Co-Targeting of BTK and TrxR as a Therapeutic Approach to the Treatment of Lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is a haematological malignancy representing the most diagnosed non-Hodgkin's lymphoma (NHL) subtype. Despite the approved chemotherapies available in clinics, some patients still suffer from side effects and relapsed disease. Recently, studies have reported the role of the Trx system and the BCR signalling pathway in cancer development and drug resistance. In this regard, we assessed a potential link between the two systems and evaluated the effects of [Au(d2pype)2]Cl (TrxR inhibitor) and ibrutinib (BTK inhibitor) alone and in combination on the cell growth of two DLBCL lymphoma cell lines, SUDHL2 and SUDHL4. In this study, we show higher expression levels of the Trx system and BCR signalling pathway in the DLBCL patient samples compared to the healthy samples. The knockdown of TrxR using siRNA reduced BTK mRNA and protein expression. A combination treatment with [Au(d2pype)2]Cl and ibrutinib had a synergistic effect on the inhibition of lymphoma cell proliferation, the activation of apoptosis, and, depending on lymphoma cell subtype, ferroptosis. Decreased BTK expression and the cytoplasmic accumulation of p65 were observed after the combination treatment in the DLBCL cells, indicating the inhibition of the NF-κB pathway. Thus, the co-targeting of BTK and TrxR may be an effective therapeutic strategy to consider for DLBCL treatment.

1.5T MR-Guided Daily-Adaptive SBRT for Prostate Cancer: Preliminary Report of Toxicity and Quality of Life of the First 100 Patients.

Purpose: The present study reports the preliminary outcomes in terms of adverse events and quality of life in the first 100 patients treated with 1.5T MR-guided daily-adaptive stereotactic body radiotherapy for prostate cancer. Methods: From October 2019 to December 2020, 100 patients, enrolled in a prospective study, received MR-guided SBRT for prostate cancer. Rectal spacer insertion was optional and administered in 37 patients. In total, 32 patients received androgen deprivation therapy in accordance with international guidelines. A prospective collection of data regarding toxicity and quality of life was performed. Results: The median age was 71 years (range, 52-84). The median total dose delivered was 35 Gy (35-36.25 Gy) in five sessions, either on alternate days (n = 25) or consecutive days (n = 75). For acute toxicity, we recorded: seven cases of acute G2 urinary pain and four cases of G2 gastrointestinal events. The median follow-up was 12 months (3-20), recording three late G2 urinary events and one G3 case, consisting of a patient who required a TURP 8 months after the treatment. For gastrointestinal toxicity, we observed 3 G ≥ 2 GI events, including one patient who received argon laser therapy for radiation-induced proctitis. Up to the last follow-up, all patients are alive and with no evidence of biochemical relapse, except for an M1 low-volume patient in distant progression two months after radiotherapy. QoL evaluation reported a substantial resolution of any discomfort within the second follow-up after radiotherapy, with the only exception being sexual items. Notably, after one year, global health items were improved compared to the baseline assessment. Conclusions: This study reports very promising outcomes in terms of adverse events and QoL, supporting the role of 1.5T MR-guided SBRT for prostate cancer. To date, this series is one of the first and largest available in the literature. Long-term results are warranted.

1.5 T MR-Guided Daily Adapted SBRT on Lymph Node Oligometastases from Prostate Cancer.

Introduction: The aim of our study was to evaluate the efficacy and toxicity of a daily adaptive MR-guided SBRT on 1.5 T MR-linac in patients affected by lymph node oligometastases from PCa. Materials and Methods: The present study is a prospective observational study conducted in a single institution (protocol n°: MRI/LINAC n. 23748). Patients with oligometastatic lymph nodes from PCa treated with daily adaptive MR-guided SBRT on 1.5 T MR-linac were included in the study. There was a minimum required follow-up of 3 months after SBRT. The primary end-point was local progression-free survival (LPFS). The secondary end-points were: nodal progression-free survival (NPFS), progression-free survival (PFS), and toxicity. Results: A total of 118 lymph node oligometastases from PCa were treated with daily adaptive 1.5 T MR-guided SBRT in 63 oligometastatic patients. Of the patients, 63.5% were oligorecurrent and 36.5% were oligoprogressive. The two-year LPFS was 90.7%. The median NPFS was 22.3 months and the 2-year NPFS was 46.5%. Receiving hormone therapy before SBRT was correlated with a lower NPFS at the multivariate analysis (1 y NPFS 87.1% versus 42.8%; p = 0.002-HR 0.199, 95% CI 0.073-0.549). Furthermore, the oligorecurrent state during ADT was correlated with a lower NPFS than was the oligoprogressive state. The median PFS was 10.3 months and the 2-year PFS was 32.4%. Patients treated with hormone therapy before SBRT had a significantly lower 1-year PFS the others (28% versus 70.4%; p = 0.01-HR 0.259, 95% CI 0.117-0.574). No acute and late toxicities occurred during treatment. Conclusions: The present study is the largest prospective study of 1.5 T lymph node SBRT on MR-linac in patients with PCa. Lymph node SBRT by 1.5 T MR-linac provides high local control rates with an excellent toxicity profile.

1.5T MR-Guided Daily Adaptive Stereotactic Body Radiotherapy for Prostate Re-Irradiation: A Preliminary Report of Toxicity and Clinical Outcomes.

Background: Prostate re-irradiation is an attractive treatment option in the case of local relapse after previous radiotherapy, either in the definitive or in the post-operative setting. In this scenario, the introduction of MR-linacs may represent a helpful tool to improve the accuracy and precision of the treatment. Methods: This study reports the preliminary data of a cohort of 22 patients treated with 1.5T MR-Linacs for prostate or prostate bed re-irradiation. Toxicity was prospectively assessed and collected according to CTCAE v5.0. Survival endpoints were measured using Kaplan-Meier method. Results: From October 2019 to October 2021, 22 patients received 1.5T MR-guided stereotactic body radiotherapy for prostate or prostate-bed re-irradiation. In 12 cases SBRT was delivered to the prostate, in 10 to the prostate bed. The median time to re-RT was 72 months (range, 12-1460). SBRT was delivered concurrently with ADT in 4 cases. Acute toxicity was: for GU G1 in 11/22 and G2 in 4/22; for GI G1 in 7/22, G2 in 4/22. With a median follow-up of 8 months (3-21), late G1 and G2 GU events were respectively 11/22 and 4/22. Regarding GI toxicity, G1 were 6/22, while G2 3/22. No acute/late G≥3 GI/GU events occurred. All patients are alive. The median PSA-nadir was 0.49 ng/ml (0.08-5.26 ng/ml), for 1-year BRFS and DPFS rates of 85.9%. Twenty patients remained free from ADT with 1-year ADT-free survival rates of 91.3%. Conclusions: Our experience supports the use of MR-linacs for prostate or prostate bed re-irradiation as a feasible and safe treatment option with minimal toxicity and encouraging results in terms of clinical outcomes.

Expanding the armory for treating lymphoma: Targeting redox cellular status through thioredoxin reductase inhibition.

Lymphoma refers to a group of cancers that arise from lymphocytes and is the most common form of hematological malignancy in adults. While the recent availability of specific chemotherapy regimes has resulted in good patient outcomes for some lymphoma subtypes, relapsed and refractory lymphoma is still a challenge that needs to be overcome. This review discusses how Nrf-2 regulated antioxidant systems such as the thioredoxin and glutathione systems are upregulated in lymphomas and have been linked with several signaling pathways involved in lymphoma development and progression, including the B cell receptor, the NF-κB, and the STAT3 signaling pathways. Thioredoxin reductase (TrxR) has been recognized as a potential anticancer target and, as a consequence, the synthesis of TrxR inhibitors, along with the discovery of inhibitors from natural resources and evaluation of their anti-cancer effects, is an ongoing active area of research. Targeting antioxidant systems, especially TrxR, may represent a new valid therapeutic approach for lymphoma, potentially in combination with existing therapies.

Post-operative hypofractionated radiotherapy for prostate cancer: a mono-institutional analysis of toxicity and clinical outcomes.

BACKGROUND: As the use of hypofractionation has spread in the setting of curative prostate radiotherapy, few data are available in the post-operative scenario. This study reports a mono-institutional experience of moderate post-operative hypofractionated radiotherapy for prostate cancer. METHODS: In February 2021, we retrospectively assessed the outcomes of 129 patients who received between April 2013 and May 2020 hypofractionated post-operative radiotherapy using Helical Tomotherapy. Toxicity was assessed using CTCAE criteria v4.0. Survival endpoints were calculated with Kaplan-Meier method. RESULTS: Median age and follow-up were, respectively, 67 years and 43 months. Adjuvant and salvage treatment were delivered to 63.5% and 36.4% of patients to a median total dose of 63.8 Gy (61.6-65.25 Gy) in 29 fractions (2.12-2.25 Gy/fraction). Pelvic lymph-nodes irradiation was performed in 67.4% of cases. ADT was added in 50%. Acute toxicity was: G1 and G2 GU events in 36% and 9.3% of cases; G1 and G2 GI events in 29.4% and 13.9%. Late GU toxicity occurred in 12.4% of cases: 3.1% G1, 7.7% G2 and 1.5% G3 events; GI toxicity consisted of 1.5% G1 and 7.7% G2 events. Biochemical relapse occurred in 26.3% of cases, recording no significant differences between adjuvant and salvage (p = 0.67), with 4- and 5-years bRFS rates of 78.7% and 75.6%. Two patients died of progressive disease and eight for non-oncological causes resulting in 3-years overall survival and cancer-specific survival rates of 98% and 98.4%. CONCLUSIONS: Our experience supports the use of moderate hypofractionation for prostate bed radiotherapy, with minimal toxicity and promising results in terms of clinical outcomes.

Application of traditional Chinese medicine as skin depigmentation agents.

Traditional Chinese medicine (TCM) has been frequently used as skin lightning agents. However, the mechanism of action of their effect is unclear. The present study aims to evaluate anti-tyrosinase activity of 10 commonly used TCM on mushroom (ab), human (hs) and mouse melanoma B16F0 (mm) tyrosinase (TYR) respectively. The results showed that at 1.0 mg/mL, extracts from Rosa rugosa Thumb, Morus alba L. and Paeonia lactiflora Pall were active against both abTYR and hsTYR (>50% inhibition), extracts from Bletilla striata (Thunb.) Rchb. F., Centella asiatica (L.) Urb, Cynanchum atratum L., Rosa canina L., Rhus chinensis Mill. and Glycyrrhiza urolensis Fisch. Ex DC. inhibited either abTYR or hsTYR (>50%), while extract from Tribulus terrestris L. had no/minimal activity (<10% inhibition). When treated with melanoma B16F0 cells, M. alba also significantly reduced mmTYR activity (70% at 250 µg/mL) and melanin content (50% at 250 µg/mL). These findings demonstrated inhibitory effects of 9 TCM against TYR and hence support their application as skin lightning agents. Our results also showed discrepancies in TYR activity from different sources, suggesting a testing regime of combining abTYR, hsTYR and mmTYR when developing depigmentation agents for human application.

Correction: Inhibition of thioredoxin 1 leads to apoptosis in drug-resistant multiple myeloma.

[This corrects the article DOI: 10.18632/oncotarget.3795.].

Modulating skin colour: role of the thioredoxin and glutathione systems in regulating melanogenesis.

Different skin colour among individuals is determined by the varying amount and types of melanin pigment. Melanin is produced in melanocytes, a type of dendritic cell located in the basal layer of the epidermis, through the process of melanogenesis. Melanogenesis consists of a series of biochemical and enzymatic reactions catalysed by tyrosinase and other tyrosinase-related proteins, leading to the formation of two types of melanin, eumelanin and pheomelanin. Melanogenesis can be regulated intrinsically by several signalling pathways, including the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA), stem cell factor (SCF)/c-kit and wingless-related integration site (Wnt)/ß-catenin signalling pathways. Ultraviolet radiation (UVR) is the major extrinsic factor in the regulation of melanogenesis, through the generation of reactive oxygen species (ROS). Antioxidants or antioxidant systems, with the ability to scavenge ROS, may decrease melanogenesis. This review focuses on the two main cellular antioxidant systems, the thioredoxin (Trx) and glutathione (GSH) systems, and discusses their roles in melanogenesis. In the Trx system, high levels/activities of thioredoxin reductase (TrxR) are correlated with melanin formation. The GSH system is linked with regulating pheomelanin formation. Exogenous addition of GSH has been shown to act as a depigmenting agent, suggesting that other antioxidants may also have the potential to act as depigmenting agents for the treatment of human hyperpigmentation disorders.

Investigating the Thioredoxin and Glutathione Systems' Response in Lymphoma Cells after Treatment with [Au(d2pype)2]CL.

Lymphoma is a blood cancer comprising various subtypes. Although effective therapies are available, some patients fail to respond to treatment and can suffer from side effects. Antioxidant systems, especially the thioredoxin (Trx) and glutathione (GSH) systems, are known to enhance cancer cell survival, with thioredoxin reductase (TrxR) recently reported as a potential anticancer target. Since the GSH system can compensate for some Trx system functions, we investigated its response in three lymphoma cell lines after inhibiting TrxR activity with [Au(d2pype)2]Cl, a known TrxR inhibitor. [Au(d2pype)2]Cl increased intracellular reactive oxygen species (ROS) levels and induced caspase-3 activity leading to cell apoptosis through inhibiting both TrxR and glutathione peroxidase (Gpx) activity. Expression of the tumour suppresser gene TXNIP increased, while GPX1 and GPX4 expression, which are related to poor prognosis of lymphoma patients, decreased. Unlike SUDHL2 and SUDHL4 cells, which exhibited a decreased GSH/GSSG ratio after treatment, in KMH2 cells the ratio remained unchanged, while glutathione reductase and glutaredoxin expression increased. Since KMH2 cells were less sensitive to treatment with [Au(d2pype)2]Cl, the GSH system may play a role in protecting cells from apoptosis after TrxR inhibition. Overall, our study demonstrates that inhibition of TrxR represents a valid therapeutic approach for lymphoma.

The regulation of bcr-abl in hypoxia is through the mTOR pathway.

Chronic myeloid leukemia (CML) is usually characterized by the formation of the fusion onco-protein bcr-abl. Therefore, the majority of CML treatments are bcr-abl specific tyrosine kinase inhibitors (TKIs). TKI resistance in CML treatment is becoming a major obstacle in managing this disease. One well-studied form of drug resistance is hypoxia-induced drug resistance, a phenomenon observed in many other cancers. This study aimed to determine the efficacy of TKIs in CML cells cultured in hypoxia. It was observed that bcr-abl translation was severely halted in hypoxia, rendering TKIs ineffective. We found that the mechanism by which bcr-abl protein levels were being suppressed in hypoxia was through the mTOR pathway, specifically via ribosomal protein S6 (RPS6). This information is vital to the improvement of CML treatments, as it can be used to determine how to best combat hypoxia-induced drug resistance in CML and subsequently to identify new targets for treatment.

Cross-talk between Bcr-abl and the Thioredoxin System in Chronic Myeloid Leukaemia: Implications for CML Treatment.

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.

Acute and late toxicity and preliminary outcomes report of moderately hypofractionated helical tomotherapy for localized prostate cancer: a mono-institutional analysis.

AIMS: To assess toxicity and clinical outcomes of moderately hypofractionated helical tomotherapy (HT) for the curative treatment of localized prostate cancer (PC). METHODS: From December 2012 to May 2018, 170 patients were treated with definitive intent for PC. Thirty-four percent were low risk, 30% intermediate risk (IR) and 36% high risk (HR). All patients received 70 Gy in 28 fractions to the prostate; 61.6 Gy were delivered to the seminal vesicles for IR; pelvic lymph nodes irradiation for a total dose of 50.4 Gy was added in the HR subgroup. Toxicity was assessed using CTCAE V4.0, and biochemical failure was defined following Phoenix criteria. Time-to-event data were analyzed using the Kaplan-Meier method and log-rank test. RESULTS: The median follow-up was 36 months (range 12-78); acute toxicity was as follows: G1 and G2 in 27.6% and 19.4% for GI; 53% and 24% for GU. No G ≥ 3 event was observed. For late toxicity, G ≥ 3 GI and GU rates were, respectively, 3% and 2.4% at 3 years and 3% and 4.8% at 4 years; no G4 occurred. A statistical correlation between acute or late G3 incidence and clinical or dosimetric parameters was not found. At the time of analysis, 2- and 3-year biochemical relapse-free survival rates were 90% and 87.5% and 2- and 3-year overall survival rates were 96.4% and 90%, respectively. The log-rank test revealed no difference between the risk groups in terms of biochemical control (p = 0.16). CONCLUSIONS: Moderately hypofractionated RT with HT for localized prostate cancer reported excellent outcomes with mild acute and late toxicity incidence, with promising biochemical control rates.

Anticancer activity of a Gold(I) phosphine thioredoxin reductase inhibitor in multiple myeloma.

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.

The Multifaceted Roles of DJ-1 as an Antioxidant.

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.

Targeted knockdown of DJ-1 induces multiple myeloma cell death via KLF6 upregulation.

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.

Expression of the thioredoxin system in an in vivo-like cancer cell environment upon auranofin treatment.

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.

TrxR1 inhibition overcomes both hypoxia-induced and acquired bortezomib resistance in multiple myeloma through NF-Ðºß inhibition.

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.

The thioredoxin system in breast cancer cell invasion and migration.

Metastasis is the most life threatening aspect of breast cancer. It is a multi-step process involving invasion and migration of primary tumor cells with a subsequent colonization of these cells at a secondary location. The aim of the present study was to investigate the role of thioredoxin (Trx1) in the invasion and migration of breast cancer cells and to assess the strength of the association between high levels of Trx1 and thioredoxin reductase (TrxR1) expression with breast cancer patient survival. Our results indicate that the expression of both Trx1 and TrxR1 are statistically significantly increased in breast cancer patient cells compared with paired normal breast tissue from the same patient. Over-expression of Trx1 in MDA-MB-231 breast cancer cell lines enhanced cell invasion in in vitro assays while expression of a redox inactive mutant form of Trx1 (designated 1SS) or the antisense mRNA inhibited cell invasion. Addition of exogenous Trx1 also enhanced cell invasion, while addition of a specific monoclonal antibody that inhibits Trx1 redox function decreased cell invasion. Over-expression of intracellular Trx1 did not increase cell migration but expression of intracellular 1SS inhibited migration. Addition of exogenous Trx1 enhanced cell migration while 1SS had no effect. Treatment with auranofin inhibited TrxR activity, cell migration and clonogenic activity of MDA-MB-231 cells, while increasing reactive oxygen species (ROS) levels. Analysis of 25 independent cohorts with 5910 patients showed that Trx1 and TrxR1 were both associated with a poor patient prognosis in terms of overall survival, distant metastasis free survival and disease free survival. Therefore, targeting the Trx system with auranofin or other specific inhibitors may provide improved breast cancer patient outcomes through inhibition of cancer invasion and migration.

Cross-talk between two antioxidants, thioredoxin reductase and heme oxygenase-1, and therapeutic implications for multiple myeloma.

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.