naphthoquinones and Prostatic-Neoplasms

Purpose Population pharmacokinetics (PK) of sepantronium bromide (YM155) was characterized in patients with non-small cell lung cancer, hormone refractory prostate cancer, or unresectable stage III or IV melanoma and enrolled in one of three phase 2 studies conducted in Europe or the U.S. Method Sepantronium was administered as a continuous intravenous infusion (CIVI) at 4.8 mg/m(2)/day over 7 days every 21 days. Population PK analysis was performed using a linear one-compartment model involving total body clearance (CL) and volume of distribution with an inter-individual random effect on CL and a proportional residual errors to describe 578 plasma sepantronium concentrations obtained from a total of 96 patients by NONMEM Version VI. The first-order conditional estimation method with interaction was applied. Results The one-compartment model with one random effect on CL and two different proportional error models provided an adequate description of the data. Creatinine clearance (CLCR), cancer type, and alanine aminotransferase (ALT) were recognized as significant covariates of CL. CLCR was the most influential covariate on sepantronium exposure and predicted to contribute to a 25 % decrease in CL for patients with moderately impaired renal function (CLCR = 40 mL/min) compared to patients with normal CLCR. Cancer type and ALT had a smaller but nonetheless significant contribution. Other patient characteristics such as age, gender, and race were not considered as significant covariates of CL. Conclusions The results provide the important information for optimizing the therapeutic efficacy and minimizing the toxicity for sepantronium in cancer therapy.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Non-Small-Cell Lung; Dose-Response Relationship, Drug; Ethnicity; Female; Follow-Up Studies; Humans; Imidazoles; Infusions, Intravenous; Inhibitor of Apoptosis Proteins; Japan; Lung Neoplasms; Male; Maximum Tolerated Dose; Melanoma; Middle Aged; Models, Biological; Naphthoquinones; Neoplasm Staging; Neoplasms, Hormone-Dependent; Prognosis; Prostatic Neoplasms; Survivin; Tissue Distribution

YM155, a small-molecule survivin suppressor, showed modest single-agent activity in a phase I study of heavily pretreated patients. This study was conducted to determine the activity of YM155 in patients with castration-resistant prostate cancer (CRPC) who received prior taxane therapy.. Patients received 4.8 mg/m(2)/day of YM155 over 168-h continuous i.v. infusion every 3 weeks. Study end points included prostate-specific antigen (PSA) response, objective tumor response, safety, progression-free survival (PFS) and overall survival (OS).. Thirty-five patients were enrolled. Two of 32 (6.2%) assessable patients had a PSA response and 2 additional patients had PSA decrements >50% but not confirmed. One of 16 (6.2%) patients also had a partial response per RECIST V1. Median PFS and OS were 3.1 and 11.2 months, respectively. The most common adverse events were fatigue (63%), nausea (40%), anorexia (31%), constipation (31%), fever (26%) and vomiting (26%).. YM155 has modest activity in taxane-pretreated CRPC with 25% of patients having prolonged stable disease (≥18 weeks). The regimen appears to be well tolerated. Based on the mechanism of action and preclinical evidence of synergy with docetaxel (Taxotere), YM155 combined with docetaxel is being evaluated in patients with CRPC.

Topics: Aged; Aged, 80 and over; Antineoplastic Agents; Bridged-Ring Compounds; Drug Resistance, Neoplasm; Humans; Imidazoles; Inhibitor of Apoptosis Proteins; Male; Middle Aged; Naphthoquinones; Prostate-Specific Antigen; Prostatic Neoplasms; Survivin; Taxoids; Treatment Outcome

Plumbagin, a bioactive naphthoquinone, has demonstrated potent antitumor potential. However, plumbagin is a sparingly water-soluble compound; therefore, clinical translation requires and will be facilitated by the development of a new pharmaceutical formulation. We have generated an oil-in-water nanoemulsion formulation of plumbagin using a low-energy spontaneous emulsification process with propylene glycol caprylate (Capryol 90) as an oil phase and Labrasol/Kolliphor RH40 as surfactant and cosurfactant excipients. Formulation studies using Capryol 90/Labrasol/Kolliphor RH40 components, based on pseudoternary diagram and analysis of particle size distribution and polydispersity determined by dynamic light scattering (DLS), identified an optimized composition of excipients for nanoparticle formulation. The nanoemulsion loaded with plumbagin as an active pharmaceutical ingredient had an average hydrodynamic diameter of 30.9 nm with narrow polydispersity. The nanoemulsion exhibited long-term stability, as well as good retention of particle size in simulated physiological environments. Furthermore, plumbagin-loaded nanoemulsion showed an augmented cytotoxicity against prostate cancer cells PTEN-P2 in comparison to free drug. In conclusion, we generated a formulation of plumbagin with high loading drug capacity, robust stability, and scalable production. Novel Capryol 90-based nanoemulsion formulation of plumbagin demonstrated antiproliferative activity against prostate cancer cells, warranting thus further pharmaceutical development.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Drug Carriers; Emulsions; Male; Mice; Nanoparticles; Naphthoquinones; Propylene Glycol; Prostatic Neoplasms

Prostate cancer (PC) metastasizes to the skeleton forming predominantly sclerotic lesions, and there is currently no cure for bone metastatic disease. The transcription factor signal transducer and activator of transcription 3 (STAT3) is implicated as a metastatic driver, but its potential as therapeutic target in bone metastasis has not been investigated. In this study, we evaluated for the first time a STAT3 inhibitor, Napabucasin, as a therapeutic option for bone metastatic PC.. Effects of STAT3 inhibitors, Stattic and Napabucasin, on metastatic potential in PC cells were studied in vitro by assessment of migration capacity, self-renewal potential, and tumorsphere formation. For evaluation of the role of STAT3 in initial skeletal establishment of PC cells as well as in progressed castration-resistant PC (CRPC) in bone, human VCaP prostate cancer cells were inoculated in the tibia of mice which subsequently were treated with the STAT3 inhibitor Napabucasin. Bone specimens were analyzed using computed tomography (CT), immunohistochemistry, and quantitative polymerase chain reaction.. The small molecule STAT3 inhibitors Stattic and Napabucasin both effectively impaired metastatic potential of PC cells in vitro. Furthermore, treatment with Napabucasin prevented metastatic establishment in tibial bones in vivo and thereby also the tumor-induced sclerotic bone response seen in vehicle-treated VCaP xenografts. In addition, treatment with Napabucasin of established bone CRPC significantly decreased both tumor burden and tumor-induced trabecular bone volume compared with effects seen in vehicle-treated animals. Anti-mitotic effects were confirmed by decreased Ki67 staining in Napabucasin-treated xenografts compared with vehicle-treated xenografts. Alterations of gene expression in the femoral bone marrow (BM) niche toward the maintenance of hematopoietic stem cells and the myeloid lineage were demonstrated by quantitative real-time polymerase chain reaction and were further reflected by a substantial increase in the number of erythrocytes in BM of Napabucasin-treated mice. Furthermore, a unique pattern of STAT3 phosphorylation in osteoblasts/stromal cells surrounding the areas of tumor cells was demonstrated immunohistochemically in bone xenograft models using several different PC cell lines.. Inhibition of STAT3 activity disrupts the bone metastatic niche and targets both the skeletal establishment of PC and advanced bone metastatic CRPC in mice, suggesting STAT3 as a candidate for molecular targeted therapies of skeletal metastatic disease.

Topics: Animals; Benzofurans; Bone Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclic S-Oxides; Male; Mice; Naphthoquinones; Prostatic Neoplasms; STAT3 Transcription Factor; Tibia

Although the biomedical sciences have achieved tremendous success in developing novel approaches to managing prostate cancer, this disease remains one of the major health concerns among men worldwide. Liposomal formulations of single drugs have shown promising results in cancer treatment; however, the use of multi drugs has shown a better therapeutic index than individual drugs. The identification of cancer-specific receptors has added value to design targeted drug delivering nanocarriers. We have developed genistein and plumbagin co-encapsulating liposomes (∼120 nm) with PSMA specific antibodies to target prostate cancer cells selectively in this work. These liposomes showed >90 % decrease in PSMA expressing prostate cancer cell proliferation without any appreciable toxicity to healthy cells and human red blood cells. Release of plumbagin and genistein was found to decrease the expression of PI3/AKT3 signaling proteins and Glut-1 receptors (inhibited glucose uptake and metabolism), respectively. The decrease in migration potential of cells and induced apoptosis established the observed anti-proliferative effect in prostate cancer cell lines. The discussed strategy of developing novel, non-toxic, and PSMA specific antibody conjugated liposomes carrying genistein and plumbagin drugs may also be used for encapsulating other drugs and inhibit the growth of different types of cancers.

Topics: Apoptosis; Cell Line, Tumor; Genistein; Glutamate Carboxypeptidase II; Humans; Liposomes; Male; Naphthoquinones; Prostatic Neoplasms

We developed a novel method for the synthesis of bis-naphthoquinones (BNQ), which are hybrids of lawsone (2-hydroxy-1,4-naphthoquinone) and 3-hydroxy-juglone (3,5-dihydroxy-1,4-naphthoquinone). The anticancer activity of three synthesized compounds, named 4 (RC10), 5 (RCDFC), and 6 (RCDOH) was evaluated in vitro against two metastatic prostate cancer (PCa) cell lines, DU145 and PC3, using MTT assays. We found that 4 (RC10) and 5 (RCDFC) induced cytotoxicity against DU145 and PC3 cells. Flow cytometry analysis revealed that these two compounds promoted cell cycle arrest in G1/S and G2/M phases, increased Sub-G1 peak and induced inhibition in cell viability. We also showed that these effects are cell-type context dependent and more selective for these tested PCa cells than for HUVEC non-tumor cells. The two BNQ compounds 4 (RC10) and 5 (RCDFC) displayed promising anticancer activity against the two tested metastatic PCa cell lines, DU145 and PC3. Their effects are mainly associated with inhibition of cell viability, possibly through apoptotic cell death, besides altering the SubG1, G1/S and G2/M phases of cell cycle. 5 (RCDFC) compound was found to be more selective than 4 (RC10), when comparing their cytotoxic effects in relation to HUVEC non-tumoral cells. Future work should also test these compounds in combination with other chemotherapeutic drugs to evaluate their effects on further sensitizing drug-resistant metastatic PCa cells.

Topics: Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Screening Assays, Antitumor; Humans; Male; Naphthoquinones; PC-3 Cells; Prostatic Neoplasms

Beta-lapachone (β-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 β-Lap loaded in polymer nanoparticles, liposome bilayers, complexed with sulfobutyl-ether (SBE)- and hydroxypropyl (HP)-β cyclodextrins, or double loaded in phospholipid vesicles. Nanoparticles led to the lowest drug loading. Encapsulation of [β-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 β-Lap concentrations. None of the studied β-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.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Survival; Cells, Cultured; Cyclodextrins; Drug Carriers; Drug Compounding; Humans; Liposomes; Male; Mice; Nanoparticles; Naphthoquinones; Phospholipids; Prostatic Neoplasms; Solubility

Despite the plethora of significant research progress made to develop novel strategies for the treatment of prostate cancer, this disease remains one of the major global health challenges among men. However, using a co-treatment approach utilizing two or more anticancer drugs has shown tremendous success in the treatment of many cancer types. Nanoliposomes are well known to encapsulate multiple drugs and deliver them at the desired site. In this work, we report the synthesis of nanoliposomes (∼100 nm) encapsulating two drugs, plumbagin, and genistein, to synergistically inhibit the growth of prostate cancer cells. The combination of plumbagin and genistein drugs was found inhibiting xenograft prostate tumor growth by ∼80 % without any appreciable toxicity. Mechanistically, the combination of plumbagin and genistein containing nanoliposomes leads to the inhibition of PI3K/AKT3 signaling pathway as well as the decreased population of Glut-1 transporters to impart the retardation in tumor growth. Decrease in proliferative cells and blood vessels are early biological processes that laid the foundation of the observed anti-tumor effect. Thus, a novel, and non-toxic liposomal formulation, containing plumbagin and genistein drugs, is reported, which can deliver anticancer agents to prostate tumors and inhibit the growth.

Topics: Animals; Antineoplastic Agents; Cell Proliferation; Cells, Cultured; Drug Delivery Systems; Drug Screening Assays, Antitumor; Genistein; Glucose Transporter Type 1; Humans; Liposomes; Male; Mice; Naphthoquinones; Neoplasms, Experimental; Particle Size; PC-3 Cells; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Surface Properties; Wound Healing

The phenomenon of high sugar consumption by tumor cells is known as Warburg effect. It results from a high glycolysis rate, used by tumors as preferred metabolic pathway even in aerobic conditions. Targeting the Warburg effect to specifically deliver sugar conjugated cytotoxic compounds into tumor cells is a promising approach to create new selective drugs. We designed, synthesized, and analyzed a library of novel 6-S-(1,4-naphthoquinone-2-yl)-d-glucose chimera molecules (SABs)-novel sugar conjugates of 1,4-naphthoquinone analogs of the sea urchin pigments spinochromes, which have previously shown anticancer properties. A sulfur linker (thioether bond) was used to prevent potential hydrolysis by human glycoside-unspecific enzymes. The synthesized compounds exhibited a Warburg effect mediated selectivity to human prostate cancer cells (including highly drug-resistant cell lines). Mitochondria were identified as a primary cellular target of SABs. The mechanism of action included mitochondria membrane permeabilization, followed by ROS upregulation and release of cytotoxic mitochondrial proteins (AIF and cytochrome C) to the cytoplasm, which led to the consequent caspase-9 and -3 activation, PARP cleavage, and apoptosis-like cell death. These results enable us to further clinically develop these compounds for effective Warburg effect targeting.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Drug Screening Assays, Antitumor; Glucose; Humans; Male; Membrane Potential, Mitochondrial; Mitochondrial Membranes; Naphthoquinones; Pigments, Biological; Prostatic Neoplasms; Sea Urchins; Warburg Effect, Oncologic

The reaction of 2-acyl-1,4-naphthoquinones with

Topics: Antineoplastic Agents; Antioxidants; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Drug Screening Assays, Antitumor; Electrochemical Techniques; Female; HEK293 Cells; Humans; Male; Naphthoquinones; Oxidation-Reduction; Prostatic Neoplasms; Structure-Activity Relationship

The imidazolium compound YM155, first discovered as a potent inhibitor of Survivin, effectively kills many carcinomas in preclinical models. However, the upstream signaling mechanism triggered by YM155 remains unclear. Here we studied early signaling responses in vitro in prostate and renal cancer cell lines in a dose-dependent manner. We found that YM155 rapidly activates the retinoblastoma protein, correlating with the loss of expression of all three Cyclin Ds. Using Western blot, various selective chemical inhibitors and q-PCR, we show that YM155-mediated decrease in protein levels of Cyclin Ds, Survivin and Mcl-1 is independent of transcription or proteasomal control mechanisms. Moreover, we provide the first evidence that YM155 changes the phosphorylation status of known mTOR-target proteins involved in translational control, namely ribosomal protein S6 (rS6) and 4E-BP1. Our data support that YM155 achieves this by blocking mTORC1 via the phosphorylation of Raptor at S792 through activated AMPKα (T172). Furthermore, we also used a polysome profile, supporting that YM155 markedly suppresses cap-dependent translation of mRNAs which include Survivin, Cyclin D1 and Mcl-1. We provide the first evidence that YM155 functions as a potent activator of AMPKα, a robust suppressor of mTORC1 and an attenuator of global protein synthesis.

Topics: Adaptor Proteins, Signal Transducing; AMP-Activated Protein Kinase Kinases; Apoptosis; Carcinoma; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin D; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Male; Mechanistic Target of Rapamycin Complex 1; Naphthoquinones; Prostate; Prostatic Neoplasms; Protein Kinases; Signal Transduction; Survivin

Topics: Animals; Antineoplastic Agents; Apoptosis; Cattle; Cell Line, Tumor; Cell Movement; Coordination Complexes; DNA; Humans; Intercalating Agents; Male; Naphthoquinones; Prostatic Neoplasms; Reactive Oxygen Species; Ruthenium; Spheroids, Cellular

The targeted induction of reactive oxygen species (ROS) is a developing mechanism for cancer therapy. LQB-118 is a pterocarpanquinone and ROS-inducing agent with proven antineoplastic activity. Here, LQB-118 efficacy and mechanism of activity, were examined in Prostate Cancer (PCa) cell and tumor models.. LQB-118 treatment triggered PCa cell death and apoptosis. Therapeutic activity was at least partially dependent upon quinone reduction and ROS generation. LQB-118 treatment caused an increase in cellular ROS and lipid peroxidation. Treated cells exhibited elevated levels of NQO1, Nrf2, and SOD1. The miRNAs miR-206, miR-1, and miR-101 targeted and reduced SOD1 expression. The knockdown of SOD1, by siRNA or miRNA, enhanced LQB-118 cytotoxicity. Orally administered LQB-118 treatment significantly reduced the growth of established PCa xenograft tumors.. LQB-118 is a developing and orally active pterocarpanquinone agent that effectively kills PCa cells through quinone reduction and ROS generation. The inhibition SOD1 expression enhances LQB-118 activity, presumably by impairing the cellular antioxidant response.

Topics: Administration, Oral; Animals; Antineoplastic Agents; Cell Line, Tumor; Disease Models, Animal; Humans; Male; Mice; Mice, Nude; Naphthoquinones; Prostate; Prostatic Neoplasms; Pterocarpans; Reactive Oxygen Species; Treatment Outcome

Prostate cancer (PCa) is the second most frequently diagnosed cancer in men worldwide. Currently available therapies for hormone-refractory PCa are only marginally effective. Plumbagin (PLB), a natural naphthoquinone isolated from the traditional folk medicine Plumbago zeylanica, is known to selectively kill tumor cells. Nevertheless, antitumor mechanisms initiated by PLB in cancer cells have not been fully defined.. MTT assay was used to evaluate the effect of PLB on the viability of cancer cells. Cell apoptosis and reactive oxygen species (ROS) production were determined by flow cytometry. Protein expression was detected by western blotting. In vivo anti-tumor effect was measured by using tumor xenoqraft model in nude mice.. In the present study, we found that PLB decreases cancer cell growth and induces apoptosis in DU145 and PC-3 cells. In addition, by increasing intracellular ROS levels, PLB induced a lethal endoplasmic reticulum stress response in PCa cells. Importantly, blockage of ROS production significantly reversed PLB-induced ER stress activation and cell apoptosis. In vivo, we found that PLB inhibits the growth of PCa xenografts without exhibiting toxicity Treatment of mice bearing human PCa xenografts with PLB was also associated with induction of ER stress activation.. Inducing ER stress by PLB thus discloses a previously unrecognized mechanism underlying the biological activity of PLB and provides an in-depth insight into the action of PLB in the treatment of hormone-refractory PCa.

Topics: Acetylcysteine; Activating Transcription Factor 4; Animals; Apoptosis; Caspase 3; Caspase 9; Cell Line, Tumor; eIF-2 Kinase; Endoplasmic Reticulum Stress; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Naphthoquinones; Prostatic Neoplasms; Reactive Oxygen Species; RNA Interference; Transcription Factor CHOP; Transplantation, Heterologous

Treatment of mice harboring PTEN-P2 tumors in the prostate or on prostate tissue in vivo with 5-hydroxy-2-methyl-1,4-naphthoquinone, also known as plumbagin, results in tumor regression in castrated mice, but not in intact mice. This suggested that dihydrotestosterone (DHT) production in the testes may prevent cell death due to plumbagin treatment, but the underlying mechanism is not understood. We performed RNA-seq analysis on cells treated with combinations of plumbagin and DHT, and analyzed differential gene expression, to gain insight into the interactions between androgen and plumbgin. DHT and plumbagin synergize to alter the expression of many genes that are not differentially regulated by either single agent when used alone. These experiments revealed that, for many genes, increases in mRNAs caused by DHT are sharply down-regulated by plumbagin, and that many transcripts change in response to plumbagin in a DHT-dependent manner. This suggests that androgen receptor mediates some of the effects of plumbagin on gene expression.

Topics: Androgens; Animals; Cell Line, Tumor; Dihydrotestosterone; Gene Expression Regulation, Neoplastic; Male; Mice; Naphthoquinones; Prostate; Prostatic Neoplasms; Receptors, Androgen; Testis; Transcriptome

Plumbagin, a medicinal plant-derived 5-hydroxy-2-methyl-1,4-naphthoquinone, is an emerging drug with a variety of pharmacological effects, including potent anticancer activity. We have previously shown that plumbagin improves the efficacy of androgen deprivation therapy (ADT) in prostate cancer and it is now being evaluated in phase I clinical trial. However, the development of formulation of plumbagin as a compound with sparing solubility in water is challenging.. We have formulated plumbagin-loaded nanoemulsion using pneumatically controlled high pressure homogenization of oleic acid dispersions with polyoxyethylene (20) sorbitan monooleate as surfactant. Nanoemulsion formulations were characterized for particle size distribution by dynamic light scattering (DLS). The kinetics of. Particle size distribution of nanoemulsions is tunable and depends on the surfactant concentration. Nanoemulsion formulations of plumbagin with 1-3.5% (w/w) of surfactant showed robust stability of size distribution over time. Plumbagin-loaded nanoemulsion with average hydrodynamic diameter of 135 nm showed exponential release of plumbagin with a half-life of 6.1 h in simulated gastric fluid, 7.0 h in simulated intestinal fluid, and displayed enhanced antiproliferative effect toward prostate cancer cells PTEN-P2 compared to free plumbagin.. High drug-loading capacity, retention of nanoparticle size, kinetics of release under simulated physiological conditions, and increased antiproliferative activity indicate that oleic-acid based nanoemulsion formulation is a suitable delivery system of plumbagin.

Topics: Animals; Cell Death; Cell Line, Tumor; Cell Proliferation; Drug Delivery Systems; Drug Liberation; Emulsions; Male; Mice; Nanoparticles; Naphthoquinones; Particle Size; Prostatic Neoplasms; Surface-Active Agents

Androgen receptor (AR) signaling is fundamental to prostate cancer and is the dominant therapeutic target in metastatic disease. However, stringent androgen deprivation therapy regimens decrease quality of life and have been largely unsuccessful in curtailing mortality. Recent clinical and preclinical studies have taken advantage of the dichotomous ability of AR signaling to elicit growth-suppressive and differentiating effects by administering hyperphysiologic levels of testosterone. In this study, high-throughput drug screening identified a potent synergy between high-androgen therapy and YM155, a transcriptional inhibitor of survivin (BIRC5). This interaction was mediated by the direct transcriptional upregulation of the YM155 transporter SLC35F2 by the AR. Androgen-mediated YM155-induced cell death was completely blocked by the overexpression of multidrug resistance transporter ABCB1. SLC35F2 expression was significantly correlated with intratumor androgen levels in four distinct patient-derived xenograft models, and with AR activity score in a large gene expression dataset of castration-resistant metastases. A subset of tumors had significantly elevated SLC35F2 expression and, therefore, may identify patients who are highly responsive to YM155 treatment.. The combination of androgen therapy with YM155 represents a novel drug synergy, and SLC35F2 may serve as a clinical biomarker of response to YM155.

Topics: Androgens; Animals; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Synergism; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Male; Membrane Transport Proteins; Mice; Naphthoquinones; Prostatic Neoplasms; Receptors, Androgen; Signal Transduction; Testosterone; Treatment Outcome; Xenograft Model Antitumor Assays

Plumbagin inhibits the growth, metastasis, and invasion of prostate cancer (PCa). However, its lower bioavailability limits biopharmaceutical properties due to insolubility in water. Prostate-specific membrane antigen (PSMA) aptamer-targeted nanoparticles (NPs) significantly enhanced cytotoxicity in prostate epithelial cells. This study aimed to investigate the effects of plumbagin-loaded prostate-specific membrane antigen (PSMA) aptamer-targeted poly D,L-lactic-co-glycolic acid-b-polyethylene glycol (PLGA-PEG) nanoparticles (NPs) on prostate cancer (PCa) in vitro.PLGA-PEG with a terminal carboxylic acid group (PLGA-PEG-COOH) was synthesized, and plumbagin was loaded on PLGA-PEG-COOH NPs using the nanoprecipitation method and characterized by field emission scanning electron microscopy (SEM), transmission electron microscopy (TEM), and laser light scattering. The uptake and distribution of plumbagin-NPs in human PCa LNCaP cells were investigated by fluorescent labeling. Subsequently, PSMA antibody-targeted PLGA-PEG-COOH NPs (targeted NPs) were prepared by covalent binding and characterized by x-ray photoelectron spectroscopy. Furthermore, the anticancer activity of plumbagin-loaded, targeted NPs was compared with that of nontargeted NPs in LNCaP cells in vitro.Plumbagin-NPs (diameter of 189.4 ± 30.6 nm and zeta potential of -17.1 ± 3.7 mV) were optimized based on theoretical drug loading of 5% and a ratio of water:acetone of 3:1. During the first 2 hours, the cumulative release rate of the drug was 66.4 ± 8.56%. Moreover, plumbagin-targeted NPs with nitrogen atoms were prepared. The uptake rate was 90% at 0.5 hours for targeted and nontargeted NPs. The IC50 of targeted NPs and nontargeted NPs was 32.59 ± 8.03 μM and 39.02 ± 7.64 μM, respectively.Plumbagin-loaded PSMA aptamer-targeted NPs can be used in targeted chemotherapy against PCa.

Topics: Antigens, Surface; Antineoplastic Agents, Phytogenic; Cell Line, Tumor; Delayed-Action Preparations; Dose-Response Relationship, Drug; Drug Carriers; Drug Evaluation, Preclinical; Drug Liberation; Glutamate Carboxypeptidase II; Humans; Male; Nanoparticles; Naphthoquinones; Particle Size; Prostatic Neoplasms

Plumbagin is a candidate drug for the treatment of prostate cancer. Previous observations indicated that it may improve the efficacy of androgen deprivation therapy (ADT). This study evaluates the effectiveness of treatment with combinations of plumbagin and alternative strategies for ADT in mouse models of prostate cancer to support its clinical use.. Plumbagin was administered per oral in a new sesame oil formulation. Standard toxicology studies were performed in rats. For tumor growth studies, mouse prostate cancer cell spheroids were placed on top of grafted prostate tissue in a dorsal chamber and allowed to form tumors. Mice were separated in various treatment groups and tumor size was measured over time by intra-vital microscopy. Survival studies were done in mice after injection of prostate cancer cells in the prostate of male animals. Androgen receptor (AR) levels were analyzed by Western blot from prostate cancer cells treated with plumbagin.. Plumbagin caused a decrease in AR levels in vitro. In mice, plumbagin at 1 mg/kg in sesame oil displayed low toxicity and caused a 50% tumor regression when combined with castration. The combination of plumbagin with various forms of chemical ADT including treatment with a GnRH receptor agonist, a GnRH receptor antagonist, or CYP17A1 inhibitors, outperformed ADT alone, increasing mouse survival compared to the standard regimen of castration alone. In contrast, the combination of plumbagin with AR antagonists, such as bicalutamide and enzalutamide, showed no improvement over AR antagonists alone. Thus, plumbagin is effective in combination with drugs that prevent the synthesis of testosterone or its conversion to dihydrotestosterone, but not with drugs that bind to AR.. Plumbagin significantly improves the effect of ADT drugs currently used in the clinic, with few side effects in mice.

Topics: Androgen Receptor Antagonists; Animals; Antineoplastic Agents, Phytogenic; Antineoplastic Combined Chemotherapy Protocols; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Female; Male; Mice; Mice, Inbred C57BL; Mice, Nude; Naphthoquinones; Prostatic Neoplasms; Rats; Rats, Sprague-Dawley; Toxicity Tests; Treatment Outcome; Tumor Burden

Eventhough the role of BRCA1/2 in hereditary prostatic cancer is being unleashed at a rapid rate; their optimal clinical management remains undefined. Cancer stem cells are thought to be responsible for cancer chemoresistance and relapse, thus they represent a significant concern for cancer prognosis and therapy. In this study, we have analyzed the effect of Plumbagin (PB) and structurally related naphthaquinones on BRCA1/2 silenced prostate cancer cells and the ability of PB to target stem cells. Our cell proliferation studies showed that both PC-3 and DU145 cells were more sensitive to PB, though all the compounds induced mitochondrial potential loss, DNA fragmentation and morphological changes which are indicative of apoptosis. Both BRCA1/2 siRNA transfected PC-3 and DU145 cells exhibited increased sensitivity to PB. Gene expression profiling post PB treatment in BRCA1/2 silenced cells revealed that PB has a putative role in tumor suppression in BRCA defective cancers. Using flow cytometric analysis we have proved that PB has the putative ability to directly target CSCs. Overall studies suggest that PB's antitumour mechanisms holds promise for novel therapeutic approaches against BRCA mutated cancers as well as CSCs.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; BRCA1 Protein; BRCA2 Protein; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Male; Naphthoquinones; Neoplastic Stem Cells; Prostate; Prostatic Neoplasms

Prostate cancer is one of the most common malignant tumors in males and it has become a major worldwide public health problem. This study characterizes the encapsulation of Nor-β-lapachone (NβL) in poly(d,l-lactide-co-glycolide) (PLGA) microcapsules and evaluates the cytotoxicity of the resulting drug-loaded system against metastatic prostate cancer cells. The microcapsules presented appropriate morphological features and the presence of drug molecules in the microcapsules was confirmed by different methods. Spherical microcapsules with a size range of 1.03 ± 0.46 μm were produced with an encapsulation efficiency of approximately 19%. Classical molecular dynamics calculations provided an estimate of the typical adsorption energies of NβL on PLGA. Finally, the cytotoxic activity of NβL against PC3M human prostate cancer cells was demonstrated to be significantly enhanced when delivered by PLGA microcapsules in comparison with the free drug.

Topics: Antineoplastic Agents; Benzofurans; Capsules; Cell Line, Tumor; Cell Survival; Delayed-Action Preparations; Drug Carriers; Drug Delivery Systems; Humans; Inhibitory Concentration 50; Lactic Acid; Male; Models, Molecular; Molecular Conformation; Molecular Structure; Naphthoquinones; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Prostatic Neoplasms; Spectrum Analysis, Raman

Plumbagin (PLB) has exhibited a potent anticancer effect in preclinical studies, but the molecular interactome remains elusive. This study aimed to compare the quantitative proteomic responses to PLB treatment in human prostate cancer PC-3 and DU145 cells using the approach of stable-isotope labeling by amino acids in cell culture (SILAC). The data were finally validated using Western blot assay. First, the bioinformatic analysis predicted that PLB could interact with 78 proteins that were involved in cell proliferation and apoptosis, immunity, and signal transduction. Our quantitative proteomic study using SILAC revealed that there were at least 1,225 and 267 proteins interacting with PLB and there were 341 and 107 signaling pathways and cellular functions potentially regulated by PLB in PC-3 and DU145 cells, respectively. These proteins and pathways played a critical role in the regulation of cell cycle, apoptosis, autophagy, epithelial to mesenchymal transition (EMT), and reactive oxygen species generation. The proteomic study showed substantial differences in response to PLB treatment between PC-3 and DU145 cells. PLB treatment significantly modulated the expression of critical proteins that regulate cell cycle, apoptosis, and EMT signaling pathways in PC-3 cells but not in DU145 cells. Consistently, our Western blotting analysis validated the bioinformatic and proteomic data and confirmed the modulating effects of PLB on important proteins that regulated cell cycle, apoptosis, autophagy, and EMT in PC-3 and DU145 cells. The data from the Western blot assay could not display significant differences between PC-3 and DU145 cells. These findings indicate that PLB elicits different proteomic responses in PC-3 and DU145 cells involving proteins and pathways that regulate cell cycle, apoptosis, autophagy, reactive oxygen species production, and antioxidation/oxidation homeostasis. This is the first systematic study with integrated computational, proteomic, and functional analyses revealing the networks of signaling pathways and differential proteomic responses to PLB treatment in prostate cancer cells. Quantitative proteomic analysis using SILAC represents an efficient and highly sensitive approach to identify the target networks of anticancer drugs like PLB, and the data may be used to discriminate the molecular and clinical subtypes, and to identify new therapeutic targets and biomarkers, for prostate cancer. Further studies are warranted to explore the p

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Autophagy; Binding Sites; Biomarkers, Tumor; Blotting, Western; Cell Cycle; Cell Line, Tumor; Computational Biology; Dose-Response Relationship, Drug; Epithelial-Mesenchymal Transition; Humans; Male; Molecular Docking Simulation; Molecular Structure; Naphthoquinones; Neoplasm Proteins; Prostatic Neoplasms; Protein Conformation; Protein Interaction Maps; Proteomics; Reproducibility of Results; Signal Transduction; Structure-Activity Relationship; Time Factors

Prostate cancer continues to remain the most common cancer and the second leading cause of cancer-related deaths in American males. The Pten deletions and/or mutations are frequently observed in both primary prostate cancers and metastatic prostate tissue samples. Pten deletion in prostate epithelium in mice results in prostatic intraepithelial neoplasia (PIN), followed by progression to invasive adenocarcinoma. The Pten conditional knockout mice [(Pten-loxp/loxp:PB-Cre4(+)) (Pten-KO)] provide a unique preclinical model to evaluate agents for efficacy for both the prevention and treatment of prostate cancer. We present here for the first time that dietary plumbagin, a medicinal plant-derived naphthoquinone (200 or 500 ppm) inhibits tumor development in intact as well as castrated Pten-KO mice. Plumbagin has shown no signs of toxicity at either of these doses. Plumbagin treatment resulted in a decrease expression of PKCε, AKT, Stat3, and COX2 compared with the control mice. Plumbagin treatment also inhibited the expression of vimentin and slug, the markers of epithelial-to-mesenchymal transition (EMT) in prostate tumors. In summary, the results indicate that dietary plumbagin inhibits growth of both primary and castration-resistant prostate cancer (CRPC) in Pten-KO mice, possibly via inhibition of PKCε, Stat3, AKT, and EMT markers (vimentin and slug), which are linked to the induction and progression of prostate cancer.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents, Phytogenic; Biomarkers; Carcinogenesis; Epithelial-Mesenchymal Transition; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Naphthoquinones; Orchiectomy; Prostatic Intraepithelial Neoplasia; Prostatic Neoplasms; Protein Kinase C-epsilon; PTEN Phosphohydrolase; Signal Transduction; STAT3 Transcription Factor

Glucose-regulated protein 78 (GRP78) is a key modulator of prostate cancer progression and therapeutic resistance. Prostate cancer is a worldwide health problem, and therapeutic resistance is a critical obstacle for the treatment of hormone-refractory prostate cancer patients. Shikonin inhibits prostate cancer proliferation and metastasis. However, the role of GRP78 in the cytotoxic effect of shikonin in prostate cancer cells remains unclear. GRP78 expression was abolished using small interfering RNA (siRNA), and the anticancer effects of shikonin were assessed using MTT assays, the XCELLigence biosensor, flow cytometric cell cycle analysis, and Annexin V-PI apoptotic assays. PC-3 cells expressed more GRP78 than DU-145 cells, and the MTT assays revealed that DU-145 cells were more sensitive to shikonin than PC-3 cells. GRP78 knockdown (GRP78KD) PC-3 cells were more sensitive to shikonin treatment than scrambled siRNA control cells. Based on cell cycle analysis and AnnexinV-PI apoptotic assays, apoptosis dramatically increased in GRP78KD cells compared with the control PC-3 in response to shikonin. Finally, in response to shikonin treatment, Mcl-1 and Bcl-2 levels increased in the scrambled control cells treated with shikonin, whereas Bcl-2 decreased and Mcl-1 slightly increased in the GRP78KD PC-3 cells. The levels of Bax and Bad did not change in the scrambled control or GRP78KD cells after shikonin treatment. These results are consistent with the increased sensitivity to shikonin after knockdown of GRP78. GRP78 expression may determine the therapeutic efficacy of shikonin against prostate cancer cells.

Topics: Apoptosis; Biosensing Techniques; Cell Line, Tumor; Cell Proliferation; Endoplasmic Reticulum Chaperone BiP; Flow Cytometry; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Heat-Shock Proteins; Humans; Male; Naphthoquinones; Prostatic Neoplasms

Stable nano-formulation of Plumbagin nanoparticles from Plumbago zeylanica root extract was explored as a potential natural drug against prostate cancer. Size and morphology analysis by DLS, SEM and AFM revealed the average size of nanoparticles prepared was 100±50nm. In vitro cytotoxicity showed concentration and time dependent toxicity on prostate cancer cells. However, plumbagin crude extract found to be highly toxic to normal cells when compared to plumbagin nanoformulation, thus confirming nano plumbagin cytocompatibility with normal cells and dose dependent toxicity to prostate cells. In vitro hemolysis assay confirmed the blood biocompatibility of the plumbagin nanoparticles. In wound healing assay, plumbagin nanoparticles provided clues that it might play an important role in the anti-migration of prostate cancer cells. DNA fragmentation revealed that partial apoptosis induction by plumbagin nanoparticles could be expected as a potent anti-cancer effect towards prostate cancer.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Cell Line, Tumor; DNA Fragmentation; Dose-Response Relationship, Drug; Humans; Hydrogen-Ion Concentration; Male; Mice; Nanoparticles; Naphthoquinones; NIH 3T3 Cells; Particle Size; Plant Extracts; Plumbaginaceae; Prostatic Neoplasms; Time Factors

Plumbagin (PLB) has been shown to have anticancer activities in animal models, but the role of PLB in prostate cancer treatment is unclear. This study aimed to investigate the effects of PLB on apoptosis and autophagy and the underlying mechanisms in human prostate cancer cell lines PC-3 and DU145. Our study has shown that PLB had potent pro-apoptotic and pro-autophagic effects on PC-3 and DU145 cells. PLB induced mitochondria-mediated apoptosis and autophagy in concentration- and time-dependent manners in both PC-3 and DU145 cells. PLB induced inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein kinase (MAPK) pathways and activation of 5'-AMP-dependent kinase (AMPK) as indicated by their altered phosphorylation, contributing to the pro-autophagic activity of PLB. Modulation of autophagy altered basal and PLB-induced apoptosis in both cell lines. Furthermore, PLB downregulated sirtuin 1 (Sirt1), and inhibition of Sirt1 enhanced autophagy, whereas the induction of Sirt1 abolished PLB-induced autophagy in PC-3 and DU145 cells. In addition, PLB downregulated pre-B cell colony-enhancing factor/visfatin, and the inhibition of pre-B cell colony-enhancing factor/visfatin significantly enhanced basal and PLB-induced apoptosis and autophagy in both cell lines. Moreover, reduction of intracellular reactive oxygen species (ROS) level attenuated the apoptosis- and autophagy-inducing effects of PLB on both PC-3 and DU145 cells. These findings indicate that PLB promotes apoptosis and autophagy in prostate cancer cells via Sirt1- and PI3K/Akt/mTOR-mediated pathways with contribution from AMPK-, p38 MAPK-, visfatin-, and ROS-associated pathways.

Topics: Apoptosis; Autophagy; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Humans; Male; Mitochondria; Molecular Structure; Naphthoquinones; Phosphatidylinositol 3-Kinases; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirtuin 1; Structure-Activity Relationship; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

Despite the recent progress in screening and therapy, a majority of prostate cancer cases eventually attain hormone refractory and chemo-resistant attributes. Conventional chemotherapeutic strategies are effective at very high doses for only palliative management of these prostate cancers. Therefore chemo-sensitization of prostate cancer cells could be a promising strategy for increasing efficacy of the conventional chemotherapeutic agents in prostate cancer patients. Recent studies have indicated that the chemo-preventive natural agents restore the pro-apoptotic protein expression and induce endoplasmic reticulum stress (ER stress) leading to the inhibition of cellular proliferation and activation of the mitochondrial apoptosis in prostate cancer cells. Therefore reprogramming ER stress-mitochondrial dependent apoptosis could be a potential approach for management of hormone refractory chemoresistant prostate cancers. We aimed to study the effects of the natural naphthoquinone Shikonin in human prostate cancer cells.. The results indicated that Shikonin induces apoptosis in prostate cancer cells through the dual induction of the endoplasmic reticulum stress and mitochondrial dysfunction. Shikonin induced ROS generation and activated ER stress and calpain activity. Moreover, addition of antioxidants attenuated these effects. Shikonin also induced the mitochondrial apoptotic pathway mediated through the enhanced expression of the pro-apoptotic Bax and inhibition of Bcl-2, disruption of the mitochondrial membrane potential (MMP) followed by the activation of caspase-9, caspase-3, and PARP cleavage.. The results suggest that shikonin could be useful in the therapeutic management of hormone refractory prostate cancers due to its modulation of the pro-apoptotic ER stress and mitochondrial apoptotic pathways.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Endoplasmic Reticulum Stress; Humans; Male; Membrane Potential, Mitochondrial; Mitochondria; Naphthoquinones; Prostatic Neoplasms

Prostate cancer initially develops in an androgen-dependent manner but, during its progression, transitions to being androgen-independent in the advanced stage. Pin1, one of the peptidyl-prolyl cis/trans isomerases, is reportedly overexpressed in prostate cancers and is considered to contribute to accelerated cell growth, which may be one of the major factors contributing to their androgen-independent growth. Thus, we investigated how Pin1 modulates the gene expressions in both androgen-dependent and androgen-independent prostate cancer cell lines using microarray analysis. In addition, the effects of Juglone, a commercially available Pin1 inhibitor were also examined.. Two prostate cancer cell-lines, LNCaP (androgen-dependent) and DU145 (androgen-independent), were treated with Pin1 siRNA and its effects on gene expressions were analyzed by microarray. Individual gene regulations induced by Pin1 siRNA or the Pin1 inhibitor Juglone were examined using RT-PCR. In addition, the effects of Juglone on the growth of LNCaP and DU145 transplanted into mice were investigated.. Microarray analysis revealed that transcriptional factors regulated by Pin1 differed markedly between LNCaP and DU145 cells, the only exception being that Nrf was regulated in the same way by Pin1 siRNA in both cell lines. Despite this marked difference in gene regulations, Pin1 siRNA and Juglone exert a strong inhibitory effect on both the LNCaP and the DU145 cell line, suppressing in vitro cell proliferation as well as tumor enlargement when transplanted into mice.. Despite Pin1-regulated gene expressions differing between these two prostate cancer cell-lines, LNCaP (androgen-dependent) and DU145 (androgen-independent), Pin1 inhibition suppresses proliferation of both cell-lines. These findings suggest the potential effectiveness of Pin1 inhibitors as therapeutic agents for prostate cancers, regardless of their androgen sensitivity.

Topics: Animals; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Mice, Nude; Naphthoquinones; Neoplasm Proteins; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Prostatic Neoplasms; Xenograft Model Antitumor Assays

Resistant cancer phenotype is a key obstacle in the successful therapy of prostate cancer. The primary aim of our study was to explore resistance mechanisms in the advanced type of prostate cancer cells (PC-3) and to clarify the role of autophagy in these processes. We performed time-lapse experiment (48 hours) with ROS generating plumbagin by using multimodal holographic microscope. Furthermore, we also performed the flow-cytometric analysis and the qRT-PCR gene expression analysis at 12 selected time points. TEM and confocal microscopy were used to verify the results. We found out that autophagy (namely mitophagy) is an important resistance mechanism. The major ROS producing mitochondria were coated by an autophagic membrane derived from endoplasmic reticulum and degraded. According to our results, increasing ROS resistance may be also accompanied by increased average cell size and polyploidization, which seems to be key resistance mechanism when connected with an escape from senescence. Many different types of cell-cell interactions were recorded including entosis, vesicular transfer, eating of dead or dying cells, and engulfment and cannibalism of living cells. Entosis was disclosed as a possible mechanism of polyploidization and enabled the long-term survival of cancer cells. Significantly reduced cell motility was found after the plumbagin treatment. We also found an extensive induction of pluripotency genes expression (NANOG, SOX2, and POU5F1) at the time-point of 20 hours. We suppose, that overexpression of pluripotency genes in the portion of prostate tumour cell population exposed to ROS leads to higher developmental plasticity and capability to faster respond to changes in the extracellular environment that could ultimately lead to an alteration of cell fate.

Topics: Autophagy; Cell Communication; Cell Line, Tumor; Cell Self Renewal; Cell Size; Cell Survival; Endoplasmic Reticulum; Entosis; Flow Cytometry; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Inhibitory Concentration 50; Male; Mitophagy; Naphthoquinones; Neoplasm Metastasis; Oxidative Stress; Principal Component Analysis; Prostatic Neoplasms; Reactive Oxygen Species; Time-Lapse Imaging

Shikonin, a natural naphthoquinone isolated from the traditional Chinese medicine Zi Cao (gromwell), has been shown to possess tumor cell killing activity. The human androgen receptor (AR) is a nuclear transcription factor that serves as a major therapeutic target for prostate cancer. However, AR regulation by shikonin has not been reported. We investigated the effects of shikonin on the growth of prostate cancer cells. We observed that shikonin decreased the expression of AR at both the mRNA and the protein levels in LNCaP and 22RV1 human prostate cancer cells. The results from a luciferase assay showed that shikonin decreased the transcriptional activity of AR. Moreover, shikonin treatment inhibited AR target gene expression, PSA and growth inhibition of prostate cancer cells. In conclusion, the present study shows for the first time that shikonin treatment causes transcriptional repression of AR and inhibition of its nuclear localization in human prostate cancer cells. We propose that shikonin, an anticancer drug extracted from natural sources, induces inhibition of cell growth through modulation of AR in androgen-responsive prostate cancer cells and is a candidate for use in cancer chemotherapy for human prostate cancer.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Humans; Male; Naphthoquinones; Prostate-Specific Antigen; Prostatic Neoplasms; Receptors, Androgen; Signal Transduction

The incidence of cancer grows annually worldwide and in Brazil it is the second cause of death. The search for anti-cancer drugs has then become urgent. It depends on the studies of natural and chemical synthesis products. The antitumor action of LQB-118, a pterocarpanquinone structurally related to lapachol, has been demonstrated to induce mechanisms linked to leukemia cell apoptosis. This work investigated some mechanisms of the in vitro antitumor action of LQB-118 on prostate cancer cells. LQB-118 reduced the expression of the c-Myc transcription factor, downregulated the cyclin D1 and cyclin B1 mRNA levels and upregulated the p21 cell cycle inhibitor. These effects resulted in cell cycle arrest in the S and G2/M phases and inhibition of tumor cell proliferation. LQB-118 also induced programmed cell death of the prostate cancer cells, as evidenced by internucleosomal DNA fragmentation and annexin-V positive cells. Except the cell cycle arrest in the S phase and enhanced c-Myc expression, all the mechanisms observed here for the in vitro antitumor action of LQB-118 were also found for Paclitaxel, a traditional antineoplastic drug. These findings suggest new molecular mechanisms for the LQB-118 in vitro antitumor action.

Topics: Apoptosis; Cell Cycle; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Male; Naphthoquinones; Prostatic Neoplasms; Proto-Oncogene Proteins c-myc; Pterocarpans; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured

Metastasis is one of the most important factors related to prostate cancer therapeutic efficacy. In previous studies, shikonin, an active naphthoquinone isolated from the Chinese medicine Zi Cao, has various anticancer activities both in vivo and in vitro. However, the mechanisms underlying shikonin's anticancer activity are not fully elucidated on prostate cancer cells. In the present study, we aimed to investigate the potential effects of shikonin on prostate cancer cells and the underlying mechanisms by which shikonin exerted its actions. With cell proliferation, flow cytometric cell cycle, migration and invasion assays, we found that shikonin potently suppressed PC-3 and DU145 cell growth by cell cycle arrest at the G2 phase and metastasis in a dose-dependent manner. Mechanically, we presented that shikonin could suppress the metastasis of PC-3 and DU145 cells via inhibiting the matrix metalloproteinase-2 (MMP-2) and MMP-9 expression and activation. In addition, shikonin significantly decreased the phosphorylation of AKT and mTOR in a dose-dependent manner while it induced extracellular signal-regulated kinase (ERK), p38 mitogen activated protein kinase (MAPK) and c-Jun N terminal kinase (JNK) phosphorylation. Further investigation of the underlying mechanism revealed that shikonin also induced the production of reactive oxygen species (ROS) that was reversed by the ROS scavenger dithiothreitol (DTT). Additionally, DTT reversed the shikonin induced activation of ERK1/2, thereby maintaining MMP-2 and MMP-9 expression and restoring cell metastasis. Together, shikonin inhibits aggressive prostate cancer cell migration and invasion by reducing MMP-2/-9 expression via AKT/mTOR and ROS/ERK1/2 pathways and presents a potential novel alternative agent for the treatment of human prostate cancer.

Topics: Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; G2 Phase; Gene Expression Regulation, Neoplastic; Humans; JNK Mitogen-Activated Protein Kinases; Male; MAP Kinase Signaling System; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Naphthoquinones; Neoplasm Metastasis; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Signal Transduction; TOR Serine-Threonine Kinases

Sepantronium bromide (YM155) exhibits time-dependent antitumor activity, although the plasma half-life of YM155 after a bolus intravenous (i.v.) administration is very short. Therefore, greater antitumor efficacy is obtained by continuous infusion than by bolus i.v. administration. In the present study, we attempted to liposomalize YM155 to obtain a longer circulation time than that achieved by bolus i.v. administration and yet retain sufficient antitumor activity. Encapsulation of YM155 in polyethylene glycol-coated liposomes extended the half-life of the drug, and high tumor accumulation of the drug was observed. Bolus i.v. administration of liposomal YM155 by a weekly administration regimen showed antitumor activity comparable to that obtained by the continuous infusion without severe toxicity in a murine xenograft model. Therefore, this liposomal formulation can be a new dosage form of YM155 that achieves sufficient efficacy and safety and is a more convenient administration regimen for users. It should be noted that liposomal YM155 showed unexpectedly high accumulation in the kidneys. This is a specific finding for liposomal YM155, offering important information for the consideration of the potential toxicity of liposomal YM155.

Topics: Animals; Antineoplastic Agents; Area Under Curve; Cell Line, Tumor; Electron Spin Resonance Spectroscopy; Humans; Imidazoles; Inhibitor of Apoptosis Proteins; Kidney; Liposomes; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Naphthoquinones; Polyethylene Glycols; Prostatic Neoplasms; Repressor Proteins; Survivin; Tissue Distribution; Xenograft Model Antitumor Assays

Sepantronium bromide (YM155) is an antitumor drug in development and is a first-in-class chemical entity, which is a survivin suppressant. We developed a radiosynthesis of [(11)C]YM155 to non-invasively evaluate its tissue and tumor distribution in mice bearing human prostate tumor xenografts.. Methods utilizing [(11)C]acetyl chloride and [(11)C]methyl triflate, both accessible with automated radiosynthesis boxes, were evaluated. The O-methylation of ethanolamine-alkolate with [(11)C]methyl triflate proved to be the key development toward a rapid and efficient process. The whole-body distribution of [(11)C]YM155 in PC-3 xenografted mice was examined using a planar positron imaging system (PPIS).. Sufficient quantities of radiopharmaceutical grade [(11)C]YM155 were produced for our PET imaging and distribution studies. The decay corrected (EOB) radiochemical yield was 16-22%, within a synthesis time of 47 min. The radiochemical purity was higher than 99%, and the specific activity was 29-60 GBq/μmol (EOS). High uptake levels of radioactivity (%ID/g, mean±SE) were observed in tumor (0.0613±0.0056), kidneys (0.0513±0.0092), liver (0.0368±0.0043) and cecum (0.0623±0.0070). The highest tumor uptake was observed at an early time point (from 10 min after) following injection. Tumor-to-blood and tumor-to-muscle uptake ratios of [(11)C]YM155, at 40 min after injection, were 26.5 (±2.9) and 25.6 (±3.6), respectively.. A rapid method for producing a radiopharmaceutical grade [(11)C]YM155 was developed. An in vivo distribution study using PPIS showed high uptake of [(11)C]YM155 in tumor tissue. Our methodology may facilitate the evaluation and prediction of response to YM155, when given as an anti-cancer agent.

Topics: Animals; Antineoplastic Agents; Carbon Radioisotopes; Cell Line, Tumor; Cell Transformation, Neoplastic; Humans; Imidazoles; Inhibitor of Apoptosis Proteins; Male; Mice; Naphthoquinones; Positron-Emission Tomography; Prostatic Neoplasms; Radiochemistry; Survivin; Tissue Distribution; Whole Body Imaging

We present here first time that Plumbagin (PL), a medicinal plant-derived 1,4-naphthoquinone, inhibits the growth and metastasis of human prostate cancer (PCa) cells in an orthotopic xenograft mouse model. In this study, human PCa PC-3M-luciferase cells (2 × 10(6)) were injected into the prostate of athymic nude mice. Three days post cell implantation, mice were treated with PL (2 mg/kg body wt. i.p. five days in a week) for 8 weeks. Growth and metastasis of PC-3M-luciferase cells was examined weekly by bioluminescence imaging of live mice. PL-treatment significantly (p = 0.0008) inhibited the growth of orthotopic xenograft tumors. Results demonstrated a significant inhibition of metastasis into liver (p = 0.037), but inhibition of metastasis into the lungs (p = 0.60) and lymph nodes (p = 0.27) was not observed to be significant. These results were further confirmed by histopathology of these organs. Results of histopathology demonstrated a significant inhibition of metastasis into lymph nodes (p = 0.034) and lungs (p = 0.028), and a trend to significance in liver (p = 0.075). None of the mice in the PL-treatment group showed PCa metastasis into the liver, but these mice had small metastasis foci into the lymph nodes and lungs. However, control mice had large metastatic foci into the lymph nodes, lungs, and liver. PL-caused inhibition of the growth and metastasis of PC-3M cells accompanies inhibition of the expression of: 1) PKCε, pStat3Tyr705, and pStat3Ser727, 2) Stat3 downstream target genes (survivin and Bcl(xL)), 3) proliferative markers Ki-67 and PCNA, 4) metastatic marker MMP9, MMP2, and uPA, and 5) angiogenesis markers CD31 and VEGF. Taken together, these results suggest that PL inhibits tumor growth and metastasis of human PCa PC3-M-luciferase cells, which could be used as a therapeutic agent for the prevention and treatment of human PCa.

Topics: Animals; Antineoplastic Agents, Phytogenic; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Mice, Nude; Naphthoquinones; Neoplasm Metastasis; Nitric Oxide Synthase Type II; Plumbaginaceae; Prostate; Prostatic Neoplasms; Protein Kinase C; STAT3 Transcription Factor

The synthesis of five 2-arylnaphtho[2,3-d]oxazole-4,9-dione derivatives was accomplished by refluxing 2-amino-3-bromo-1,4-naphthoquinone with appropriate benzoyl chloride analogs at elevated temperatures. In vitro anticancer evaluation of these compounds was performed on androgen-dependent, LNCaP, and androgen-independent, PC3, human prostate cancer cell lines. In general, these compounds displayed slightly stronger cytotoxicity on the androgen-dependent LNCaP than on the androgen-independent PC3 prostate cancer cell lines. The meta-substituted 2-(3-Chloro-phenyl)-naphtho[2,3-d]oxazole-4,9-dione (10) appear to display the best cytotoxicity on both cell lines with an IC(50) of 0.03 μM on LNCaP and 0.08 μM on PC3 after 5 days of exposure.

Topics: Androgens; Benzoxazoles; Cell Death; Cell Line, Tumor; Crystallography, X-Ray; Humans; Inhibitory Concentration 50; Male; Naphthoquinones; Oxazoles; Prostatic Neoplasms

Plumbagin (PL), 5-hydroxy-2-methyl-1,4-naphthoquinone, is a quinoid constituent isolated from the roots of the medicinal plant Plumbago zeylanica L. (also known as chitrak). PL has also been found in Juglans regia (English Walnut), Juglans cinerea (whitenut) and Juglans nigra (blacknut). The roots of P. zeylanica have been used in Indian and Chinese systems of medicine for more than 2500 years for the treatment of various types of ailments. We were the first to report that PL inhibits the growth and invasion of hormone refractory prostate cancer (PCa) cells [Aziz,M.H. et al. (2008) Plumbagin, a medicinal plant-derived naphthoquinone, is a novel inhibitor of the growth and invasion of hormone-refractory prostate cancer. Cancer Res., 68, 9024-9032.]. Now, we present that PL inhibits in vivo PCa development in the transgenic adenocarcinoma of mouse prostate (TRAMP). PL treatment (2 mg/kg body weight i.p. in 0.2 ml phosphate-buffered saline, 5 days a week) to FVB-TRAMP resulted in a significant (P < 0.01) decrease in prostate tumor size and urogenital apparatus weights at 13 and 20 weeks. Histopathological analysis revealed that PL treatment inhibited progression of prostatic intraepithelial neoplasia (PIN) to poorly differentiated carcinoma (PDC). No animal exhibited diffuse tumor formation in PL-treated group at 13 weeks, whereas 75% of the vehicle-treated mice elicited diffuse PIN and large PDC at this stage. At 20 weeks, 25% of the PL-treated animals demonstrated diffuse PIN and 75% developed small PDC, whereas 100% of the vehicle-treated mice showed large PDC. PL treatment inhibited expression of protein kinase C epsilon (PKCε), signal transducers and activators of transcription 3 phosphorylation, proliferating cell nuclear antigen and neuroendocrine markers (synaptophysin and chromogranin-A) in excised prostate tumor tissues. Taken together, these results further suggest PL could be a novel chemopreventive agent against PCa.

Topics: Adenocarcinoma; Animals; Anticarcinogenic Agents; Antigens, Polyomavirus Transforming; Chromogranin A; Disease Models, Animal; Male; Mice; Mice, Transgenic; Naphthoquinones; Phosphorylation; Proliferating Cell Nuclear Antigen; Prostatic Neoplasms; Protein Kinase C-epsilon; STAT3 Transcription Factor; Synaptophysin

• To evaluate the cytotoxicity of dimeric naphthoquinones (BiQs) in prostate cancer cells. • To assess the interaction of dimeric naphthoquinones with common therapies including radiation and docetaxel.. • The cytotoxicity of 12 different dimeric naphthoquinones was assessed in androgen-independent (PC-3, DU-145) and androgen-responsive (LNCaP, 22RV1) prostate cancer cell lines and in prostate epithelial cells (PrECs). • BiQ2 and BiQ11 were selected for determination of dose response, effects on colony formation and initial exploration into mechanism of action. • Synergistic effects with radiation and docetaxel were explored using colony-forming and MTT assays.. • At concentrations of 15µM, BiQ2, BiQ3, BiQ11, BiQ12, and BiQ15 demonstrated cytotoxicity in all prostate cancer cell lines. • Treatment with BiQs limited the ability of prostate cancer cells to form colonies in clonogenic assays. • Exposure of prostate cancer to BiQs increased cellular reactive oxygen species (ROS), decreased ATP production, and promoted apoptosis. • BiQ cytotoxicity was independent of NADP(H):quinone oxidoreductase 1 (NQO1) activity in PrECs, PC-3 and 22RV1, but not DU-145 cells. • Exposure of prostate cancer cells to radiation before treatment with BiQs increased their activity allowing for inhibitory effects well below the IC(50) s of these compounds in PrECs. • Co-administration of BiQs with docetaxel had minimal additive effects.. • Dimeric naphthoquinones represent a new class of compounds with prostate cancer cytotoxicity and synergistic effects with radiation. The cytotoxic effect of these agents is probably contributed to by the accumulation of ROS and mitochondrial dysfunction. • Further studies are warranted to better characterize this class of potential chemo-therapeutics.

Topics: Adenosine Triphosphate; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Survival; Drug Synergism; Humans; Male; Naphthoquinones; Prostatic Neoplasms; Radiation, Ionizing; Reactive Oxygen Species

In this study, we demonstrated that YM155, a novel survivin suppressant, induced both apoptosis, and autophagy that was shown by conversion of cytosolic-associated protein light chain 3 (LC3I) into autophagosome-associated form (LC3II) and a punctate fluorescence pattern of an ectopic GFP-LC3 protein. The lysosomal inhibitor chloroquine further accumulated YM155-induced LC3II, indicating an increase of autophagic flux. Ectopic expression of survivin significantly attenuated YM155-induced apoptosis and autophagy, whereas survivin siRNA induced autophagy. Furthermore, inhibition of either early or late events of autophagy attenuated YM155-induced apoptosis, demonstrating that induction of autophagy proceeds apoptosis. In conclusion, suppression of survivin by YM155 induces autophagy-dependent apoptosis, and YM155-induced autophagy plays a pro-apoptotic role thereby unveiling a novel mechanism of YM155 in prostate cancer cells.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Autophagy-Related Protein 5; Beclin-1; Blotting, Western; Cell Line, Tumor; Chloroquine; Flow Cytometry; Green Fluorescent Proteins; Humans; Imidazoles; Inhibitor of Apoptosis Proteins; Lysosomal Membrane Proteins; Lysosomal-Associated Membrane Protein 2; Male; Membrane Proteins; Microscopy, Confocal; Microtubule-Associated Proteins; Naphthoquinones; Prostatic Neoplasms; RNA Interference; Survivin

Worldwide among men, prostate cancer ranks third in cancer occurrence and sixth in cancer mortality. A number of 1, 4-naphthoquinone derivatives have been identified that possess significant pharmacological effects associated with antitumor activities. In this study, the in vitro effects of N-(3-chloro-1,4-dioxo 1,4-dihydro-naphthalen-2-yl)-benzamide (NCDDNB) were evaluated on androgen-dependent (CWR-22) and androgen-independent (PC-3, DU-145) human prostate cancer cell lines, and on a normal bone marrow cell line (HS-5). Specifically, the in vitro activity of this compound on cell cycle regulation and apoptosis was evaluated.. Established methods of cell viability, cell cycle, Western blot and apoptosis were used.. The effect of NCDDNB on CWR-22, PC-3, DU-145 and HS-5 cells revealed significant anti-tumor activities with IC(50)s, of 2.5, 2.5, 6.5, and 25 muM respectively. The results of cell cycle analysis showed that NCDDNB arrested PC-3, DU-145, and CWR-22 cells in the G(1)-phase of the cell cycle. The compound showed no effect on the cell cycle progression in the HS-5 bone marrow cell line. These findings were further validated using Western blot analysis. NCDDNB showed the greatest amount of apoptosis in the androgen-independent PC-3 cells in a time-dependent manner with the apoptotic apex at day 5 of treatment. Furthermore, NCDDNB induced-apoptosis in DU-145 and CWR-22 cells peaked at day 3 of treatment.. Although the mechanism of action of this compound has not been completely elucidated, the effect on the cell cycle and the induction of apoptosis in different prostate cancer cell lines prompted us to carry out a more in-depth preclinical evaluation. This study suggests that NCDDNB may have an impact on treatment of prostate cancer while protecting the bone marrow.

Topics: Antineoplastic Agents; Apoptosis; Benzamides; Blotting, Western; Cell Cycle; Cell Proliferation; Enzyme-Linked Immunosorbent Assay; Humans; Immunoenzyme Techniques; Male; Naphthoquinones; Neoplasms, Hormone-Dependent; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured

The clinical experimental agent, β-lapachone (β-lap; Arq 501), can act as a potent radiosensitizer in vitro through an unknown mechanism. In this study, we analyzed the mechanism to determine whether β-lap may warrant clinical evaluation as a radiosensitizer. β-Lap killed prostate cancer cells by NAD(P)H:quinone oxidoreductase 1 (NQO1) metabolic bioactivation, triggering a massive induction of reactive oxygen species, irreversible DNA single-strand breaks (SSB), poly(ADP-ribose) polymerase-1 (PARP-1) hyperactivation, NAD(+)/ATP depletion, and μ-calpain-induced programmed necrosis. In combination with ionizing radiation (IR), β-lap radiosensitized NQO1(+) prostate cancer cells under conditions where nontoxic doses of either agent alone achieved threshold levels of SSBs required for hyperactivation of PARP-1. Combination therapy significantly elevated SSB level, γ-H2AX foci formation, and poly(ADP-ribosylation) of PARP-1, which were associated with ATP loss and induction of μ-calpain-induced programmed cell death. Radiosensitization by β-lap was blocked by the NQO1 inhibitor dicoumarol or the PARP-1 inhibitor DPQ. In a mouse xenograft model of prostate cancer, β-lap synergized with IR to promote antitumor efficacy. NQO1 levels were elevated in ∼60% of human prostate tumors evaluated relative to adjacent normal tissue, where β-lap might be efficacious alone or in combination with radiation. Our findings offer a rationale for the clinical utilization of β-lap (Arq 501) as a radiosensitizer in prostate cancers that overexpress NQO1, offering a potentially synergistic targeting strategy to exploit PARP-1 hyperactivation.

Topics: Animals; Apoptosis; Cell Death; Colony-Forming Units Assay; Comet Assay; Dicumarol; DNA Damage; DNA, Neoplasm; Enzyme Activation; Enzyme Inhibitors; Glutathione; Humans; In Situ Nick-End Labeling; Male; Mice; Mice, Nude; Naphthoquinones; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Radiation-Sensitizing Agents; Regression Analysis

beta-Lapachone (ARQ 501, a formulation of beta-lapachone complexed with hydroxypropyl-beta-cyclodextrin) is a novel anticancer agent with selectivity against prostate cancer cells overexpressing the NAD(P)H:quinone oxidoreductase-1 enzyme. Lack of solubility and an efficient drug delivery strategy limits this compound in clinical applications. In this study, we aimed to develop beta-lapachone-containing polymer implants (millirods) for direct implantation into prostate tumors to test the hypothesis that the combination of a tumor-specific anticancer agent with site-specific release of the agent will lead to significant antitumor efficacy.. Survival assays in vitro were used to test the killing effect of beta-lapachone in different prostate cancer cells. beta-Lapachone release kinetics from millirods was determined in vitro and in vivo. PC-3 prostate tumor xenografts in athymic nude mice were used for antitumor efficacy studies in vivo.. beta-Lapachone killed three different prostate cancer cell lines in an NAD(P)H:quinone oxidoreductase-1-dependent manner. Upon incorporation of solid-state inclusion complexes of beta-lapachone with hydroxypropyl-beta-cyclodextrin into poly(D,L-lactide-co-glycolide) millirods, beta-lapachone release kinetics in vivo showed a burst release of approximately 0.5 mg within 12 hours and a subsequently sustained release of the drug ( approximately 0.4 mg/kg/d) comparable with that observed in vitro. Antitumor efficacy studies showed significant tumor growth inhibition by beta-lapachone millirods compared with controls (P < 0.0001; n = 10 per group). Kaplan-Meier survival curves showed that tumor-bearing mice treated with beta-lapachone millirods survived nearly 2-fold longer than controls, without observable systemic toxicity.. Intratumoral delivery of beta-lapachone using polymer millirods showed the promising therapeutic potential for human prostate tumors.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Drug Carriers; Drug Implants; Humans; Male; Mice; Mice, Nude; Naphthoquinones; Polymers; Prostatic Neoplasms; Xenograft Model Antitumor Assays

2-Piperazinyl naphthoquinones (2) and 2-piperidinyl naphthoquinones (3) were designed and synthesized as new cytotoxic and apoptosis inducing agents by utilizing the anti-parasite drug atovaquone as lead compound. Several compounds displayed significantly improved cytotoxic activities against a panel of cancer cell lines than that of atovaquone. These compounds also induced apoptosis through activating pro-apoptotic caspases 9 and 3.

Topics: Anti-Infective Agents; Antineoplastic Agents; Apoptosis; Atovaquone; Caspase 3; Caspase 9; Cell Line, Tumor; Crystallography, X-Ray; Drug Design; Drug Screening Assays, Antitumor; Humans; Male; Molecular Conformation; Naphthoquinones; Prostatic Neoplasms

Through cytotoxicity screening with naphthoquinone derivatives, a novel compound 6-(1-oxoallkyl)-5,8-dimethoxy-1,4-naphthoquinone-S17 (DMNQ-S17) showed its potency against human myeloid leukemia U937 cells. Thus, to elucidate the apoptotic mechanism of DMNQ-S17, this study was performed in myeloid leukemia U937 cells by 2,3-bis [2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) assay, eletrophoretic mobility shift assay (EMSA), terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay, 4',6-diamidino-2-phenylindole (DAPI) staining, and Western blotting. In the present study, DMNQ-S17 inhibited constitutive NF kappaB activation and its transcriptional activity in U937 cells. In addition, DMNQ-S17 induced apoptotic features such as apoptotic bodies, cell shrinkage and chromatin condensation in U937 cells. Consistently, flow cytometric analysis showed that DMNQ-S17 increased sub-G1 portion and TUNEL positive cells in a concentration-dependent manner. Furthermore, DMNQ-S17 effectively attenuated mitochondrial membrane potential, released cytochrome C, activated caspase-3 expression, and cleaved poly (ADP-ribose) polymerase (PARP). Reversely, caspase-3 and -9 inhibitors also blocked the DMNQ-S17 induced caspase-3 activation and PARP cleavage in U937 cells. Taken together, these findings suggest that DMNQ-S17 can be a potent anticancer candidate for myeloid leukemias by the suppression of NF-kappaB activation leading to the activation of caspase-3 in human myeloid leukemia U937 cells.

Topics: Antineoplastic Agents; Apoptosis; Caspase 3; Caspase Inhibitors; Cytochromes c; Drug Screening Assays, Antitumor; Enzyme Inhibitors; Fluorescent Dyes; Humans; In Situ Nick-End Labeling; Indoles; Leukemia, Myeloid; Lymphoma, Large B-Cell, Diffuse; Male; Membrane Potential, Mitochondrial; Naphthoquinones; NF-kappa B; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; U937 Cells

Beta-lapachone, an o-naphthoquinone, induces various carcinoma cells to undergo apoptosis, but the mechanism is poorly understood. In the present study, we found that the beta-lapachone-induced apoptosis of DU145 human prostate carcinoma cells was associated with endoplasmic reticulum (ER) stress, as shown by increased intracellular calcium levels and induction of GRP-78 and GADD-153 proteins, suggesting that the endoplasmic reticulum is a target of beta-lapachone. Beta-Lapachone-induced DU145 cell apoptosis was dose-dependent and accompanied by cleavage of procaspase-12 and phosphorylation of p38, ERK, and JNK, followed by activation of the executioner caspases, caspase-7 and calpain. However, pretreatment with the general caspase inhibitor, z-VAD-FMK, or calpain inhibitors, including ALLM or ALLN, failed to prevent beta-lapachone-induced apoptotic cell death. Blocking the enzyme activity of NQO1 with dicoumarol, a known NQO1 inhibitor, or preventing an increase in intracellular calcium levels using BAPTA-AM, an intracellular calcium chelator, substantially inhibited MAPK phosphorylation, abolished the activation of calpain, caspase-12 and caspase-7, and provided significant protection of beta-lapachone-treated cells. These findings show that beta-lapachone-induced ER stress and MAP kinase phosphorylation is a novel signaling pathway underlying the molecular mechanism of the anticancer effect of beta-lapachone.

Topics: Antineoplastic Agents; Apoptosis; Calcium; Calpain; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cell Survival; Chelating Agents; Dose-Response Relationship, Drug; Endoplasmic Reticulum; Enzyme Activation; Enzyme Inhibitors; Homeostasis; Humans; Male; Mitogen-Activated Protein Kinases; NAD(P)H Dehydrogenase (Quinone); Naphthoquinones; Phosphorylation; Prostatic Neoplasms; Signal Transduction; Stress, Physiological; Time Factors

Prostate cancer (PCa) is the second leading cause of cancer-related deaths in men. Hormone-refractory invasive PCa is the end stage and accounts for the majority of PCa patient deaths. We present here that plumbagin (PL), a quinoid constituent isolated from the root of the medicinal plant Plumbago zeylanica L., may be a potential novel agent in the control of hormone-refractory PCa. Specific observations are the findings that PL inhibited PCa cell invasion and selectively induced apoptosis in PCa cells but not in immortalized nontumorigenic prostate epithelial RWPE-1 cells. In addition, i.p. administration of PL (2 mg/kg body weight), beginning 3 days after ectopic implantation of hormone-refractory DU145 PCa cells, delayed tumor growth by 3 weeks and reduced both tumor weight and volume by 90%. Discontinuation of PL treatment in PL-treated mice for as long as 4 weeks did not result in progression of tumor growth. PL, at concentrations as low as 5 micromol/L, inhibited in both cultured PCa cells and DU145 xenografts (a) the expression of protein kinase Cepsilon (PKCepsilon), phosphatidylinositol 3-kinase, phosphorylated AKT, phosphorylated Janus-activated kinase-2, and phosphorylated signal transducer and activator of transcription 3 (Stat3); (b) the DNA-binding activity of transcription factors activator protein-1, nuclear factor-kappaB, and Stat3; and (c) Bcl-xL, cdc25A, and cyclooxygenase-2 expression. The results indicate for the first time, using both in vitro and in vivo preclinical models, that PL inhibits the growth and invasion of PCa. PL inhibits multiple molecular targets including PKCepsilon, a predictive biomarker of PCa aggressiveness. PL may be a novel agent for therapy of hormone-refractory PCa.

Topics: Animals; Apoptosis; Base Sequence; Cell Division; DNA Primers; Electrophoretic Mobility Shift Assay; Humans; Immunohistochemistry; Male; Mice; Mice, Nude; Naphthoquinones; Neoplasm Invasiveness; Plants, Medicinal; Prostatic Neoplasms

To investigate the mechanism of human prostate cancer cell growth inhibition by plumbagin, a constituent of the widely used medicinal herb Plumbago zeylanica L.. Cell viability was determined by trypan blue dye exclusion assay. Apoptosis induction was assessed by analysis of cytoplasmic histone-associated DNA fragmentation. Cell cycle distribution and generation of reactive oxygen species (ROS) were determined by flow cytometry. The effect of plumbagin treatment on cellular redox status was determined by analysis of intracellular glutathione (GSH) levels and expression of genes involved in ROS metabolism.. Plumbagin treatment decreased viability of human prostate cancer cells (PC-3, LNCaP, and C4-2) irrespective of their androgen responsiveness or p53 status. Plumbagin-mediated decrease in cell viability correlated with apoptosis induction, which was accompanied by ROS generation and depletion of intracellular GSH levels. Pretreatment of cells with the antioxidant N-acetylcysteine inhibited plumbagin-mediated ROS generation and apoptosis. Plumbagin treatment also resulted in altered expression of genes responsible for ROS metabolism, including superoxide dismutase 2 (Mn-SOD).. The present study points towards an important role of ROS in plumbagin-induced apoptosis in human prostate cancer cells.

Topics: Acetylcysteine; Anticarcinogenic Agents; Antioxidants; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Glutathione; Humans; Male; Naphthoquinones; Oxidation-Reduction; Peroxidases; Prostatic Neoplasms; Reactive Oxygen Species; Superoxide Dismutase

Various accumulating evidence suggests that survivin, a member of the inhibitor of apoptosis (IAP) family, plays an important role in drug resistance and cancer cell survival in many types of cancer, including hormone-refractory prostate cancer (HRPC). Here, we characterized YM155, a novel small-molecule survivin suppressant, using a survivin gene promoter activity assay. YM155 suppressed expression of survivin and induced apoptosis in PC-3 and PPC-1 human HRPC cell lines at 10 nmol/L. In contrast, YM155 up to 100 nmol/L showed little effect on expression levels of other IAP- or Bcl-2-related proteins. In a s.c. xenografted PC-3 tumor model in mice, 3-day continuous infusions of YM155 at 3 to 10 mg/kg induced massive tumor regression accompanied by suppression of intratumoral survivin. YM155 also completely inhibited the growth of orthotopically xenografted PC-3 tumors. No significant decreases in body weight were observed in mice treated with YM155 during the experimental period. Pharmacokinetic analyses indicated that YM155 is highly distributed to tumors and at concentrations approximately 20-fold higher than those in plasma. Our findings represent the first attempt to show tumor regression and suppression of survivin in p53-deficient human HRPC cells by a single small molecular compound treatment. Further extensive investigation of YM155 in many types of cancer, including HRPC, seems to be worthwhile to develop this novel therapeutic approach.

Topics: Animals; Antineoplastic Agents; Antineoplastic Agents, Hormonal; Carcinoma; CHO Cells; Cricetinae; Cricetulus; Drug Evaluation, Preclinical; Drug Resistance, Neoplasm; HeLa Cells; Humans; Imidazoles; Inhibitor of Apoptosis Proteins; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Microtubule-Associated Proteins; Models, Biological; Naphthoquinones; Neoplasm Proteins; Prostatic Neoplasms; Remission Induction; Survivin; Treatment Failure; Tumor Burden; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

1-(1,4-dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enylfuran-2-caroxylate (SH-7), a new naphthoquinone compound, derived from shikonin, exhibited obvious inhibitory actions on topoisomerase II (Topo II) and topoisomerase I (Topo I), which were stronger than its mother compound shikonin. Notably, the SH-7's inhibitory potency on Topo II was much stronger than that on Topo I. In addition, SH-7 significantly stabilized Topo II-DNA cleavable complex and elevated the expression of phosphorylated-H2AX. The in vitro cell-based investigation demonstrated that SH-7 displayed wide cytotoxicity in diversified cancer cell lines with the mean IC(50) value of 7.75 microM. One important finding is SH-7 displayed significant cytotoxicity in the 3 MDR cell lines, with an average IC(50) value nearly equivalent to that of the corresponding parental cell lines. The average resistance factor (RF) of SH-7 was 1.74, which was much lower than those of reference drugs VP-16 (RF 145.92), ADR (RF 105.97) and VCR (RF 197.39). Further studies illustrated that SH-7 had the marked apoptosis-inducing function on leukemia HL-60 cells, which was validated to be of mitochondria-dependence. The in vivo experiments showed that SH-7 had inhibitory effects on S-180 sarcoma implanted to mice, SMMC-7721, BEL-7402 human hepatocellular carcinoma and PC-3 human prostate cancer implanted to nude mice. Taken together, these results suggest that SH-7 induces DSBs as a Topo II inhibitor, which was crucial to activate the apoptotic process, and subsequently accounts for its both in vitro and in vivo antitumor activities. The well-defined Topo II inhibitory activity, antitumor effects particularly with its obvious anti-MDR action, better solubility and less toxicity make SH-7 as a potential antitumor drug candidate for further research and development.

Topics: Animals; Antineoplastic Agents; Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Electrophoresis, Agar Gel; Female; Flow Cytometry; Humans; Leukemia; Liver Neoplasms; Male; Mice; Mice, Nude; Naphthoquinones; Neoplasms; Prostatic Neoplasms; Sarcoma; Topoisomerase II Inhibitors; Transplantation, Heterologous

It has been reported that beta-lapachone (beta-lap), a bioreductive anti-cancer drug, synergistically interacts with ionizing radiation and that the sensitivity of cells to beta-lap is closely related to the activity of NAD(P)H:quinone oxidoreductase 1 (NQO1). Here we report the results of our studies of mechanisms underlying the synergistic interaction of beta-lap and radiation in killing cancer cells using the DU-145 human prostate cancer cell line. The clonogenic cell death caused by the combination of radiation and beta-lap was synergistic when beta-lap was administered 0-10 h after irradiation but not when it was given before irradiation. The expression and activity of NQO1 increased significantly and remained elevated for longer than 12 h after 4 Gy irradiation, suggesting that the long-lasting elevation of NQO1 sensitized the cells to beta-lap. Studies with split-dose irradiation demonstrated that beta-lap given immediately after irradiation effectively inhibited sublethal radiation damage (SLD) repair. Taken together, these results lead us to conclude that the synergistic interaction between beta-lap and radiation in killing cells is the result of two distinct mechanisms: First, radiation sensitizes cells to beta-lap by up-regulating NQO1, and second, beta-lap sensitizes cells to radiation by inhibiting SLD repair. The combination of beta-lap and radiotherapy is potentially promising modality for the treatment of cancer in humans.

Topics: Cell Line, Tumor; Cell Survival; Cesium Radioisotopes; Combined Modality Therapy; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Humans; Male; NAD(P)H Dehydrogenase (Quinone); Naphthoquinones; Prostatic Neoplasms; Radiation Tolerance; Radiation-Sensitizing Agents

Beta-lapachone, the product of a tree Tabebuia avellanedae from South America, is known to exhibit various pharmacologic properties, the mechanisms of which are poorly understood. In the present study, we investigated further possible mechanisms by which beta-lapachone exerts its anti-proliferative action in cultured human prostate carcinoma DU145 cells. Exposure of DU145 cells to beta-lapachone resulted in growth inhibition and induction of apoptosis in a dose-dependent manner as measured by MTT assay, fluorescent microscopy, and flow-cytometry analysis. The increase in apoptosis was associated with a dose-dependent up-regulation in pro-apoptotic Bax expression, down-regulation of anti-apoptotic Bcl-2, and proteolytic activation of caspase-3 protease. We found beta-lapachone decreased the levels of cyclooxygenase (COX)-2 mRNA and protein expression without significant changes in the levels of COX-1, which was correlated with a decrease in prostaglandin E2 (PGE2) synthesis. Furthermore, beta-lapachone treatment markedly inhibited the activity of telomerase in a dose-dependent fashion. Additionally, the expression of human telomerase reverse transcriptase (hTERT), a main determinant of the telomerase enzymatic activity, was progressively down-regulated by beta-lapachone treatment. Taken together, these findings provide important new insights into the possible molecular mechanisms of the anti-cancer activity of beta-lapachone.

Topics: Cell Line, Tumor; Cyclooxygenase 2 Inhibitors; Down-Regulation; Humans; Male; Naphthoquinones; Prostatic Neoplasms; Telomerase

The serine/threonine kinase Raf-1 is a major regulator of the mitogen activated protein kinase (MAPK) pathway, which has been associated with the progression of prostate cancer to the more advanced and androgen-independent disease. Cdc25A phosphatase has been implicated in the regulation of Raf-1 and the MAPK pathway.. We used a novel and potent Cdc25A inhibitor, 2,3-bis-[2-hydroxyethylsulfonyl]-[1,4] naphthoquinone (NSC 95397), and its congener (2-mercaptoethanol)-3-methyl-1, 4-naphthoquinone (NSC 672121) to study the role of Cdc25A on the MAPK pathway in human prostate cancer cells.. We found Raf-1 physically interacted with Cdc25A in PC-3 and LNCap cells and inhibitors of Cdc25A induced both extracellular signal-regulated kinase (Erk) activation and Raf-1 tyrosine phosphorylation. NSC 95397 attenuated Cdc25A and Raf-1 interactions due to accelerated degradation of Cdc25A, which was mediated by proteasome degradation. The MAPK kinase (MEK) inhibitor U0126 completely inhibited Erk activation by NSC 95397 and NSC 672121.. These results indicate Cdc25A phosphatase regulates Raf-1/MEK/Erk kinase activation in human prostate cancer cells.

Topics: Blotting, Western; Butadienes; cdc25 Phosphatases; Cell Line, Tumor; Enzyme Inhibitors; Humans; Leupeptins; Male; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Naphthoquinones; Nitriles; Phosphorylation; Prostatic Neoplasms; Proto-Oncogene Proteins c-raf

Diosquinone [1], a naphthoquinone epoxide previously isolated from the root bark of Diospyros mespiliformis (Hostch) and D. tricolor [Ebenaceae] is been assessed for cytotoxicity activity against ten cancer cell lines by standard NIH method. The ethno-pharmacological claim of this plant and the previously observed good antibacterial activity of this compound among the others isolated from this plant suggest its probable cytotoxicity activity. Diosquinone was observed to be very active against most of the cancer cell lines. It shows very good activity against all the cell lines tested with ED50 value ranging between 0.18 microg/ml. against Human Glioblastoma (U373) to 4.5 microg/ml. against Hormone dependent human prostrate cancer( LNCaP).

Topics: Antineoplastic Agents; Breast Neoplasms; Colonic Neoplasms; Diospyros; Drug Resistance, Multiple; Female; Fibrosarcoma; Humans; Lung Neoplasms; Male; Naphthoquinones; Nasopharyngeal Neoplasms; Neoplasms, Hormone-Dependent; Phytotherapy; Plant Extracts; Prostatic Neoplasms; Tumor Cells, Cultured

The product of a tree (Tabebuia avellanedae) from South America, beta-lapachone, is known to exhibit various pharmacological properties, the mechanisms of which are poorly understood. The aim of the present study was to further elucidate the possible mechanisms by which beta-lapachone exerts its anti-proliferative action in cultured human prostate cancer cells. We observed that the proliferation-inhibitory effect of beta-lapachone was due to the induction of apoptosis, which was confirmed by observing the morphological changes and cleavage of the poly(ADP-ribose) polymerase protein. A DNA flow cytometric analysis also revealed that beta-lapachone arrested the cell cycle progression at the G1 phase. The effects were associated with the down-regulation of the phosphorylation of the retinoblastoma protein (pRB) as well as the enhanced binding of pRB and the transcription factor E2F-1. Also, beta-lapachone suppressed the cyclin-dependent kinases (Cdks) and cyclin E-associated kinase activity without changing their expressions. Furthermore, this compound induced the levels of the Cdk inhibitor p21(WAF1/CIP1) expression in a p53-independent manner, and the p21 proteins that were induced by beta-lapachone were associated with Cdk2. beta-lapachone also activated the reporter construct of a p21 promoter. Overall, our results demonstrate a combined mechanism that involves the inhibition of pRB phosphorylation and induction of p21 as targets for beta-lapachone. This may explain some of its anticancer effects.

Topics: Antineoplastic Agents; Apoptosis; CDC2-CDC28 Kinases; Cell Cycle Proteins; Cell Division; Cell Line, Tumor; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; DNA-Binding Proteins; Down-Regulation; E2F Transcription Factors; E2F1 Transcription Factor; Humans; Male; Naphthoquinones; Phosphorylation; Prostatic Neoplasms; Retinoblastoma Protein; Transcription Factors

A wide spectrum of anti-cancer activity of genistein and beta-lapachone in various tumors has been reported in single treatments. In this study the combined effects of genistein and beta-lapachone on the chemosensitivity of LNCaP and PC3 human prostate cancer cells was determined in vitro, using 3-[4,5-dimethylthiazol-2-yl]-2-,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) to study treatment-induced growth inhibition and cytotoxicity and, annexin V-fluoresceine (FI) and terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-propidium iodide (PI) assays to determine potential treatment-induced apoptosis and/or necrosis. The results showed: i) that both PC3 and LNCaP are sensitive to single and combination treatments regardless of hormone sensitivity status, ii) that treatment induced dual death pathways (apoptosis and necrosis) in both cell types, iii) that growth inhibition in both cell types correlated positively with cell death via apoptosis at lower drug concentrations and necrosis at higher concentrations, iv) that combination of genistein and beta-lapachone had synergistic inhibitory effects on growth and proliferation in both cell types. The synergistic inhibitory effect was correlated positively with treatment-induced cell death via apoptosis and necrosis. The overall results indicate that combination treatments with beta-lapachone and genistein are more potent in killing both PC3 and LNCaP cancer cells than treatment with either genistein or beta-lapachone alone. beta-lapachone acts at the G1 and S phase checkpoints in the cell cycle, while genistein induces cell cycle arrest at the G2-M stage. The current results are therefore in agreement with the hypothesis that drug combinations that target cell cycles at different critical checkpoints would be more effective in causing cell death. This result provides a rationale for in vivo studies to determine whether beta-lapachone-genistein combination will provide effective chemotherapy for prostate cancer, regardless of the tumor sensitivity to hormone.

Topics: Adenocarcinoma; Antibiotics, Antineoplastic; Apoptosis; Cell Division; Drug Synergism; Drug Therapy, Combination; Estrogens, Non-Steroidal; Humans; Isoflavones; Male; Naphthoquinones; Necrosis; Prostatic Neoplasms; Tumor Cells, Cultured

The hollow fiber test has been developed for the preliminary in vivo assessment of cancer chemotherapeutic efficacy of selected natural products. Using this model, we have established growth conditions for HL-60, HUVEC, Ishikawa, KB, KB-V1, LNCaP, Lu1, MCF-7, Mel2, P-388, and SW626 cells implanted at the intraperitoneal (i.p.) and subcutaneous (s.c.) compartments of athymic mice. Five cytotoxic natural product isolates (2-6) were tested in this model, along with paclitaxel (taxol) (1). Among the compounds tested, dioscin (2) and 13-methoxy-15-oxozoapatlin (3) were found to be active, indicating their potential to function as cancer chemotherapeutic agents. On the other hand, ochraceolide A (4), alpha-lapachone (5), and 2-(1-hydroxyethyl)naphtha[2,3-b]furan-4,9-quinone (6), all of which were significantly cytotoxic to cultured mammalian cells, did not mediate significant responses with the hollow fiber model. In further xenograft studies using KB cells implanted at the subcutaneous site, compound 3 mediated a statistically significant response which was consistent with the response observed at the subcutaneous compartment in the hollow fiber tests. In sum, these studies illustrate the usefulness of the hollow fiber model in natural product drug discovery programs. Preliminary indications of potential therapeutic efficacy can be provided quickly at relatively low expense. Agents capable of mediating a response at the subcutaneous site would appear to warrant greatest attention.

Topics: Animals; Biological Factors; Colonic Neoplasms; Diosgenin; Disease Models, Animal; Diterpenes; Drug Screening Assays, Antitumor; Female; Heterocyclic Compounds, 3-Ring; HL-60 Cells; Humans; Inhibitory Concentration 50; KB Cells; Leukemia P388; Male; Melanoma; Mice; Molecular Structure; Naphthoquinones; Ovarian Neoplasms; Paclitaxel; Polymers; Prostatic Neoplasms; Triterpenes; Tumor Cells, Cultured

beta-Lapachone, a novel anti-neoplastic drug, induces various cancer cells to undergo apoptosis. In a previous report, we showed that beta-lapachone-induced apoptosis of HL-60 cells is mediated by oxidative stress. However, in the present study, we found that beta-lapachone-induced apoptosis of human prostate cancer (HPC) cells may be independent of oxidative stress. In contrast to the 10-fold beta-lapachone-induced increase in H(2)O(2) production seen in HL-60 cells, only a 2- to 4-fold increase was observed in HPC cells. N-acetyl-L-cysteine (NAC), a thiol antioxidant, inhibited the apoptosis in DU145 cells after 12 h exposure to beta-lapachone. Nonetheless, NAC, along with other antioxidants, failed to exert similar effect in HPC cells subjected to beta-lapachone treatment for 24 h. Under this premise, we suggest that the oxidative stress may not play a crucial role in beta-lapachone-mediated HPC cell apoptosis. Here we demonstrate that damage to genomic DNA is the trigger for the apoptosis of HPC cells induced by beta-lapachone. According to our results, beta-lapachone stimulates DNA dependent kinase expression and poly(ADP-ribose) polymerase cleavage in advance of significant morphological changes. beta-Lapachone promotes the expression of cyclin-dependent kinase (cdk) inhibitors (p21(WAF1) and p27(Kip1)), induces bak expression, and subsequently stimulates the activation of caspase-7 but not of caspase-3 or caspase-8 during the apoptosis of HPC cells. Taken together, these results suggest that the signaling pathway involving the beta-lapachone-induced apoptosis of HPC cell may be by DNA damage, induction of cdk inhibitors (p21 and p27), and then subsequent stimulation of caspase-7 activation.

Topics: Antibiotics, Antineoplastic; Antioxidants; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; Caspases; Cell Cycle; Cell Cycle Proteins; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cyclins; DNA-Activated Protein Kinase; DNA-Binding Proteins; Enzyme Inhibitors; Fluoresceins; Fluorescent Dyes; Genes, Tumor Suppressor; Humans; Male; Membrane Proteins; Microtubule-Associated Proteins; Naphthoquinones; Nuclear Proteins; Phenanthridines; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Reactive Oxygen Species; Tumor Cells, Cultured; Tumor Suppressor Proteins

beta-Lapachone (beta-Lap) triggers apoptosis in a number of human breast and prostate cancer cell lines through a unique apoptotic pathway that is dependent upon NQO1, a two-electron reductase. Downstream signaling pathway(s) that initiate apoptosis following treatment with beta-Lap have not been elucidated. Since calpain activation was suspected in beta-Lap-mediated apoptosis, we examined alterations in Ca(2+) homeostasis using NQO1-expressing MCF-7 cells. beta-Lap-exposed MCF-7 cells exhibited an early increase in intracellular cytosolic Ca(2+), from endoplasmic reticulum Ca(2+) stores, comparable to thapsigargin exposures. 1,2-Bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester, an intracellular Ca(2+) chelator, blocked early increases in Ca(2+) levels and inhibited beta-Lap-mediated mitochondrial membrane depolarization, intracellular ATP depletion, specific and unique substrate proteolysis, and apoptosis. The extracellular Ca(2+) chelator, EGTA, inhibited later apoptotic end points (observed >8 h, e.g. substrate proteolysis and DNA fragmentation), suggesting that later execution events were triggered by Ca(2+) influxes from the extracellular milieu. Collectively, these data suggest a critical, but not sole, role for Ca(2+) in the NQO1-dependent cell death pathway initiated by beta-Lap. Use of beta-Lap to trigger an apparently novel, calpain-like-mediated apoptotic cell death could be useful for breast and prostate cancer therapy.

Topics: 4-Nitroquinoline-1-oxide; Adenosine Triphosphate; Apoptosis; Blotting, Western; Breast Neoplasms; Calcium; Cell Death; Cell Division; Chelating Agents; Cytosol; DNA Fragmentation; Dose-Response Relationship, Drug; Egtazic Acid; Endoplasmic Reticulum; Female; Flow Cytometry; Humans; In Situ Nick-End Labeling; Male; Membrane Potentials; Microscopy, Confocal; Mitochondria; Models, Biological; Naphthoquinones; Prostatic Neoplasms; Quinolones; Signal Transduction; Time Factors; Tumor Cells, Cultured

beta-Lapachone (beta-lap) induces apoptosis in various cancer cells, and its intracellular target has recently been elucidated in breast cancer cells. Here we show that NAD(P)H:quinone oxidoreductase (NQO1/xip3) expression in human prostate cancer cells is a key determinant for apoptosis and lethality after beta-lap exposures. beta-Lap-treated, NQO1-deficient LNCaP cells were significantly more resistant to apoptosis than NQO1-expressing DU-145 or PC-3 cells after drug exposures. Formation of an atypical 60-kDa PARP cleavage fragment in DU-145 or PC-3 cells was observed after 10 microM beta-lap treatment and correlated with apoptosis. In contrast, LNCaP cells required 25 microM beta-lap to induce similar responses. Atypical PARP cleavage in beta-lap-treated cells was not affected by 100 microM zVAD-fmk; however, coadministration of dicoumarol, a specific inhibitor of NQO1, reduced beta-lap-mediated cytotoxicity, apoptosis, and atypical PARP cleavage in NQO1-expressing cells. Dicoumarol did not affect the more beta-lap-resistant LNCaP cells. Stable transfection of LNCaP cells with NQO1 increased their sensitivity to beta-lap, enhancing apoptosis compared to parental LNCaP cells or vector-alone transfectants. Dicoumarol increased survival of beta-lap-treated NQO1-expressing LNCaP transfectants. NQO1 activity, therefore, is a key determinant of beta-lap-mediated apoptosis and cytotoxicity in prostate cancer cells.

Topics: Antibiotics, Antineoplastic; Apoptosis; Camptothecin; Caspases; Dicumarol; Enzyme Inhibitors; Humans; Male; NAD(P)H Dehydrogenase (Quinone); Naphthoquinones; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Transformation, Genetic; Tumor Cells, Cultured; Tumor Suppressor Protein p53

beta-Lapachone and certain of its derivatives directly bind and inhibit topoisomerase I (Topo I) DNA unwinding activity and form DNA-Topo I complexes, which are not resolvable by SDS-K+ assays. We show that beta-lapachone can induce apoptosis in certain cells, such as in human promyelocytic leukemia (HL-60) and human prostate cancer (DU-145, PC-3, and LNCaP) cells, as also described by Li et al. (Cancer Res., 55: 0000-0000, 1995). Characteristic 180-200-bp oligonucleosome DNA laddering and fragmented DNA-containing apoptotic cells via flow cytometry and morphological examinations were observed in 4 h in HL-60 cells after a 4-h, > or = 0.5 microM beta-lapachone exposure. HL-60 cells treated with camptothecin or topotecan resulted in greater apoptotic DNA laddering and apoptotic cell populations than comparable equitoxic concentrations of beta-lapachone, although beta-lapachone was a more effective Topo I inhibitor. beta-Lapachone treatment (4 h, 1-5 microM) resulted in a block at G0/G1, with decreases in S and G2/M phases and increases in apoptotic cell populations over time in HL-60 and three separate human prostate cancer (DU-145, PC-3, and LNCaP) cells. Similar treatments with topotecan or camptothecin (4 h, 1-5 microM) resulted in blockage of cells in S and apoptosis. Thus, beta-lapachone causes a block in G0/G1 of the cell cycle and induces apoptosis in cells before, or at early times during, DNA synthesis. These events are p53 independent, since PC-3 and HL-60 cells are null cells, LNCaP are wild-type, and DU-145 contain mutant p53, yet all undergo apoptosis after beta-lapachone treatment. Interestingly, beta-lapachone treatment of p53 wild type-containing prostate cancer cells (i.e., LNCaP) did not result in the induction of nuclear levels of p53 protein, as did camptothecin-treated cells. Like other Topo I inhibitors, beta-lapachone may induce apoptosis by locking Topo I onto DNA, blocking replication fork movement, and inducing apoptosis in a p53-independent fashion. beta-Lapachone and its derivatives, as well as other Topo I inhibitors, have potential clinical utility alone against human leukemia and prostate cancers.

Topics: Apoptosis; Breast Neoplasms; Camptothecin; Cell Division; Dimethyl Sulfoxide; Drug Screening Assays, Antitumor; G1 Phase; Humans; Leukemia, Promyelocytic, Acute; Male; Naphthoquinones; Prostatic Neoplasms; Resting Phase, Cell Cycle; Time Factors; Topoisomerase I Inhibitors; Tumor Cells, Cultured; Tumor Stem Cell Assay

beta-Lapachone, a plant product, has been shown to be a novel inhibitor of DNA topoisomerase I, with a mode of action different from camptothecin and a chemical structure distinct from those of current anti-cancer drugs. We observed that beta-lapachone, at concentrations of less than 8 microM, induces cell death with characteristics of apoptosis in human prostate cancer cell lines. This effect of beta-lapachone was also observed in a human promyelocytic leukemia cell line (HL-60). beta-Lapachone-induced apoptosis is independent of p53 expression, and ectopic overexpression of bcl-2 did not confer significant resistance to beta-lapachone. Among other human carcinoma and adenoma cell lines tested, human breast and ovary carcinoma showed sensitivity to the cytotoxic effect of beta-lapachone without manifesting signs of apoptosis. These results suggest that beta-lapachone is a potential compound to be added to cancer chemotherapy, particularly for prostate cancer.

Topics: Apoptosis; Cell Survival; Drug Screening Assays, Antitumor; Flow Cytometry; Humans; Leukemia, Promyelocytic, Acute; Male; Naphthoquinones; Prostatic Neoplasms; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured; Tumor Stem Cell Assay; Tumor Suppressor Protein p53