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BioRxiv : the Preprint Server For... Jun 2024Lysine Specific Demethylase 1 (KDM1A / LSD1) regulates mitochondrial respiration and stabilizes HIF-1A (hypoxia-inducible factor 1A). HIF-1A modulates reactive oxygen...
Lysine Specific Demethylase 1 (KDM1A / LSD1) regulates mitochondrial respiration and stabilizes HIF-1A (hypoxia-inducible factor 1A). HIF-1A modulates reactive oxygen species (ROS) levels by increasing cellular glucose uptake, glycolysis, and endogenous antioxidants. The role of KDM1A in cellular ROS response has not previously been described. We determined the role of KDM1A in regulating the ROS response and the utility of KDM1A inhibitors in combination with ROS-inducing cancer therapies. Our results show that KDM1A inhibition sensitized cells to oxidative stress and increased total cellular ROS, which was mitigated by treatment with the antioxidant N-acetyl cysteine. KDM1A inhibition decreased basal mitochondrial respiration and impaired induction of HIF-1A after ROS exposure. Overexpression of HIF-1A salvaged cells from KDM1A inhibition enhanced sensitivity to ROS. Thus we found that increased sensitivity of ROS after KDM1A inhibition was mediated by HIF-1A and depletion of endogenous glutathione. We also show that KDM1A-specific inhibitor bizine synergized with antioxidant-depleting therapies, buthionine sulfoximine, and auranofin in rhabdomyosarcoma cell lines (Rh28 and Rh30). In this study, we describe a novel role for KDM1A in regulating HIF- 1A functions under oxidative stress and found that dual targeting of KDM1A and antioxidant systems may serve as an effective combination anticancer strategy.
PubMed: 38915482
DOI: 10.1101/2024.06.12.597953 -
Redox Biology Jun 2024Since the survival of lymphoma patients who experience disease progression or relapse remains very poor, new therapeutic approaches and effective drugs are urgently...
Since the survival of lymphoma patients who experience disease progression or relapse remains very poor, new therapeutic approaches and effective drugs are urgently needed. Here we show that auranofin (AF), an anti-rheumatoid drug thought to inhibit thioredoxin reductases (TXNRDs) as its mechanism of action, exhibited potent activity against multiple cancer types, especially effective against B cell lymphoma. Surprisingly, a knockdown of TXNRD1 and TXNRD2 did not cause significant cytotoxicity, suggesting that abrogation of TXNRD enzyme per se was insufficient to cause cancer cell death. Further mechanistic study showed that the interaction of AF with TXNRD could convert this antioxidant enzyme to a ROS-generating molecule via disrupting its electron transport, leading to a leak of electrons that interact with molecular oxygen to form superoxide. AF also suppressed energy metabolism by inhibiting both mitochondria complex II and the glycolytic enzyme GAPDH, leading to a significant depletion of ATP and inhibition of cancer growth in vitro and in vivo. Importantly, we found that the AF-mediated ROS stress could induce PD-L1 expression, revealing an unwanted effect of AF in causing immune suppression. We further showed that a combination of AF with anti-PD-1 antibody could enhance the anticancer activity in a syngeneic immune-competent mouse B-cell lymphoma model. Our study suggests that AF could be a potential drug for lymphoma treatment, and its combination with immune checkpoint inhibitors would be a logical strategy to increase the therapeutic activity.
PubMed: 38909408
DOI: 10.1016/j.redox.2024.103245 -
Nature Communications Jun 2024Radio-immunotherapy exploits the immunostimulatory features of ionizing radiation (IR) to enhance antitumor effects and offers emerging opportunities for treating...
Radio-immunotherapy exploits the immunostimulatory features of ionizing radiation (IR) to enhance antitumor effects and offers emerging opportunities for treating invasive tumor indications such as melanoma. However, insufficient dose deposition and immunosuppressive microenvironment (TME) of solid tumors limit its efficacy. Here we report a programmable sequential therapeutic strategy based on multifunctional fusogenic liposomes (Lip@AUR-ACP-aptPD-L1) to overcome the intrinsic radio-immunotherapeutic resistance of solid tumors. Specifically, fusogenic liposomes are loaded with gold-containing Auranofin (AUR) and inserted with multivariate-gated aptamer assemblies (ACP) and PD-L1 aptamers in the lipid membrane, potentiating melanoma-targeted AUR delivery while transferring ACP onto cell surface through selective membrane fusion. AUR amplifies IR-induced immunogenic death of melanoma cells to release antigens and damage-associated molecular patterns such as adenosine triphosphate (ATP) for triggering adaptive antitumor immunity. AUR-sensitized radiotherapy also upregulates matrix metalloproteinase-2 (MMP-2) expression that combined with released ATP to activate ACP through an "and" logic operation-like process (AND-gate), thus triggering the in-situ release of engineered cytosine-phosphate-guanine aptamer-based immunoadjuvants (eCpG) for stimulating dendritic cell-mediated T cell priming. Furthermore, AUR inhibits tumor-intrinsic vascular endothelial growth factor signaling to suppress infiltration of immunosuppressive cells for fostering an anti-tumorigenic TME. This study offers an approach for solid tumor treatment in the clinics.
Topics: Liposomes; Aptamers, Nucleotide; Animals; Mice; Cell Line, Tumor; Immunotherapy; Melanoma; Humans; Tumor Microenvironment; Matrix Metalloproteinase 2; Gold; Mice, Inbred C57BL; Female; B7-H1 Antigen; Adenosine Triphosphate
PubMed: 38866788
DOI: 10.1038/s41467-024-49482-9 -
Nature Communications May 2024Checkpoint kinase 1 (CHK1) is critical for cell survival under replication stress (RS). CHK1 inhibitors (CHK1i's) in combination with chemotherapy have shown promising...
Checkpoint kinase 1 (CHK1) is critical for cell survival under replication stress (RS). CHK1 inhibitors (CHK1i's) in combination with chemotherapy have shown promising results in preclinical studies but have displayed minimal efficacy with substantial toxicity in clinical trials. To explore combinatorial strategies that can overcome these limitations, we perform an unbiased high-throughput screen in a non-small cell lung cancer (NSCLC) cell line and identify thioredoxin1 (Trx1), a major component of the mammalian antioxidant-system, as a determinant of CHK1i sensitivity. We establish a role for redox recycling of RRM1, the larger subunit of ribonucleotide reductase (RNR), and a depletion of the deoxynucleotide pool in this Trx1-mediated CHK1i sensitivity. Further, the TrxR inhibitor auranofin, an approved anti-rheumatoid arthritis drug, shows a synergistic interaction with CHK1i via interruption of the deoxynucleotide pool. Together, we show a pharmacological combination to treat NSCLC that relies on a redox regulatory link between the Trx system and mammalian RNR activity.
Topics: Checkpoint Kinase 1; Humans; Oxidation-Reduction; Thioredoxins; Cell Line, Tumor; Auranofin; Carcinoma, Non-Small-Cell Lung; Lung Neoplasms; Protein Kinase Inhibitors; Ribonucleoside Diphosphate Reductase; Ribonucleotide Reductases; Drug Synergism; Animals
PubMed: 38821952
DOI: 10.1038/s41467-024-48076-9 -
BioRxiv : the Preprint Server For... May 2024Novel therapeutic approaches are needed for the treatment of Ewing sarcoma tumors. We previously identified that Ewing sarcoma cell lines are sensitive to drugs that...
Novel therapeutic approaches are needed for the treatment of Ewing sarcoma tumors. We previously identified that Ewing sarcoma cell lines are sensitive to drugs that inhibit protein translation. However, translational and therapeutic approaches to inhibit protein synthesis in tumors are limited. In this work, we identified that reactive oxygen species, which are generated by a wide range of chemotherapy and other drugs, inhibit protein synthesis and reduce the level of critical proteins that support tumorigenesis in Ewing sarcoma cells. In particular, we identified that both hydrogen peroxide and auranofin, an inhibitor of thioredoxin reductase and regulator of oxidative stress and reactive oxygen species, activate the repressor of protein translation 4E-BP1 and reduce the levels of the oncogenic proteins RRM2 and PLK1 in Ewing and other sarcoma cell lines. These results provide novel insight into the mechanism of how ROS-inducing drugs target cancer cells via inhibition of protein translation and identify a mechanistic link between ROS and the DNA replication (RRM2) and cell cycle regulatory (PLK1) pathways.
PubMed: 38798568
DOI: 10.1101/2024.05.13.593567 -
Neoplasia (New York, N.Y.) Jul 2024Thioredoxin reductases are frequently overexpressed in various solid tumors as a protective mechanism against heightened oxidative stress. Inhibitors of this system,...
Thioredoxin reductases are frequently overexpressed in various solid tumors as a protective mechanism against heightened oxidative stress. Inhibitors of this system, such as Auranofin, are effective in eradicating cancer cells. However, the clinical significance of thioredoxin reductase 1 (TrxR1) in lung cancer, as well as the potential for its antagonist as a treatment option, necessitated further experimental validation. In this study, we observed significant upregulation of TrxR1 specifically in non-small cell lung cancer (NSCLC), rather than small cell lung cancer. Moreover, TrxR1 expression exhibited associations with survival rate, tumor volume, and histological classification. We developed a novel TrxR1 inhibitor named LW-216 and assessed its antitumor efficacy in NSCLC. Our results revealed that LW-216 is effectively bound with intracellular TrxR1 at sites R371 and G442, facilitating TrxR1 ubiquitination and suppressing TrxR1 expression, while not affecting TrxR2 expression. Treatment of LW-216-induced DNA damage and cell apoptosis in NSCLC cells through the generation of reactive oxygen species (ROS). Importantly, supplementation with N-acetylcysteine (NAC) or ectopic TrxR1 expression reversed LW-216-induced apoptosis. Furthermore, LW-216 displayed potent tumor growth inhibition in NSCLC cell-implanted mice, reducing TrxR1 expression in xenografts. Remarkably, LW-216 exhibited superior antitumor activity compared to Auranofin in vivo. Collectively, our research provides compelling evidence supporting the potential of targeting TrxR1 by LW-216 as a promising therapeutic strategy for NSCLC.
Topics: Carcinoma, Non-Small-Cell Lung; Humans; Thioredoxin Reductase 1; Reactive Oxygen Species; Lung Neoplasms; Apoptosis; Animals; Mice; Ubiquitination; Xenograft Model Antitumor Assays; Cell Line, Tumor; Proteolysis; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Disease Models, Animal; Male; Antineoplastic Agents
PubMed: 38733769
DOI: 10.1016/j.neo.2024.101004 -
Research Square Apr 2024HO is a key oxidant in mammalian biology and a pleiotropic signaling molecule at the physiological level, and its excessive accumulation in conjunction with decreased...
HO is a key oxidant in mammalian biology and a pleiotropic signaling molecule at the physiological level, and its excessive accumulation in conjunction with decreased cellular reduction capacity is often found to be a common pathological marker. Here, we present a red fluorescent Genetically Encoded HO Indicator (GEHI) allowing versatile optogenetic dissection of redox biology. Our new GEHI, oROS-HT, is a chemigenetic sensor utilizing a HaloTag and Janelia Fluor (JF) rhodamine dye as fluorescent reporters. We developed oROS-HT through a structure-guided approach aided by classic protein structures and recent protein structure prediction tools. Optimized with JF, oROS-HT is a sensor with 635 nm excitation and 650 nm emission peaks, allowing it to retain its brightness while monitoring intracellular HO dynamics. Furthermore, it enables multi-color imaging in combination with blue-green fluorescent sensors for orthogonal analytes and low auto-fluorescence interference in biological tissues. Other advantages of oROS-HT over alternative GEHIs are its fast kinetics, oxygen-independent maturation, low pH sensitivity, lack of photo-artifact, and lack of intracellular aggregation. Here, we demonstrated efficient subcellular targeting and how oROS-HT can map inter and intracellular HO diffusion at subcellular resolution. Lastly, we used oROS-HT with other green fluorescence reporters to investigate the transient effect of the anti-inflammatory agent auranofin on cellular redox physiology and calcium levels via multi-parametric, dual-color imaging.
PubMed: 38699332
DOI: 10.21203/rs.3.rs-3974015/v1 -
Proceedings of the National Academy of... May 2024HIV latency regulation in monocytes and macrophages can vary according to signals directing differentiation, polarization, and function. To investigate these processes,...
HIV latency regulation in monocytes and macrophages can vary according to signals directing differentiation, polarization, and function. To investigate these processes, we generated an HIV latency model in THP-1 monocytes and showed differential levels of HIV reactivation among clonal populations. Monocyte-to-macrophage differentiation of HIV-infected primary human CD14+ and THP-1 cells induced HIV reactivation and showed that virus production increased concomitant with macrophage differentiation. We applied the HIV-infected THP-1 monocyte-to-macrophage (MLat) model to assess the biological mechanisms regulating HIV latency dynamics during monocyte-to-macrophage differentiation. We pinpointed protein kinase C signaling pathway activation and Cyclin T1 upregulation as inherent differentiation mechanisms that regulate HIV latency reactivation. Macrophage polarization regulated latency, revealing proinflammatory M1 macrophages suppressed HIV reactivation while anti-inflammatory M2 macrophages promoted HIV reactivation. Because macrophages rely on reactive-oxygen species (ROS) to exert numerous cellular functions, we disrupted redox pathways and found that inhibitors of the thioredoxin (Trx) system acted as latency-promoting agents in T-cells and monocytes, but opposingly acted as latency-reversing agents in macrophages. We explored this mechanism with Auranofin, a clinical candidate for reducing HIV reservoirs, and demonstrated Trx reductase inhibition led to ROS induced NF-κB activity, which promoted HIV reactivation in macrophages, but not in T-cells and monocytes. Collectively, cell type-specific differences in HIV latency regulation could pose a barrier to HIV eradication strategies.
Topics: Humans; Virus Latency; Macrophages; Monocytes; Cell Differentiation; Oxidation-Reduction; HIV-1; HIV Infections; Virus Activation; Homeostasis; Reactive Oxygen Species; THP-1 Cells; Signal Transduction; Protein Kinase C
PubMed: 38683980
DOI: 10.1073/pnas.2313823121 -
BioRxiv : the Preprint Server For... Apr 2024Ferroptosis is an iron-dependent, non-apoptotic form of cell death resulting from the accumulation of lipid peroxides. Colorectal cancer (CRC) accumulates high levels of...
Ferroptosis is an iron-dependent, non-apoptotic form of cell death resulting from the accumulation of lipid peroxides. Colorectal cancer (CRC) accumulates high levels of intracellular iron and reactive oxygen species (ROS), thereby sensitizing cells to ferroptosis. The selenoprotein glutathione peroxidase (GPx4) is a key enzyme in the detoxification of lipid peroxides and can be inhibited by the compound (S)-RSL3 ([1S,3R]-RSL3). However, the stereoisomer (R)-RSL3 ([1R,3R]-RSL3), which does not inhibit GPx4, exhibits equipotent activity to (S)-RSL3 across a panel of CRC cell lines. Utilizing CRC cell lines with an inducible knockdown of GPx4, we demonstrate that (S)-RSL3 sensitivity does not align with GPx4 dependency. Subsequently, a biotinylated (S)-RSL3 was then synthesized to perform affinity purification-mass spectrometry (AP-MS), revealing that (S)-RSL3 acts as a pan-inhibitor of the selenoproteome, targeting both the glutathione and thioredoxin peroxidase systems as well as multiple additional selenoproteins. To investigate the therapeutic potential of broadly disrupting the selenoproteome as a therapeutic strategy in CRC, we employed further chemical and genetic approaches to disrupt selenoprotein function. The findings demonstrate that the selenoprotein inhibitor Auranofin can induce ferroptosis and/or oxidative cell death both and . Consistent with this data we observe that AlkBH8, a tRNA-selenocysteine methyltransferase required for the translational incorporation of selenocysteine, is essential for CRC growth. In summary, our research elucidates the complex mechanisms underlying ferroptosis in CRC and reveals that modulation of the selenoproteome provides multiple new therapeutic targets and opportunities in CRC.
PubMed: 38617233
DOI: 10.1101/2024.03.29.587381 -
Biomedicine & Pharmacotherapy =... May 2024Thioredoxin reductase 1 (TrxR1) has emerged as a promising target for cancer therapy. In our previous research, we discovered several new TrxR1 inhibitors and found that...
Thioredoxin reductase 1 (TrxR1) has emerged as a promising target for cancer therapy. In our previous research, we discovered several new TrxR1 inhibitors and found that they all have excellent anti-tumor activity. At the same time, we found these TrxR1 inhibitors all lead to an increase in AKT phosphorylation in cancer cells, but the detailed role of AKT phosphorylation in TrxR1 inhibitor-mediated cell death remains unclear. In this study, we identified the combination of AKT and TrxR1 inhibitor displayed a strong synergistic effect in colon cancer cells. Furthermore, we demonstrated that the synergistic effect of auranofin (TrxR1 inhibitor) and MK-2206 (AKT inhibitor) was caused by ROS accumulation. Importantly, we found that ATM inhibitor KU-55933 can block the increase of AKT phosphorylation caused by auranofin, and exhibited a synergistic effect with auranofin. Taken together, our study demonstrated that the activation of ATM/AKT pathway is a compensatory mechanism to cope with ROS accumulation induced by TrxR1 inhibitor, and synergistic targeting of TrxR1 and ATM/AKT pathway is a promising strategy for treating colon cancer.
Topics: Humans; Colonic Neoplasms; Proto-Oncogene Proteins c-akt; Thioredoxin Reductase 1; Drug Synergism; Auranofin; Ataxia Telangiectasia Mutated Proteins; Reactive Oxygen Species; Signal Transduction; Heterocyclic Compounds, 3-Ring; Cell Line, Tumor; Phosphorylation; Morpholines; HCT116 Cells; Pyrones
PubMed: 38565059
DOI: 10.1016/j.biopha.2024.116507