Beta-lapachone ameliorates the progression of primary sclerosing cholangitis pathogenesis in rodent models.

AIMS: Primary sclerosing cholangitis (PSC) is a rare cholestatic liver disease characterized by chronic inflammation and severe fibrosis for which effective treatment options are currently lacking. In this study, we explored the potential of beta-lapachone (ßL) as a drug candidate for PSC therapy. MATERIALS AND METHODS: We employed an animal model fed a diet containing 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) to assess the preventive and therapeutic effects of ßL. The beneficial effects of ßL on PSC pathogenic characteristics, including blood biomarkers, inflammation, and fibrosis, were determined by assessing relevant parameters. Differential gene expression between each group was analyzed by RNA sequencing of liver tissues. Mdr2-/- mice were utilized to explore the involvement of Abcb4 in the ßL-induced improvement of PSC pathogenesis. KEY FINDINGS: ßL effectively inhibited key features of PSC pathogenesis, as demonstrated by reduced blood biomarkers and improved pathogenic characteristics. Treatment with ßL significantly mitigated DDC-induced apoptosis, cell proliferation, inflammation, and fibrosis. Analysis of differential gene expression confirmed a new insight that ßL could stimulate the expression of genes related to NAD synthesis and Abcb4. Indeed, ßL-induced NAD exhibited effective functioning, as evidenced by enhanced sirt1/3 and acetyl-lysine levels, leading to improved mitochondrial stability. The role of Abcb4 in response to ßL was confirmed in Mdr2/Abcb4 KO mice, where the beneficial effects of ßL were abolished. SIGNIFICANCE: This study provided a new concept for PSC treatment, suggesting that pharmacological stimulation of the NAD synthetic pathway and Abcb4 via ßL ameliorates PSC pathogenesis.

Tailored Beta-Lapachone Nanomedicines for Cancer-Specific Therapy.

Nanotechnology shows the power to improve efficacy and reduce the adverse effects of anticancer agents. As a quinone-containing compound, beta-lapachone (LAP) is widely employed for targeted anticancer therapy under hypoxia. The principal mechanism of LAP-mediated cytotoxicity is believed due to the continuous generation of reactive oxygen species with the aid of NAD(P)H: quinone oxidoreductase 1 (NQO1). The cancer selectivity of LAP relies on the difference between NQO1 expression in tumors and that in healthy organs. Despite this, the clinical translation of LAP faces the problem of narrow therapeutic window that is challenging for dose regimen design. Herein, the multifaceted anticancer mechanism of LAP is briefly introduced, the advance of nanocarriers for LAP delivery is reviewed, and the combinational delivery approaches to enhance LAP potency in recent years are summarized. The mechanisms by which nanosystems boost LAP efficacy, including tumor targeting, cellular uptake enhancement, controlled cargo release, enhanced Fenton or Fenton-like reaction, and multidrug synergism, are also presented. The problems of LAP anticancer nanomedicines and the prospective solutions are discussed. The current review may help to unlock the potential of cancer-specific LAP therapy and speed up its clinical translation.

Beta-Lapachone Attenuates BMSC-Mediated Neuroblastoma Malignant Transformation by Inhibiting Gal-3/Gal-3BP/IL6 Axis.

The inflammatory factor IL6 secreted by bone marrow mesenchymal stem cells (BMSCs) in the tumor microenvironment (TME) facilitates the survival and therapeutic resistance of neuroblastoma (NB). Here, we found that IL6 expression in primary tumor tissues or bone marrow (BM) metastases was closely associated with the disease risk and prognosis of NB patients. IL6 secretion from immortalized BMSC (iBMSC) was directly regulated by NB cells and is involved in promoting the proliferation and metastasis of NB cells. Beta-Lapachone (ARQ-501, LPC), an ortho-naphthoquinone natural product, significantly prevented the iBMSC-induced malignant transformation effect on NB cells through suppressing the expression and secretion of IL6 from iBMSC in vitro and in vivo. Mechanistically, LPC disrupted the crosstalk between NB cells and iBMSC in an NQO1-dependent manner through blocking the Gal-3/Gal-3BP/IL6 axis. Our results reveal the effect of iBMSC-derived IL6 on TME-induced malignant transformation of NB cells, and provide theoretical basis for the clinical application of LPC as a potential IL6 inhibitor in high-risk refractory NB patients.

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.

The possibility of low isomerization of ß-lapachone in the human body.

ß-Lapachone has been reported to have anticancer and various other therapeutic effects, but is limited in clinical applications by its low bioavailability. pH-Dependent isomerization can be suggested as one plausible factor influencing its low bioavailability. Since it is known that ß-lapachone is converted to its isomer, α-lapachone in hydrochloric acid (HCl) solution, isomerization in the human body may be driven by HCl in the gastric fluid. The purpose of this study was to evaluate the possibility of isomerization of ß-lapachone in the human body. Chemical reactions were conducted using simulated gastric fluid (SGF, pH 1.2) and simulated intestinal fluid (SIF, pH 7.5) at 37°C. ß-Lapachone was observed in SGF at 37°C for 1 hour and SIF for 3 hours. In addition, biofluid analysis was performed on plasma samples 1 hour and 4 hours, and on urine sample 12 hours after oral administration of 100 mg MB12066, a synthetic ß-lapachone, in healthy adult male. All samples were analyzed using liquid chromatography-tandem mass spectrometry. Only ß-lapachone peaks existed in the spectra obtained from SGF and SIF. No isomerization of ß-lapachone was observed in the analysis of any of the human samples. In the current study, the possibility of pH-dependent isomerization of ß-lapachone in the human body was not confirmed.

Cytotoxicity assessment of beta-lapachone in endothelial cells

The selective activity of an antineoplastic drug is related to its ability to promote cytotoxic action on tumor cells and preserve the integrity of non-neoplastic cells. Beta-lapachone is extracted from the sawdust of Ipe wood, a thick bark tree from the Ipe wood found in the Brazilian Cerrado biome. This study aimed to evaluate the cytotoxic action of beta-lapachone in an endothelial cell line. The EA.hy926 cells were seeded in two groups, G1 and G2, cultured and exposed to beta-lapachone at concentrations of 0.0, 0.01, 0.03, 0.1, 0.3, 1 and 3 & 956;M for 24 hours. G1 remained under normal cultivation conditions and G2 was subjected to oxidative stress through an ischemia and reperfusion assay, in a deoxygenated sealed chamber. The cytotoxicity assay was performed using the tetrazolium reduction method. In G1, the cytotoxicity ranged from 0.0 to 10.0%; and in G2 between 0.0 and 6.3%. No statistically significant difference was observed between the obtained values. Moreover, we found no cytotoxic action of beta-lapachone on endothelial cells, and the results point out that the drug might have preserved the cell’s integrity against oxidative stress under the conditions of this experiment. This promising result suggests the possibility of beta-lapachone as a chemotherapy drug with selective activity.

Naphthoquinones from Handroanthus impetiginosus promote skin wound healing through Sirt3 regulation.

OBJECTIVES: Lapachone is a natural naphthoquinone-derived compound found in Tabebuia avellanedae. It is well-known for its analgesic, anti-inflammatory, anti-microbial, diuretic, and anti-cancerous effects. However, the wound-healing effects of this compound are not known yet. The aim of this study was to investigate the wound healing activity of naphthoquinones (α-lapachone and ß-lapachone) from Handroanthus impetiginosus. MATERIALS AND METHODS: Expression of Sirt3, migration-related proteins (Rac1, Cdc42, α-Pak) and angiogenesis-related protein of vascular endothelial growth factor (VEGF) was monitored using western blot analysis. Blood vessel formation and tissue development were monitored by angiogenesis assay and hematoxylin & eosin (H & E) staining, respectively on mouse skin tissue samples. Both α-lapachone and ß-lapachone increased Sirt3 expression in vivo, but only ß-lapachone increased Sirt3 expression in vitro. RESULTS: Both the compounds accelerated wound healing in cultured skin cells as well as mouse skin; however, ß-lapachone was more effective at lower concentrations. Both of the compounds increased the expression of migration-related proteins both in vitro and in vivo. Similarly, α-lapachone and ß-lapachone increased VEGF expression, tissue development and blood vessel formation in mouse skin. CONCLUSION: These findings indicated that α-lapachone and ß-lapachone are novel Sirt3 activators, and Sirt3 has a role in wound healing. Thus, Sirt3 and its regulators come out as a novel target and potential drug candidates, respectively in the important field of cutaneous wound healing.

Therapeutic Strategies and Biomarkers to Modulate PARP Activity for Targeted Cancer Therapy.

Poly-(ADP-ribose) polymerase 1 (PARP1) is commonly known for its vital role in DNA damage response and repair. However, its enzymatic activity has been linked to a plethora of physiological and pathophysiological transactions ranging from cellular proliferation, survival and death. For instance, malignancies with BRCA1/2 mutations heavily rely on PARP activity for survival. Thus, the use of PARP inhibitors is a well-established intervention in these types of tumors. However, recent studies indicate that the therapeutic potential of attenuating PARP1 activity in recalcitrant tumors, especially where PARP1 is aberrantly overexpressed and hyperactivated, may extend its therapeutic utility in wider cancer types beyond BRCA-deficiency. Here, we discuss treatment strategies to expand the tumor-selective therapeutic application of PARP inhibitors and novel approaches with predictive biomarkers to perturb NAD+ levels and hyperPARylation that inactivate PARP in recalcitrant tumors. We also provide an overview of genetic alterations that transform non-BRCA mutant cancers to a state of "BRCAness" as potential biomarkers for synthetic lethality with PARP inhibitors. Finally, we discuss a paradigm shift for the use of novel PARP inhibitors outside of cancer treatment, where it has the potential to rescue normal cells from severe oxidative damage during ischemia-reperfusion injury induced by surgery and radiotherapy.

Assessment of various formulation approaches for the application of beta-lapachone in prostate cancer therapy.

Beta-lapachone (ß-Lap) is an anticancer drug activated by the NAD(P)H:quinone oxidoreductase (NQO1), an enzyme over-expressed in a large variety of tumors. B-Lap is poorly soluble in water and in most biocompatible solvents. Micellar systems, liposomes and cyclodextrins (CDs) have been proposed for its solubilization. In this work, we analyzed the properties and in vitro efficacy of ß-Lap loaded in polymer nanoparticles, liposome bilayers, complexed with sulfobutyl-ether (SBE)- and hydroxypropyl (HP)-ß cyclodextrins, or double loaded in phospholipid vesicles. Nanoparticles led to the lowest drug loading. Encapsulation of [ß-Lap:CD] complexes in vesicles made it possible to slightly increase the encapsulation rate of the drug in liposomes, however at the cost of poor encapsulation efficiency. Cytotoxicity tests generally showed a higher sensitivity of NIH 3T3 and PNT2 cells to the treatment compared to PC-3 cells, but also a slight resistance at high ß-Lap concentrations. None of the studied ß-Lap delivery systems showed significant enhanced cytotoxicity against PC-3 cells compared to the free drug. Cyclodextrins and double loaded vesicles, however, appeared more efficient drug delivery systems than liposomes and nanoparticles, combining both good solubilizing and cytotoxic properties. Ligand-functionalized double loaded liposomes might allow overcoming the lack of selectivity of the drug.

Potent Cytotoxicity of Novel L-Shaped Ortho-Quinone Analogs through Inducing Apoptosis.

Twenty-seven L-shaped ortho-quinone analogs were designed and synthesized using a one pot double-radical synthetic strategy followed by removing methyl at C-3 of the furan ring and introducing a diverse side chain at C-2 of the furan ring. The synthetic derivatives were investigated for their cytotoxicity activities against human leukemia cells K562, prostate cancer cells PC3, and melanoma cells WM9. Compounds TB1, TB3, TB4, TB6, TC1, TC3, TC5, TC9, TC11, TC12, TC14, TC15, TC16, and TC17 exhibited a better broad-spectrum cytotoxicity on three cancer cells. TB7 and TC7 selectively displayed potent inhibitory activities on leukemia cells K562 and prostate cancer cells PC3, respectively. Further studies indicated that TB3, TC1, TC3, TC7, and TC17 could significantly induce the apoptosis of PC3 cells. TC1 and TC17 significantly induced apoptosis of K562 cells. TC1, TC11, and TC14 induced significant apoptosis of WM9 cells. The structure-activity relationships evaluation showed that removing methyl at C-3 of the furan ring and introducing diverse side chains at C-2 of the furan ring is an effective strategy for improving the anticancer activity of L-shaped ortho-quinone analogs.

Beta-lapachone attenuates immobilization-induced skeletal muscle atrophy in mice.

Skeletal muscle atrophy reduces quality of life and increases morbidity and mortality in patients with chronic conditions. Oxidative stress is a key factor contributing to skeletal muscle atrophy by altering both protein synthesis and protein degradation pathways. Beta-lapachone (Beta-L) is known to act as a pro-oxidant in cancer cells but suppresses oxidative stress in normal cells and tissues. In the present study, we examined whether Beta-L (100 mg/kg body weight) prevents immobilization-induced skeletal muscle atrophy in male C57BL/6N mice. Skeletal muscle atrophy was induced by immobilization of left hindlimbs for two weeks, and right hindlimbs were used as controls. The muscle weights of gastrocnemius (0.132 ±â€¯0.003 g vs. 0.115 ±â€¯0.003 g in Beta-L and SLS, respectively, p < 0.01) and tibialis anterior (0.043 ±â€¯0.001 vs. 0.027 ±â€¯0.002 in Beta-L and SLS, respectively, p < 0.001) were significantly heavier in Beta-L-treated mice than that in SLS-treated mice in immobilization group, which was accompanied by improved skeletal muscle function as tested by treadmill exhaustion and grip strength test. Immobilization increased H2O2 levels, while Beta-L treatment normalized such levels (1.6 ±â€¯0.16 µM vs. 2.7 ±â€¯0.44 µM in Beta-L and vehicle, respectively, p < 0.05). Oxidative stress makers were also normalized by Beta-L treatment. Protein synthesis signaling pathways were unaltered in the case of both immobilization and Beta-L treatment. However, protein catabolic, ubiquitin-proteasomal, and autophagy-lysosomal pathways were stimulated by immobilization and were normalized by Beta-L treatment. Upregulation of transforming growth factor ß and Smad 2/3 after immobilization was significantly diminished by Beta-L treatment. These results suggest that Beta-L attenuates the loss of muscle weight and function induced by immobilization through suppression of oxidative stress.

Hybrid Lipid Polymer Nanoparticles for Combined Chemo- and Photodynamic Therapy.

Retinoblastoma is a malignant tumor of the retina in infants. Conventional therapies are associated to severe side effects and some of them induce secondary tumors. Photodynamic therapy (PDT) thus appears as a promising alternative as it is nonmutagenic and generates minimal side effects. The effectiveness of PDT requires the accumulation of a photosensitizer (PS) in the tumor. However, most porphyrins are hydrophobic and aggregate in aqueous medium. Their incorporation into a nanocarrier may improve their delivery to the cell cytoplasm. In this work, we designed biodegradable liponanoparticles (LNPs) consisting of a poly(d,l)-lactide (PDLLA) nanoparticle coated with a phospholipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-3-trimethylammonium-propane) bilayer. An anticancer drug, beta-lapachone (ß-Lap) and a PS, m-THPC, were co-encapsulated for combined chemo- and PDT because it has been suggested that they may have a synergistic effect based on the activation of ß-Lap by PDT-induced over-expression of the enzyme NQO1. Using dynamic light scattering measurements, cryogenic transmission electron microscopy, and fluorescence confocal microscopy, we selected the appropriate conditions for the encapsulation of the compounds. LNPs were internalized in retinoblastoma cells within few hours. No obvious synergistic effect related to the activation of ß-Lap by PDT was observed. Conversely, the LNPs were cytotoxic at lower doses of the two encapsulated compounds as compared to the single therapies. Analysis of the combinatorial treatment showed that PDT and chemotherapy had an additive effect on the viability of retinoblastoma cells.

Beta Lapachone blocks the cell cycle and induces apoptosis in canine osteosarcoma cells / Beta Lapachona bloqueia o ciclo celular e induz apoptose em células de osteossarcoma canino

Osteosarcoma is a malignant tumor of primitive bone cells with a high incidence in dogs and humans. The need for more effective drugs with less adverse consequences has pushed the development of chemotherapeutic agents from plants and other natural sources. The aim of this study was to verify the cytotoxic effects of beta-lapachone, a compound present in the sawdust of Tabebuia sp. (popularly known as ipê) wood, on canine osteosarcoma cells subcultured and treated in different concentrations (0.1µm, 0.3µm e 1.0µm) and exposure times (24h, 48h e 72h). Results were obtained through Trypan blue dye exclusion, tetrazolium reducing method, cell survival assay, Annexin V-FITC and Propidium Iodine labeling, JC-1 dye labeling and cell cycle kinetics e analysis. The group treated with 0.3µm beta-lapachone presented higher decrease in cell viability (80.27%, 24h, 47.41%, 48h and 35.19%, 72h) and greater progression of cytotoxicity (19.73%, 24h, 52.59%, 48h and 64.81%, 72h). The lower IC50 (0.180µm) was verified in the group treated for 72 hours. Cell growth after treatment decreased as concentration and time of exposure increased, with 0.50% survival fraction at the concentration of 1.0µm. Initial apoptosis was the most frequent type of cell death in all groups, reaching bottom in the 24-hour group treated with 0.1µm (4.26%) and peaking in the 72-hour group treated with 1.0µm (85.89%). Mitochondrial depolarization demonstrated a dose-dependent phenomenon, indicating the intrinsic apoptosis. Cell growth inhibition by blocking cell cycle in the G0/G1 phase related to the exposure the time. β-lapachone is cytotoxic for canine osteosarcoma cells, induces apoptosis and promotes cell cycle arrest in G0/G1 phase.(AU)

O osteossarcoma é o tumor maligno das células ósseas primitivas, com alta incidência em cães e humanos. A necessidade de medicamentos mais efetivos, com menor consequência adversa, tem gerado esforços para o desenvolvimento de agentes quimioterápicos compostos por plantas e outras fontes naturais. O objetivo deste estudo foi verificar os efeitos citotóxicos da beta lapachona, um composto presente na serragem da madeira do ipê, sobre células de osteossarcoma canino subcultivadas e submetidas ao tratamento, de acordo com as diferentes concentrações (0.1µm, 0.3µm e 1.0µm) e tempo de exposição (24h, 48h e 72h). Os resultados foram obtidos por meio dos métodos de exclusão do corante azul de Tripan e de redução do tetrazólio, além dos ensaios de sobrevivência celular, de dupla marcação com Anexina V-FITC e Iodeto de Propídio, de marcação com o corante JC-1 e pela análise da cinética do ciclo celular. O grupo tratado com 0.3µm de beta lapachona apresentou melhor regressão da viabilidade celular (80,27%, 24h; 47,41%, 48h e 35,19%, 72h) e maior progressão da citotoxicidade (19,73%, 24h; 52,59%, 48h e 64,81%, 72h). O menor IC50 (0.180µm) ocorreu no grupo tratado por 72 horas. O crescimento celular após o tratamento foi menor, de acordo com o aumento da concentração e tempo de exposição, apresentando 0,50% de fração de sobrevivência na concentração de 1.0µm. A apoptose inicial foi o tipo de morte celular mais frequente em todos os grupos, menor no grupo de 24 horas tratado com 0.1µm (4,26%) e maior no grupo de 72 horas tratado com 1.0µm (85,89%). A despolarização mitocondrial ocorreu de maneira dose dependente, indicando a ocorrência de apoptose intrínseca. A β lapachona possui efeitos citotóxicos em células de osteossarcoma canino, induz apoptose e promove o bloqueio do ciclo celular na fase G0/G1.(AU)

A Highly Sensitive Chemiluminometric Assay for Real-Time Detection of Biological Hydrogen Peroxide Formation.

Hydrogen peroxide (H2O2) is a major reactive oxygen species (ROS) produced by various cellular sources, especially mitochondria. At high levels, H2O2 causes oxidative stress, leading to cell injury, whereas at low concentrations, this ROS acts as an important second messenger to participate in cellular redox signaling. Detection and measurement of the levels or rates of production of cellular H2O2 are instrumental in studying the biological effects of this major ROS. While a number of assays have been developed over the past decades for detecting and/or quantifying biological H2O2formation, none has been shown to be perfect. Perhaps there is no perfect assay for sensitively and accurately quantifying H2O2 as well as other ROS in cells, wherein numerous potential reactants are present to interfere with the reliable measurement of the specific ROS. In this context, each assay has its own advantages and intrinsic limitations. This article describes a highly sensitive assay for real-time detection of H2O2 formation in cultured cells and isolated mitochondria. This assay is based on the luminol/horseradish peroxidase-dependent chemiluminescence that is inhibitable by catalase. The article discusses the usefulness and shortcomings of this chemiluminometric assay in detecting biological H2O2 formation induced by beta-lapachone redox cycling with both cells and isolated mitochondria.

Preclinical Pharmacokinetic Evaluation of beta-Lapachone: Characteristics of Oral Bioavailability and First-Pass Metabolism in Rats

beta-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 beta-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 beta-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 beta-lapachone at doses of 40 mg/kg and 1.5 mg/kg, respectively. The measured oral bioavailability of beta-lapachone was 15.5%. The considerable degradation of beta-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 beta-lapachone may be resulted from the first-pass metabolic degradation of beta-lapachone in the liver, small and large intestinal tracts and its low aqueous solubility.

Mechanistic studies of cancer cell mitochondria- and NQO1-mediated redox activation of beta-lapachone, a potentially novel anticancer agent.

Beta-lapachone (beta-Lp) derived from the Lapacho tree is a potentially novel anticancer agent currently under clinical trials. Previous studies suggested that redox activation of beta-Lp catalyzed by NAD(P)H: quinone oxidoreductase 1 (NQO1) accounted for its killing of cancer cells. However, the exact mechanisms of this effect remain largely unknown. Using chemiluminescence and electron paramagnetic resonance (EPR) spin-trapping techniques, this study for the first time demonstrated the real-time formation of ROS in the redox activation of beta-lapachone from cancer cells mediated by mitochondria and NQO1 in melanoma B16-F10 and hepatocellular carcinoma HepG2 cancer cells. ES936, a highly selective NQO1 inhibitor, and rotenone, a selective inhibitor of mitochondrial electron transport chain (METC) complex I were found to significantly block beta-Lp meditated redox activation in B16-F10 cells. In HepG2 cells ES936 inhibited beta-Lp-mediated oxygen radical formation by ~80% while rotenone exerted no significant effect. These results revealed the differential contribution of METC and NQO1 to beta-lapachone-induced ROS formation and cancer cell killing. In melanoma B16-F10 cells that do not express high NQO1 activity, both NOQ1 and METC play a critical role in beta-Lp redox activation. In contrast, in hepatocellular carcinoma HepG2 cells expressing extremely high NQO1 activity, redox activation of beta-Lp is primarily mediated by NQO1 (METC plays a minor role). These findings will contribute to our understanding of how cancer cells are selectively killed by beta-lapachone and increase our ability to devise strategies to enhance the anticancer efficacy of this potentially novel drug while minimizing its possible adverse effects on normal cells.

beta-lapachone-Induced Apoptosis of Human Gastric Carcinoma AGS Cells Is Caspase-Dependent and Regulated by the PI3K/Akt Pathway

beta-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 beta-lapachone on the induction of apoptosis in human gastric carcinoma AGS cells. beta-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 beta-lapachone-treated AGS cells. Treatment with beta-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 beta-lapachone-induced cell death and inhibited growth. Further analysis demonstrated that the induction of apoptosis by beta-lapachone was accompanied by inactivation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. The PI3K inhibitor LY29004 significantly increased beta-lapachone-induced apoptosis and growth inhibition. Taken together, these findings indicate that the apoptotic activity of beta-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 beta-lapachone-mediated AGS cell growth inhibition and apoptosis induction.

The Tumor-Selective Cytotoxic Agent ß-Lapachone is a Potent Inhibitor of IDO1.

ß-lapachone is a naturally occurring 1,2-naphthoquinone-based compound that has been advanced into clinical trials based on its tumor-selective cytotoxic properties. Previously, we focused on the related 1,4-naphthoquinone pharmacophore as a basic core structure for developing a series of potent indoleamine 2,3-dioxygenase 1 (IDO1) enzyme inhibitors. In this study, we identified IDO1 inhibitory activity as a previously unrecognized attribute of the clinical candidate ß-lapachone. Enzyme kinetics-based analysis of ß-lapachone indicated an uncompetitive mode of inhibition, while computational modeling predicted binding within the IDO1 active site consistent with other naphthoquinone derivatives. Inhibition of IDO1 has previously been shown to breach the pathogenic tolerization that constrains the immune system from being able to mount an effective anti-tumor response. Thus, the finding that ß-lapachone has IDO1 inhibitory activity adds a new dimension to its potential utility as an anti-cancer agent distinct from its cytotoxic properties, and suggests that a synergistic benefit can be achieved from its combined cytotoxic and immunologic effects.

Enhancement of radiation effect using beta-lapachone and underlying mechanism.

Beta-lapachone (ß-Lap; 3,4-dihydro-2, 2-dimethyl-2H-naphthol[1, 2-b]pyran-5,6-dione) is a novel anti-cancer drug under phase I/II clinical trials. ß-Lap has been demonstrated to cause apoptotic and necrotic death in a variety of human cancer cells in vitro and in vivo. The mechanisms underlying the ß-Lap toxicity against cancer cells has been controversial. The most recent view is that ß-Lap, which is a quinone compound, undergoes two-electron reduction to hydroquinone form utilizing NAD(P)H or NADH as electron source. This two-electron reduction of ß-Lap is mediated by NAD(P)H:quinone oxidoreductase (NQO1), which is known to mediate the reduction of many quinone compounds. The hydroquinone forms of ß-Lap then spontaneously oxidizes back to the original oxidized ß-Lap, creating futile cycling between the oxidized and reduced forms of ß-Lap. It is proposed that the futile recycling between oxidized and reduced forms of ß-Lap leads to two distinct cell death pathways. First one is that the two-electron reduced ß-Lap is converted first to one-electron reduced ß-Lap, i.e., semiquinone ß-Lap (SQ)(·-) causing production of reactive oxygen species (ROS), which then causes apoptotic cell death. The second mechanism is that severe depletion of NAD(P)H and NADH as a result of futile cycling between the quinone and hydroquinone forms of ß-Lap causes severe disturbance in cellular metabolism leading to apoptosis and necrosis. The relative importance of the aforementioned two mechanisms, i.e., generation of ROS or depletion of NAD(P)H/NADH, may vary depending on cell type and environment. Importantly, the NQO1 level in cancer cells has been found to be higher than that in normal cells indicating that ß-Lap may be preferentially toxic to cancer cells relative to non-cancer cells. The cellular level of NQO1 has been found to be significantly increased by divergent physical and chemical stresses including ionizing radiation. Recent reports clearly demonstrated that ß-Lap and ionizing radiation kill cancer cells in a synergistic manner. Indications are that irradiation of cancer cells causes long-lasting elevation of NQO1, thereby sensitizing the cells to ß-Lap. In addition, ß-Lap has been shown to inhibit the repair of sublethal radiation damage. Treating experimental tumors growing in the legs of mice with irradiation and intraperitoneal injection of ß-Lap suppressed the growth of the tumors in a manner more than additive. Collectively, ß-Lap is a potentially useful anti-cancer drug, particularly in combination with radiotherapy.

Impact of Micellar Vehicles on in situ Intestinal Absorption Properties of Beta-Lapachone in Rats.

The aim of the present study was to examine the effect of micellar systems on the absorption of beta-lapachone (b-lap) through different intestinal segments using a single-pass rat intestinal perfusion technique. B-lap was solubilized in mixed micelles composed of phosphatidylcholine and sodium deoxycholate, and in sodium lauryl sulfate (SLS)-based conventional micelles. Both mixed micelles and SLS micelles improved the in situ permeability of b-lap in all intestinal segments tested although the mixed micellar formulation was more effective in increasing the intestinal absorption of b-lap. The permeability of b-lap was greatest in the large intestinal segments. Compared with SLS micelles, the effective permeability coefficient values measured with mixed micelles were 5- to 23-fold higher depending on the intestinal segment. Our data suggest that b-lap should be delivered to the large intestine using a mixed micellar system for improved absorption.