Inhibitor Dicoumarol Research Articles

DPP9 regulates NQO1 and ROS to promote resistance to chemotherapy in liver cancer cells

Chemotherapy has been the standard treatment for liver cancer. However, intrinsic or acquired drug resistance remains a major barrier to successful treatment. At present, the underlying molecular mechanisms of chemoresistance in liver cancer have not been elucidated. Dipeptidyl peptidase 9 (DPP9) is a member of the dipeptidyl peptidase IV family that has been found to be highly expressed in a variety of tumors, including liver cancer. It is unclear whether DPP9 affects chemoresistance in liver cancer. In this study, we find that DPP9 weakens the responses of liver cancer cells to chemotherapy drugs by up-regulating NQO1 and inhibiting intracellular ROS levels. In terms of mechanism, DPP9 inhibits ubiquitin-mediated degradation of NRF2 protein by binding to KEAP1, up-regulates NRF2 protein levels, promotes mRNA transcription of NQO1, and inhibits intracellular ROS levels. In addition, the NQO1 inhibitor dicoumarol can enhance the efficacy of chemotherapy drugs in liver cancer cells. Collectively, our findings suggest that inhibiting DPP9/NQO1 signaling can serve as a potential therapeutic strategy for liver cancer.

Skullcapflavone II, a novel NQO1 inhibitor, alleviates aristolochic acid I-induced liver and kidney injury in mice.

Aristolochic acid I (AAI) is a well established nephrotoxin and human carcinogen. Cytosolic NAD(P)H quinone oxidoreductase 1 (NQO1) plays an important role in the nitro reduction of aristolochic acids, leading to production of aristoloactam and AA-DNA adduct. Application of a potent NQO1 inhibitor dicoumarol is limited by its life-threatening side effect as an anticoagulant and the subsequent hemorrhagic complications. As traditional medicines containing AAI remain available in the market, novel NQO1 inhibitors are urgently needed to attenuate the toxicity of AAI exposure. In this study, we employed comprehensive 2D NQO1 biochromatography to screen candidate compounds that could bind with NQO1 protein. Four compounds, i.e., skullcapflavone II (SFII), oroxylin A, wogonin and tectochrysin were screened out from Scutellaria baicalensis. Among them, SFII was the most promising NQO1 inhibitor with a binding affinity (KD = 4.198 μmol/L) and inhibitory activity (IC50 = 2.87 μmol/L). In human normal liver cell line (L02) and human renal proximal tubular epithelial cell line (HK-2), SFII significantly alleviated AAI-induced DNA damage and apoptosis. In adult mice, oral administration of SFII dose-dependently ameliorated AAI-induced renal fibrosis and dysfunction. In infant mice, oral administration of SFII suppressed AAI-induced hepatocellular carcinoma initiation. Moreover, administration of SFII did not affect the coagulation function in short term in adult mice. In conclusion, SFII has been identified as a novel NQO1 inhibitor that might impede the risk of AAI to kidney and liver without obvious side effect.

Osthole Induces Apoptosis and Caspase-3/GSDME-Dependent Pyroptosis via NQO1-Mediated ROS Generation in HeLa Cells.

Osthole is a natural coumarin which has been proved to inhibit growth of cancer cells by inducing cell death, while its mechanism was considered to be just caused by apoptosis. In our study, we found that osthole activated not just apoptosis, but also pyroptosis which is a form of regulated cell death accompanied by loss of cell membrane integrity and lactate dehydrogenase (LDH) release. Caspase-3 is a key protein of apoptosis as well as pyroptosis. The apoptosis and pyroptosis induced by osthole were all inhibited by irreversible caspase-3 inhibitor Z-DEVD-FMK. Meanwhile, knockdown of gasdermin E (GSDME) only reduced the osthole-induced pyroptosis but did not affect the occurrence of apoptosis. Our proteomic analysis revealed that the expression of NAD(P)H: quinone oxidoreductase 1 (NQO1) was decreased in osthole-treated cells. Moreover, NQO1 inhibition by osthole induced the overproduction of reactive oxygen species (ROS), as well as apoptosis and pyroptosis. ROS inhibitor N-Acetyl-L-cysteine (NAC) not only reduced osthole-induced apoptosis but also reversed its effect on the pyroptosis. Meanwhile, knockdown of NQO1 by si-NQO1 or its inhibitor dicoumarol (DIC) not only enhanced ROS generation but also strengthened the GSDME-mediated pyroptosis. Finally, we demonstrated that osthole inhibited tumor growth and the expression of NQO1 in a HeLa xenograft mode. Similar to the results in vitro, osthole stimulated the activation of caspase-3, PARP, and GSDME in vivo. Taken together, all these data suggested that osthole induced apoptosis and caspase-3/GSDME-mediated pyroptosis via NQO1-mediated ROS accumulation.

Virtual Screening for Potential Phytobioactives as Therapeutic Leads to Inhibit NQO1 for Selective Anticancer Therapy.

NAD(P)H:quinone acceptor oxidoreductase-1 (NQO1) is a ubiquitous flavin adenine dinucleotide-dependent flavoprotein that promotes obligatory two-electron reductions of quinones, quinonimines, nitroaromatics, and azo dyes. NQO1 is a multifunctional antioxidant enzyme whose expression and deletion are linked to reduced and increased oxidative stress susceptibilities. NQO1 acts as both a tumor suppressor and tumor promoter; thus, the inhibition of NQO1 results in less tumor burden. In addition, the high expression of NQO1 is associated with a shorter survival time of cancer patients. Inhibiting NQO1 also enables certain anticancer agents to evade the detoxification process. In this study, a series of phytobioactives were screened based on their chemical classes such as coumarins, flavonoids, and triterpenoids for their action on NQO1. The in silico evaluations were conducted using PyRx virtual screening tools, where the flavone compound, Orientin showed a better binding affinity score of −8.18 when compared with standard inhibitor Dicumarol with favorable ADME properties. An MD simulation study found that the Orientin binding to NQO1 away from the substrate-binding site induces a potential conformational change in the substrate-binding site, thereby inhibiting substrate accessibility towards the FAD-binding domain. Furthermore, with this computational approach we are offering a scope for validation of the new therapeutic components for their in vitro and in vivo efficacy against NQO1.

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.

\u03b2-Lapachone Induces Acute Oxidative Stress in Rat Primary Astrocyte Cultures that is Terminated by the NQO1-Inhibitor Dicoumarol

β-lapachone (β-lap) is reduced in tumor cells by the enzyme NAD(P)H: quinone acceptor oxidoreductase 1 (NQO1) to a labile hydroquinone which spontaneously reoxidises to β-lap, thereby generating reactive oxygen species (ROS) and oxidative stress. To test for the consequences of an acute exposure of brain cells to β-lap, cultured primary rat astrocytes were incubated with β-lap for up to 4 h. The presence of β-lap in concentrations of up to 10 µM had no detectable adverse consequences, while higher concentrations of β-lap compromised the cell viability and the metabolism of astrocytes in a concentration- and time-dependent manner with half-maximal effects observed for around 15 µM β-lap after a 4 h incubation. Exposure of astrocytes to β-lap caused already within 5 min a severe increase in the cellular production of ROS as well as a rapid oxidation of glutathione (GSH) to glutathione disulfide (GSSG). The transient cellular accumulation of GSSG was followed by GSSG export. The β-lap-induced ROS production and GSSG accumulation were completely prevented in the presence of the NQO1 inhibitor dicoumarol. In addition, application of dicoumarol to β-lap-exposed astrocytes caused rapid regeneration of the normal high cellular GSH to GSSG ratio. These results demonstrate that application of β-lap to cultured astrocytes causes acute oxidative stress that depends on the activity of NQO1. The sequential application of β-lap and dicoumarol to rapidly induce and terminate oxidative stress, respectively, is a suitable experimental paradigm to study consequences of a defined period of acute oxidative stress in NQO1-expressing cells.

A Dynamic Core in Human NQO1 Controls the Functional and Stability Effects of Ligand Binding and Their Communication across the Enzyme Dimer.

Human NAD(P)H:quinone oxidoreductase 1 (NQO1) is a multi-functional protein whose alteration is associated with cancer, Parkinson’s and Alzheimer´s diseases. NQO1 displays a remarkable functional chemistry, capable of binding different functional ligands that modulate its activity, stability and interaction with proteins and nucleic acids. Our understanding of this functional chemistry is limited by the difficulty of obtaining structural and dynamic information on many of these states. Herein, we have used hydrogen/deuterium exchange monitored by mass spectrometry (HDXMS) to investigate the structural dynamics of NQO1 in three ligation states: without ligands (NQO1apo), with FAD (NQO1holo) and with FAD and the inhibitor dicoumarol (NQO1dic). We show that NQO1apo has a minimally stable folded core holding the protein dimer, with FAD and dicoumarol binding sites populating binding non-competent conformations. Binding of FAD significantly decreases protein dynamics and stabilizes the FAD and dicoumarol binding sites as well as the monomer:monomer interface. Dicoumarol binding further stabilizes all three functional sites, a result not previously anticipated by available crystallographic models. Our work provides an experimental perspective into the communication of stability effects through the NQO1 dimer, which is valuable for understanding at the molecular level the effects of disease-associated variants, post-translational modifications and ligand binding cooperativity in NQO1.

Activation of NQO-1 mediates the augmented contractions of isolated arteries due to biased activity of soluble guanylyl cyclase in their smooth muscle.

Earlier studies on isolated arteries demonstrated that the para-quinone thymoquinone, like acute hypoxia, induces augmentation of contractions, depending on biased activity of soluble guanylyl cyclase (sGC), generating inosine-3',5'-cyclic monophosphate (cyclic IMP) rather than guanosine-3',5'-cyclic monophosphate (cyclic GMP). NAD(P)H:quinone oxidoreductase 1 (NQO-1), the enzyme responsible for biotransformation of quinones into hydroquinones, was examined for its involvement in these endothelium-dependent augmentations, establishing a link between the metabolism of quinones by NQO-1 and biased sGC activity. Isolated arteries of Sprague-Dawley rats (aortae and mesenteric arteries) and farm pigs (coronary arteries) were studied for measurement of changes in tension and collected to measure NQO-1 activity or its protein level. β-lapachone, an ortho-quinone and hence substrate of NQO-1, increased the activity of the enzyme and augmented contractions in arteries with endothelium. This augmentation was inhibited by endothelium removal and inhibitors of endothelial NO synthase (eNOS), sGC, or NQO-1; in preparations without endothelium or treated with an eNOS inhibitor, it was restored by the NO donor DETA NONOate and by ITP and cyclic IMP, revealing biased sGC activity as the underlying mechanism, as with thymoquinone. Hydroquinone, the end product of quinone metabolism by NQO-1, augmented contractions depending on sGC activation but in an endothelium-independent manner. In coronary arteries, repeated acute hypoxia caused similar augmentations as those to quinones that were inhibited by the NQO-1 inhibitor dicoumarol. Augmentations of contraction observed with different naturally occurring quinones and with acute hypoxia are initiated by quinone metabolism by NQO-1, in turn interfering with the NO/biased sGC pathway, suggesting a possibly detrimental role of this enzyme in ischemic cardiovascular disorders.

Upregulation of Enzymatic Idebenone Reduction Activity after Proinflammatory Stimulation of Microglia

Mitochondrial bioenergetic dysfunction is a component of nearly all neurodegenerative diseases. One strategy to overcome this dysfunction is to replace the loss of mitochondrial reducing equivalents using exogenous electron donors. The drug was recently approved in Europe for the treatment of Leber’s Hereditary Optic Neuropathy, a mitochondrial disease due to deficient Complex I activity. For efficacy, it is believed that idebenone requires enzymatic reduction to idebenol. Previously, quinone reduction activity was mostly attributed to NAD(P)H quinone oxidoreductases, particularly NQO1. In the brain, NQO1 is primarily expressed in astrocytes; we therefore hypothesized that astrocytes would display higher NAD(P)H-dependent idebenone reduction activity compared to other brain cell types. Using lysates derived from primary cells, we found that rat cortical astrocytes have the highest capacity for idebenone reduction, with little to no activity observed in rat cortical neurons or microglia. As expected, idebenone reduction in astrocytes was fully inhibited by the NQO1 inhibitor dicoumarol. Interestingly, we found a significant increase in idebenone reduction potential in lysates derived from microglia treated with the pro-inflammatory stimulus lipopolysaccharide (LPS) plus interferon-gamma (IFN-γ) for 18 hours. This activity was not sensitive to dicoumarol and was not observed in rat bone marrow-derived macrophages similarly stimulated with LPS/IFN-γ. Preliminary results suggest that it may be a multi-subunit flavoprotein. Identification of the enzyme responsible for this novel idebenone-reducing activity may lead to improved therapeutic strategies using idebenone as well as suggest other diseases wherein idebenone may have a beneficial effect.

Pressurized Hot Water Extraction as a Viable Bioprospecting Tool: Isolation of Coumarin Natural Products from Previously Unexamined Correa (Rutaceae) Species

AbstractThe potential of a recently developed pressurized hot water extraction (PHWE) method employing an unmodified household espresso machine to facilitate rapid bioprospecting was investigated. A survey of some 16 Australian native plant species identified three Correa (Rutaceae) species containing significant quantities of coumarin natural products. As part of this study, seselin, (+)‐epoxysuberosin, alosin, (–)‐meranzin and heraclenin were isolated and, subsequently, used to synthesize the related natural products (+)‐trachypleuranin A and (–)‐yuehgesin B. In addition, seselin, (+)‐epoxysuberosin, alosin, and (–)‐meranzin were found to inhibit NQO1 activity. Interestingly, (–)‐meranzin showed inhibitory activity comparable to the known potent NQO1 inhibitor dicoumarol.

On the Role of DT-Diaphorase Inhibition in Aminochrome-Induced Neurotoxicity In Vivo.

Dopamine oxidation in the pathway leading to neuromelanin formation generates the ortho-quinone aminochrome, which is potentially neurotoxic but normally rapidly converted by DT-diaphorase to nontoxic leukoaminochrome. However, when administered exogenously into rat striatum, aminochrome is able to produce damage to dopaminergic neurons. Because of a recent report that substantia nigra pars compacta (SNpc) tyrosine hydroxylase (T-OH) levels were unaltered by aminochrome when there was cell shrinkage of dopaminergic neurons along with a reduction in striatal dopamine release, the following study was conducted to more accurately determine the role of DT-diaphorase in aminochrome neurotoxicity. In this study, a low dose of aminochrome (0.8nmol) with or without the DT-diaphorase inhibitor dicoumarol (0.2nmol) was injected into the left striatum of rats. Intrastriatal 6-hydroxydopamine (6-OHDA, 32nmol) was used as a positive neurotoxin control in other rats. Two weeks later, there was significant loss in numbers of T-OH immunoreactive fibers in SNpc, also a loss in cell density in SNpc, and prominent apomorphine (0.5mg/kg sc)-induced contralateral rotations in rats that had been treated with aminochrome, with aminochrome/dicoumarol, or with 6-OHDA. Findings demonstrate that neurotoxic aminochrome is able to exert neurotoxicity only when DT-diaphorase is suppressed-implying that DT-diaphorase is vital in normally suppressing toxicity of in vivo aminochrome, generated in the pathway towards neuromelanin formation.

NQO1 and CYP450 reductase decrease the systemic exposure of rifampicin-quinone and mediate its redox cycle in rats

Rifampicin (RIF) is used in regimens for infections caused by Mycobacteria accompanied by serious adverse reactions. Rifampicin-quinone (RIF-Q) is a major autoxidation product of RIF. It is not clear whether RIF-Q plays a role in RIF induced adverse reactions. Investigation of the systemic exposure of RIF-Q is helpful to better understand the role of RIF-Q in RIF induced adverse reactions. In this study, a simple and reproducible high performance liquid chromatography-mass spectrometry (LC–MS) method involving a procedure to prevent the RIF from oxidation for simultaneous quantification of RIF and RIF-Q in rat plasma has been developed and validated, and applied to elucidate the systemic exposure of RIF-Q in rats. The pharmacokinetics data showed that the systemic exposure of RIF-Q was very low (0.67% of RIF, AUC0-24) in rats after oral administration of RIF. However, RIF-Q may undergo the redox cycle in vivo by the evidence that the majority of RIF-Q was reduced to RIF after an oral dose of RIF-Q. Pretreatment with the NAD(P)H: quinone oxidoreductase 1 (NQO1) specific inhibitor dicoumarol and/or cytochrome P450 reductase (CPR) inhibitor diphenyleneiodonium suppressed the redox cycle and significantly increased the systemic exposure of RIF-Q. The inhibitors also attenuated the redox cycle induced reactive oxygen species formation and cytotoxicity in RIF-Q-treated HepG2 cells. These results indicate that NQO1 and CPR play an important role in redox cycle of RIF-Q and may thus contribute to RIF-induced adverse reactions.

Conformational dynamics is key to understanding loss-of-function of NQO1 cancer-associated polymorphisms and its correction by pharmacological ligands.

Protein dynamics is essential to understand protein function and stability, even though is rarely investigated as the origin of loss-of-function due to genetic variations. Here, we use biochemical, biophysical, cell and computational biology tools to study two loss-of-function and cancer-associated polymorphisms (p.R139W and p.P187S) in human NAD(P)H quinone oxidoreductase 1 (NQO1), a FAD-dependent enzyme which activates cancer pro-drugs and stabilizes several oncosuppressors. We show that p.P187S strongly destabilizes the NQO1 dimer in vitro and increases the flexibility of the C-terminal domain, while a combination of FAD and the inhibitor dicoumarol overcome these alterations. Additionally, changes in global stability due to polymorphisms and ligand binding are linked to the dynamics of the dimer interface, whereas the low activity and affinity for FAD in p.P187S is caused by increased fluctuations at the FAD binding site. Importantly, NQO1 steady-state protein levels in cell cultures correlate primarily with the dynamics of the C-terminal domain, supporting a directional preference in NQO1 proteasomal degradation and the use of ligands binding to this domain to stabilize p.P187S in vivo. In conclusion, protein dynamics are fundamental to understanding loss-of-function in p.P187S, and to develop new pharmacological therapies to rescue this function.

Synthesis and evaluation of (±)-dunnione and its ortho-quinone analogues as substrates for NAD(P)H:quinone oxidoreductase 1 (NQO1)

Natural product (±)-dunnione (2) and its ortho-quinone analogues (3-8) were synthesized and found to be substrates for NQO1. The structure-activity relationship study revealed that the biological activity was favored by the presence of methyl group at the C ring and methoxy group at the A ring. The docking studies supported the rationalization of the metabolic studies. Deeper location in the active site of NQO1, interactions with hydrophobic pocket and C-H…π interactions with the adjacent Phe178 residue contributed to the better catalytic efficiency and specificity to NQO1. Cytotoxicity studies and determination of superoxide (O2(-)) production in the presence and absence of the NOQ1 inhibitor dicoumarol confirmed that the ortho-quinones exerted their antitumor activity through NQO1-mediated ROS production by redox cycling.

2-Substituted 3-methylnaphtho[1,2-b]furan-4,5-diones as novel L-shaped ortho-quinone substrates for NAD(P)H:quinone oxidoreductase (NQO1)

A series of L-shaped ortho-quinone analogs were designed by analyzing the binding mode with NQO1. Metabolic studies demonstrated that compounds 2m, 2n and 2q exhibited higher metabolic rates than β-lapachone. The docking studies, which supported the rationalization of the metabolic studies, constituted a prospective rational basis for the development of optimized ortho-quinone analogs. Besides, good substrates (2m, 2n and 2r) for NQO1 showed higher selective toxicity than β-lapachone toward A549 (NQO1-rich) cancer cells versus H596 (NQO1-deficient) cells. Determination of superoxide (O2•-) production and in vitro cytotoxicity evaluation in the presence of the NQO1 inhibitor dicoumarol confirmed that the ortho-quinones exerted their antitumor activity through NQO1-mediated ROS production by redox cycling. It was suggested that the L-shaped quinone substrates for NQO1 possessed better specificity and safety than β-lapachone.

Arsenic Trioxide Induces Apoptosis in Human Platelets via C-Jun NH2-Terminal Kinase Activation

Arsenic trioxide (ATO), one of the oldest drugs in both Western and traditional Chinese medicine, has become an effective anticancer drug, especially in the treatment of acute promyelocytic leukemia (APL). However, thrombocytopenia occurred in most of ATO-treated patients with APL or other malignant diseases, and the pathogenesis remains unclear. Here we show that ATO dose-dependently induces depolarization of mitochondrial inner transmembrane potential (ΔΨm), up-regulation of Bax and down-regulation of Bcl-2 and Bcl-XL, caspase-3 activation, and phosphotidylserine (PS) exposure in platelets. ATO did not induce surface expression of P-selectin and PAC-1 binding, whereas, obviously reduced collagen, ADP, and thrombin induced platelet aggregation. ATO dose-dependently induced c-Jun NH2-terminal kinase (JNK) activation, and JNK specific inhibitor dicumarol obviously reduced ATO-induced ΔΨm depolarization in platelets. Clinical therapeutic dosage of ATO was intraperitoneally injected into C57 mice, and the numbers of circulating platelets were significantly reduced after five days of continuous injection. The data demonstrate that ATO induces caspase-dependent apoptosis via JNK activation in platelets. ATO does not incur platelet activation, whereas, it not only impairs platelet function but also reduces circulating platelets in vivo, suggesting the possible pathogenesis of thrombocytopenia in patients treated with ATO.

Arsenic Trioxide Induces Platelet Apoptosis

Background Arsenic trioxide (ATO), a component of Traditional Chinese Medicine, is known as an effective anticancer drug especially in the treatment of acute promyelocytic leukaemia (APL). APL has emerged as the most curable subtype of acute myeloid leukaemia since the widely use of arsenic trioxide-based chemotherapy. However, recent researches showed that thrombocytopenia occurred in 79% of the relapsed or refractory APL patients treated with arsenic trioxide, and part of the APL patients had to be stopped treatment because of catastrophic bleeding, such as intracranial and pulmonary haemorrhage. Thrombocytopenia also occurred in 43% of the myelodysplastic syndrome patients treated with arsenic trioxide. Recently, arsenic trioxide has been proved to have a pro-apoptotic effect on various kinds of nucleated tumour cells or non-tumour cells. The effect of ATO on enucleated platelet, however, still remains unclear. The aim of current study is to investigate whether ATO induces platelet apoptosis. Methods Washed platelets (3 × 108/ml) were incubated with different concentrations of ATO or vehicle at 37°C for 4 hours. Then, mitochondrial inner transmembrane potential (ΔΨm), phosphatidylserine (PS) exposure, P-selection and PAC-1 binding were tested by flow cytometry. In the mean time, the treated platelets were analyzed by western blot for the expression levels of pro-apoptotic protein (Bax), and anti-apoptotic proteins (Bcl-2 and Bcl-XL). Activation of caspase-3 and c-jun NH2-terminal kinase (JNK) was also examined by western blot. To test whether inhibition of JNK can attenuate ATO-induced platelet apoptosis, JNK specific inhibitor dicumarol was used and ΔΨm was analysis by flow cytometry. For platelet aggregation analysis, PRP was incubated with ATO (2 μM) for 1 hr and then subjected to platelet aggregation assay by collagen. To further investigate whether ATO incurs thrombocytopenia in vivo, clinical therapeutic dosage of ATO (or vehicle (0.9% NS) control) was intraperitoneally injected into C57 mice, and the numbers of circulating platelets were counted. Results and Conclusions ATO dose-dependently induces depolarization of mitochondrial inner ΔΨm, up-regulation of Bax and down-regulation of Bcl-2 and Bcl-XL, caspase-3 activation, and PS exposure in platelets. ATO did not induce surface expression of P-selectin and PAC-1 binding, whereas, obviously reduced collagen-induced platelet aggregation. ATO dose-dependently induced JNK activation, and JNK specific inhibitor dicumarol obviously reduced ATO-induced ΔΨm depolarization in platelets. Clinical therapeutic dosage of ATO was intraperitoneally injected into C57 mice, and the numbers of circulating platelets were significantly reduced after 12 and 24 hours of injection. Taken together, the data demonstrate that ATO induces caspase-dependent apoptosis via mitochondria-mediated intrinsic pathway in platelets. ATO does not incur platelet activation, whereas, it not only impairs platelet function but also incurs thrombocytopenia in vivo, suggesting the possible pathogenesis of thrombocytopenia in patients treated with ATO. Disclosures: No relevant conflicts of interest to declare.

Sunitinib enhances neuronal survival in vitro via NF-κB-mediated signaling and expression of cyclooxygenase-2 and inducible nitric oxide synthase

BackgroundAngiogenesis is tightly linked to inflammation and cancer. Regulation of angiogenesis is mediated primarily through activation of receptor tyrosine kinases, thus kinase inhibitors represent a new paradigm in anti-cancer therapy. However, these inhibitors have broad effects on inflammatory processes and multiple cell types. Sunitinib is a multitarget receptor tyrosine kinase inhibitor, which has shown promise for the treatment of glioblastoma, a highly vascularized tumor. However, there is little information as to the direct effects of sunitinib on brain-derived neurons. The objective of this study is to explore the effects of sunitinib on neuronal survival as well as on the expression of inflammatory protein mediators in primary cerebral neuronal cultures.MethodsPrimary cortical neurons were exposed to various doses of sunitinib. The drug-treated cultures were assessed for survival by MTT assay and cell death by lactate dehydrogenase release. The ability of sunitinib to affect NF-κB, COX2 and NOS2 expression was determined by western blot. The NF-κB inhibitors dicoumarol, SN50 and BAY11-7085 were employed to assess the role of NF-κB in sunitinib-mediated effects on neuronal survival as well as COX2 and NOS2 expression.ResultsTreatment of neuronal cultures with sunitinib caused a dose-dependent increase in cell survival and decrease in neuronal cell death. Exposure of neurons to sunitinib also induced an increase in the expression of NF-κB, COX2 and NOS2. Inhibiting NF-κB blunted the increase in cell survival and decrease in cell death evoked by sunitinib. Treatment of cell cultures with both sunitinib and NF-κB inhibitors mitigated the increase in COX2 and NOS2 caused by sunitinib.ConclusionsSunitinib increases neuronal survival and this neurotrophic effect is mediated by NF-κB. Also, the inflammatory proteins COX2 and NOS2 are upregulated by sunitinib in an NF-κB-dependent manner. These data are in agreement with a growing literature suggesting beneficial effects for inflammatory mediators such as NF-κB, COX2 and NOS2 in neurons. Further work is needed to fully explore the effects of sunitinib in the brain and its possible use as a treatment for glioblastoma. Finally, sunitinib may be useful for the treatment of a range of central nervous system diseases where neuronal injury is prominent.

Abstract 589: NQO1-dependent anticancer action with combination β-lapachone and ionizing radiation in the head and neck cancer.

Abstract The number of new cases of oral cavity, pharynx and larynx cancers is estimated be 52,610 in 2012 with an expected 11,500 deaths. Despite progressive improvements in outcome, current treatment approaches still result in an overall survival rate of approximately 50% at five years for locally advanced head and neck cancer (HNC). Additional therapeutic approaches are needed to further enhance outcomes. NAD(P)H:quinone oxidoreductase 1 (NQO1) is a two-electron oxidoreductase that reduces quinones to hydroquinones and has been shown to be overexpressed in a number of solid tumors. For the first time, our studies in 45 HNC cell lines identified NQO1 overexpression in more than 70% of HNC cell lines. Furthermore over-expression of NQO1 has been shown to be a critical determinant for tumor-selective therapy using β-lapachone (β-lap), a substrate of NQO1 that undergoes partial reduction and spontaneous reversion, creating a futile cycle that produces very high levels of reactive oxygen species. HNC cell lines that overexpress NQO1 are significantly killed by treatment with β-lap. The anti-cancer action of β-lap in HNC cells is initiated and processed through five essential downstream mediators: (i) reactive oxygen species (ROS), particularly hydrogen peroxide (H2O2); (ii) DNA single strand breaks (SSBs) induced by ROS; (iii) hyperactivation of poly(ADP-ribose)polymerase-1 (PARP1) and accompanying poly(ADP-ribose)-PARP1 (PAR) formation; (iv) dramatic NAD+/ATP depletion; and (v) programmed necrosis (necroptosis). In combination with ionizing radiation (IR), β-Lap radiosensitized NQO1(+) HNC cells under conditions where nontoxic doses of either agent alone did not achieve threshold levels of DNA lesions and /or SSBs required for PARP-1 hyper-activation. Furthermore, combination therapy with β-lap and IR significantly enhanced γ-H2AX and 53BP1 foci formation and PARP-1 hyper-activation which was associated with ATP loss and induction of programmed cell death. Furthermore, the anticancer effects of combination β-Lap and IR were blocked by the NQO1 inhibitor dicoumarol. Our findings offer a strategy for the clinical utilization of β-lap as a radiosensitizer in HNC. Citation Format: Long Shan Li, Srilakshmi Reddy, Jinming Gao, David A. Boothman, Baran Sumer, John S. Yordy. NQO1-dependent anticancer action with combination β-lapachone and ionizing radiation in the head and neck cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 589. doi:10.1158/1538-7445.AM2013-589

The metabolism of 9-chloro-β-lapachone and its effects in isolated hepatocytes. The involvement of NAD(P)H:quinone oxidoreductase 1 (NQO1)

A β-lapachone analogue (3,4-dihydro-2,2-dimethyl-9-chloro-2H-naphtho[1,2b]pyran-5,6-dione) (9-chloro β-lapachone), named CGQ, with antitumoral, antiviral and antitrypanocidal activities was assayed for cytotoxic effects on isolated rat hepatocytes. The incubation of hepatocytes with this o-naphthoquinone showed (a) decreased adenylate energy charge, as a result of a decrease in ATP, and an increase in AMP levels; (b) increased NADP+ content, with a concomitant decrease of NADPH, NADH and NAD+ content; (c) decreased GSH content, accompanied by an increase in GSSG formation; (d) stimulated oxygen uptake as well as increased superoxide anion production and hydrogen peroxide formation; (e) inhibited lipid peroxidation; (f) hepatocyte viability was not reduced unless the NQO1 inhibitor dicoumarol was present. We hypothesize that the cytotoxicity of CGQ in dicoumarol-treated hepatocytes was the result of inhibition of the NQO1 detoxification pathway, thus allowing more quinone to be metabolized towards the one-electron pathway to form reactive semiquinones and/or reactive oxygen species. The results obtained indicate a protective role of NQO1 in preventing CGQ cytotoxicity in isolated rat hepatocytes.