negative breast cancer) has limited treatment options and still lacks viable molecular targets, leading to poor outcomes. Recently, RNA-binding proteins (RBPs) have been shown to play crucial roles in human cancers, including BC, by modulating a number of oncogenic phenotypes. This suggests that RBPs represent potential molecular targets for BC therapy. Methods: We employed genomic data to identify RBPs specifically expressed in TNBC. NONO was silenced in TNBC cell lines to examine cell growth, colony formation, invasion, and migration. Gene expression profiles in NONO-silenced cells were generated and analyzed. A high-throughput screening for NONO-targeted drugs was performed using an FDA-approved library. Results: We found that the NONO RBP is highly expressed in TNBC and is associated with poor patient outcomes. NONO binds to STAT3 mRNA, increasing STAT3 mRNA levels in TNBC. Surprisingly, NONO directly interacts with STAT3 protein increasing its stability and transcriptional activity, thus contributing to its oncogenic function. Importantly, high-throughput drug screening revealed that auranofin is a potential NONO inhibitor and inhibits cell growth in TNBC. Conclusions: NONO is an RBP upstream regulator of both STAT3 RNA and protein levels and function. It represents an important and clinically relevant promoter of growth and resistance of TNBCs. NONO is also therefore a potential therapeutic target in TNBC.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , DNA-Binding Proteins/metabolism , Drug Resistance, Neoplasm/genetics , RNA-Binding Proteins/metabolism , STAT3 Transcription Factor/metabolism , Triple Negative Breast Neoplasms/genetics , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Apoptosis/drug effects , Apoptosis/genetics , Cell Line, Tumor , Cell Movement/drug effects , Cell Movement/genetics , Cell Proliferation/drug effects , Cell Proliferation/genetics , DNA-Binding Proteins/antagonists & inhibitors , Drug Carriers/chemistry , Drug Resistance, Neoplasm/drug effects , Drug Screening Assays, Antitumor , Female , Gene Expression Regulation, Neoplastic/drug effects , Genomics , Humans , Nanoparticles/chemistry , Precision Medicine/methods , RNA-Binding Proteins/antagonists & inhibitors , Signal Transduction/drug effects , Signal Transduction/genetics , Theranostic Nanomedicine/methods , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/pathologyABSTRACT
Purpose: Lacking effective targeted therapies, triple-negative breast cancer (TNBCs) is highly aggressive and metastatic disease, and remains clinically challenging breast cancer subtype to treat. Despite the survival dependency on the proteasome pathway genes, FDA-approved proteasome inhibitors induced minimal clinical response in breast cancer patients due to weak proteasome inhibition. Hence, developing effective targeted therapy using potent proteasome inhibitor is required. Methods: We evaluated anti-cancer activity of a potent proteasome inhibitor, marizomib, in vitro using breast cancer lines and in vivo using 4T1.2 murine syngeneic model, MDA-MB-231 xenografts, and patient-derived tumor xenografts. Global proteome profiling, western blots, and RT-qPCR were used to investigate the mechanism of action for marizomib. Effect of marizomib on lung and brain metastasis was evaluated using syngeneic 4T1BR4 murine TNBC model in vivo. Results: We show that marizomib inhibits multiple proteasome catalytic activities and induces a better anti-tumor response in TNBC cell lines and patient-derived xenografts alone and in combination with the standard-of-care chemotherapy. Mechanistically, we show that marizomib is a dual inhibitor of proteasome and oxidative phosphorylation (OXPHOS) in TNBCs. Marizomib reduces lung and brain metastases by reducing the number of circulating tumor cells and the expression of genes involved in the epithelial-to-mesenchymal transition. We demonstrate that marizomib-induced OXPHOS inhibition upregulates glycolysis to meet the energetic demands of TNBC cells and combined inhibition of glycolysis with marizomib leads to a synergistic anti-cancer activity. Conclusions: Our data provide a strong rationale for a clinical evaluation of marizomib in primary and metastatic TNBC patients.
Subject(s)
Antineoplastic Agents/therapeutic use , Lactones/therapeutic use , Proteasome Endopeptidase Complex/metabolism , Pyrroles/therapeutic use , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/metabolism , Animals , Apoptosis/drug effects , Apoptosis/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/genetics , Epithelial-Mesenchymal Transition/drug effects , Epithelial-Mesenchymal Transition/genetics , Female , Humans , Mice , Oxidative Phosphorylation/drug effects , Proteasome Endopeptidase Complex/drug effects , Proteasome Inhibitors/therapeutic use , Triple Negative Breast Neoplasms/genetics , Xenograft Model Antitumor AssaysABSTRACT
Chronic myeloid leukaemia (CML) is currently treated with inhibitors of the CML specific oncoprotein, bcr-abl. While this strategy is initially successful, drug resistance can become a problem. Therefore, new targets need to be identified to ensure the disease can be appropriately managed. The thioredoxin (Trx) system, comprised of Trx, thioredoxin reductase (TrxR), and NADPH, is an antioxidant system previously identified as a target for therapies aimed at overcoming drug resistance in other cancers. We assessed the effectiveness of TrxR inhibitors on drug resistant CML cells and examined links between TrxR and the bcr-abl cell-signalling pathway. Two TrxR inhibitors, auranofin and [Au(d2pype)2]Cl, increased intracellular ROS levels and elicited apoptosis in both sensitive and imatinib resistant CML cells. Inhibition of TrxR activity by these pharmacological inhibitors, or by specific siRNA, also resulted in decreased bcr-abl mRNA and protein levels, and lower bcr-abl downstream signalling activity, potentially enhancing the effectiveness of TrxR inhibitors as CML therapies. In addition, imatinib resistant CML cell lines showed upregulated expression of the Trx system. Furthermore, analysis of datasets showed that CML patients who did not respond to imatinib had higher Trx mRNA levels than patients who responded to treatment. Our study demonstrates a link between the Trx system and the bcr-abl protein and highlights the therapeutic potential of targeting the Trx system to improve CML patients' outcomes.
ABSTRACT
Triple-negative breast cancer (TNBCs) is a very aggressive and lethal form of breast cancer with no effective targeted therapy. Neoadjuvant chemotherapies and radiotherapy remains a mainstay of treatment with only 25-30% of TNBC patients responding. Thus, there is an unmet clinical need to develop novel therapeutic strategies for TNBCs. TNBC cells have increased intracellular oxidative stress and suppressed glutathione, a major antioxidant system, but still, are protected against higher oxidative stress. We screened a panel of antioxidant genes using the TCGA and METABRIC databases and found that expression of the thioredoxin pathway genes is significantly upregulated in TNBC patients compared to non-TNBC patients and is correlated with adverse survival outcomes. Treatment with auranofin (AF), an FDA-approved thioredoxin reductase inhibitor caused specific cell death and impaired the growth of TNBC cells grown as spheroids. Furthermore, AF treatment exerted a significant in vivo antitumor activity in multiple TNBC models including the syngeneic 4T1.2 model, MDA-MB-231 xenograft and patient-derived tumor xenograft by inhibiting thioredoxin redox activity. We, for the first time, showed that AF increased CD8+Ve T-cell tumor infiltration in vivo and upregulated immune checkpoint PD-L1 expression in an ERK1/2-MYC-dependent manner. Moreover, combination of AF with anti-PD-L1 antibody synergistically impaired the growth of 4T1.2 primary tumors. Our data provide a novel therapeutic strategy using AF in combination with anti-PD-L1 antibody that warrants further clinical investigation for TNBC patients.
Subject(s)
Antibodies/therapeutic use , Auranofin/therapeutic use , B7-H1 Antigen/immunology , Enzyme Inhibitors/therapeutic use , Thioredoxin-Disulfide Reductase/antagonists & inhibitors , Triple Negative Breast Neoplasms/drug therapy , Animals , Auranofin/pharmacology , Cell Death/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Enzyme Inhibitors/pharmacology , Female , Humans , Mice , Mice, Inbred BALB C , Reactive Oxygen Species/metabolism , Triple Negative Breast Neoplasms/metabolism , Triple Negative Breast Neoplasms/pathology , Xenograft Model Antitumor AssaysABSTRACT
Multiple myeloma (MM), the second most common haematological malignancy, is a clonal plasma B-cell neoplasm that forms within the bone marrow. Despite recent advancements in treatment, MM remains an incurable disease. Auranofin, a linear gold(I) phosphine compound, has previously been shown to exert a significant anti-myeloma activity by inhibiting thioredoxin reductase (TrxR) activity. A bis-chelated tetrahedral gold(I) phosphine complex [Au(d2pype)2]Cl (where d2pype is 1,2-bis(di-2-pyridylphosphino)ethane) was previously designed to improve the gold(I) compound selectivity towards selenol- and thiol-containing proteins, such as TrxR. In this study, we show that [Au(d2pype)2]Cl significantly inhibited TrxR activity in both bortezomib-sensitive and resistant myeloma cells, which led to a significant reduction in cell proliferation and induction of apoptosis, both of which were dependent on ROS. In clonogenic assays, treatment with [Au(d2pype)2]Cl completely abrogated the tumourigenic capacity of MM cells, whereas auranofin was less effective. We also show that [Au(d2pype)2]Cl exerted a significant anti-myeloma activity in vivo in human RPMI8226 xenograft model in immunocompromised NOD/SCID mice. The MYC oncogene, known to drive myeloma progression, was downregulated in both in vitro and in vivo models when treated with [Au(d2pype)2]Cl. This study highlights the "proof of concept" that improved gold(I)-based compounds could potentially be used to not only treat MM but as an alternative tool to understand the role of the Trx system in the pathogenesis of this blood disease.
Subject(s)
Gold/chemistry , Multiple Myeloma/drug therapy , Phosphines/administration & dosage , Thioredoxin-Disulfide Reductase/antagonists & inhibitors , Animals , Cell Proliferation/drug effects , Cell Survival/drug effects , Down-Regulation , Gene Expression Regulation, Neoplastic/drug effects , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Multiple Myeloma/enzymology , Multiple Myeloma/genetics , Phosphines/chemistry , Phosphines/pharmacology , Proto-Oncogene Proteins c-myc/genetics , Xenograft Model Antitumor AssaysABSTRACT
The DJ-1 protein was originally linked with Parkinson's disease and is now known to have antioxidant functions. The protein has three redox-sensitive cysteine residues, which are involved in its dimerisation and functional properties. A mildly oxidised form of DJ-1 is the most active form and protects cells from oxidative stress conditions. DJ-1 functions as an antioxidant through a variety of mechanisms, including a weak direct antioxidant activity by scavenging reactive oxygen species. DJ-1 also regulates a number of signalling pathways, including the inhibition of apoptosis signal-regulating kinase 1 (ASK1)-induced apoptosis under oxidative stress conditions. Other proteins regulated by DJ-1 include enzymes, chaperones, the 20S proteasome and transcription factors, including Nrf2. Once activated by oxidative stress, Nrf2 upregulates antioxidant gene expression including members of the thioredoxin and glutathione pathways, which in turn mediate an antioxidant protective function. Crosstalk between DJ-1 and both the thioredoxin and glutathione systems has also been identified. Thioredoxin reduces a cysteine residue on DJ-1 to modulate its activity, while glutaredoxin1 de-glutathionylates DJ-1, preventing degradation of DJ-1 and resulting in its accumulation. DJ-1 also regulates the activity of glutamate cysteine ligase, which is the rate-limiting step for glutathione synthesis. These antioxidant functions of DJ-1 are key to its role in protecting neurons from oxidative stress and are hypothesised to protect the brain from the development of neurodegenerative diseases such as Parkinson's disease (PD) and to protect cardiac tissues from ischaemic-reperfusion injury. However, DJ-1, as an antioxidant, also protects cancer cells from undergoing oxidative stress-induced apoptosis.
Subject(s)
Antioxidants/metabolism , Glutathione/metabolism , Protein Deglycase DJ-1/metabolism , Thioredoxins/metabolism , Animals , Humans , MAP Kinase Kinase Kinase 5/metabolism , Models, Biological , NF-E2-Related Factor 2/metabolism , Oxidation-Reduction , Reactive Oxygen Species/metabolism , Signal TransductionABSTRACT
Multiple myeloma (MM) is an incurable plasma B cell malignancy. Despite recent advancements in anti-MM therapies, development of drug resistance remains a major clinical hurdle. DJ-1, a Parkinson's disease-associated protein, is upregulated in many cancers and its knockdown suppresses tumor growth and overcomes chemoresistance. However, the role of DJ-1 in MM remains unknown. Using gene expression databases we found increased DJ-1 expression in MM patient cells, which correlated with shorter overall survival and poor prognosis in MM patients. Targeted DJ-1 knockdown using siRNAs induced necroptosis in myeloma cells. We found that Krüppel-like factor 6 (KLF6) is expressed at lower levels in myeloma cells compared to PBMCs, and DJ-1 knockdown increased KLF6 expression in myeloma cells. Targeted knockdown of KLF6 expression in DJ-1 knockdown myeloma cells rescued these cells from undergoing cell death. Higher DJ-1 levels were observed in bortezomib-resistant myeloma cells compared to parent cells, and siRNA-mediated DJ-1 knockdown reversed bortezomib resistance. DJ-1 knockdown increased KLF6 expression in bortezomib-resistant myeloma cells, and subsequent siRNA-mediated KLF6 knockdown rescued bortezomib-resistant myeloma cells from undergoing cell death. We also demonstrated that specific siRNA-mediated DJ-1 knockdown reduced myeloma cell growth under a hypoxic microenvironment. DJ-1 knockdown reduced the expression of HIF-1α and its target genes in hypoxic-myeloma cells, and overcame hypoxia-induced bortezomib resistance. Our findings demonstrate that elevated DJ-1 levels correlate with myeloma cell survival and acquisition of bortezomib resistance. Thus, we propose that inhibiting DJ-1 may be an effective therapeutic strategy to treat newly diagnosed as well as relapsed/refractory MM patients.
Subject(s)
Apoptosis , Kruppel-Like Factor 6/genetics , Multiple Myeloma/physiopathology , Antineoplastic Agents/pharmacology , Bortezomib/pharmacology , Cell Line, Tumor , Drug Resistance, Neoplasm/drug effects , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Humans , Kruppel-Like Factor 6/metabolism , Multiple Myeloma/genetics , Multiple Myeloma/metabolism , Protein Deglycase DJ-1/metabolism , Up-RegulationABSTRACT
As essential elements of the tumor microenvironment, the variable oxygenation state of the tumor tissue, the extracellular matrix (ECM) and different cell types are important determinants of carcinogenesis. These elements may also influence how tumor cells respond to therapeutic treatments. In the present study, we assessed the anti-cancer activity of auranofin and its effect on the thioredoxin (Trx) system under conditions that closely resemble the in vivo tumor microenvironment with respect to the oxygen levels and tissue architecture. We utilised an oxygen scheme involving growth of cancer cells under normoxia (20%) and hypoxia (0.1%). We also preconditioned cells with intermittent hypoxia (IH) prior to a prolonged hypoxic incubation. This oxygen scheme did not affect the cytotoxicity of auranofin; however, IH preconditioned cells were less sensitive towards the inhibition of thioredoxin reductase (TrxR) specific activity upon treatment with auranofin. IH preconditioning also upregulated Trx protein levels in auranofin treated cells. We also compared the activity of auranofin against cancer cells cultured in 2D monolayer and 3D spheroid-based culture models. Auranofin was less potent against cells grown under a more in vivo-like 3D environment. The results presented in this paper implicate the importance of the tumor oxygen environment and tissue architecture in influencing the response of cancer cells towards auranofin.
Subject(s)
Auranofin/pharmacology , Breast Neoplasms/drug therapy , Lung Neoplasms/drug therapy , Thioredoxins/biosynthesis , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Hypoxia/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Doxorubicin/pharmacology , Female , Humans , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Thioredoxin-Disulfide Reductase/metabolism , Thioredoxins/antagonists & inhibitorsABSTRACT
Multiple myeloma (MM) is a B-cell malignancy characterized by an accumulation of abnormal clonal plasma cells in the bone marrow. Introduction of the proteasome-inhibitor bortezomib has improved MM prognosis and survival; however hypoxia-induced or acquired bortezomib resistance remains a clinical problem. This study highlighted the role of thioredoxin reductase 1 (TrxR1) in the hypoxia-induced and acquired bortezomib resistance in MM. Higher TrxR1 gene expression correlated with high-risk disease, adverse overall survival, and poor prognosis in myeloma patients. We demonstrated that hypoxia induced bortezomib resistance in myeloma cells and increased TrxR1 protein levels. Inhibition of TrxR1 using auranofin overcame hypoxia-induced bortezomib resistance and restored the sensitivity of hypoxic-myeloma cells to bortezomib. Hypoxia increased NF-Ðºß subunit p65 nuclear protein levels and TrxR1 inhibition decreased hypoxia-induced NF-Ðºß p65 protein levels in the nucleus and reduced the expression of NF-кß-regulated genes. In addition, higher TrxR1 protein levels were observed in bortezomib-resistant myeloma cells compared to the naïve cells, and its inhibition using either auranofin or TrxR1-specific siRNAs reversed bortezomib resistance. TrxR1 inhibition reduced p65 mRNA and protein expression in bortezomib-resistant myeloma cells, and also decreased the expression of NF-кß-regulated anti-apoptotic and proliferative genes. Thus, TrxR1 inhibition overcomes both hypoxia-induced and acquired bortezomib resistance by inhibiting the NF-Ðºß signaling pathway. Our findings demonstrate that elevated TrxR1 levels correlate with the acquisition of bortezomib resistance in MM. We propose considering TrxR1-inhibiting drugs, such as auranofin, either for single agent or combination therapy to circumvent bortezomib-resistance and improve survival outcomes of MM patients.
Subject(s)
Drug Resistance, Neoplasm/genetics , Multiple Myeloma/drug therapy , Multiple Myeloma/genetics , Thioredoxin Reductase 1/genetics , Apoptosis/drug effects , Auranofin/administration & dosage , Bortezomib/administration & dosage , Cell Hypoxia/drug effects , Cell Hypoxia/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Humans , Multiple Myeloma/pathology , NF-kappa B/genetics , Proteasome Inhibitors/administration & dosage , Signal Transduction/drug effects , Thioredoxin Reductase 1/biosynthesisABSTRACT
Multiple myeloma (MM) is characterized by an accumulation of abnormal clonal plasma cells in the bone marrow. Despite recent advancements in anti-myeloma therapies, MM remains an incurable disease. Antioxidant molecules are upregulated in many cancers, correlating with tumor proliferation, survival, and chemoresistance and therefore, have been suggested as potential therapeutic targets. This study investigated the cross-talk between two antioxidant molecules, thioredoxin reductase (TrxR) and heme oxygenase-1 (HO-1), and their therapeutic implications in MM. We found that although auranofin, a TrxR inhibitor, significantly inhibited TrxR activity by more than 50% at lower concentrations, myeloma cell proliferation was only inhibited at higher concentrations of auranofin. Inhibition of TrxR using lower auranofin concentrations induced HO-1 protein expression in myeloma cells. Using a sub-lethal concentration of auranofin to inhibit TrxR activity in conjunction with HO-1 inhibition significantly decreased myeloma cell growth and induced apoptosis. TrxR was shown to regulate HO-1 via the Nrf2 signaling pathway in a ROS-dependent manner. Increased HO-1 mRNA levels were observed in bortezomib-resistant myeloma cells compared to parent cells and HO-1 inhibition restored the sensitivity to bortezomib in bortezomib-resistant myeloma cells. These findings indicate that concurrent inhibition of HO-1 with either a TrxR inhibitor or with bortezomib would improve therapeutic outcomes in MM patients. Hence, our findings further support the need to target multiple antioxidant systems alone or in combination with other therapeutics to improve therapeutic outcomes in MM patients.
Subject(s)
Antioxidants/metabolism , Heme Oxygenase-1/metabolism , Multiple Myeloma/metabolism , Oxidation-Reduction , Signal Transduction , Thioredoxin-Disulfide Reductase/metabolism , Antineoplastic Agents/pharmacology , Antioxidants/pharmacology , Apoptosis/drug effects , Auranofin/pharmacology , Bortezomib/pharmacology , Cell Line, Tumor , Drug Resistance, Neoplasm , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Female , Gene Expression Regulation, Neoplastic/drug effects , Heme Oxygenase-1/antagonists & inhibitors , Heme Oxygenase-1/genetics , Humans , Male , Middle Aged , Molecular Targeted Therapy , Multiple Myeloma/drug therapy , Multiple Myeloma/genetics , NF-E2-Related Factor 2/metabolism , Oxidation-Reduction/drug effects , Protein Binding , Reactive Oxygen Species/metabolism , Signal Transduction/drug effects , Thioredoxin-Disulfide Reductase/antagonists & inhibitorsABSTRACT
Multiple myeloma (MM) is a hematological malignancy characterized by the aberrant accumulation of clonal plasma cells in the bone marrow. Despite recent advancement in anti-myeloma treatment, MM remains an incurable disease. This study showed higher intrinsic oxidative stress and higher Trx1 and TrxR1 protein levels in MM cells compared to normal cells. Drug-induced Trx1 (PX-12) and TrxR1 (Auranofin) inhibition disrupted redox homeostasis resulting in ROS-induced apoptosis in MM cells and a reduction in clonogenic activity. Knockdown of either Trx1 or TrxR1 reduced MM cell viability. Trx1 inhibition by PX-12 sensitized MM cells to undergo apoptosis in response to the NF-κß inhibitors, BAY 11-7082 and curcumin. PX-12 treatment decreased the expression of the NF-κß subunit p65 in MM cells. Bortezomib-resistant MM cells contained higher Trx1 protein levels compared to the parental cells and PX-12 treatment resulted in apoptosis. Thus, increased Trx1 enhances MM cell growth and survival and exerts resistance to NF-κß inhibitors. Therefore inhibiting the thioredoxin system may be an effective therapeutic strategy to treat newly diagnosed as well as relapsed/refractory MM.
Subject(s)
Apoptosis/physiology , Multiple Myeloma/drug therapy , Reactive Oxygen Species/metabolism , Thioredoxin Reductase 1/antagonists & inhibitors , Thioredoxins/antagonists & inhibitors , Auranofin/pharmacology , Bortezomib/pharmacology , Caspase 3/metabolism , Cell Line, Tumor , Cell Proliferation , Cell Survival/genetics , Curcumin/pharmacology , Disulfides/pharmacology , Drug Resistance, Neoplasm , Humans , Imidazoles/pharmacology , Leukocytes, Mononuclear , Multiple Myeloma/pathology , Nitriles/pharmacology , Oxidative Stress/physiology , Sulfones/pharmacology , Thioredoxin Reductase 1/genetics , Thioredoxin Reductase 1/metabolism , Thioredoxins/genetics , Thioredoxins/metabolism , Transcription Factor RelA/antagonists & inhibitors , Transcription Factor RelA/biosynthesisABSTRACT
The neutral beam injector of steady state superconducting tokamak (SST1-NBI) at IPR is designed for injecting upto 1.7 MW of neutral beam (Hº, 30-55 keV) power to the tokamak plasma for heating and current drive. Operations of the positive ion source (PINI or Plug-In-Neutral-Injector) of SST1-NBI were carried out on the NBI test stand. The PINI was operated at reduced gas feed rate of 2-3 Torr l/s, without using the high speed cryo pumps. Experiments were conducted to achieve a stable beam extraction by optimizing operational parameters namely, the arc current (120-300 A), acceleration voltage (16-40 kV), and a suitable control sequence. The beam divergence, power density profiles, and species fractions (H(+):H2(+):H3(+)) were measured by using the diagnostics such as thermal calorimetry, infrared thermography, and Doppler shift spectroscopy. The maximum extracted beam current was about 18 A. A further increase of beam current was found to be limited by the amount of gas feed rate to the ion source.
ABSTRACT
Oxidative stress, which is associated with an increased concentration of reactive oxygen species (ROS), is involved in the pathogenesis of numerous diseases including cancer. In response to increased ROS levels, cellular antioxidant molecules such as thioredoxin, peroxiredoxins, glutaredoxins, DJ-1, and superoxide dismutases are upregulated to counteract the detrimental effect of ROS. However, cancer cells take advantage of upregulated antioxidant molecules for protection against ROS-induced cell damage. This review focuses on two antioxidant systems, Thioredoxin and DJ-1, which are upregulated in many human cancer types, correlating with tumour proliferation, survival, and chemo-resistance. Thus, both of these antioxidant molecules serve as potential molecular targets to treat cancer. However, targeting one of these antioxidants alone may not be an effective anti-cancer therapy. Both of these antioxidant molecules are interlinked and act on similar downstream targets such as NF-κß, PTEN, and Nrf2 to exert cytoprotection. Inhibiting either thioredoxin or DJ-1 alone may allow the other antioxidant to activate downstream signalling cascades leading to tumour cell survival and proliferation. Targeting both thioredoxin and DJ-1 in conjunction may completely shut down the antioxidant defence system regulated by these molecules. This review focuses on the cross-talk between thioredoxin and DJ-1 and highlights the importance and consequences of targeting thioredoxin and DJ-1 together to develop an effective anti-cancer therapeutic strategy.
ABSTRACT
Using squid axoplasm as a model system, we have visualized the fast transport of non-filamentous neurofilament protein particles along axonal microtubules. This transport occurs at speeds of 0.5-1.0 microm/second and the majority of neurofilament particles stain with kinesin antibody. These observations demonstrate, for the first time, that fast (0.5-1.0 microm/second) transport of neurofilament proteins occurs along microtubules. In addition, our studies suggest that neurofilament protein can be transported as non-membrane bound, nonfilamentous subunits along axons, and that the transport is kinesin-dependent. Microtubule-based fast transport might therefore provide a mechanism for the distribution and turnover of neurofilament, and perhaps other cytoskeletal proteins, throughout neurons.
Subject(s)
Axons/physiology , Microtubules/physiology , Neurofilament Proteins/metabolism , Amino Acid Sequence , Animals , Axonal Transport , Decapodiformes , Epitopes/chemistry , Fluorescent Antibody Technique , Kinesins/analysis , Kinesins/chemistry , Kinesins/metabolism , Kinetics , Molecular Sequence Data , Protein Subunits , Protein Transport , Time FactorsABSTRACT
BHK-21 fibroblasts contain type III vimentin/desmin intermediate filament (IF) proteins that typically co-isolate and co-cycle in in vitro experiments with certain high molecular weight proteins. Here, we report purification of one of these and demonstrate that it is in fact the type VI IF protein nestin. Nestin is expressed in several fibroblastic but not epithelioid cell lines. We show that nestin forms homodimers and homotetramers but does not form IF by itself in vitro. In mixtures, nestin preferentially co-assembles with purified vimentin or the type IV IF protein alpha-internexin to form heterodimer coiled-coil molecules. These molecules may co-assemble into 10 nm IF provided that the total amount of nestin does not exceed about 25%. However, nestin does not dimerize with types I/II keratin IF chains. The bulk of the nestin protein consists of a long carboxyl-terminal tail composed of various highly charged peptide repeats. By analogy with the larger neurofilament chains, we postulate that these sequences serve as cross-bridgers or spacers between IF and/or other cytoskeletal constituents. In this way, we propose that direct incorporation of modest amounts of nestin into the backbone of cytoplasmic types III and IV IFs affords a simple yet flexible method for the regulation of their dynamic supramolecular organization and function in cells.
Subject(s)
Carrier Proteins/metabolism , Intermediate Filament Proteins/chemistry , Nerve Tissue Proteins , Vimentin/metabolism , Animals , Antibodies, Monoclonal , Cell Line , Cricetinae , Desmin/metabolism , Fibroblasts/metabolism , Intermediate Filament Proteins/metabolism , Kidney/metabolism , Microscopy, Electron , Molecular Sequence Data , Molecular Weight , Nestin , Rats , Sequence Homology, Amino Acid , SoftwareSubject(s)
Cytoskeleton/physiology , Intermediate Filaments/physiology , Animals , Cell Movement , Humans , Molecular MimicryABSTRACT
The motile properties of intermediate filament (IF) networks have been studied in living cells expressing vimentin tagged with green fluorescent protein (GFP-vimentin). In interphase and mitotic cells, GFP-vimentin is incorporated into the endogenous IF network, and accurately reports the behavior of IF. Time-lapse observations of interphase arrays of vimentin fibrils demonstrate that they are constantly changing their configurations in the absence of alterations in cell shape. Intersecting points of vimentin fibrils, or foci, frequently move towards or away from each other, indicating that the fibrils can lengthen or shorten. Fluorescence recovery after photobleaching shows that bleach zones across fibrils rapidly recover their fluorescence. During this recovery, bleached zones frequently move, indicating translocation of fibrils. Intriguingly, neighboring fibrils within a cell can exhibit different rates and directions of movement, and they often appear to extend or elongate into the peripheral regions of the cytoplasm. In these same regions, short filamentous structures are also seen actively translocating. All of these motile properties require energy, and the majority appear to be mediated by interactions of IF with microtubules and microfilaments.