Β-lapachone Treatment Research Articles

β-Lapachone induces ferroptosis of colorectal cancer cells via NCOA4-mediated ferritinophagy by activating JNK pathway

β-Lapachone is a natural product that can promote ROS generation and ultimately triggers tumor cells death by inducing DNA damage. Recent studies have indicated that the targeting of ferroptosis or iron metabolism is a feasible strategy for treating cancer. In this study, bulk RNA-seq analysis suggested that β-Lapachone might induce ferroptosis in CRC cells. We further tested this hypothesis using a xenograft model of human colorectal cancer as an animal model and in SW620 and DLD-1 of CRC cell lines. Western blot was used to determine the key proteins of ferroptosis (SLC7A11, GPX4), autophagy (LC3B, P62, ATG7), ferritinophagy (NCOA4, FTH1, TFRC), and JNK pathway (p-JNK, JNK, p-c-Jun, c-Jun). The levels of MDA, GSH/GSSG, lipid ROS, and intracellular ferrous iron were determined after β-Lapachone treatment, and inhibitors of various pathways, including NAC, Ferrostatin-1, DFO, 3-MA, and SP600125 were utilized to explore the molecular mechanism underlying β-Lapachone-mediated ferroptosis. As the result, we identified that β-Lapachone inhibited cell proliferation and induced apoptosis, autophagy, and ROS generation. In addition, β-Lapachone induced ferroptosis as demonstrated by intra-cellular iron overload, increased levels of lipid ROS and MDA. Mechanistically, JNK signaling pathway was involved in β-Lapachone-induced xCT/GPX4-mediated ferroptosis and NCOA4-mediated ferritinophagy in CRC cells. In vivo experiments in nude mice demonstrated that β-Lapachone significantly inhibited CRC growth and induced ferroptosis and NCOA4-mediated ferritinophagy. These findings not only identify a novel role for β-Lapachone in ferroptosis but also indicate that β-Lapachone may be a valuable candidate for the research and development of anti-cancer therapeutic agents.

Single-Cell Kinetic Modeling of β-Lapachone Metabolism in Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) cells are highly heterogeneous in their metabolism and typically experience elevated reactive oxygen species (ROS) levels such as superoxide and hydrogen peroxide (H2O2) in the tumor microenvironment. Tumor cells survive under these chronic oxidative conditions by upregulating antioxidant systems. To investigate the heterogeneity of cellular responses to chemotherapeutic H2O2 generation in tumor and healthy tissue, we leveraged single-cell RNA-sequencing (scRNA-seq) data to perform redox systems-level simulations of quinone-cycling β-lapachone treatment as a source of NQO1-dependent rapid superoxide and hydrogen peroxide (H2O2) production. Transcriptomic data from 10 HNSCC patient tumors was used to populate over 4000 single-cell antioxidant enzymatic network models of drug metabolism. The simulations reflected significant systems-level differences between the redox states of healthy and cancer cells, demonstrating in some patient samples a targetable cancer cell population or in others statistically indistinguishable effects between non-malignant and malignant cells. Subsequent multivariate analyses between healthy and malignant cellular models pointed to distinct contributors of redox responses between these phenotypes. This model framework provides a mechanistic basis for explaining mixed outcomes of NAD(P)H:quinone oxidoreductase 1 (NQO1)-bioactivatable therapeutics despite the tumor specificity of these drugs as defined by NQO1/catalase expression and highlights the role of alternate antioxidant components in dictating drug-induced oxidative stress.

Synergistic Effect of β-Lapachone and Aminooxyacetic Acid on Central Metabolism in Breast Cancer.

The compound β-lapachone, a naturally derived naphthoquinone, has been utilized as a potent medicinal nutrient to improve health. Over the last twelve years, numerous reports have demonstrated distinct associations of β-lapachone and NAD(P)H: quinone oxidoreductase 1 (NQO1) protein in the amelioration of various diseases. Comprehensive research of NQO1 bioactivity has clearly confirmed the tumoricidal effects of β-lapachone action through NAD+-keresis, in which severe DNA damage from reactive oxygen species (ROS) production triggers a poly-ADP-ribose polymerase-I (PARP1) hyperactivation cascade, culminating in NAD+/ATP depletion. Here, we report a novel combination strategy with aminooxyacetic acid (AOA), an aspartate aminotransferase inhibitor that blocks the malate-aspartate shuttle (MAS) and synergistically enhances the efficacy of β-lapachone metabolic perturbation in NQO1+ breast cancer. We evaluated metabolic turnover in MDA-MB-231 NQO1+, MDA-MB-231 NQO1−, MDA-MB-468, and T47D cancer cells by measuring the isotopic labeling of metabolites from a [U-13C]glucose tracer. We show that β-lapachone treatment significantly hampers lactate secretion by ~85% in NQO1+ cells. Our data demonstrate that combinatorial treatment decreases citrate, glutamate, and succinate enrichment by ~14%, ~50%, and ~65%, respectively. Differences in citrate, glutamate, and succinate fractional enrichments indicate synergistic effects on central metabolism based on the coefficient of drug interaction. Metabolic modeling suggests that increased glutamine anaplerosis is protective in the case of MAS inhibition.

Investigating β‐Lapachone Mediated Metabolic Disruption Using Stable Isotope Tracers

β‐lapachone has been found to be a highly potent anti‐cancer agent in phase 1 clinical trials, but has dose limiting toxicity in red blood cells [Gerber et. al Br J Cancer. 2018 Oct;119(8):928‐936]. Identifying a safe and quantitative method of monitoring treatment efficacy will be highly beneficial to establishing an adaptive dosing platform. Adaptative dosing can allow personalized treatments that significantly reduce off target effects while maintaining an effective therapeutic regimen. In this study we present the use of a safe and noninvasive tracer strategy utilizing both [2H7]glucose and [U‐13C]glucose tracers to assess the metabolic effects caused by β‐lapachone treatment in vitro. Treating cancers with β‐lapachone bioactivates NAD(P)H: quinone oxidoreductase 1 (NQO1) to produce a highly unstable hydroquinone intermediate that reestablishes itself through an aggressive futile cycle, generating reactive oxygen species (ROS) in the form of hydrogen peroxide. Accelerated accumulation of ROS causes large amounts of DNA damage, triggers poly‐ADP‐ribose polymerase‐I hyperactivation, induces global depletion of NAD+ and ATP, and hinders overall glycolytic flux [Moore et. al Cell Death Dis. 2015 Jan; 6(1): e159]. Cancer cells that overexpress NQO1 subsequently die through NAD+‐keresis. Measuring changes in glycolysis and downstream metabolism caused by the bioactivation of NQO1 would establish a platform for assessing the efficacy of treatment, potentially allowing the reduction of chemotherapeutic dosage thereby diminishing off‐target toxicities. Changes in central carbon metabolism caused by NQO1 bioactivation were detected and assessed in various cancer cells using both tracer strategies. Deuterated lactate and HDO (deuterated water) were easily quantified, and the production of these metabolic products were linearly correlated with [2H7]glucose consumption. Mass isotopologue distribution analysis by gas chromatography‐mass spectrometry demonstrated downregulated energy metabolism with a reduction in 2H and 13C labeling in NQO1+ cancer cells treated with β‐lapachone. These methods are highly beneficial for measuring rates of glycolytic metabolism, identifying potential therapeutic synergies, the efficacy of chemotherapeutic agents that hamper central carbon metabolism, and can be potentially translated to in vivo models.

β-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. β-Lapachone (ARQ761 in clinical form) selectively kill NADPH: quinone oxidoreductase 1 (NQO1)-overexpressing cancer cells. However, the effect of β-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. β-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 β-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 β-Lapachone. This study provides novel preclinical evidence for β-Lapachone as a new promising chemotherapeutic agent for use in NQO1-positive HCC patients.

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

β-Lapachone is a classic quinone-containing antitumor NQO1-bioactivatable drug that directly kills NQO1-overexpressing cancer cells. However, the clinical applications of β-lapachone are primarily limited by its high toxicity and modest lethality. To overcome this side effect and expand the therapeutic utility of β-lapachone, we demonstrate the effects of a novel combination therapy including β-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 β-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 β-lapachone treatment with PCNA inhibition that is highly effective in treating NQO1+ solid tumor cells.

Measuring NQO1 Bioactivation Using [2H7]Glucose.

Simple SummaryCancer is often characterized by profound changes in metabolism, some of which are appropriate targets for therapeutic intervention. Many cancers overexpress the two-electron reductase NQO1, which can bioactivate the drug β-lapachone, inducing a futile redox cycle that liberates large amounts of reactive oxygen species and results in subsequent cell death. However, β-lapachone has off-target toxicities in red blood cells, which makes minimal dosing for chemotherapeutic response desirable. Here, we show that magnetic resonance-based detection of [2H7]glucose metabolism provides a robust metric of NQO1 activation, as the redox perturbation causes downregulation of glycolytic flux that is detectable in the HDO and lactate signals. Imaging of either metabolic product could provide constraints for a continual reassessment model for controlling therapeutic dosing levels.Treatment of cancers with β-lapachone causes NAD(P)H: quinone oxidoreductase 1 (NQO1) to generate an unstable hydroquinone that regenerates itself in a futile cycle while producing reactive oxygen species (ROS) in the form of superoxide and subsequently hydrogen peroxide. Rapid accumulation of ROS damages DNA, hyperactivates poly-ADP-ribose polymerase-I, causes massive depletion of NAD+/ATP, and hampers glycolysis. Cells overexpressing NQO1 subsequently die rapidly through an NAD+-keresis mechanism. Assessing changes in glycolytic rates caused by NQO1 bioactivation would provide a means of assessing treatment efficacy, potentially lowering the chemotherapeutic dosage, and reducing off-target toxicities. NQO1-mediated changes in glycolytic flux were readily detected in A549 (lung), MiaPaCa2 (pancreatic), and HCT-116 (colon) cancer cell lines by 2H-NMR after administration of [2H7]glucose. The deuterated metabolic products 2H-lactate and HDO were quantified, and linear relationships with glucose consumption for both products were observed. The higher concentration of HDO compared to 2H-lactate allows for more sensitive measurement of the glycolytic flux in cancer. Gas chromatography-mass spectrometry analysis agreed with the NMR results and confirmed downregulated energy metabolism in NQO1+ cells after β-lapachone treatment. The demonstrated method is ideal for measuring glycolytic rates, the effects of chemotherapeutics that target glycolysis, and has the potential for in vivo translation.

Inhibition of TXNRD or SOD1 overcomes NRF2-mediated resistance to β-lapachone

Alterations in the NRF2/KEAP1 pathway result in the constitutive activation of NRF2, leading to the aberrant induction of antioxidant and detoxification enzymes, including NQO1. The NQO1 bioactivatable agent β-lapachone can target cells with high NQO1 expression but relies in the generation of reactive oxygen species (ROS), which are actively scavenged in cells with NRF2/KEAP1 mutations. However, whether NRF2/KEAP1 mutations influence the response to β-lapachone treatment remains unknown. To address this question, we assessed the cytotoxicity of β-lapachone in a panel of NSCLC cell lines bearing either wild-type or mutant KEAP1. We found that, despite overexpression of NQO1, KEAP1 mutant cells were resistant to β-lapachone due to enhanced detoxification of ROS, which prevented DNA damage and cell death. To evaluate whether specific inhibition of the NRF2-regulated antioxidant enzymes could abrogate resistance to β-lapachone, we systematically inhibited the four major antioxidant cellular systems using genetic and/or pharmacologic approaches. We demonstrated that inhibition of the thioredoxin-dependent system or copper-zinc superoxide dismutase (SOD1) could abrogate NRF2-mediated resistance to β-lapachone, while depletion of catalase or glutathione was ineffective. Interestingly, inhibition of SOD1 selectively sensitized KEAP1 mutant cells to β-lapachone exposure. Our results suggest that NRF2/KEAP1 mutational status might serve as a predictive biomarker for response to NQO1-bioactivatable quinones in patients. Further, our results suggest SOD1 inhibition may have potential utility in combination with other ROS inducers in patients with KEAP1/NRF2 mutations.

The NQO1 bioactivatable drug, β-lapachone, alters the redox state of NQO1+ pancreatic cancer cells, causing perturbation in central carbon metabolism

Many cancer treatments, such as those for managing recalcitrant tumors like pancreatic ductal adenocarcinoma, cause off-target toxicities in normal, healthy tissue, highlighting the need for more tumor-selective chemotherapies. β-Lapachone is bioactivated by NAD(P)H:quinone oxidoreductase 1 (NQO1). This enzyme exhibits elevated expression in most solid cancers and therefore is a potential cancer-specific target. β-Lapachone's therapeutic efficacy partially stems from the drug's induction of a futile NQO1-mediated redox cycle that causes high levels of superoxide and then peroxide formation, which damages DNA and causes hyperactivation of poly(ADP-ribose) polymerase, resulting in extensive NAD+/ATP depletion. However, the effects of this drug on energy metabolism due to NAD+ depletion were never described. The futile redox cycle rapidly consumes O2, rendering standard assays of Krebs cycle turnover unusable. In this study, a multimodal analysis, including metabolic imaging using hyperpolarized pyruvate, points to reduced oxidative flux due to NAD+ depletion after β-lapachone treatment of NQO1+ human pancreatic cancer cells. NAD+-sensitive pathways, such as glycolysis, flux through lactate dehydrogenase, and the citric acid cycle (as inferred by flux through pyruvate dehydrogenase), were down-regulated by β-lapachone treatment. Changes in flux through these pathways should generate biomarkers useful for in vivo dose responses of β-lapachone treatment in humans, avoiding toxic side effects. Targeting the enzymes in these pathways for therapeutic treatment may have the potential to synergize with β-lapachone treatment, creating unique NQO1-selective combinatorial therapies for specific cancers. These findings warrant future studies of intermediary metabolism in patients treated with β-lapachone.

Genome-Scale Modeling of NADPH-Driven β-Lapachone Sensitization in Head and Neck Squamous Cell Carcinoma.

The purpose of this study was to investigate differential nicotinamide adenine dinucleotide phosphate, reduced (NADPH) production between radiation-sensitive and -resistant head and neck squamous cell carcinoma (HNSCC) cell lines and whether these differences are predictive of sensitivity to the chemotherapeutic β-lapachone. We have developed a novel human genome-scale metabolic modeling platform that combines transcriptomic, kinetic, thermodynamic, and metabolite concentration data. Upon incorporation of this information into cell line-specific models, we observed that the radiation-resistant HNSCC model redistributed flux through several major NADPH-producing reactions. Upon RNA interference of canonical NADPH-producing genes, the metabolic network can further reroute flux through alternate NADPH biosynthesis pathways in a cell line-specific manner. Model predictions of perturbations in cellular NADPH production after gene knockdown match well with experimentally verified effects of β-lapachone treatment on NADPH/NADP+ ratio and cell viability. This computational approach accurately predicts HNSCC-specific oxidoreductase genes that differentially affect cell viability between radiation-responsive and radiation-resistant cancer cells upon β-lapachone treatment. Quantitative genome-scale metabolic models that incorporate multiple levels of biological data are applied to provide accurate predictions of responses to a NADPH-dependent redox cycling chemotherapeutic drug under a variety of perturbations. Our modeling approach suggests differences in metabolism and β-lapachone redox cycling that underlie phenotypic differences in radiation-sensitive and -resistant cancer cells. This approach can be extended to investigate the synergistic action of NAD(P)H: quinone oxidoreductase 1 bioactivatable drugs and radiation therapy. Antioxid. Redox Signal. 29, 937-952.

Abstract 4483: TGF-β downregulates osteopontin isoforms and induce and epithelial plasticity in PC3 metastatic prostate cancer cells

Abstract Total osteopontin (OPN) has been described as a master regulator of epithelial-mesenchymal transition (EMT), including in prostate cancer (PCa). However, the contribution of each OPN splicing isoform (OPN-SI), named OPNa, OPNb, and OPNc in the epithelial plasticity dynamic process is currently unknown. Besides, the contribution of alternative splicing regulation in this scenario is poorly understood. Here we aimed to investigate the expression of OPN-SI and the associated splicing pattern regulation in response to metastatic prostate cancer (PCa) cells treatment with TGF-β . Further, we aimed to investigate the resulting cell phenotypic features. To achieve this, we treated PC3 cells with TGF-β 10ng/ml for 48 h as a model to induce epithelial plasticity in these cells. Total RNA was extracted and the corresponding cDNA was tested for transcriptional levels of OPN-SI, SR and HnRNP splicing regulators as well as classical (EMT) markers by using real time PCR. EMT markers and cell morphology have also been analyzed by immunofluoresce. PC3 cell phenotype has also been evaluated by using cell migration and viabilitity assays in response to β-lapachone cytotoxic reagent. PC3 cells treated with TGF-β displayed an intermediate EMT phenotype, as evidenced by a significant downregulation of both epithelial (E-cadherin, claudin-3 andcytokeratin 18) and mesenchymal markers (vimentin, N-cadherin, snailandslug). These cells presented a higher cytoplasmic E-cadherin expression, as well as a lower vimentin cytoplasmic staining. A major cortical distribution of actin filaments beneath the cell cortex has been observed. Further, a significant downregulation of OPNa and mainly, OPNb and OPNc isoforms, as well as most tested splicing regulators has been shown. TGF-β treatment also promoted a significant inhibition on cell migration as well as higher sensitivity to β-lapachone treatment, as compared to control cells. Altogether, our data evidence that in our in vitro experimental model, TGF-β promoted a an intermediate EMT phenotype, with a predominance of epithelial features, associated to downregulation of OPN-SI and most tested splicing regulators. We then hypothesize that TGF-β may also be an EMT-like inducer, in which metastatic PCa cells could acquire major epithelial features that facilitate cell anchorage and adhesion in distant tumor sites. OPN-SI dowregulation in this process could favour epithelial-like features, as apposed to OPN-SI overexpression during tumor progression towards metastic circulating cells typically displaying mesechymal phenotypes. Citation Format: Durval Marques, Rodrigo A. Peres, Paula P. de Freitas, Abigail C. Resende, Nataly dos Santos Melo, Waldemir Fernandes de Souza, José A. Morgado-Diaz, Etel Rodrigues Pereira Gimba. TGF-β downregulates osteopontin isoforms and induce and epithelial plasticity in PC3 metastatic prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4483. doi:10.1158/1538-7445.AM2017-4483

β-Lapachone Inhibits Lung Metastasis of Colorectal Cancer by Inducing Apoptosis of CT26 Cells.

Background: β-Lapachone is a quinone-containing compound found in red lapacho (Tabebuia impetiginosa, syn. T avellanedae) trees. Lapacho has been used in traditional medicine by several South and Central American indigenous people to treat various types of cancer. The purpose of this study was to investigate the antimetastatic properties of β-lapachone and the underlying mechanisms using colon cancer cells. Methods: This research used metastatic murine colon cancer cell lines, colon 26 (CT26) and colon 38 (MC38). A WST assay, annexin V assay, cell cycle analysis, wound healing assay, invasion assay, western blot analysis, and real-time reverse transcription–polymerase chain reaction were performed to examine the effects of β-lapachone on metastatic phenotypes and molecular mechanisms. The effect of β-lapachone on lung metastasis was assessed in a mouse experimental metastasis model. Results: We found that the inhibition of proliferation of the colon cancer cell lines by β-lapachone was due to the induction of apoptosis and cell cycle arrest. β-Lapachone induced the apoptosis of CT26 cells through caspase-3, -8, and -9 activation; poly(ADP-ribose) polymerase cleavage; and downregulation of the Bcl-2 family in a dose- and time-dependent manner. In addition, a low concentration of β-lapachone decreased the cell migration and invasion by decreasing the expression of matrix metalloproteinases-2 and -9, and increased the expression of tissue inhibitors of metalloproteinases-1 and -2. Moreover, β-lapachone treatment regulated the expression of epithelial-mesenchymal transition markers such as E- and N-cadherin, vimentin, β-catenin, and Snail in CT26 cells. In the mouse experimental metastasis model, β-lapachone significantly inhibited the lung metastasis of CT26 cells. Conclusions: Our results demonstrated the inhibitory effect of β-lapachone on colorectal lung metastasis. This compound may be useful for developing therapeutic agents to treat metastatic cancer.

β-Lapachone Regulates the Transforming Growth Factor-β-Smad Signaling Pathway Associated with Collagen Biosynthesis in Human Dermal Fibroblasts.

The transforming growth factor (TGF)-β-Smad signaling pathway regulates collagen biosynthesis in human dermal fibroblasts. We found that β-lapachone stimulated type I collagen expression in human dermal fibroblasts. In this study, we evaluated whether the β-lapachone-induced upregulation of collagen biosynthesis in human dermal fibroblasts is associated with the TGF-β-Smad signaling pathway. In cultured human dermal fibroblasts, both Smad 2 and Smad 3 (Smad 2/3) were phosphorylated by β-lapachone treatment in a concentration-dependent manner. SB431542, a specific inhibitor of TGF-β receptor I kinase, inhibited the β-lapachone-mediated Smad 2/3 phosphorylation and type I collagen expression, suggesting that β-lapachone stimulates collagen production via the TGF-β receptor I kinase-dependent pathway. β-Lapachone did not increase TGF-β1 synthesis in human dermal fibroblasts, suggesting that the molecular mechanism of β-lapachone for the upregulation of collagen synthesis is due to the extracellular regulation of availability and activities of TGF-β. This study provides new insights into the role of β-lapachone in collagen synthesis in human dermal fibroblasts and suggests that β-lapachone can be used as a pharmacological tool to study collagen homeostasis associated with TGF-β-Smad signaling.

β-Lapachone enhances Mre11-Rad50-Nbs1 complex expression in cisplatin-induced nephrotoxicity.

Recent studies suggest a potential involvement of the Mre11-Rad50-Nbs1 (MRN) complex, a DNA double-strand breaks (DSBs) sensor, in the development of nephrotoxicity following cisplatin administration. β-Lapachone is a topoisomerase I inhibitor known to reduce cisplatin-induced nephrotoxicity. In this study, by assessing MRN complex expression, we explored whether β-lapachone was involved in DNA damage response in the context of cisplatin-induced nephrotoxicity. Male Balb/c mice were randomly allocated to 4 groups: control, β-lapachone alone, cisplatin alone, and β-lapachone+cisplatin. β-Lapachone was administered with the diet (0.066%) for 2 weeks prior to cisplatin injection (18mg/kg). All mice were sacrificed 3 days after cisplatin treatment. In the cisplatin-alone group, renal function was disrupted and MRN complex expression increased. As expected, β-lapachone co-treatment attenuated cisplatin-induced pathologic alterations. Notably, although β-lapachone markedly decreased cisplatin-induced renal cell apoptosis and DSBs formation, the β-lapachone+cisplatin group showed the highest MRN complex expression. Moreover, β-lapachone treatment increased the basal expression level of the MRN complex, which was accompanied by enhanced basal expression of SIRTuin1, which is known to regulate Nbs1 acetylation. Although, it remains unclear how β-lapachone induces MRN complex expression, our findings suggest that β-lapachone might affect MRN complex expression and participate in DNA damage recovery in cisplatin-induced nephrotoxicity.

Sulindac Compounds Facilitate the Cytotoxicity of β-Lapachone by Up-Regulation of NAD(P)H Quinone Oxidoreductase in Human Lung Cancer Cells

β-lapachone, a major component in an ethanol extract of Tabebuia avellanedae bark, is a promising potential therapeutic drug for various tumors, including lung cancer, the leading cause of cancer-related deaths worldwide. In the first part of this study, we found that apoptotic cell death induced in lung cancer cells by high concentrations of β-lapachone was mediated by increased activation of the pro-apoptotic factor JNK and decreased activation of the cell survival/proliferation factors PI3K, AKT, and ERK. In addition, β-lapachone toxicity was positively correlated with the expression and activity of NAD(P)H quinone oxidoreductase 1 (NQO1) in the tumor cells. In the second part, we found that the FDA-approved non-steroidal anti-inflammatory drug sulindac and its metabolites, sulindac sulfide and sulindac sulfone, increased NQO1 expression and activity in the lung adenocarcinoma cell lines CL1-1 and CL1-5, which have lower NQO1 levels and lower sensitivity to β-lapachone treatment than the A549 cell lines, and that inhibition of NQO1 by either dicoumarol treatment or NQO1 siRNA knockdown inhibited this sulindac-induced increase in β-lapachone cytotoxicity. In conclusion, sulindac and its metabolites synergistically increase the anticancer effects of β-lapachone primarily by increasing NQO1 activity and expression, and these two drugs may provide a novel combination therapy for lung cancers.

Enhanced activation of NAD(P)H

Guanosine 5'-triphosphate cyclohydrolase-1 (GTPCH-1) is a rate-limiting enzyme in de-novo synthesis of tetrahydrobiopterin (BH4), an essential cofactor for endothelial nitric oxide synthase (eNOS) coupling. Adenosine 5'-monophosphate-activated protein kinase (AMPK) is crucial for GTPCH-1 preservation, and tumor suppressor kinase liver kinase B1 (LKB1), an upstream kinase of AMPK, is activated by NAD-dependent class III histone deacetylase sirtuin 1 (SIRT1)-mediated deacetylation. β-Lapachone has been shown to increase cellular NAD/NADH ratio via quinone oxidoreductase 1 (NQO1) activation. In this study, we have evaluated whether β-lapachone-induced NQO1 activation modulates blood pressure (BP) through preservation of GTPCH-1 in a hypertensive animal model. Spontaneously hypertensive rats (SHRs), primary aortic endothelial cells, and endothelial cell line were used to investigate the hypotensive effect of β-lapachone and its action mechanism. β-Lapachone treatment dramatically lowered BP and vascular tension in SHRs and induced eNOS activation in endothelial cells. Consistent with these effects, β-lapachone treatment also elevated levels of both aortic cGMP and plasma nitric oxide in SHRs. Meanwhile, β-lapachone-treated SHRs showed significantly increased levels of aortic NAD, LKB1 deacetylation, and AMPK Thr phosphorylation followed by increased GTPCH-1 and tetrahydrobiopterin/dihydrobiopterin ratio. In-vitro study revealed that AMPK inhibition by overexpression of dominant-negative AMPK nearly abolished GTPCH-1 protein conservation. Enhanced LKB1 deacetylation and AMPK activation were also elicited by β-lapachone in endothelial cells. However, inhibition of LKB1 deacetylation by blocking of NQO1 or SIRT1 blunted AMPK activation by β-lapachone. This is the first study demonstrating that eNOS coupling can be regulated by NQO1 activation via LKB1/AMPK/GTPCH-1 modulation, which is possibly correlated with relieving hypertension. These findings provide strong evidence to suggest that NQO1 might be a new therapeutic target for hypertension.

Synergistic enhancement of antitumor effect of β-Lapachone by photodynamic induction of quinone oxidoreductase (NQO1)

β-Lapachone is a phytochemotherapeutic originally isolated from Lapacho tree whose extract has been used medicinally for centuries. It is well known that NAD(P)H:quinone oxidoreductase (NQO1) activity is the principal determinant of β-Lapachone cytotoxicity. As NQO1 is overexpressed in most common carcinomas, recent investigations suggest its potential application against cancer. Photodynamic therapy (PDT) is a clinically approved and rapidly developing cancer treatment. PDT involves the administration of photosensitizer (PS) followed by local illumination with visible light of specific wavelength. In the presence of oxygen molecules, the light illumination of PS can lead to a series of photochemical reactions and consequently the generation of cytotoxic reactive oxygen species (ROS). It has been reported that β-Lapachone synergistically interacts with ionizing radiation, hyperthermia and cisplatin and that the sensitivity of cells to β-Lapachone is closely related to the activity of NQO1. So, the present study aimed to investigate the feasibility of PDT to increase the anticancer effect of β-Lapachone by up-regulating NQO1 expression on breast cancer MCF-7c3 cells. NQO1 expression was evaluated by Western blot analysis at different times after PDT using ME-ALA as PS. The cytotoxicity of the photodynamic treatment and β-Lapachone alone or in combination was determined by MTT assay and the combination index (CI)-isobologram method and the dose reduction index (DRI) analysis were used to assess the effect of drug combinations. Our studies for the first time demonstrated that the expression of NQO1 is induced 24h after photodynamic treatment. The sensitivity of cancer cells to β-Lapachone treatment increased 24h after PDT and a synergistic inhibitory effect on MCF-7c3 cells was showed. Taken together, these results lead us to conclude that the synergistic interaction between β-Lapachone and PDT in killing cells was consistent with the up-regulation of NQO1. The combination of β-Lapachone and PDT is a potentially promising modality for the treatment of cancer.

β-lapachone accelerates the recovery of burn-wound skin.

β-lapachone is a quinone of lapachol extracted from the bark of lapacho tree. Recent findings demonstrated that punched skin wounds of mice healed faster with β-lapachone treatment. The present study investigates the effects of β-lapachone on burn-wound skin of C57BL/6 mice injured by a 100 °C iron stick. Our results indicated that wounds treated with β-lapachone recovered faster than those treated with control ointment containing no β-lapachone. On the third day after burning, the area of β-lapachone treated-wound was 30% smaller than wound treated with control ointment. H&E and immunohistochemistry staining showed that burn-wound skin treated with ointment containing β-lapachone healed faster in its epidermis, dermis, and underlying connective tissues with more macrophages appeared than those treated with control ointment alone. RAW264.7 cell, a macrophage-like cell line derived from BALB/C mice, was used as a model for scrutinizing the effect of β-lapachone on macrophages. We found that the proliferation and the secretion of EGF and VEGF by macrophages were higher in cultures treated with β-lapachone and that ß-lapachone can also increase the release of EGF with TNF-α pretreatment. We conclude that β-lapachone plays an important role in accelerating burn wound healing, and that β-lapachone not only can raise the proliferation of macrophages but also increase the release of VEGF from macrophages.

Endoplasmic Reticulum Stress-Induced JNK Activation Is a Critical Event Leading to Mitochondria-Mediated Cell Death Caused by β-Lapachone Treatment

Backgroundβ-lapachone (β-lap) is a bioreductive agent that is activated by the two-electron reductase NAD(P)H quinone oxidoreductase 1 (NQO1). Although β-lap has been reported to induce apoptosis in various cancer types in an NQO1-dependent manner, the signaling pathways by which β-lap causes apoptosis are poorly understood.Methodology/Principal Findingsβ-lap-induced apoptosis and related molecular signaling pathways in NQO1-negative and NQO1-overexpressing MDA-MB-231 cells were investigated. Pharmacological inhibitors or siRNAs against factors involved in β-lap-induced apoptosis were used to clarify the roles played by such factors in β-lap-activated apoptotic signaling pathways. β-lap leads to clonogenic cell death and apoptosis in an NQO1- dependent manner. Treatment of NQO1-overexpressing MDA-MB-231 cells with β-lap causes rapid disruption of mitochondrial membrane potential, nuclear translocation of AIF and Endo G from mitochondria, and subsequent caspase-independent apoptotic cell death. siRNAs targeting AIF and Endo G effectively attenuate β-lap-induced clonogenic and apoptotic cell death. Moreover, β-lap induces cleavage of Bax, which accumulates in mitochondria, coinciding with the observed changes in mitochondria membrane potential. Pretreatment with Salubrinal (Sal), an endoplasmic reticulum (ER) stress inhibitor, efficiently attenuates JNK activation caused by β-lap, and subsequent mitochondria-mediated cell death. In addition, β-lap-induced generation and mitochondrial translocation of cleaved Bax are efficiently blocked by JNK inhibition.Conclusions/SignificanceOur results indicate that β-lap triggers induction of endoplasmic reticulum (ER) stress, thereby leading to JNK activation and mitochondria-mediated apoptosis. The signaling pathways that we revealed in this study may significantly contribute to an improvement of NQO1-directed tumor therapies.

β-lapachone Activates a Mre11p-Tel1p G1/S Checkpoint in Budding Yeast

β-lapachone is an anticancer agent that selectively induces cell death in several human cancer cells. The mechanism of β-lapachone cytotoxicity is not yet fully understood. Here we report that β-lapachone treatment delayed cell cycle progression at the G1/S transition, incremented phosphorylation of the Rad53p checkpoint kinase and decreased cell survival in the budding yeast Saccharomyces cerevisiae. Furthermore, β-lapachone induced phosphorylation of histone H2A at serine 129. These checkpoint responses were regulated by Mec1p and Tel1p kinases. Mec1p was required for Rad53p/histone H2A phosphorylation and cell survival following β-lapachone treatment in asynchronous cultures, but not for the G1 delay. The tel1Δ mutation increased sensitivity to β-lapachone in a mec1 defective strain and compromised checkpoint responses in G1. Both Rad53p phosphorylation and G1 delay were fully dependent on a functional Mre11p-Rad50p-Xrs2p (XMR) complex, and mutants in the XMR complex were hypersensitive to β-lapachone treatment. Finally, XRS2 and TEL1 worked epistatically regarding β-lapachone sensitivity and Xrs2p was phosphorylated in a Tel1p dependent-manner after β-lapachone treatment. Taken together, these findings indicate that β-lapachone activates a Mre11p-Tel1p checkpoint pathway in budding yeast. Given the conserved nature of the Mre11p-Tel1p pathway, these results suggest that activation of the Mre11-Tel1p checkpoint could be of significance for β-lapachone antitumour activity.