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.

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)

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.

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.

Enhancement of radiation effect using beta-lapachone and underlying mechanism

Beta-lapachone (beta-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. beta-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 beta-Lap toxicity against cancer cells has been controversial. The most recent view is that beta-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 beta-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 beta-Lap then spontaneously oxidizes back to the original oxidized beta-Lap, creating futile cycling between the oxidized and reduced forms of beta-Lap. It is proposed that the futile recycling between oxidized and reduced forms of beta-Lap leads to two distinct cell death pathways. First one is that the two-electron reduced beta-Lap is converted first to one-electron reduced beta-Lap, i.e., semiquinone beta-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 beta-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 beta-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 beta-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 beta-Lap. In addition, beta-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 beta-Lap suppressed the growth of the tumors in a manner more than additive. Collectively, beta-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.

Antifungal activity of the naphthoquinone beta-lapachone against disseminated infection with Cryptococcus neoformans var. neoformans in dexamethasone-immunosuppressed Swiss mice

The in vivo antifungal activity of the naphthoquinone beta-lapachone against disseminated infection by Cryptococcus neoformans was investigated. Swiss mice were immunosuppressed daily with dexamethasone (0.5 mg per mouse) intraperitoneally for 3 days, the procedure was repeated 4 days later, and the animals were then challenged intravenously with C. neoformans (10(6) CFU/mL) 1 week later. Seven days after infection, the mice were divided into groups and treated daily with beta-lapachone (10 mg/kg, iv) for 7 (N = 6) and 14 days (N = 10). Amphotericin B (0.5 mg/kg) was used as comparator drug and an additional group received PBS. Treatment with beta-lapachone cleared the yeast from the spleen and liver, and the fungal burden decreased approximately 10(4) times in the lungs and brain 14 days after infection when compared to the PBS group (P < 0.05). This result was similar to that of the amphotericin B-treated group. Protection was suggestively due to in vivo antifungal activity of this drug and apparently not influenced by activation of the immune response, due to similar leukocyte cell counts among all groups. This study highlights the prospective use of beta-lapachone for treatment of disseminated cryptococcosis.

beta-Lapachone suppresses radiation-induced activation of nuclear factor-kappaB

Anticancer effects of beta-lapachone (beta-lap) are due to generation of ROS and metabolic catastrophes as a result of NAD(P)H:quinone oxidoreductase (NQO1)-mediated futile cycling between the oxidized and reduced forms of beta-lap. It has been shown that NQO1 is also essential for the TNF-induced activation of NF-kappaB and that beta-lap suppresses the TNF-induced NF-kappaB activation. We investigated whether or not NQO1 is involved and beta-lap suppresses the radiation-induced NF-kappaB activation using A549 human lung cancer cells and NQO1-knock down A549 cells (shNQO1 A549 cells). Irradiation with 4 Gy markedly increased the DNA binding activity of NF-kappaB in A549 cells, but not in the shNQO1 A549 cells, thus demonstrating that NQO1 plays a pivotal role in irradiation-induced NF-kappaB activation. Treatment with 10 micrometer beta-lap for 4 h almost completely abrogated the radiation-induced increase in NF-kappaB activation and the transcription of NF-kappaB target genes such as bcl2, gadd45beta and cyclinD1. Moreover, beta-lap markedly suppressed the activation of IkappaB kinase gamma (IKKgamma) and the subsequent phosphorylation of IkappaBalpha, thereby inhibiting NF-kappaB activation. It is concluded that beta-lap suppresses the radiation-induced activation of NF-kappaB by interrupting the involvement of NQO1 in the activation of NF-kappaB, thereby inhibiting the transcription of survival signals. The radiosensitization caused by beta-lap may, in part, be attributed to beta-lap-induced suppression of NF-kappaB activation.

In vitro synergic effect of beta-lapachone and isoniazid on the growth of Mycobacterium fortuitum and Mycobacterium smegmatis

Nontuberculous mycobacteria are ubiquitous and saprophytic organisms that have been implicated in a wide spectrum of diseases due to an increasing number of immunocompromised patients. The natural resistance of atypical mycobacteria to classical antituberculous drugs has encouraged research into new chemotherapeutic agents and drug combinations. The aim of this study was to determine the in vitro antimycobacterial activities of ²-lapachone alone and in combination with isoniazid against Mycobacterium fortuitum and Mycobacterium smegmatis via the Time-Kill Curve method. A 2 log10 CFU/mL reduction in the M. smegmatis culture was observed 72 h after adding ²-lapachone at its minimum inhibitory concentration. This drug sterilised the culture in 120 h. For M. fortuitum, a reduction of 1.55 log10 CFU/mL occurred in 24 h, but regrowth was seen in contact with ²-lapachone. Both microorganisms were resistant to isoniazid. Regrowth of M. fortuitum and M. smegmatis was observed at 48 h and 72 h, respectively. In combination, these two drugs had a bactericidal effect and sterilised both cultures in 96 h. These results are valuable because antibiotic-resistant bacteria are a major public health problem.

Spasmolytic activity of lapachol and its derivatives, alpha and beta-lapachone, on the guinea-pig ileum involves blockade of voltage-gated calcium channels / Atividade espasmolítica do lapachol e seus derivados, alfa e beta-lapachona, em íleo de cobaia envolve bloqueio dos canais de cálcio dependentes de voltagem

O lapachol, alfa e beta-lapachona são o naftoquinonas obtidas de espécies de Tabebuia, apresentam propriedades antiinflamatória, antibacteriana, anticâncer e tripanossomicida. O objetivo deste trabalho foi investigar um possível efeito espasmolítico destas naftoquinonas em íleo de cobaia, uma vez que, outras naftoquinonas inibem a atividade contrátil de músculos lisos. O lapachol, alfa e beta-lapachona inibiram as contrações fásicas induzidas tanto por carbacol (CI50 = 1,5 ± 0,2 x 10-4; 7,3 ± 0,9 x 10-5 e 3,2 ± 0,5 x 10-5 M, respectivamente) quanto por histamina (CI50 = 3,6± 0,5; 3,6 ± 0,7 e 3,3 ± 0,6 x 10-5 M, respectivamente). Estes compostos também relaxaram o íleo pré-contraído com KCl (CE50 = 1,2 ± 0,4; 4,3 ± 0,8 e 2,7 ± 0,2 x 10-5 M, respectivamente); carbacol (CE50 = 2,6 ± 0,7; 3,5 ± 0,5 e 2,2 ± 0,7 x 10-5 M, respectivamente) ou histamina (CE50 = 3,0 ± 0,8; 1,1 ± 0,3 e 3,3 ± 0,6 x 10-5 M, respectivamente) de maneira dependente de concentração. Este efeito é provavelmente devido à inibição do influxo de Ca2+ através dos canais de Ca2+ dependentes de voltagem (CaV). Beta-lapachona antagonizou (pD'2 = 5,73 ± 0,12; "slope" = 1,51 ± 0,05) as contrações induzidas por CaCl2 em meio despolarizante nominalmente sem Ca2+. O achado de que a beta-lapachona inibiu as contrações tônicas induzidas por S-(-)-Bay K8644 (CE50 = 1,4 ± 0,1 x 10-5 M) é sugestivo que o CaV envolvido é o do tipo L. Em conclusão lapachol, alfa e beta-lapachona apresentam atividade espasmolítica não seletiva em íleo de cobaia, e beta-lapachona exerce este efeito pelo bloqueio dos canais CaV tipo L.

Lapachol, alpha and beta-lapachone are naphthoquinones extracted from species of Tabebuia that have shown antiinflammatory, antibacterial, anticancer and trypanosomicidal properties. The aim of this work was to investigate the spasmolytic effect of these naphthoquinones on the guinea-pig ileum, since other naphthoquinones are known to depress the contractile activity of smooth muscles. Lapachol, alpha and beta-lapachone inhibited the phasic contractions induced by both carbachol (IC50 = 1.5 ± 0.2 x 10-4; 7.3 ± 0.9 x 10-5 and 3.2 ± 0.5 x 10-5 M, respectively) and histamine (IC50 = 3.6± 0.5; 3.6 ± 0.7 and 3.3 ± 0.6 x 10-5 M, respectively). These compounds also relaxed the ileum pre-contracted with KCl (EC50 = 1.2 ± 0.4; 4.3 ± 0.8 and 2.7 ± 0.2 x 10-5 M, respectively); carbachol (EC50 = 2.6 ± 0.7; 3.5 ± 0.5 and 2.2 ± 0.7 x 10-5 M, respectively) or histamine (EC50 = 3.0 ± 0.8; 1.1 ± 0.3 and 3.3 ± 0.6 x 10-5 M, respectively) in a concentration-dependent manner. This effect is probably due to inhibition of calcium influx through voltage-gated calcium channels (Ca v). Beta-lapachone antagonized (pD'2 = 5.73 ± 0.12; slope = 1.51 ± 0.05) CaCl2-induced contractions in depolarizing medium nominally without Ca2+. The finding that Beta-lapachone inhibited the tonic contractions induced by S-(-)-Bay K8644 (EC50 = 1.4 ± 0.1 x 10-5 M) is suggestive that the L-type CaV is involved. In conclusion, lapachol, alpha and beta-lapachone showed non-selective spasmolytic activity in guinea-pig ileum, and beta-lapachone exerts this effect by to blockade of L-type CaV channels.

Synergistic Effect of Ionizing Radiation and beta-lapachone against RKO Human Colon Adenocarcinoma Cells / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: To reveal the interaction between beta-Lapachone (beta-lap) and ionizing radiation in causing cell death in RKO human colon adenocarcinoma cells, and to elucidate the potential usefulness of combined beta-lap treatment and radiotherapy for cancer treatment. MATERIALS AND METHODS: The cytotoxicities of various treatments were determined in vitro using clonogenic and apoptotic cell death. The changes in cell cycle distribution were studied using flow cytometry and an in vitro kinase assay. The tumor growth was studied using RKO tumors grown s.c. in the hind leg BALB/c- nuslc nude mice. RESULTS: beta-lap caused clonogenic cell death and rapid apoptosis in RKO cells in vitro, in a dose dependent manner. The repair of sublethal radiation damage was almost completely inhibited when cells were maintained in beta-lap during the interval between the two-dose irradiation. Flow cytometry study demonstrated that beta-lap induced apoptosis, independent of the cell cycle phase, and completely prohibited the induction of radiation- induced G2 arrest in irradiated cells. The prohibition of radiation-induced G2 arrest is unclear, but may be related to the profound suppression of the p53, p21 and cyclin B1-Cdc2 kinase activities observed in cells treated with beta-lap. The combination of beta-lap and radiation markedly enhanced the radiation-induced growth suppression of tumors. CONCLUSION: beta-lap is cytotoxic against RKO cells, both in vitro and in vivo, and also sensitized cells to ionizing radiation by inhibiting sublethal radiation damage repair. beta-lap is potentially useful as a potent anti-cancer chemotherapy drug and potent radiosensitizer against caner cells.