Modulation of HIF-2α PAS-B domain contributes to physiological responses.

Hypoxia-inducible factors (HIFs) are transcription factors in the basic helix-loop-helix PER-ARNT-SIM (bHLH-PAS) protein family that contain internal hydrophobic cavities within their PAS-A and PAS-B domains. Among HIFs, the HIF-2α PAS-B domain contains a relatively large cavity exploited for the development of specific artificial ligands such as PT2399. Administration of PT2399 could suppress HIF-2α target gene expression without affecting HIF-1 activity in mice under hypoxia conditions. A single mutation (S305M) within the HIF-2α PAS-B domain suppressed HIF-2α activity while conferring resistance to PT2399 in vivo, indicating the vital role of PAS-B domain in HIF-2α hypoxia response. In contrast, the mutant mice did not phenocopy PT2399 intervention in wild-type mice under metabolic stress. Under a high-fat diet (HFD), the mutant mice exert enhanced adipogenesis and obtain larger adipose mass and body weight gain compared to wild type. However, administration of PT2399 along with HFD feeding sufficiently suppressed HFD-induced body weight and adipose mass increase through suppression of adipogenesis and lipogenesis. The accompanying decreased lipid accumulation in the liver and improved glucose tolerance in wild-type mice were not observed in the mutant mice indicating negative regulation of HIF-2α on obesity and a complex role for the PAS-B domain in metabolic regulation. Notably, short-term administration of PT2399 to obese mice decreased adipose mass and improved metabolic condition. These results indicate a regulatory role for HIF-2α in obesity progression and suggest a therapeutic opportunity for PT2399 in obesity and associated metabolic disorders.

A novel colorimetric immunoassay based on enzyme-regulated instant generation of Turnbull's blue for the sensitive determination of ochratoxin A.

The aim of this study was to develop a novel colorimetric sensing method based on enzyme-regulated instant generation of Turnbull's blue, serving as a chromogenic agent, for a sensitive immunoassay for the determination of ochratoxin A (OTA). Unlike the traditional enzyme-linked immunosorbent assay (ELISA), the chromogenic reaction reported herein relies on the immediate formation of Turnbull's blue. K3[Fe(CN)6] rapidly forms a coordinate bond with iron(ii), yielding a blue product. Meanwhile, glucose oxidase (GOx) catalyzes glucose hydrolysis to produce hydrogen peroxide (H2O2), which was used to inhibit the formation of Turnbull's blue by oxidizing iron(ii) to iron(iii). Thus, Turnbull's blue was generated in an enzyme-regulated manner. Accordingly, a competitive-type colorimetric enzyme immunoassay was established using a GOx based nanolabel. Under optimal conditions, the absorbance increased upon increasing the target OTA concentration in the range of 0.01-10 ng mL-1 with a detection limit of 8.3 pg mL-1 estimated at the 3Sblank level. The assay accuracy was validated by analyzing spiked wine samples. The present results potentially provide novel insights into the development of Turnbull's blue-based biological detection methods and colorimetric immunoassay strategies.

MIR196A2 rs11614913 C > T polymorphism correlates with an increased risk of hepatopulmonary syndrome in liver cirrhosis: a case-control study in China.

OBJECTIVE: This case-control study is designed to explore the relationship between microRNA-196a2 (MIR196A2) rs11614913 C > T polymorphism and the risk of hepatopulmonary syndrome (HPS) in liver cirrhosis. METHODS: From January 2013 to January 2015, 163 liver cirrhosis patients with HPS (case group), 264 liver cirrhosis patients without HPS (control group), and 195 healthy people (normal group) were selected. A DNA extraction kit was used to extract plasma DNA from peripheral blood. Polymerase chain reaction-restriction fragment length polymorphism was used to detect the allele and genotype frequencies of MIR196A2 C > T polymorphism. Real-time quantitative polymerase chain reaction was adopted to detect the relative expression of MIR196A. RESULTS: The frequencies of C allele in the case group were higher than those in the control and normal groups (all P < 0.05), whereas no significant difference was found between the control and normal groups, which indicated that MIR196A2 C > T polymorphism was closely associated with an increased risk of HPS in patients with liver cirrhosis. Compared with the normal group, the relative expression of MIR196A in the case group was significantly increased (P < 0.05), but there was no significant difference in the control group (P > 0.05). In the case group, compared with patients carrying the TT genotype, the relative expression of MIR196A of patients carrying the C allele (CT + CC) evidently increased (P < 0.05). CONCLUSIONS: The MIR196A2 rs11614913 C > T polymorphism may contribute to an increased risk of HPS in liver cirrhosis patients.

Linc-ROR induces epithelial-mesenchymal transition and contributes to drug resistance and invasion of breast cancer cells.

We aimed to investigate the role of large intergenic noncoding RNA regulator of reprogramming (linc-ROR) in the chemotherapy resistance of human breast cancer (BC) cells and its mechanism. A total of 142 patients diagnosed with BC in the First Affiliated Hospital, Zhejiang University between January 2012 and January 2014 were enrolled in our study. The BC tissues and the adjacent normal tissues (5 cm away from tumor tissue) of the enrolled patients were selected, and human BC cell lines (MCF10A, SK-BR-3, MCF-7, Bcap-37, MDA-MB-231, and T47D) were also selected. Quantitative real-time polymerase chain reaction (qRT-PCR), Western blot, 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay, and Transwell were applied in our study. Expression level of linc-ROR messenger RNA (mRNA) in BC tissues was clearly higher than that in adjacent normal tissues, and significant difference was found between expression level of linc-ROR mRNA and lymph node metastasis (all P < 0.05). Linc-ROR was highly expressed in others BC cell lines compared with that in immortalized mammary epithelial cells (MECs) MCF10A (both P < 0.05), while MDA-MB231 cell presented the higher expression (P < 0.001). Under different concentrations of 5-FU and paclitaxel in MDA-MB231 cell, E-cadherin mRNA and protein expressions increased gradually with the increase of concentrations, and Vimentin and N-cadherin mRNA and protein expressions decreased gradually with the decrease of concentrations (all P < 0.05). Compared with shCtrl group, MDA-MB231 cell in shROR group presented higher sensibility of 5-FU and paclitaxel with increased E-cadherin expression, decreased Vimentin and N-cadherin expression and invasion ability (all P < 0.05). Compared with vector cell, overexpressed linc-ROR cell presented decreased sensibility of 5-FU and paclitaxel with decreased E-cadherin expression, increased Vimentin, N-cadherin expression, and invasion ability (all P < 0.05). Our study demonstrated that linc-ROR is an important marker for multidrug resistance of BC, and its up-regulation is important for chemotherapy tolerance and invasion of BC.

Hsp90 chaperone inhibitor 17-AAG attenuates Aß-induced synaptic toxicity and memory impairment.

The excessive accumulation of soluble amyloid peptides (Aß) plays a crucial role in the pathogenesis of Alzheimer's disease (AD), particularly in synaptic dysfunction. The role of the two major chaperone proteins, Hsp70 and Hsp90, in clearing misfolded protein aggregates has been established. Despite their abundant presence in synapses, the role of these chaperones in synapses remains elusive. Here, we report that Hsp90 inhibition by 17-AAG elicited not only a heat shock-like response but also upregulated presynaptic and postsynaptic proteins, such as synapsin I, synaptophysin, and PSD95 in neurons. 17-AAG treatment enhanced high-frequency stimulation-evoked LTP and protected neurons from synaptic damage induced by soluble Aß. In AD transgenic mice, the daily administration of 17-AAG over 7 d resulted in a marked increase in PSD95 expression in hippocampi. 17-AAG treatments in wild-type C57BL/6 mice challenged by soluble Aß significantly improved contextual fear memory. Further, we demonstrate that 17-AAG activated synaptic protein expression via transcriptional mechanisms through the heat shock transcription factor HSF1. Together, our findings identify a novel function of Hsp90 inhibition in regulating synaptic plasticity, in addition to the known neuroprotective effects of the chaperones against Aß and tau toxicity, thus further supporting the potential of Hsp90 inhibitors in treating neurodegenerative diseases.

ß-Lapachone promotes the recruitment and polarization of tumor-associated neutrophils (TANs) toward an antitumor (N1) phenotype in NQO1-positive cancers.

NAD(P)H:quinone oxidoreductase 1 (NQO1) is overexpressed in most solid cancers, emerging as a promising target for tumor-selective killing. ß-Lapachone (ß-Lap), an NQO1 bioactivatable drug, exhibits significant antitumor effects on NQO1-positive cancer cells by inducing immunogenic cell death (ICD) and enhancing tumor immunogenicity. However, the interaction between ß-Lap-mediated antitumor immune responses and neutrophils, novel antigen-presenting cells (APCs), remains unknown. This study demonstrates that ß-Lap selectively kills NQO1-positive murine tumor cells by significantly increasing intracellular ROS formation and inducing DNA double strand breaks (DSBs), resulting in DNA damage. Treatment with ß-Lap efficiently eradicates immunocompetent murine tumors and significantly increases the infiltration of tumor-associated neutrophils (TANs) into the tumor microenvironment (TME), which plays a crucial role in the drug's therapeutic efficacy. Further, the presence of ß-Lap-induced antigen medium leads bone marrow-derived neutrophils (BMNs) to directly kill murine tumor cells, aiding in dendritic cells (DCs) recruitment and significantly enhancing CD8+ T cell proliferation. ß-Lap treatment also drives the polarization of TANs toward an antitumor N1 phenotype, characterized by elevated IFN-ß expression and reduced TGF-ß cytokine expression, along with increased CD95 and CD54 surface markers. ß-Lap treatment also induces N1 TAN-mediated T cell cross-priming. The HMGB1/TLR4/MyD88 signaling cascade influences neutrophil infiltration into ß-Lap-treated tumors. Blocking this cascade or depleting neutrophil infiltration abolishes the antigen-specific T cell response induced by ß-Lap treatment. Overall, this study provides comprehensive insights into the role of tumor-infiltrating neutrophils in the ß-Lap-induced antitumor activity against NQO1-positive murine tumors.

Alzheimer's ß-secretase (BACE1) regulates the cAMP/PKA/CREB pathway independently of ß-amyloid.

ß-Amyloid protein (Aß), the major component of neuritic plaques in Alzheimer's disease (AD), is derived from proteolytic cleavages of the amyloid precursor protein (APP) by ß-site APP-cleaving enzyme 1 (BACE1) and the γ-secretase complex. BACE1 is the rate-limiting enzyme for Aß production, and an increase in BACE1 level/activity contributes to the pathogenesis of sporadic AD. In addition to cleaving APP for Aß generation, BACE1 plays multiple physiological roles including the regulation of synaptic functions. Here, we found that overexpression of BACE1 reduces cAMP response element binding protein (CREB) phosphorylation, protein kinase A (PKA) activity, and cAMP levels, whereas downregulation of BACE1 has the opposite effect. We showed that BACE1's effect is independent of its activity for Aß production, which is corroborated by the observation that BACE1 transgenic mice have impaired learning/memory in the absence of neurotoxic human Aß. Furthermore, we demonstrated that BACE1 interacts via its transmembrane domain with adenylate cyclase, resulting in reduction of cellular cAMP levels and thus PKA inactivation and reduced CREB phosphorylation. Our study suggests that in addition to its function as the ß-secretase to produce Aß, BACE1 may contribute to the memory and cognitive deficits typical of AD by regulating the cAMP/PKA/CREB pathway, which is important for memory functions.

Appoptosin is a novel pro-apoptotic protein and mediates cell death in neurodegeneration.

Apoptosis is an essential cellular process in multiple diseases and a major pathway for neuronal death in neurodegeneration. The detailed signaling events/pathways leading to apoptosis, especially in neurons, require further elucidation. Here we identify a ß-amyloid precursor protein (APP)-interacting protein, designated as appoptosin, whose levels are upregulated in brain samples from Alzheimer's disease and infarct patients, and in rodent stroke models, as well as in neurons treated with ß-amyloid (Aß) and glutamate. We further demonstrate that appoptosin induces reactive oxygen species release and intrinsic caspase-dependent apoptosis. The physiological function of appoptosin is to transport/exchange glycine/5-amino-levulinic acid across the mitochondrial membrane for heme synthesis. Downregulation of appoptosin prevents cell death and caspase activation caused by glutamate or Aß insults. APP modulates appoptosin-mediated apoptosis through interaction with appoptosin. Our study identifies appoptosin as a crucial player in apoptosis and a novel pro-apoptotic protein involved in neuronal cell death, providing a possible new therapeutic target for neurodegenerative disorders.

Upregulation of the E3 ligase NEDD4-1 by oxidative stress degrades IGF-1 receptor protein in neurodegeneration.

The importance of ubiquitin E3 ligases in neurodegeneration is being increasingly recognized. The crucial role of NEDD4-1 in neural development is well appreciated; however, its role in neurodegeneration remains unexplored. Herein, we report increased NEDD4-1 expression in the degenerated tissues of several major neurodegenerative diseases. Moreover, its expression is upregulated in cultured neurons in response to various neurotoxins, including zinc and hydrogen superoxide, via transcriptional activation likely mediated by the reactive oxygen species (ROS)-responsive FOXM1B. Reduced protein levels of the insulin-like growth factor receptor (IGF-1Rß) were observed as a consequence of upregulated NEDD4-1 via the ubiquitin-proteasome system. Overexpression of a familial mutant form of superoxide dismutase 1 (SOD1) (G93A) in neuroblastoma cells resulted in a similar reduction of IGF-1Rß protein. This inverse correlation between NEDD4-1 and IGF-1Rß was also observed in the cortex and spinal cords of mutant (G93A) SOD1 transgenic mice at a presymptomatic age, which was similarly induced by in vivo-administered zinc in wild-type C57BL/6 mice. Furthermore, histochemistry reveals markedly increased NEDD4-1 immunoreactivity in the degenerating/degenerated motor neurons in the lumbar anterior horn of the spinal cord, suggesting a direct causative role for NEDD4-1 in neurodegeneration. Indeed, downregulation of NEDD4-1 by shRNA or overexpression of a catalytically inactive form rescued neurons from zinc-induced cell death. Similarly, neurons with a NEDD4-1 haplotype are more resistant to apoptosis, largely due to expression of higher levels of IGF-1Rß.Together, our work identifies a novel molecular mechanism for ROS-upregulated NEDD4-1 and the subsequently reduced IGF-1Rß signaling in neurodegeneration.

Pathological complete response rate and clinical outcome after neoadjuvant therapy of HER2-low breast cancer: A National Cancer Database Analysis.

BACKGROUND: The interest in breast cancer with low HER2 expression as a distinct subtype is increasing. We aimed to explore the differences between HER2-low and HER2-zero breast cancer in their prognosis and rate of pathological complete response (pCR) after neoadjuvant therapy. METHODS: The National Cancer Database (NCDB) was used to select patients with breast cancer who received neoadjuvant therapy from 2004 to 2017. Logistic regression model was constructed for analysis of pCR. Cox proportional hazards regression model and Kaplan-Meier method were used for survival analysis. RESULTS: A total of 41500 breast cancer patients were included, among which 14814 (35.7%) had HER2-zero tumors and 26686 (64.3%) had HER2-low. HER2-low tumors were more commonly HR-positive in comparison with HER2-zero (66.3% versus 47.1%, P < 0.001). A lower rate of pCR was observed in HER2-low tumors than in HER2-zero tumors after neoadjuvant therapy in the total cohort (OR = 0.90; 95% CI [0.86-0.95]; P < 0.001) and in the subset of HR-positive (OR = 0.87; 95% CI [0.81-0.94]; P < 0.001). Patients with HER2-low tumors had a significantly superior survival than those with HER2-zero tumors (HR = 0.90; 95% CI [0.86-0.94]; P < 0.001), regardless of the HR status. Additionally, a marginal survival difference was also observed between HER2 IHC1+ and HER2 IHC2+/ISH-negative (HR = 0.91; 95% CI [0.85-0.97]; P = 0.003) cohorts. CONCLUSION: HER2-low tumors are a clinically relevant breast cancer subtype that is distinct from HER2-zero tumors. These findings may provide clues to appropriate therapeutic strategies for this subtype in the future.

IP-DNQ induces mitochondrial dysfunction and G2/M phase cell cycle arrest to selectively kill NQO1-positive pancreatic cancer cells.

Pancreatic cancer is among the top five leading causes of cancer-related deaths worldwide, with low survival rates. Current therapies for pancreatic cancer lack tumor specificity, resulting in harmful effects on normal tissues. Therefore, developing tumor-specific agents for the treatment of pancreatic cancer is critical. NAD(P)H:quinone oxidoreductase 1 (NQO1), highly expressed in pancreatic cancers but not in normal tissues, makes NQO1 bioactivatable drugs a potential therapy for selectively killing NQO1-positive cancer cells. Our previous studies have revealed that novel NQO1 bioactivatable drug deoxynyboquinone (DNQ) is ten-fold more potent than the prototypic NQO1 bioactivatable drug ß-lapachone in killing of NQO1-positive cancer cells. However, DNQ treatment results in high-grade methemoglobinemia, a significant side effect that limits clinical development. Here, we report for the first time on a DNQ derivative, isopentyl-deoxynboquinone (IP-DNQ), which selectively kills pancreatic ductal adenocarcinoma cells in an NQO1-dependent manner with equal potency to the parent DNQ. IP-DNQ evokes massive ROS production and oxidative DNA lesions that results in PARP1 hyperactivation, mitochondrial catastrophe and G2/M-phase arrest, leading to apoptotic and necrotic programmed cell death. Importantly, IP-DNQ treatment causes mild methemoglobinemia in vivo, with a three-fold improvement in the maximum tolerated dose compared to DNQ, while significantly suppresses tumor growth and extends the lifespan of mice in subcutaneous and orthotopic pancreatic cancer xenograft models. Our study demonstrates that IP-DNQ is a promising therapy for NQO1-positive pancreatic cancers and may enhance the efficacy of other anticancer drugs. IP-DNQ represents a novel approach to treating pancreatic cancer with the potential to improve patient outcomes.

Augmented Concentration of Isopentyl-Deoxynyboquinone in Tumors Selectively Kills NAD(P)H Quinone Oxidoreductase 1-Positive Cancer Cells through Programmed Necrotic and Apoptotic Mechanisms.

Lung and breast cancers rank as two of the most common and lethal tumors, accounting for a substantial number of cancer-related deaths worldwide. While the past two decades have witnessed promising progress in tumor therapy, developing targeted tumor therapies continues to pose a significant challenge. NAD(P)H quinone oxidoreductase 1 (NQO1), a two-electron reductase, has been reported as a promising therapeutic target across various solid tumors. ß-Lapachone (ß-Lap) and deoxynyboquinone (DNQ) are two NQO1 bioactivatable drugs that have demonstrated potent antitumor effects. However, their curative efficacy has been constrained by adverse effects and moderate lethality. To enhance the curative potential of NQO1 bioactivatable drugs, we developed a novel DNQ derivative termed isopentyl-deoxynyboquinone (IP-DNQ). Our study revealed that IP-DNQ treatment significantly increased reactive oxygen species generation, leading to double-strand break (DSB) formation, PARP1 hyperactivation, and catastrophic energy loss. Notably, we discovered that this novel drug induced both apoptosis and programmed necrosis events, which makes it entirely distinct from other NQO1 bioactivatable drugs. Furthermore, IP-DNQ monotherapy demonstrated significant antitumor efficacy and extended mice survival in A549 orthotopic xenograft models. Lastly, we identified that in mice IP-DNQ levels were significantly elevated in the plasma and tumor compared with IB-DNQ levels. This study provides novel preclinical evidence supporting IP-DNQ efficacy in NQO1+ NSCLC and breast cancer cells.

The Rps23rg gene family originated through retroposition of the ribosomal protein s23 mRNA and encodes proteins that decrease Alzheimer's beta-amyloid level and tau phosphorylation.

Retroposition is an important mechanism for gene origination. However, studies to elucidate the functions of new genes originated through retroposition, especially the functions related to diseases, are limited. We recently identified a mouse gene, Rps23 retroposed gene 1 (Rps23rg1), that regulates beta-amyloid (Abeta) level and tau phosphorylation, two major pathological hallmarks of Alzheimer's disease (AD), and found that Rps23rg1 originated through retroposition of the mouse ribosomal protein S23 (Rps23) mRNA. Here we show that retroposition of Rps23 mRNA occurred multiple times in different species but only generated another functionally expressed Rps23rg1-homologous gene, Rps23rg2, in mice, whereas humans may not possess functional Rps23rg homologs. Both Rps23rg1 and Rps23rg2 are reversely transcribed relative to the parental Rps23 gene, expressed in various tissues and encode proteins that interact with adenylate cyclases. Similar to the RPS23RG1 protein, RPS23RG2 can upregulate protein kinase A activity to reduce the activity of glycogen synthase kinase-3, Abeta level and tau phosphorylation. However, the effects of RPS23RG2 are weaker than those of RPS23RG1 and such a difference could be attributed to the extra carboxyl-terminal region of RPS23RG2, which may have an inhibitory effect. In addition, we show that the transmembrane domain of RPS23RG1 is important for its function. Together, our results present a new gene family, whose products and associated signaling pathways might prevent mice from developing AD-like pathologies.

Antidiabetic drug metformin (GlucophageR) increases biogenesis of Alzheimer's amyloid peptides via up-regulating BACE1 transcription.

Epidemiological, clinical and experimental evidence suggests a link between type 2 diabetes and Alzheimer's disease (AD). Insulin modulates metabolism of beta-amyloid precursor protein (APP) in neurons, decreasing the intracellular accumulation of beta-amyloid (Abeta) peptides, which are pivotal in AD pathogenesis. The present study investigates whether the widely prescribed insulin-sensitizing drug, metformin (Glucophage(R)), affects APP metabolism and Abeta generation in various cell models. We demonstrate that metformin, at doses that lead to activation of the AMP-activated protein kinase (AMPK), significantly increases the generation of both intracellular and extracellular Abeta species. Furthermore, the effect of metformin on Abeta generation is mediated by transcriptional up-regulation of beta-secretase (BACE1), which results in an elevated protein level and increased enzymatic activity. Unlike insulin, metformin exerts no effect on Abeta degradation. In addition, we found that glucose deprivation and various tyrphostins, known inhibitors of insulin-like growth factors/insulin receptor tyrosine kinases, do not modulate the effect of metformin on Abeta. Finally, inhibition of AMP-activated protein kinase (AMPK) by the pharmacological inhibitor Compound C largely suppresses metformin's effect on Abeta generation and BACE1 transcription, suggesting an AMPK-dependent mechanism. Although insulin and metformin display opposing effects on Abeta generation, in combined use, metformin enhances insulin's effect in reducing Abeta levels. Our findings suggest a potentially harmful consequence of this widely prescribed antidiabetic drug when used as a monotherapy in elderly diabetic patients.

KP372-1-Induced AKT Hyperactivation Blocks DNA Repair to Synergize With PARP Inhibitor Rucaparib via Inhibiting FOXO3a/GADD45α Pathway.

Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) have exhibited great promise in the treatment of tumors with homologous recombination (HR) deficiency, however, PARPi resistance, which ultimately recovers DNA repair and cell progress, has become an enormous clinical challenge. Recently, KP372-1 was identified as a novel potential anticancer agent that targeted the redox enzyme, NAD(P)H:quinone oxidoreductase 1 (NQO1), to induce extensive reactive oxygen species (ROS) generation that amplified DNA damage, leading to cancer cell death. To overcome PARPi resistance and expand its therapeutic utility, we investigated whether a combination therapy of a sublethal dose of KP372-1 with a nontoxic dose of PARPi rucaparib would synergize and enhance lethality in NQO1 over-expressing cancers. We reported that the combination treatment of KP372-1 and rucaparib induced a transient and dramatic AKT hyperactivation that inhibited DNA repair by regulating FOXO3a/GADD45α pathway, which enhanced PARPi lethality and overcame PARPi resistance. We further found that PARP inhibition blocked KP372-1-induced PARP1 hyperactivation to reverse NAD+/ATP loss that promoted Ca2+-dependent autophagy and apoptosis. Moreover, pretreatment of cells with BAPTA-AM, a cytosolic Ca2+ chelator, dramatically rescued KP372-1- or combination treatment-induced lethality and significantly suppressed PAR formation and γH2AX activation. Finally, we demonstrated that this combination therapy enhanced accumulation of both agents in mouse tumor tissues and synergistically suppressed tumor growth in orthotopic pancreatic and non-small-cell lung cancer xenograft models. Together, our study provides novel preclinical evidence for new combination therapy in NQO1+ solid tumors that may broaden the clinical utility of PARPi.

PCNA inhibition enhances the cytotoxicity of ß-lapachone in NQO1-Positive cancer cells by augmentation of oxidative stress-induced DNA damage.

ß-Lapachone is a classic quinone-containing antitumor NQO1-bioactivatable drug that directly kills NQO1-overexpressing cancer cells. However, the clinical applications of ß-lapachone are primarily limited by its high toxicity and modest lethality. To overcome this side effect and expand the therapeutic utility of ß-lapachone, we demonstrate the effects of a novel combination therapy including ß-lapachone and the proliferating cell nuclear antigen (PCNA) inhibitor T2 amino alcohol (T2AA) on various NQO1+ cancer cells. PCNA has DNA clamp processivity activity mediated by encircling double-stranded DNA to recruit proteins involved in DNA replication and DNA repair. In this study, we found that compared to monotherapy, a nontoxic dose of the T2AA synergized with a sublethal dose of ß-lapachone in an NQO1-dependent manner and that combination therapy prevented DNA repair, increased double-strand break (DSB) formation and promoted programmed necrosis and G1 phase cell cycle arrest. We further determined that combination therapy enhanced antitumor efficacy and prolonged survival in Lewis lung carcinoma (LLC) xenografts model. Our findings show novel evidence for a new therapeutic approach that combines of ß-lapachone treatment with PCNA inhibition that is highly effective in treating NQO1+ solid tumor cells.

ß-Lapachone Selectively Kills Hepatocellular Carcinoma Cells by Targeting NQO1 to Induce Extensive DNA Damage and PARP1 Hyperactivation.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death globally. Currently there is a lack of tumor-selective and efficacious therapies for hepatocellular carcinoma. ß-Lapachone (ARQ761 in clinical form) selectively kill NADPH: quinone oxidoreductase 1 (NQO1)-overexpressing cancer cells. However, the effect of ß-Lapachone on HCC is virtually unknown. In this study, we found that relatively high NQO1 and low catalase levels were observed in both clinical specimens collected from HCC patients and HCC tumors from the TCGA database. ß-Lapachone treatment induced NQO1-selective killing of HCC cells and caused ROS formation and PARP1 hyperactivation, resulting in a significant decrease in NAD+ and ATP levels and a dramatic increase in double-strand break (DSB) lesions over time in vitro. Administration of ß-Lapachone significantly inhibited tumor growth and prolonged survival in a mouse xenograft model in vivo. Our data suggest that NQO1 is an ideal potential biomarker, and relatively high NQO1:CAT ratios in HCC tumors but low ratios in normal tissues offer an optimal therapeutic window to use ß-Lapachone. This study provides novel preclinical evidence for ß-Lapachone as a new promising chemotherapeutic agent for use in NQO1-positive HCC patients.

Mitofusin-2 protects against cold stress-induced cell injury in HEK293 cells.

Mitochondrial impairment is hypothesized to contribute to cell injury during cold stress. Mitochondria fission and fusion are closely related in the function of the mitochondria, but the precise mechanisms whereby these processes regulate cell injury during cold stress remain to be determined. HEK293 cells were cultured in a cold environment (4.0+/-0.1 degrees C) for 2, 4, 8, or 12h. Western blot analyses showed that these cells expressed decreased fission-related protein Drp1 and increased fusion-related protein Mfn2 at 4h; meanwhile, electron microscopy analysis revealed large and long mitochondrial morphology within these cells, indicating increased mitochondrial fusion. With silencing of Mfn2 but not of Mfn1 by siRNA promoted cold-stress-induced cell death with decreased ATP production in HEK293 cells. Our results show that increased expression of Mfn2 and mitochondrial fusion are important for mitochondrial function as well as cell survival during cold stress. These findings have important implications for understanding the mechanisms of mitochondrial fusion and fission in cold-stress-induced cell injury.

Retinoid X receptor-alpha mediates (R )-flurbiprofen's effect on the levels of Alzheimer's beta-amyloid.

Alzheimer's disease (AD) is characterized by the formation of extracellular senile plaques in the brain, whose major component is a small peptide called beta-amyloid (Abeta). Long-term use of non-steroidal anti-inflammatory drugs (NSAIDs) has been found beneficial for AD and several reports suggest that NSAIDs reduce the generation of Abeta, especially the more amyloidogenic form Abeta42. However, the exact mechanism underlying NSAIDs' effect on AD risk remains largely inconclusive and all clinical trials using NSAIDs for AD treatment show negative results so far. Recent studies have shown that some NSAIDs can bind to certain nuclear receptors, suggesting that nuclear receptors may be involved in NSAID's effect on AD risk. Here we find that (R)-flurbiprofen, the R-enantiomer of the racemate NSAID flurbiprofen, can significantly reduce Abeta secretion, but at the same time, increases the level of intracellular Abeta. In addition, we find that a nuclear receptor, retinoid X receptor alpha (RXRalpha), can regulate Abeta generation and that down-regulation of RXRalpha significantly increases Abeta secretion. We also show that (R)-flurbiprofen can interfere with the interaction between RXRalpha and 9-cis-retinoid acid, and that 9-cis-retinoid acid decreases (R)-flurbiprofen's reduction of Abeta secretion. Moreover, the modulation effect of (R)-flurbiprofen on Abeta is abolished upon RXRalpha down-regulation. Together, these results suggest that RXRalpha can regulate Abeta generation and is also required for (R)-flurbiprofen-mediated Abeta generation.

Promotional effect of microRNA-194 on breast cancer cells via targeting F-box/WD repeat-containing protein 7.

Breast cancer is the most common type of malignant cancer in females. An increasing number of studies have revealed that microRNAs (miR), which belong to a class of small non-coding RNAs, serve an important role in a number of human cancer subtypes. In the present study, the role of miR-194 in breast cancer cells and its underlying mechanisms were investigated. The results demonstrated that the serum levels of miR-194 were significantly higher in patients of the poorly differentiated and well-differentiated groups, compared with in healthy adults. Additionally, the serum level of miR-194 was significantly higher in the poorly differentiated group compared with in the well-differentiated group. In order to further investigate the role of miR-194 in breast cancer cells, the present study transfected two breast cancer cell lines, MCF-7 and MDA-MB-231, with an empty vector (control), miR-194 (overexpression), antagomiR-194 (inhibitor, functional knock down) or antagomiR-194 and miR-194. An MTT assay was performed in order to detect the proliferation of breast cancer cells in the various groups. The results revealed that the overexpression of miR-194 significantly accelerated cell proliferation, whereas the inhibition of miR-194 significantly decelerated the proliferation of MCF-7 and MDA-MB-231 cells. Furthermore, the expression levels of cyclin D and cyclin E were significantly upregulated in miR-194 overexpressing cells, and the expression levels of cyclin D and cyclin E were significantly downregulated in miR-194 inhibited cells, as compared with in control cells. No significant change was observed in the level of proliferation of cells co-transfected with miR-194 and antagomiR-194, compared with in the control cells. According to the hypothesis suggesting possible target genes of miR-194, the present study proposed that F-box/WD repeat-containing protein 7 (Fbxw-7) may be a direct target of miR-194, which was confirmed by a luciferase reporter assay. The present study suggested that miR-194 expression promoted the proliferation of breast cancer cells by targeting Fbxw-7, and may serve as a biomarker and a novel target for breast cancer therapy.