Fighting drug-resistant lung cancer by induction of NAD(P)H:quinone oxidoreductase 1 (NQO1)-mediated ferroptosis.

Drug resistance is a major challenge in cancer treatment. The substrates of NAD(P)H:quinone oxidoreductase 1 (NQO1) show a promising anticancer effect in clinical trials. We previously identified a natural NQO1 substrate 2-methoxy-6-acetyl-7-methyljuglone (MAM) with a potent anticancer effect. The present study was designed to explore the efficacy of MAM in fighting against drug-resistant non-small cell lung cancer (NSCLC). The anticancer effect of MAM was evaluated in cisplatin-resistant A549 and AZD9291-resistant H1975 cells. The interaction of MAM with NQO1 was measured by cellular thermal shift assay and drug affinity responsive target stability assay. The NQO1 activity and expression were measured using NQO1 recombinant protein, Western blotting, and immunofluorescence staining assay. The roles of NQO1 were examined by NQO1 inhibitor, small interfering RNA (siRNA), and short hairpin RNA (shRNA). The roles of reactive oxygen species (ROS), labile iron pool (LIP), and lipid peroxidation were determined. MAM induced significant cell death in drug-resistant cells with similar potency to that of parental cells, which were completely abolished by NQO1 inhibitor, NQO1 siRNA, and iron chelators. MAM activates and binds to NQO1, which triggers ROS generation, LIP increase, and lipid peroxidation. MAM significantly suppressed tumor growth in the tumor xenograft zebrafish model. These results showed that MAM induced ferroptosis by targeting NQO1 in drug-resistant NSCLC cells. Our findings provided a novel therapeutic strategy for fighting against drug resistance by induction of NQO1-mediated ferroptosis.

Nitrovin (difurazone), an antibacterial growth promoter, induces ROS-mediated paraptosis-like cell death by targeting thioredoxin reductase 1 (TrxR1).

Glioblastoma multiforme (GBM) is one of the most lethal malignant tumors in the human brain, with only a few chemotherapeutic drugs available after surgery. Nitrovin (difurazone) is widely used as an antibacterial growth promoter in livestock. Here, we reported that nitrovin might be a potential anticancer lead. Nitrovin showed significant cytotoxicity to a panel of cancer cell lines. Nitrovin induced cytoplasmic vacuolation, reactive oxygen species (ROS) generation, MAPK activation, and Alix inhibition but had no effect on caspase-3 cleavage and activity, suggesting paraptosis activation. Nitrovin-induced cell death of GBM cells was significantly reversed by cycloheximide (CHX), N-acetyl-l-cysteine (NAC), glutathione (GSH), and thioredoxin reductase 1 (TrxR1) overexpression. Vitamins C and E, inhibitors of pan-caspase, MAPKs, and endoplasmic reticulum (ER) stress failed to do so. Nitrovin-triggered cytoplasmic vacuolation was reversed by CHX, NAC, GSH, and TrxR1 overexpression but not by Alix overexpression. Furthermore, nitrovin interacted with TrxR1 and significantly inhibited its activity. In addition, nitrovin showed a significant anticancer effect in a zebrafish xenograft model, which was reversed by NAC. In conclusion, our results showed that nitrovin induced non-apoptotic and paraptosis-like cell death mediated by ROS through targeting TrxR1. Nitrovin might be a promising anticancer lead for further development.

Fighting cancer by triggering non-canonical mitochondrial permeability transition-driven necrosis through reactive oxygen species induction.

Non-apoptotic necrosis shows therapeutic potential for the treatment of various diseases, especially cancer. Mitochondrial permeability transition (MPT)-driven necrosis is a form of non-apoptotic cell death triggered by oxidative stress and cytosolic Ca2+ overload, and relies on cyclophilin D (CypD). Previous reports demonstrated that isobavachalcone (IBC), a natural chalcone, has anticancer effect by apoptosis induction. Here, we found that IBC induced regulated necrosis in cancer cells. IBC triggered non-apoptotic cell death in lung and breast cancer cells mediated by reactive oxygen species (ROS). IBC caused mitochondrial injury and dysfunction as evidenced by mitochondrial Ca2+ overload, the opening of MPT pore, mitochondrial membrane potential collapse, and structural damages. IBC-triggered cell death could be remarkably reversed by the ROS scavengers, cyclosporin A (CsA) and hemin, whereas CypD silence and heme oxygenase-1 overexpression failed to do so. Protein kinase B, dihydroorotate dehydrogenase, and mitogen-activated protein kinases were not involved in IBC-induced necrosis as well. In addition, IBC showed an anticancer effect in a 4T1 breast cancer cell-derived allograft mouse model, and this effect was considerably reversed by CsA. Collectively, our results showed that IBC triggered non-canonical MPT-driven necrosis mediated by ROS in cancer cells, which might provide a novel strategy for fighting against cancer.

Exogenous iron impairs the anti-cancer effect of ascorbic acid both in vitro and in vivo.

INTRODUCTION: The anti-cancer effect of high concentrations of ascorbic acid (AA) has been well established while its underlying mechanisms remain unclear. The association between iron and AA has attracted great attention but was still controversial due to the complicated roles of iron in tumors. OBJECTIVES: Our study aims to explore the anti-cancer mechanisms of AA and the interaction between AA and iron in cancer. METHODS: The MTT and ATP assays were used to evaluate the cytotoxicity of AA. Reactive oxygen species (ROS) generation, calcium (Ca2+), and lipid peroxidation were monitored with flow cytometry. Mitochondrial dysfunction was assessed by mitochondrial membrane potential (MMP) detection with JC-1 or tetramethylrhodamine methyl ester (TMRM) staining. Mitochondrial swelling was monitored with MitoTracker Green probe. FeSO4 (Fe2+), FeCl3 (Fe3+), Ferric ammonium citrate (Fe3+), hemin chloride (Fe3+) were used as an iron donor to investigate the effects of iron on AA's anti-tumor activity. The in vivo effects of AA and iron were analyzed in xenograft zebrafish and allograft mouse models. RESULTS: High concentrations of AA exhibited cytotoxicity in a panel of cancer cells. AA triggered ROS-dependent non-apoptotic cell death. AA-induced cell death was essentially mediated by the accumulated intracellular Ca2+, which was partly originated from endoplasmic reticulum (ER). Surprisingly, exogenous iron could significantly reverse AA-induced ROS generation, Ca2+ overloaded, and cell death. Especially, the iron supplements significantly impaired the in vivo anti-tumor activity of AA. CONCLUSIONS: Our study elucidated the protective roles of iron in ROS/Ca2+ mediated necrosis triggered by AA both in vitro and in vivo, which might shed novel insight into the anti-cancer mechanisms and provide clinical application strategies for AA in cancer treatment.

Nannocystin ax, an eEF1A inhibitor, induces G1 cell cycle arrest and caspase-independent apoptosis through cyclin D1 downregulation in colon cancer in vivo.

Colorectal cancer (CRC) is one of the most common causes of cancer-related death worldwide. Nannocystin ax (NAN), a 21-membered cyclodepsipeptide initially isolated from myxobacteria of the Nannocystis genus, was found to target the eukaryotic elongation factor 1A (eEF1A). The current study was designed to evaluate the anticancer effect and underlying mechanisms of NAN with in vitro and in vivo models. Results showed that NAN induced G1 phase cell cycle arrest and caspase-independent apoptosis in HCT116 and HT29 human CRC cells. NAN significantly downregulated cyclin D1 level in a short time, but NAN did not affect the transcription level and ubiquitin-dependent degradation of cyclin D1. Furthermore, NAN treatment directly targeted eEF1A and partially decreased the synthesis of new proteins, contributing to the downregulation of cyclin D1. Besides, NAN significantly suppressed tumor growth in the zebrafish xenograft model. In conclusion, NAN triggered G1 phase cell cycle arrest through cyclin D1 downregulation and eEF1A-targeted translation inhibition and promoted caspase-independent apoptosis in CRC cells.

Therapeutic potential of isobavachalcone, a natural flavonoid, in murine experimental colitis by inhibiting NF-κB p65.

The incidence of ulcerative colitis (UC), one of the two types of inflammatory bowel disease, is increasing in many countries. Various natural products have been demonstrated with therapeutic potentials for UC. Herein, the therapeutic effects and mechanisms of isobavachalcone (IBC), a natural chalcone, were evaluated in dextran sulfate sodium (DSS)-induced colitis mice and lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. The results demonstrated that IBC treatment significantly improved the clinical symptoms, assessed by the disease activity index (DAI) scores and the histological changes of the colon. The levels of myeloperoxidase (MPO), TNF-α, IL-6, IL-1ß, and prostaglandin E2 (PGE2) in colon tissues were suppressed by IBC. The upregulation of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and NF-κB p65 in colon tissues were reversed by IBC as well. Furthermore, IBC significantly inhibited LPS-triggered secretion of TNF-α, IL-6, and nitrite, and nuclear translocation of NF-κB p65, in RAW264.7 cells. The luciferase reporter assay indicated that IBC significantly inhibited LPS-triggered transcription of toll-like receptor 4 (TLR4). Molecular docking results showed that the binding pocket of IBC was adjacent to Ser276 of p65-p50 heterodimer and IBC could form H-bond with Thr191. Collectively, these results demonstrated that IBC ameliorated colitis in mice possibly through inhibition of NF-κB p65.

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) inhibitors Necrostatin-1 (Nec-1) and 7-Cl-O-Nec-1 (Nec-1s) are potent inhibitors of NAD(P)H: Quinone oxidoreductase 1 (NQO1).

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) has been identified as a critical mediator of cell death (necroptosis and apoptosis) and inflammation. Necrostatin-1 (Nec-1) and 7-Cl-O-Nec-1 (Nec-1s) are widely used as selective small-molecule inhibitors of RIPK1 in various culture cells and disease models. NAD(P)H: quinone oxidoreductase 1 (NQO1) is a ubiquitous flavoenzyme that catalyzes the reduction and detoxification of quinones and other organic compounds. Here, we showed that Nec-1 and Nec-1s could bind and inhibit NQO1 activity. Similar to dicoumarol, the specific inhibitor of NQO1, both Nec-1 and Nec-1s significantly suppress NQO1-dependent cell death. However, dicoumarol failed to reverse necroptosis induced by TNFα/BV6/Z-VAD-FMK (TBZ) in HT29 cells. These findings suggest that besides RIPK1, NQO1 might be another target for Nec-1 and Nec-1s and provide new insights for the interpretation of Nec-1-based experimental results.

A novel strategy for glioblastoma treatment by induction of noptosis, an NQO1-dependent necrosis.

Glioblastoma (GBM) is one of the most prevalent malignant primary tumors in the human brain. Temozolomide (TMZ), the chemotherapeutic drug for GBM treatment, induces apoptosis. Unfortunately, apoptosis-resistance to TMZ results in treatment failure. GBM shows enhanced expression of NAD(P)H: quinone oxidoreductase 1 (NQO1). Recently, noptosis, a type of NQO1-dependent necrosis, was proposed. Here, we identified that tanshindiol B (TSB) inhibits GBM growth by induction of noptosis. TSB triggered significant cell death, which did not fit the criteria of apoptosis but oxidative stress-induced necrosis. Molecular docking, cellular thermal shift assay, and NQO1 activity assay revealed that TSB bind to and promptly activated NQO1 enzyme activity. As the substrate of NQO1, TSB induced oxidative stress, which resulted in dramatic DNA damage, poly (ADP-ribose) polymerase 1 (PARP1) hyperactivation, and NAD+ depletion, leading to necrotic cell death. These effects of TSB were completely abolished by specific NQO1 inhibitor dicoumarol (DIC). Furthermore, the c-Jun N-terminal kinase 1/2 (JNK1/2) plays an essential role in mediating TSB-induced cell death. Besides, TSB significantly suppressed tumor growth in a zebrafish xenograft model mediated by NQO1. In conclusion, these results showed that TSB was an NQO1 substrate and triggered noptosis of GBM. TSB exhibited anti-tumor potentials in GBM both in vitro and in vivo. This study provides a novel strategy for fighting GBM through the induction of noptosis.

Induction of programmed necrosis: A novel anti-cancer strategy for natural compounds.

Cell death plays a critical role in organism development and the pathogenesis of diseases. Necrosis is considered a non-programmed cell death in an extreme environment. Recent advances have provided solid evidence that necrosis could be programmed and quite a few types of programmed necrosis, such as necroptosis, ferroptosis, pyroptosis, paraptosis, mitochondrial permeability transition-driven necrosis, and oncosis, have been identified. The specific biomarkers, detailed signaling, and precise pathophysiological importance of programmed necrosis are yet to be clarified, but these forms of necrosis provide novel strategies for the treatment of various diseases, including cancer. Natural compounds are a unique source of lead compounds for the discovery of anti-cancer drugs. Natural compounds can induce both apoptosis and programmed necrosis. In this review, we summarized the recent progress of programmed necrosis and introduced their natural inducers. Noptosis, which is a novel type of programmed necrosis that is strictly dependent on NAD(P)H: quinone oxidoreductase 1-derived oxidative stress was proposed. Furthermore, the anti-cancer strategies that take advantage of programmed necrosis and the main concerns from the scientific community in this regard were discussed.

2-Methoxy-6-acetyl-7-methyljuglone (MAM) induced programmed necrosis in glioblastoma by targeting NAD(P)H: Quinone oxidoreductase 1 (NQO1).

Glioblastoma (GBM) are the most malignant brain tumors in humans and have a very poor prognosis. Temozolomide (TMZ), the only chemotherapeutic drug for GBM treatment, induced apoptosis but frequently developed resistance. Non-apoptotic cell death offers an alternative strategy to fight cancers. Our previous studies showed that 2-methoxy-6-acetyl-7-methyljuglone (MAM), a natural product, induced necroptosis in lung and colon cancer cells. The current study is designed to investigate its therapeutic potentials for GBM with in vitro and in vivo models. The protein expression of NAD(P)H: quinone oxidoreductase 1 (NQO1) in human GBM specimens were detected by immunohistochemistry. Effect of MAM on NQO1 was measured by recombinant protein and cellular thermal shift assay. The roles of NQO1 activation, superoxide (O2-) generation, calcium (Ca2+) accumulation, and c-Jun N-terminal kinase (JNK1/2) activation in MAM-induced cell death in U87 and U251 glioma cells were investigated. The effect of MAM on tumor growth was tested with a U251 tumor xenograft zebrafish model. Results showed that the NQO1 expression is positively correlated with the degree of malignancy in GBM tissues. MAM could directly bind and activate NQO1. Furthermore, MAM treatment induced rapid O2- generation, cytosolic Ca2+ accumulation, and sustained JNK1/2 activation. In addition, MAM significantly suppressed tumor growth in the zebrafish model. In conclusion, MAM induced GBM cell death by triggering an O2-/Ca2+/JNK1/2 dependent programmed necrosis. NQO1 might be the potential target for MAM and mediated its anticancer effect. This non-apoptotic necrosis might have therapeutic potentials for GBM treatment.

Pharmacological activities of dihydrotanshinone I, a natural product from Salvia miltiorrhiza Bunge.

Salvia miltiorrhiza Bunge (Danshen), a famous traditional Chinese herb, has been used clinically for the treatment of various diseases for centuries. Document data showed that tanshinones, a class of lipophilic abietane diterpenes rich in this herb, possess multiple biological effects in vitro and in vivo models. Among which, 15,16-dihydrotanshinone I (DHT) has received much attention in recent years. In this systematical review, we carefully selected, analyzed, and summarized high-quality publications related to pharmacological effects and the underlying mechanisms of DHT. DHT has anti-cancer, cardiovascular protective, anti-inflammation, anti-Alzheimer's disease, and other effects. Furthermore, several molecules such as hypoxia-inducible factor (HIF-1α), human antigen R (HuR), acetylcholinesterase (AchE), etc. have been identified as the potential targets for DHT. The diverse pharmacological activities of DHT provide scientific evidence for the local and traditional uses of Salvia miltiorrhiza Bunge. We concluded that DHT might serve as a lead compound for drug discovery in related diseases while further in-depth investigations are still needed.

A magic drug target: Androgen receptor.

Androgen receptor (AR) is closely associated with a group of hormone-related diseases including the cancers of prostate, breast, ovary, pancreas, etc and anabolic deficiencies such as muscle atrophy and osteoporosis. Depending on the specific type and stage of the diseases, AR ligands including not only antagonists but also agonists and modulators are considered as potential therapeutics, which makes AR an extremely interesting drug target. Here, we at first review the current understandings on the structural characteristics of AR, and then address why and how AR is investigated as a drug target for the relevant diseases and summarize the representative antagonists and agonists targeting five prospective small molecule binding sites at AR, including ligand-binding pocket, activation function-2 site, binding function-3 site, DNA-binding domain, and N-terminal domain, providing recent insights from a target and drug development view. Further comprehensive studies on AR and AR ligands would bring fruitful information and push the therapy of AR relevant diseases forward.