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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.

Background: Bacterial infections constantly have a large impact on public health, because of increased rates of resistance and reduced frequency of development of novel antibiotics. The utility of conventional antibiotics for treating bacterial infections has become increasingly challenging. The aim of the study was to assess the antibacterial effect of β-Lapachone (β-Lap), a novel synthetic compound. Methods: The antibacterial activity of the β-Lap compound was examined against laboratory strains by agar well diffusion method and broth dilution assay. Growth kinetics in presence of β-Lap on Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa (ATCC 27853) were assessed by microplate alamarBlue assay. Crystal violet blue assay was used for biofilm inhibition and biofilm eradication. P. aeruginosa catalase (KatA) complexed with β-Lap was modeled using molecular docking approach. Results: β-Lap exhibited potent antimicrobial activity against laboratory strains of bacteria with MIC of 0.2 mM for S. saprophyticus and Staphylococcus aureus, and 0.04 mM for Staphylococcus epidermidis and Pseudomonas aeruginosa ATCC 27853. The inhibition of catalase enzyme was found to be the cause for its antibacterial activity. Bioinformatics analysis suggests that β-Lap can inhibit KatA activity by interacting with catalase proximal active site and heme binding site. The activity of some commercial antibiotics was enhanced in association with β-Lap. In addition, β-Lap inhibited the biofilm formation and eradicated the already formed and ultra-mature biofilms of aforesaid bacterial strains. Conclusion: These observations indicated that β-Lap could be a promising antibacterial agent for the treatment and prevention of infectious diseases.

Although the enzyme catalytic nanoreactors reported so far have achieved excellent therapeutic efficacy, how to accurately exert enzyme activity in the tumor microenvironment to specifically kill tumor cells and avoid systemic oxidative damage would be an inevitable challenge for catalytic nanomedicine. At the present study, we fabricate an advanced biomimetic nanoreactor, SOD-Fe0@Lapa-ZRF for tumor multi-enzyme cascade delivery that combined specifically killing tumor cells and protect cells from oxidative stress. Methods: We first synthesized the FeNP-embedded SOD (SOD-Fe0) by reduction reaction using sodium borohydride. Next, SOD-Fe0 and Lapa cargo were encapsulated in ZIF-8 by self-assembly. In order to protect the cargo enzyme from digestion by protease and prolong blood circulating time, SOD-Fe0@Lapa-Z was further cloaked with RBC membrane and functionalized with folate targeting, resulting in the final advanced biomimetic nanoreactor SOD-Fe0@Lapa-ZRF. Results: Once internalized, ZIF-8 achieves pH-triggered disassembly in weakly acidic tumor microenvironment. The released SOD-Fe0 and Lapa were further endocytosed by tumor cells and the Lapa produces superoxide anion (O2-•) through the catalysis of NQO1 that is overexpressed in tumor cells, while O2-• is converted to H2O2 via SOD. At this time, the released ferrous ions from SOD-Fe0 and H2O2 are further transformed to highly toxic hydroxyl radicals (•OH) for specifically killing tumor cells, and there was no obvious toxicological response during long-term treatment. Importantly, SOD-Fe0@Lapa-ZRF enhanced the normal cell's anti-oxidation ability, and thus had little effect on the secretion of TNF-α, IL-6 and IL-1β pro-inflammatory cytokines, while effectively reversed the decreased activity of T-SOD and GSH-Px and remained stable MDA content after tumor treatment. In vitro and in vivo results indicate that the tumor microenvironment-responsive release multi-enzyme cascade have high tumor specificity and effective anti-tumor efficacy, and can protect cells from oxidative stress damage. Conclusion: The biomimetic nanoreactor will have a great potential in cancer nanomedicine and provide a novel strategy to regulate oxidative stress.

SUMMARY Introduction: Leishmaniasis is a disease caused by protozoa of the genus Leishmania and is considered endemic in 98 countries. Treatment with pentavalent antimonials has a high toxicity, which motivates the search for effective and less toxic drugs. α- and β-lapachones have shown different biological activities, including antiprotozoa. In recent studies, the isonicotinoylhydrazone and phthalazinylhydrazone groups were considered innovative in the development of antileishmania drugs. Molecular hybridization is a strategy for the rational development of new prototypes, where the main compound is produced through the appropriate binding of pharmacophoric subunits. Aims: To synthesize four hybrids of α- and β-lapachones, together with the isonicotinoylhydrazone and phthalazinylhydrazone groups and to determine the antileishmania activity against the promastigotic forms of L. amazonensis, L. infantum and L. major. Results: β-lapachone derivatives were more active against all tested leishmania species. βACIL (IC50 0.044μM) and βHDZ (IC50 0.023μM) showed 15-fold higher activity than amphotericin B. The high selectivity index exhibited by the compounds indicates greater safety for vertebrate host cells. Conclusion: The results of this work show that the hybrids βACIL and (3HDZ are promising molecules for the development of new antileishmania drugs. RESUMEN Introducción: Leishmaniasis es una enfermedad causada por protozoos del género Leishmania y se considera endémica en 98 países. El tratamiento con antimoniales pentavalentes tiene una alta toxicidad, lo que motiva la búsqueda de fármacos eficaces y menos tóxico. α- y β-lapachones han mostrado diferentes actividades biológicas, incluido los antiprotozoarios. En estudios recientes, los grupos isonicotinoilhidra-zona y ftalazinilhidrazona se consideraron innovadores en el desarrollo de fármacos antileishmania. La hibridación molecular es una estrategia para el desarrollo racional de nuevos prototipos, donde el compuesto principal se produce a través de la unión apropiada de subunidades farmacofóricas. Objetivos: Sintetizar cuatro híbridos de α- y β-lapachones, junto con los grupos isonicotinoilhidrazona y ftalazinilhidrazona y determinar la actividad antileishmania frente a las formas promastigotas de L. amazonensis, L. infantum y L. major. Resultados: Los derivados de β-lapachone fueron más activos contra todas las especies de leishmania probadas. La βACIL (CI50 0,044μM) y βHDZ (CI50 0,023μM) mostraron actividad 15 veces mayor que la anfotericina B. El alto índice de selectividad que presentan los compuestos indica una mayor seguridad para las células huésped del vertebrado. Conclusión: Los resultados de este trabajo demuestran que los híbridos (ACIL y (HDZ son moléculas prometodoras para el desarrollo de nuevos fármacos antileishmania. RESUMO Introdução: A leishmaniose é uma doença causada por protozoários do género Leishmania e é considerada endémica em 98 países. O tratamento com antimoniais pentavalentes apresenta alta toxicidade, o que motiva a pesquisa por medicamentos eficazes e menos tóxicos. α- e β-lapachones tém mostrado diferentes atividades biológicas, incluindo antiprotozoários. Em estudos recentes, os grupos isonicotinoilhi-drazona e ftalazinilhidrazona foram considerados inovadores no desenvolvimento de drogas antileishmania. A hibridização molecular é uma estratégia para o desenvolvimento racional de novos protótipos, onde o composto principal é produzido através da ligação apropriada de subunidades farmacofóricas. Objetivos: Sintetizar quatro híbridos de α- e β-lapachones, juntamente com os grupos isonicotinoil-hidra-zona e ftalazinilhidrazona e determinar a atividade antileishmania contra as formas promastigóticas de L. amazonensis, L. infantum e L. major. Resultados: Os derivados de β-lapachona foram mais ativos contra todas as espécies de leishmania testadas. BACIL (IC50 0,044 μM) e βHDZ (IC50 0,023 μM) apresentaram atividade 15 vezes maior do que a anfotericina B. O alto índice de seletividade dos compostos indica maior segurança para células hospedeiras de vertebrados. Conclusaõ: Os resultados deste trabalho mostram que os híbridos βACIL e βHDZ são moléculas promissoras para o desenvolvimento de novos fármacos antileishmania.

Simple Summary Cancer 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. Abstract 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.

The dopamine precursor 3,4-dihydroxyphenyl- l-alanine (L-DOPA) is the most widely used symptomatic treatment for Parkinson's disease (PD); however, its prolonged use is associated with L-DOPA-induced dyskinesia in more than half of patients after 10 years of treatment. The present study investigated whether co-treatment with β-Lapachone, a natural compound, and L-DOPA has protective effects in a 6-hydroxydopamine (6-OHDA)-induced mouse model of PD. Unilateral 6-OHDA-lesioned mice were treated with vehicle or β-Lapachone (10 mg/kg/day) and L-DOPA for 11 days. Abnormal involuntary movements (AIMs) were scored on days 5 and 10. β-Lapachone (10 mg/kg) co-treatment with L-DOPA decreased the AIMs score on both days 5 and 10. β-Lapachone was demonstrated to have a beneficial effect on the axial and limb AIMs scores on day 10. There was no significant suppression in dopamine D1 receptor-related and ERK1/2 signaling in the DA-denervated striatum by β-Lapachone-cotreatment with L-DOPA. Notably, β-Lapachone-cotreatment with L-DOPA increased phosphorylation at the Ser9 site of glycogen synthase kinase 3β (GSK-3β), indicating suppression of GSK-3β activity in both the unlesioned and 6-OHDA-lesioned striata. In addition, astrocyte activation was markedly suppressed by β-Lapachone-cotreatment with L-DOPA in the striatum and substantia nigra of the unilateral 6-OHDA model. These findings suggest that β-Lapachone cotreatment with L-DOPA therapy may have therapeutic potential for the suppression or management of the development of L-DOPA-induced dyskinesia in patients with PD.

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.

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.

Parkinson's disease is a neurodegenerative disease characterized by the progressive loss of dopaminergic neurons within the substantia nigra pars compacta. In the present study, we investigated whether β-Lapachone (β-LAP), a natural naphthoquinone compound isolated from the lapacho tree (Tabebuia avellanedae), elicits neuroprotective effects in a 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP)-induced Parkinson's disease mouse model. β-LAP reduced the tyrosine hydroxylase (TH)-immunoreactive fiber loss induced by MPTP in the dorsolateral striatum, and alleviated motor dysfunction as determined by the rotarod test. In addition, β-LAP protected against MPTP-induced loss of TH positive neurons, and upregulated B-cell lymphoma 2 protein (Bcl-2) expression in the substantia nigra. Based on previous reports on the neuroprotective role of nuclear factor-E2-related factor-2 (Nrf2) in neurodegenerative diseases, we investigated whether β-LAP induces upregulation of the Nrf2-hemeoxygenae-1 (HO-1) signaling pathway molecules in MPTP-injected mouse brains. Western blot and immunohistochemical analyses indicated that β-LAP increased HO-1 expression in glial fibrillary acidic protein-positive astrocytes. Moreover, β-LAP increased the nuclear translocation and DNA binding activity of Nrf2, and the phosphorylation of upstream adenosine monophosphate-activated protein kinase (AMPK). β-LAP also increased the localization of p-AMPK and Nrf2 in astrocytes. Collectively, our data suggest that β-LAP exerts neuroprotective effect in MPTP-injected mice by upregulating the p-AMPK/Nrf2/HO-1 signaling pathways in astrocytes.

The treatment of infections caused by resistant microorganisms is limited, and vancomycin (VAN) treatment failures for methicillin-resistant Staphylococcus aureus (MRSA) bacteremia are not uncommon, even when MRSA clinical isolates are susceptible to VAN. Thus, this study proposed the association of VAN with usnic acid and β-lapachone encapsulated into liposomes as a novel therapeutic option for infections caused by MRSA. Liposomes containing β-lap (β-lap-lipo) or usnic acid (UA-lipo) were prepared by the thin lipid film hydration method followed by sonication. Antimicrobial activity against MRSA clinical isolates was investigated by the microdilution method according to the Clinical and Laboratory Standards Institute (CLSI). The interaction studies were carried out using the checkerboard method and epsilometer test (Etest). The interaction between VAN and β-lap or β-lap-lipo was synergistic (FICI = 0.453 and FICI = 0.358, respectively). An additive interaction between VAN and UA (FICI = 0.515) was found. UA-lipo resulted in synergism with VAN (FICI = 0.276). The Etest reproduced the results obtained by the checkerboard method for approximately 82% of the analysis. Thus, the present study demonstrated that VAN in combination with UA-lipo, β-lap or β-lap-lipo synergistically enhanced antibacterial activity against MRSA.

β-Lapachone has drawn increasing attention as an anti-inflammatory and anti-cancer drug. However, its oral bioavailability has not been yet assessed, which might be useful to develop efficient dosage forms possibly required for non-clinical and clinical studies and future market. The aim of the present study was thus to investigate pharmacokinetic properties of β-lapachone as well as its first-pass metabolism in the liver, and small and large intestines after oral administration to measure the absolute bioavailability in rats. A sensitive HPLC method was developed to evaluate levels of β-lapachone in plasma and organ homogenates. The drug degradation profiles were examined in plasma to assess the stability of the drug and in liver and intestinal homogenates to evaluate first-pass metabolism. Pharmacokinetic profiles were obtained after oral and intravenous administration of β-lapachone at doses of 40 mg/kg and 1.5 mg/kg, respectively. The measured oral bioavailability of β-lapachone was 15.5%. The considerable degradation of β-lapachone was seen in the organ homogenates but the drug was quite stable in plasma. In conclusion, we suggest that the fairly low oral bioavailability of β-lapachone may be resulted from the first-pass metabolic degradation of β-lapachone in the liver, small and large intestinal tracts and its low aqueous solubility.

The aim of this study was to evaluate the antimicrobial activity of lapachol, α-lapachone, β-lapachone and six antimicrobials (ampicillin, amoxicillin/clavulanic acid, cefoxitin, gentamicin, ciprofloxacin and meropenem) against twelve strains of Staphylococcus aureus from which resistance phenotypes were previously determined by the disk diffusion method. Five S. aureus strains (LFBM 01, LFBM 26, LFBM 28, LFBM 31 and LFBM 33) showed resistance to all antimicrobial agents tested and were selected for the study of the interaction between β-lapachone and antimicrobial agents, busing checkerboard method. The criteria used to evaluate the synergistic activity were defined by the Fractional Inhibitory Concentration Index (FICI). Among the naphthoquinones, β-lapachone was the most effective against S. aureus strains. FICI values ranged from 0.07 to 0.5, suggesting a synergistic interaction against multidrug resistant S. aureus (MRSA) strains. An additive effect was observed with the combination β-lapachone/ciprofloxacin against the LFBM 33 strain. The combination of β-lapachone with cefoxitin showed no added benefit against LFBM 31 and LFBM 33 strains. This study demonstrated that, in general, β-lapachone combined with beta lactams antimicrobials, fluoroquinolones and carbapenems acts synergistically inhibiting MRSA strains.

Patients affected by cutaneous leishmaniasis need a topical treatment which cures lesions without leaving scars. Lesions are produced not only by the parasite but also by an uncontrolled and persistent inflammatory immune response. In this study, we proposed the loading of β-lapachone (β-LP) in lecithin-chitosan nanoparticles (NP) for targeting the drug to the dermis, where infected macrophages reside, and promote wound healing. Although the loading of β-LP in NP did not influence the drug antileishmanial activity it was critical to achieve important drug accumulation in the dermis and permeation through the skin. When topically applied in Leishmania major infected BALB/c mice, β-LP NP achieved no parasite reduction but they stopped the lesion progression. Immuno-histopathological assays in CL lesions and quantitative mRNA studies in draining lymph nodes confirmed that β-LP exhibited anti-inflammatory activity leading to the down-regulation of IL-1β and COX-2 expression and a decrease of neutrophils infiltrate. From the Clinical Editor Cutaneous leishmaniasis often leaves patients with unsightly scars due to the body's inflammatory response to the infection. The authors in this paper described topical treatment using β-lapachone (β- LP) loaded in lecithin-chitosan nanoparticles (NP) in an animal model. Results confirmed the reduction of inflammatory response without affecting the parasite killing efficacy. These findings would pave way for further clinical testing in the near future.

β-lapachone is a naturally occurring quinone that selectively induces apoptotic cell death in a variety of human cancer cells in vitro and in vivo; however, its mechanism of action needs to be further elaborated. In this study, we investigated the effects of β-lapachone on the induction of apoptosis in human gastric carcinoma AGS cells. β-lapachone significantly inhibited cellular proliferation, and some typical apoptotic characteristics such as chromatin condensation and an increase in the population of sub-G1 hypodiploid cells were observed in β-lapachone-treated AGS cells. Treatment with β-lapachone caused mitochondrial transmembrane potential dissipation, stimulated the mitochondria-mediated intrinsic apoptotic pathway, as indicated by caspase-9 activation, cytochrome c release, Bcl-2 downregulation and Bax upregulation, as well as death receptor-mediated extrinsic apoptotic pathway, as indicated by activation of caspase-8 and truncation of Bid. This process was accompanied by activation of caspase-3 and concomitant with cleavage of poly(ADP-ribose) polymerase. The general caspase inhibitor, z-VAD-fmk, significantly abolished β-lapachone-induced cell death and inhibited growth. Further analysis demonstrated that the induction of apoptosis by β-lapachone was accompanied by inactivation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. The PI3K inhibitor LY29004 significantly increased β-lapachone-induced apoptosis and growth inhibition. Taken together, these findings indicate that the apoptotic activity of β-lapachone is probably regulated by a caspase-dependent cascade through activation of both intrinsic and extrinsic signaling pathways, and that inhibition of the PI3K/Akt signaling may contribute to β-lapachone-mediated AGS cell growth inhibition and apoptosis induction.

beta-Lapachone activates multiple cell death mechanisms including apoptosis, autophagy and necrotic cell death in cancer cells. In this study, we investigated beta-lapachone-induced cell death and the underlying mechanisms in human hepatocellular carcinoma SK-Hep1 cells. beta-Lapachone markedly induced cell death without caspase activation. beta-Lapachone increased PI uptake and HMGB-1 release to extracellular space, which are markers of necrotic cell death. Necrostatin-1 (a RIP1 kinase inhibitor) markedly inhibited beta-lapachone-induced cell death and HMGB-1 release. In addition, beta-lapachone activated poly (ADP-ribosyl) polymerase-1(PARP-1) and promoted AIF release, and DPQ (a PARP-1 specific inhibitor) or AIF siRNA blocked beta-lapachone-induced cell death. Furthermore, necrostatin-1 blocked PARP-1 activation and cytosolic AIF translocation. We also found that beta-lapachone-induced reactive oxygen species (ROS) production has an important role in the activation of the RIP1-PARP1-AIF pathway. Finally, beta-lapachone-induced cell death was inhibited by dicoumarol (a NQO-1 inhibitor), and NQO1 expression was correlated with sensitivity to beta-lapachone. Taken together, our results demonstrate that beta-lapachone induces programmed necrosis through the NQO1-dependent ROS-mediated RIP1-PARP1-AIF pathway.

In our continued search for novel trypanocidal compounds, twenty-six derivatives of para- and ortho-naphthoquinones coupled to 1,2,3-triazoles were synthesized. These compounds were evaluated against the infective bloodstream form of Trypanosoma cruzi, the etiological agent of Chagas disease. Compounds 17–24, 28–30 and 36–38 are described herein for the first time. Three of these novel compounds (28–30) were found to be more potent than the standard drug benznidazole, with IC50/24h values between 6.8 and 80.8μM. Analysis of the toxicity to heart muscle cells led to LC50/24h of

Superparamagnetic iron oxide nanoparticles (SPION) are an important and versatile nano- platform with broad biological applications. Despite extensive studies, the biological and pharmacological activities of SPION have not been exploited in therapeutic applications. Recently, β-lapachone (β-lap), a novel anticancer drug, has shown considerable cancer specificity by selectively increasing reactive oxygen species (ROS) stress in cancer cells. In this study, we report that pH-responsive SPION-micelles can synergize with β-lap for improved cancer therapy. These SPION-micelles selectively release iron ions inside cancer cells, which interact with hydrogen peroxide (H(2)O(2)) generated from β-lap in a tumor-specific, NQO1-dependent manner. Through Fenton reactions, these iron ions escalate the ROS stress in β-lap-exposed cancer cells, thereby greatly enhancing the therapeutic index of β-lap. More specifically, a 10-fold increase in ROS stress was detected in β-lap-exposed cells pretreated with SPION-micelles over those treated with β-lap alone, which also correlates with significantly increased cell death. Catalase treatment of cells or administration of an iron chelator can block the therapeutic synergy. Our data suggest that incorporation of SPION-micelles with ROS-generating drugs can potentially improve drug efficacy during cancer treatment, thereby provides a synergistic strategy to integrate imaging and therapeutic functions in the development of theranostic nanomedicine.

AMP-activated protein kinase (AMPK), a crucial regulator of energy metabolic homeostasis, is suggested to regulate inflammatory responses, but its precise mechanisms are not fully understood. It has been reported that pharmacological activation of AMPK induces heme oxygenase-1 (HO-1) expression. β-Lapachone (BL), a well-known substrate of NAD(P)H:quinone oxidoreductase (NQO1), has been demonstrated to stimulate AMPK activation via NQO1 activation, and to exert anti-inflammatory effects in macrophages. Here we examined whether AMPK activation by BL would be linked to HO-1 expression in RAW264.7 macrophages and whether HO-1 expression could mediate the anti-inflammatory effects of BL. BL treatment induced concentration- and time-dependent AMPK phosphorylation and HO-1 expression. 5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside, an AMPK activator, also induced HO-1 expression. In contrast, compound C (CC), an inhibitor of AMPK activation, prevented the increase in BL-induced HO-1 expression. BL pretreatment reduced lipopolysaccharide-induced production of tumor necrosis factor-α, a pro-inflammatory cytokine, and expression of inducible nitric oxide synthase, a pro-inflammatory enzyme. These inhibitory effects BL were almost completely abolished by CC and partly by tin protoporphyrin-IX, a competitive inhibitor of HO-1. Accordingly, the present results indicate that BL induces anti-inflammatory HO-1 expression in macrophages via AMPK activation, providing one of possible mechanisms by which BL can exert anti-inflammatory effects.

Background β-Lapachone has antitumor and wound healing-promoting activities. To address the potential influences of various chemicals on heart development of zebrafish embryos, we previously treated zebrafish embryos with chemicals from a Sigma LOPAC1280™ library and found several chemicals including β-lapachone that affected heart morphogenesis. In this study, we further evaluated the effects of β-lapachone on zebrafish embryonic heart development. Methods Embryos were treated with β-lapachone or dimethyl sulfoxide (DMSO) at 24 or 48 hours post fertilization (hpf) for 4 h at 28°C. Heart looping and valve development was analyzed by whole-mount in situ hybridization and histological analysis. For fractional shortening and wall shear stress analyses, AB and Tg (gata1:DsRed) embryos were recorded for their heart pumping and blood cell circulations via time-lapse fluorescence microscopy. Dextran rhodamine dye injection into the tail reticular cells was used to analyze circulation. Reactive oxygen species (ROS) was analyzed by incubating embryos in 5-(and 6-)-chloromethyl-2',7'-dichloro-dihydrofluorescein diacetate (CM-H2DCFDA) and recorded using fluorescence microscopy. o-Dianisidine (ODA) staining and whole mount in situ hybridization were used to analyze erythrocytes. TUNEL assay was used to examine DNA fragmentation. Results We observed a linear arrangement of the ventricle and atrium, bradycardia arrhythmia, reduced fractional shortening, circulation with a few or no erythrocytes, and pericardial edema in β-lapachone-treated 52-hpf embryos. Abnormal expression patterns of cmlc2, nppa, BMP4, versican, and nfatc1, and histological analyses showed defects in heart-looping and valve development of β-lapachone-treated embryos. ROS production was observed in erythrocytes and DNA fragmentation was detected in both erythrocytes and endocardium of β-lapachone-treated embryos. Reduction in wall shear stress was uncovered in β-lapachone-treated embryos. Co-treatment with the NQO1 inhibitor, dicoumarol, or the calcium chelator, BAPTA-AM, rescued the erythrocyte-deficiency in circulation and heart-looping defect phenotypes in β-lapachone-treated embryos. These results suggest that the induction of apoptosis of endocardium and erythrocytes by β-lapachone is mediated through an NQO1- and calcium-dependent pathway. Conclusions The novel finding of this study is that β-lapachone affects heart morphogenesis and function through the induction of apoptosis of endocardium and erythrocytes. In addition, this study further demonstrates the importance of endocardium and hemodynamic forces on heart morphogenesis and contractile performance.

Naphthoquinones are compounds present in several families of higher plants. Their molecular structures confer redox properties, and they are involved in multiple biological oxidative processes. In folk medicine, especially among Indian populations, plants containing naphthoquinones have been employed for the treatment of various diseases. The biological redox cycle of quinones can be initiated by one electron reduction leading to the formation of semiquinones, unstable intermediates that react rapidly with molecular oxygen, generating free radicals. Alternatively, the reduction by two electrons, mediated by DT-diphorase, leads to the formation of hydroquinone. Lapachol, alpha-lapachone and beta-lapachone, which are isolated from the heartwood of trees of the Bignoniaceae family, are examples of bioactive naphthoquinones. In this review, we will discuss studies investigating the activity of these natural products and their derivatives in the context of the search for alternative drugs for Chagas disease, caused by Trypanosoma cruzi, a neglected illness that is endemic in Latin America.