Seleno-aspirin compound AS-10 promotes histone acetylation ahead of suppressing androgen receptor transcription, G1 arrest, and apoptosis of prostate cancer cells.

BACKGROUND: The novel selenium-aspirin compound AS-10 was recently reported by us with a cancer cell killing potency three orders of magnitude greater than aspirin in pancreatic cancer cell lines with caspase-mediated apoptosis and a reasonable selectivity against malignant cells. Although we also observed its cytocidal activity against PC-3 and DU145 androgen receptor (AR)-negative and P53-null/mutant aggressive human prostate cancer (PCa) cell lines in NCI-60 screen, the potential involvement and targeting of AR and P53 pathways that are intact in early-stage prostate carcinogenesis has not been examined, nor its primary molecular signaling after exposure. METHODS: Human LNCaP PCa cells with functional AR and intact P53 were used to examine their cell cycle and cell fate responses to AS-10 exposure and upstream molecular signaling events including histone acetylation as a known aspirin effect. The AR-positive 22Rv1 human PCa cells were used to validate key findings. RESULTS: In addition to confirming AS-10's superior cytocidal potency than aspirin against all four PCa cell lines, we report a rapid (within 5 min) promotion of histone acetylation several hours ahead of the suppression of AR and prostate-specific antigen (PSA, coded by KLK3 gene) in LNCaP and 22Rv1 cells. AS-10 decreased AR and KLK3 mRNA levels without impacting pre-existing AR protein degradation or nuclear translocation in LNCaP cells. Sustained exposure to AS-10 arrested cells predominantly in G1 , and induced caspase-mediated apoptosis without necrosis. The death induced by AS-10 in LNCaP cells was attenuated by nontranscriptional activation of P53 protein or Jun N-terminal Kinase cellular stress signaling and was mitigated modestly by glutathione-boosting antioxidant N-acetylcysteine. AS-10 synergized with histone deacetylase inhibitor SAHA to suppress AR/PSA abundance and kill LNCaP cells. RNA-seq confirmed AR suppression at the transcriptional level and suggested multiple oncogene, cyclin, and CDK/CKI transcriptional actions to contribute to the cellular consequences. CONCLUSIONS: AS-10 promotes histone acetylation as its probable primary mechanism of action to induce PCa cell-cycle arrest and apoptosis, regardless of AR and P53 status. Nevertheless, the inhibition of AR signaling through mechanisms distinct from canonical AR antagonists may hold promise for combinatorial use with androgen deprivation therapy regimens or AR-axis targeting drugs.

Opportunities and Challenges of Kava in Lung Cancer Prevention.

Lung cancer is the leading cause of cancer-related deaths due to its high incidence, late diagnosis, and limited success in clinical treatment. Prevention therefore is critical to help improve lung cancer management. Although tobacco control and tobacco cessation are effective strategies for lung cancer prevention, the numbers of current and former smokers in the USA and globally are not expected to decrease significantly in the near future. Chemoprevention and interception are needed to help high-risk individuals reduce their lung cancer risk or delay lung cancer development. This article will review the epidemiological data, pre-clinical animal data, and limited clinical data that support the potential of kava in reducing human lung cancer risk via its holistic polypharmacological effects. To facilitate its future clinical translation, advanced knowledge is needed with respect to its mechanisms of action and the development of mechanism-based non-invasive biomarkers in addition to safety and efficacy in more clinically relevant animal models.

Suppressing the activation of protein kinase A as a DNA damage-independent mechanistic lead for dihydromethysticin prophylaxis of NNK-induced lung carcinogenesis.

Our earlier work demonstrated varying potency of dihydromethysticin (DHM) as the active kava phytochemical for prophylaxis of tobacco carcinogen nicotine-derived nitrosamine ketone (NNK)-induced mouse lung carcinogenesis. Efficacy was dependent on timing of DHM gavage ahead of NNK insult. In addition to DNA adducts in the lung tissues mitigated by DHM in a time-dependent manner, our in vivo data strongly implicated the existence of DNA damage-independent mechanism(s) in NNK-induced lung carcinogenesis targeted by DHM to fully exert its anti-initiation efficacy. In the present work, RNA seq transcriptomic profiling of NNK-exposed (2 h) lung tissues with/without a DHM (8 h) pretreatment revealed a snap shot of canonical acute phase tissue damage and stress response signaling pathways as well as an activation of protein kinase A (PKA) pathway induced by NNK and the restraining effects of DHM. The activation of the PKA pathway by NNK active metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) at a concentration incapable of promoting DNA adduct was confirmed in a lung cancer cell culture model, potentially through NNAL binding to and activation of the ß-adrenergic receptor. Our in vitro and in vivo data overall support the hypothesis that DHM suppresses PKA activation as a key DNA damage-independent mechanistic lead, contributing to its effective prophylaxis of NNK-induced lung carcinogenesis. Systems biology approaches with a detailed temporal dissection of timing of DHM intake versus NNK exposure are warranted to fill the knowledge gaps concerning the DNA damage-driven mechanisms and DNA damage-independent mechanisms to optimize the implementation strategy for DHM to achieve maximal lung cancer chemoprevention.

Aqueous metabolome of tissue-specific conditional Pten-knockout mouse prostate cancer and TRAMP neuroendocrine carcinoma.

BACKGROUND: Metabolic reprograming is now a recognized hallmark of cancer. The prostate-specific phosphatase and tensin homolog deleted on chromosome 10 (Pten) gene-conditional knockout (KO) mouse carcinogenesis model is highly desirable for studying prostate cancer biology and prevention due to its close resemblance of primary molecular defects and histopathological features of human prostate cancer. We have recently published macromolecular profiling of this model by proteomics and transcriptomics, denoting a preeminence of inflammation and myeloid suppressive immune cell features. Here, we performed metabolomic analyses of Pten-KO prostate versus wild type (WT) counterpart for discernable changes in the aqueous metabolites and contrasted to those in the TRAMP neuroendocrine carcinoma (NECa). METHODS: Three matched pairs of tissue-specific conditional Pten-KO mouse prostate and WT prostate of litter/cage-mates at 20-22 weeks of age and three pairs of TRAMP NECa versus WT (28-31 weeks) were profiled for their global aqueous metabolite changes, using hydrophilic interaction liquid chromatography-tandem mass spectrometry. RESULTS: The Pten-KO prostate increased purine nucleotide pools, cystathionine, and both reduced and oxidized glutathione (GSH, GSSG), and gluconate/glucuronate species in addition to cholesteryl sulfate and polyamine precursor ornithine. On the contrary, Pten-KO prostate contained diminished pools of glycolytic intermediates and phosphorylcholine derivatives, select amino acids, and their metabolites. Bioinformatic integration revealed a significant shunting of glucose away from glycolysis-citrate cycle and glycerol-lipid genesis to pentose phosphate cycle for NADPH/GSH/GSSG redox and pentose moieties for purine and pyrimidine nucleotides, and glycosylation/glucuronidation. Implicit arginine catabolism to ornithine was consistent with immunosuppression in Pten-KO model. While also increased in cystathionine-GSH/GSSG, purine, and pyrimidine nucleotide pools and glucuronidation at the expense of glycolysis-citrate cycle, the TRAMP NECa increased abundance of many amino acids, methyl donor S-adenosyl-methionine, and intermediates for phospholipids without increasing cholesteryl sulfate or ornithine. CONCLUSIONS: The aqueous metabolomic patterns in Pten-KO prostate and TRAMP NECa shared similarities in the greater pools of cystathionine, GSH/GSSG redox pair, and nucleotides and shunting away from glycolysis-citrate cycle in both models. Remarkable metabolic distinctions between them included metabolisms of many amino acids (protein synthesis; arginine-ornithine/immune suppression) and cholesteryl sulfate and methylation donor for epigenetic regulations.

Effect of Dietary Methylseleninic Acid and Se-Methylselenocysteine on Carcinogen-Induced, Androgen-Promoted Prostate Carcinogenesis in Rats.

Selenomethionine (SeMet) did not prevent prostate cancer in the SELECT trial and in two hormone-driven rat models. However, we have shown that daily oral bolus administration of next-generation selenium forms, methylseleninic acid (MSeA) and Se-methylselenocysteine (MSeC) at 3 mg Se/kg body weight, inhibits prostate carcinogenesis in the TRAMP and pten-deficient mouse models and In Vivo growth of human prostate cancer cells. Here, we determined whether these Se forms prevent prostate cancer in a chemically induced-androgen promoted carcinogenesis rat model in which SeMet was not preventive. WU rats were treated with methylnitrosourea, and one week later, slow-release testosterone implants when they were randomized to groups fed AIN-93M diet supplemented with 3 ppm selenium as MSeA or MSeC or control diet. Mean survival, tumor incidence in all accessory sex glands combined (dorsolateral and anterior prostate plus seminal vesicle) and the incidence of tumors confined to dorsolateral and/or anterior prostate were not statistically significantly different among the groups. Thus, MSeA and MSeC feeding was not preventive in this model. The contrast with the inhibitory effects of MSeA and MSeC in mouse models may be due to differences in carcinogenic mechanisms, selenium dosage, delivery mode, and pharmacokinetics or fundamental rat-mouse differences in selenium metabolism.

Novel Seleno-Aspirinyl Compound AS-10 Induces Apoptosis, G1 Arrest of Pancreatic Ductal Adenocarcinoma Cells, Inhibits Their NF-κB Signaling, and Synergizes with Gemcitabine Cytotoxicity.

Current available therapies for pancreatic ductal adenocarcinoma (PDAC) provide minimal overall survival benefits and cause severe adverse effects. We have identified a novel molecule AS-10, a selenazolidine-bis-aspirinyl derivative, that was two to three orders of magnitude more potent than aspirin and at least one to two orders of magnitude more potent than gemcitabine in inhibiting PDAC cancer cell growth/viability against three PDAC cell lines while sparing mouse embryonic fibroblasts in the same exposure range. In Panc-1 cells, AS-10 induced apoptosis without necrosis, principally through caspase-3/7 cascade and reactive oxygen species, in addition to an induction of G1 cell cycle block. Transcriptomic profiling with RNA-seq indicated the top responses to AS-10 exposure as CDKN1A (P21Cip1), CCND1, and nuclear transcription factor-kappa B (NF-κB) complex and the top functions as cell cycle, cell death, and survival without inducing the DNA damage gene signature. AS-10 pretreatment (6 h) decreased cytokine tumor necrosis factor-alpha (TNF-α)-stimulated NF-κB nuclear translocation, DNA binding activity, and degradation of cytosolic inhibitor of κB (IκB) protein. As NF-κB activation in PDAC cells confers resistance to gemcitabine, the AS-10 combination with gemcitabine increased the in vitro cytotoxicity more than the additivity of both compounds. Overall, our results suggest AS-10 may be a promising drug lead for PDAC, both as a single agent and in combination therapy.

Proteomic and transcriptomic profiling of Pten gene-knockout mouse model of prostate cancer.

BACKGROUND: The prostate-specific phosphatase and tensin homolog deleted on chromosome 10 (Pten) gene-conditional knockout (KO) mouse carcinogenesis model is highly desirable for studies of prostate cancer biology and chemoprevention due to its close resemblance of primary molecular defect and many histopathological features of human prostate cancer including androgen response and disease progression from prostatic intraepithelial neoplasia to invasive adenocarcinoma. Here, we profiled the proteome and transcriptome of the Pten-KO mouse prostate tumors for global macromolecular expression alterations for signaling changes and biomarker signatures. METHODS: For proteomics, four pairs of whole prostates from tissue-specific conditional knockout Pten-KO mice (12-15 weeks of age) and their respective wild-type littermates housed in the same cages were analyzed by 8-plex isobaric tags for relative and absolute quantitation iTRAQ. For microarray transcriptomic analysis, three additional matched pairs of prostate/tumor specimens from respective mice at 20 to 22 weeks of age were used. Real-time quantitative reverse transcription-polymerase chain reaction was used to verify the trends of protein and RNA expression changes. Gene Set Enrichment Analysis and Ingenuity Pathway Analysis were carried out for bioinformatic characterizations of pathways and networks. RESULTS: At the macromolecular level, proteomic and transcriptomic analyses complement and cross-validate to reveal overexpression signatures including inflammation and immune alterations, in particular, neutrophil/myeloid lineage suppressor cell features, chromatin/histones, ion and nutrient transporters, and select glutathione peroxidases and transferases in Pten-KO prostate tumors. Suppressed expression patterns in the Pten-KO prostate tumors included glandular differentiation such as secretory proteins and androgen receptor targets, smooth muscle features, and endoplasmic reticulum stress proteins. Bioinformatic analyses identified immune and inflammation responses as the most profound macromolecular landscape changes, and the predicted key nodal activities through Akt, nuclear factor-kappaB, and P53 in the Pten-KO prostate tumor. Comparison with other genetically modified mouse prostate carcinogenesis models revealed notable molecular distinctions, especially the dominance of immune and inflammation features in the Pten-KO prostate tumors. CONCLUSIONS: Our work identified prominent macromolecular signatures and key nodal molecules that help to illuminate the patho- and immunobiology of Pten-loss driven prostate cancer and can facilitate the choice of biomarkers for chemoprevention and interception studies in this clinically relevant mouse prostate cancer model.

Oral Dosing of Dihydromethysticin Ahead of Tobacco Carcinogen NNK Effectively Prevents Lung Tumorigenesis in A/J Mice.

Our early studies demonstrated an impressive chemopreventive efficacy of dihydromethysticin (DHM), unique in kava, against tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis in A/J mice in which DHM was supplemented in the diet. The current work was carried out to validate the efficacy, optimize the dosing schedule, and further elucidate the mechanisms using oral bolus dosing of DHM. The results demonstrated a dose-dependent chemopreventive efficacy of DHM (orally administered 1 h before each of the two NNK intraperitoneal injections, 1 week apart) against NNK-induced lung adenoma formation. Temporally, DHM at 0.8 mg per dose (∼32 mg per kg body weight) exhibited 100% lung adenoma inhibition when given 3 and 8 h before each NNK injection and attained >93% inhibition when dosed at either 1 or 16 h before each NNK injection. The simultaneous treatment (0 h) or 40 h pretreatment (-40 h) decreased lung adenoma burden by 49.8% and 52.1%, respectively. However, post-NNK administration of DHM (1-8 h after each NNK injection) was ineffective against lung tumor formation. In short-term experiments for mechanistic exploration, DHM treatment reduced the formation of NNK-induced O6-methylguanine (O6-mG, a carcinogenic DNA adduct in A/J mice) in the target lung tissue and increased the urinary excretion of NNK detoxification metabolites as judged by the ratio of urinary NNAL-O-gluc to free NNAL, generally in synchrony with the tumor prevention efficacy outcomes in the dose scheduling time-course experiment. Overall, these results suggest DHM as a potential chemopreventive agent against lung tumorigenesis in smokers, with O6-mG and NNAL detoxification as possible surrogate biomarkers.

Optimizing live-animal bioluminescence imaging prediction of tumor burden in human prostate cancer xenograft models in SCID-NSG mice.

BACKGROUND: Noninvasive live-animal longitudinal monitoring of xenograft tumor growth and metastasis by bioluminescent imaging (BLI) has been widely reported in cancer biology and preclinical therapy literature, mainly in athymic nude mice. Our own experience at calibrating BLI readout with tumor weight/volume in human prostate cancer xenograft models in haired, SCID-NSG mice through intraprostatic (orthotopic) and subcutaneous (SC) inoculations revealed either nonexistent or poor correlation (coefficient of determination, R 2 = ~0.01-0.3). The present work examined several technical and biological factors to improve BLI utility. METHODS: After ruling out promoter-luciferase (luc) specificity and luc gene loss in the cell inoculum with LNCaP-AR-luc cells expressing an androgen receptor (AR) and tagged with AR-responsive probasin promoter-luc gene, we evaluated different routes of d-luciferin administration, imaging time during the day, charge-coupled device camera image acquisition settings, and hair removal methods to improve the imaging protocol. For most imaging sessions, BLI was carried out within the same day of tumor volume measurement. After necropsy, histological and immunohistochemical (IHC) analyses were performed on the tumors to evaluate necrosis and expression of luciferase and AR, respectively. RESULTS: Injection of d-luciferin by SC route, robust image-capture setting (30 000 counts and autoexposure), imaging in the morning and thorough hair removal resulted in a substantial improvement of R2 to ~0.6. Histological analyses confirmed the lack of BLI signal in necrotic tumor masses consistent with luciferase-mediated light emission only in oxygenated adenosine triphosphate-producing viable cells. IHC staining detected heterogeneous expression of luciferase tracking generally with AR expression in nonnecrotic tumor tissues. CONCLUSIONS: Our body of work highlighted a framework to validate imaging protocols to ensure the acquisition of interpretable BLI data as an indicator of xenograft tumor burden. The vast tissue heterogeneity in prostate tumor xenografts and variable luciferase expression constrained this technology from achieving a high correlation.

Phenylbutyl isoselenocyanate induces reactive oxygen species to inhibit androgen receptor and to initiate p53-mediated apoptosis in LNCaP prostate cancer cells.

Previous studies have established the in vivo bioavailability and efficacious dosages of phenylbutyl isoselenocyanate (ISC-4), a selenium-substituted isothiocyanate, against mouse xenograft models of human melanoma and colorectal cancer. To explore its potential attributes against prostate cancer, we treated human LNCaP prostate cancer cells with ISC-4 and examined their apoptosis responses, and interrogated the signaling mechanisms through pharmacological and siRNA knockdown approaches. Our results show that ISC-4 was more potent at inducing apoptosis than its sulfur analog phenylbutyl isothiocyanate (PBITC) without suppressing protein kinase AKT Ser473 phosphorylation. ISC-4 induced apoptosis in concentration- and time-dependent manners, and the apoptosis execution was attenuated by pre-incubation with a pan caspase inhibitor. ISC-4 decreased the abundance of androgen receptor (AR) and its best known target prostate specific antigen (PSA) without decreasing their steady state mRNA. ISC-4 upregulated the abundance of p53 protein and its Ser15 -phosphorylative activation, and that of DNA double strand break marker Ser139 -p-H2A.X coincident with apoptotic exposure. Similar to the rapid induction of reactive oxygen species (ROS) by isothiocyanates, ISC-4 increased dihydroethidium-detectable signals in LNCaP cells. Pre-incubation with ROS scavenger N-acetyl-l-cysteine preserved AR and PSA abundance, markedly reduced ISC-4-induced apoptosis and attenuated p53 Ser phosphorylation, p21Cip1, and p-H2A.X. Furthermore, siRNA knockdown of p53 did not suppress ROS production, but decreased ISC-4-induced apoptosis. Knocking down p53-targets PUMA and Bax exerted greater protective effect on ISC-4-induced apoptosis than depleting p21Cip1. In summary, ISC-4 inhibited LNCaP cell growth and survival with ROS-mediated suppression of AR axis signaling and induction of p53-PUMA-Bax mitochondrial apoptosis.

Gene expression signatures associated with suppression of TRAMP prostate carcinogenesis by a kavalactone-rich Kava fraction.

Kava (Piper methysticum Forster) extract and its major kavalactones have been shown to block chemically induced lung tumor initiation in mouse models. Here we evaluated the chemopreventive effect of a kavalactone-rich Kava fraction B (KFB), free of flavokavains, on carcinogenesis in a transgenic adenocarcinoma of mouse prostate (TRAMP) model and characterized the prostate gene expression signatures. Male C57BL/6 TRAMP mice were fed AIN93M diet with or without 0.4% KFB from 8 wk of age. Mice were euthanized at 16 or 28 wk. The growth of the dorsolateral prostate (DLP) lobes in KFB-treated TRAMP mice was inhibited by 66% and 58% at the respective endpoint. Anterior and ventral prostate lobes in KFB-treated TRAMP mice were suppressed by 40% and 49% at 28 wk, respectively. KFB consumption decreased cell proliferation biomarker Ki-67 and epithelial lesion severity in TRAMP DLP, without detectable apoptosis enhancement. Real time qRT-PCR detection of mRNA from DLP at 28 wk showed decreased expression of cell cycle regulatory genes congruent with Ki-67 suppression. Microarray profiling of DLP mRNA indicated that "oncogene-like" genes related to angiogenesis and cell proliferation were suppressed by KFB but tumor suppressor, immunity, muscle/neuro, and metabolism-related genes were upregulated by KFB in both TRAMP and WT DLP. TRAMP mice fed KFB diet developed lower incidence of neuroendocrine carcinomas (NECa) (2 out of 14 mice) than those fed the basal diet (8 out of 14 mice, χ2 = 5.6, P < 0.025). KFB may, therefore, inhibit not only TRAMP DLP epithelial lesions involving multiple molecular pathways, but also NECa. © 2016 Wiley Periodicals, Inc.

Cancer chemoprevention research with selenium in the post-SELECT era: Promises and challenges.

The negative efficacy outcomes of double-blinded, randomized, placebo-controlled Phase III human clinical trials with selenomethionine (SeMet) and SeMet-rich selenized-yeast (Se-yeast) for prostate cancer prevention and Se-yeast for prevention of nonsmall cell lung cancer (NSCLC) in North America lead to rejection of SeMet/Se-yeast for cancer prevention in Se-adequate populations. We identify 2 major lessons from the outcomes of these trials: 1) the antioxidant hypothesis was tested in wrong subjects or patient populations, and 2) the selection of Se agents was not supported by cell culture and preclinical animal efficacy data as is common in drug development. We propose that next-generation forms of Se (next-gen Se), such as methylselenol precursors, offer biologically appropriate approaches for cancer chemoprevention but these are faced with formidable challenges. Solid mechanism-based preclinical efficacy assessments and comprehensive safety studies with next-gen Se will be essential to revitalize the idea of cancer chemoprevention with Se in the post-SELECT era. We advocate smaller mechanism-driven Phase I/II trials with these next-gen Se to guide and justify future decisions for definitive Phase III chemoprevention efficacy trials.

Characterization of the Fluorescence Properties of 4-Dialkylaminochalcones and Investigation of the Cytotoxic Mechanism of Chalcones.

Understanding the mechanisms responsible for the various biological activities of chalcones, particularly the direct cellular targets, presents an unmet challenge. Here, we prepared a series of fluorescent chalcone derivatives as chemical probes for their mechanistic investigation. Upon systematic physicochemical characterization, we explored their potential to elucidate the mode of action of chalcones' cytotoxicity. The fluorescence of the chalcones was found to be highly sensitive to structural and environmental factors. Structurally, a 4-dialkylamino group on the B ring, suitable electronic properties of the A ring substituents, and the planar conformation of the chalcone's core structure were essential for optimal fluorescence. Environmental factors influencing fluorescence included solvent polarity, pH, and the interactions of the chalcones with proteins and detergents. It was found that 18 chalcones showed a fluorescent brightness greater than 6000 M(-1) cm(-1) in DMSO. However, water dramatically quenched the fluorescence, although it could be partially recovered in the presence of BSA or detergents. As expected, these fluorescent chalcones showed a sharp structure-activity relationship in their cellular cytotoxicity, leading to the identification of structurally similar cytotoxic and non-cytotoxic fluorescent chalcones as chemical probes. Confocal microscopy results revealed the co-localization of the cytotoxic probe C8 and tubulin in cells, supporting tubulin as the direct cellular target responsible for the cytotoxicity of chalcones.

Chemopreventive effect of Korean Angelica root extract on TRAMP carcinogenesis and integrative "omic" profiling of affected neuroendocrine carcinomas.

Angelica gigas Nakai (AGN) root ethanol extract exerts anti-cancer activity in several allograft and xenograft models. Here we examined its chemopreventive efficacy through gavage administration against primary carcinogenesis in the transgenic adenocarcinoma of mouse prostate (TRAMP) model. Male C57BL/6 TRAMP mice and wild type littermates were given a daily gavage (5 mg/mouse, Monday-Friday) of AGN or vehicle, beginning at 8 wk of age (WOA). All mice were terminated at 24 WOA, unless earlier euthanasia was necessitated by large tumors. Whereas AGN-treated TRAMP mice decreased dorsolateral prostate lesion growth by 30% (P = 0.009), they developed fewer and smaller neuroendocrine-carcinomas (NE-Ca) (0.12 g/mouse) than vehicle-treated counterparts (0.81 g/mouse, P = 0.037). We analyzed the proteome and transcriptome of banked NE-Ca to gain molecular insights. Angiogenesis-antibody array detected a substantial reduction in AGN-treated NE-Ca of basic fibroblast growth factor (FGF2), an angiogenesis stimulator. iTRAQ proteomics plus data mining suggested changes of genes upstream and downstream of FGF2 functionally consistent with AGN inhibiting FGF2/FGFR1 signaling at different levels of the transduction cascade. Moreover, AGN upregulated mRNA of genes related to immune responses, restored expression of many tumor suppressor genes, and prostate function and muscle differentiation genes. On the other hand, AGN down-regulated mRNA of genes related to neuron signaling, oncofetal antigens, inflammation, and mast cells, Wnt signaling, embryonic morphogenesis, biosynthesis, cell adhesion, motility, invasion, and angiogenesis. These changes suggest not only multiple cancer cell targeting actions of AGN but also impact on the tumor microenvironments such as angiogenesis, inflammation, and immune surveillance.

Dihydromethysticin from kava blocks tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumorigenesis and differentially reduces DNA damage in A/J mice.

We have previously shown that kava and its flavokavain-free Fraction B completely blocked 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis in A/J mice with a preferential reduction in NNK-induced O (6)-methylguanine (O (6)-mG). In this study, we first identified natural (+)-dihydromethysticin (DHM) as a lead compound through evaluating the in vivo efficacy of five major compounds in Fraction B on reducing O (6)-mG in lung tissues. (+)-DHM demonstrated outstanding chemopreventive activity against NNK-induced lung tumorigenesis in A/J mice with 97% reduction of adenoma multiplicity at a dose of 0.05mg/g of diet (50 ppm). Synthetic (±)-DHM was equally effective as the natural (+)-DHM in these bioassays while a structurally similar analog, (+)-dihydrokavain (DHK), was completely inactive, revealing a sharp in vivo structure-activity relationship. Analyses of an expanded panel of NNK-induced DNA adducts revealed that DHM reduced a subset of DNA adducts in lung tissues derived from 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL, the active metabolite of NNK). Preliminary 17-week safety studies of DHM in A/J mice at a dose of 0.5mg/g of diet (at least 10× its minimum effective dose) revealed no adverse effects, suggesting that DHM is likely free of kava's hepatotoxic risk. These results demonstrate the outstanding efficacy and promising safety margin of DHM in preventing NNK-induced lung tumorigenesis in A/J mice, with a unique mechanism of action and high target specificity.

Methylseleninic acid suppresses pancreatic cancer growth involving multiple pathways.

As a potential novel agent for treating pancreatic cancer, methylseleninic acid (MSeA) was evaluated in cell culture and xenograft models. Results showed that MSeA induced G1 cell cycle arrest and apoptosis in a majority of human and mouse pancreatic cancer cell lines, but G2 arrest in human PANC-1 and PANC-28 cell lines. In contrast to our previous finding in human prostate cancer LNCaP cells having a lack of P53 activation by MSeA, induction of G2 arrest in PANC-1 cells was accompanied by increased mutant P53 Ser15 phosphorylation, upregulation of P53-targets P21Cip1 and GADD45 and G2 checkpoint kinase (Chk2) activation, suggestive of DNA damage responses. A rapid inhibition of AKT phosphorylation was followed by reduced mTOR signaling and increased autophagy in PANC-1 cells attenuating caspase-mediated apoptosis execution. Furthermore, daily oral treatment with MSeA (3 mg Se/kg body weight) significantly suppressed growth of subcutaneously inoculated PANC-1 xenograft in SCID mice. Immunohistochemical analyses detected increased p-Ser15 P53, P21Cip1, pS139-H2AX (DNA damage responses), and caspase-3 cleavage and decreased pSer473AKT and Ki67 proliferative index and reduced intratumor vascular density in MSeA-treated xenograft. These results provide impetus for further research of MSeA in the therapy and/or chemoprevention of pancreatic cancer.

2-Deoxyglucose and hydroxychloroquine HPLC-MS-MS analytical methods and pharmacokinetic interactions after oral co-administration in male rats.

Our previous work has shown a synergistic tumoricidal efficacy of combining the hexokinase (HK) inhibitor 2-deoxyglucose (2-DG) and the autophagy inhibitor chloroquine (CQ) through intraperitoneal injections on HK2-addicted prostate cancers in animal models. The pharmacokinetic (PK) behaviors of these oral drugs after simultaneous oral administration have not been reported. We developed high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS) analytical methods for 2-DG and the clinically favored drug hydroxychloroquine (HCQ) for sera samples. Using a jugular vein-cannulated male rat model with serial blood collection before and after a single gavage dose of each drug alone or in combination, we examined their PK metrics for drug-drug interactions. The data demonstrated a rapid and complete separation of 2-DG from common monosaccharides by HPLC-MS-MS multi-reaction monitoring. Application of the HPLC-MS-MS 2-DG and HCQ methods to sera samples of nine rats showed a peak time (Tmax ) for 2-DG of 0.5 h after 2-DG alone or with HCQ and identical post-peak half-life of approximately 1 h. With a seemingly bi-modal time course for HCQ, the Tmax for HCQ alone (1.2 h) was faster than that for the combination (2 h; p = .017). After combination dosing, the peak concentration (Cmax ) and area under the curve (AUC0-4h ) of 2-DG were decreased by 53.8% (p = .0004) and 53.7% (p = .0001), whereas AUC0-8h for HCQ was decreased by 30.8% (p = .0279) from the respective single dosing. Without changing the mean residence time (MRT0-∞ ) of each drug, the combination affected the apparent volume of distribution (Vd ) and clearance (CL) of 2-DG, and CL for HCQ without affecting its Vd . We observed significant negative PK interactions, probably at the intestinal absorption level, between 2-DG and HCQ taken simultaneously by mouth. Future optimization efforts are warranted for their combination regimen for clinical translation.

Library Screening and Preliminary Characterization of Synthetic Cannabinoids Against Prostate and Pancreatic Cancer Cell Lines.

Background: Our previous screening efforts with colorectal cancer cell lines suggested potential cannabinoid therapeutic leads for other solid cancers. Objectives: The aim of this study was to identify cannabinoid lead compounds that have cytostatic and cytocidal activities against prostate and pancreatic cancer cell lines and profile cellular responses and molecular pathways of select leads. Materials and Methods: A library of 369 synthetic cannabinoids was screened against 4 prostate and 2 pancreatic cancer cell lines with 48 h of exposure at 10 µM in medium with 10% fetal bovine serum using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) viability assay. Concentration titration of the top 6 hits was carried out to identify their concentration-response patterns and calculate IC50 values. Three select leads were examined for cell cycle, apoptosis, and autophagy responses. The role of cannabinoid receptors (CB1 and CB2) and noncanonical receptors in apoptosis signaling was examined with selective antagonists. Results: Two independent screening experiments in each cell line detected growth inhibitory activities against all six or a majority of cancer cell lines for HU-331 (a known cannabinoid topoisomerase II inhibitor), (±)5-epi-CP55,940, and PTI-2, each previously identified in our colorectal cancer study. 5-Fluoro NPB-22, FUB-NPB-22, and LY2183240 were novel hits. Morphologically and biochemically, (±)5-epi-CP55,940 elicited caspase-mediated apoptosis of PC-3-luc2 (a PC-3 subline with luciferase) prostate cancer and Panc-1 pancreatic cancer cell lines, each the most aggressive of the respective organ site. The apoptosis induced by (±)5-epi-CP55,940 was abolished by the CB2 antagonist, SR144528, but not modulated by the CB1 antagonist, rimonabant, and GPR55 antagonist, ML-193, nor TRPV1 antagonist, SB-705498. In contrast, 5-fluoro NPB-22 and FUB-NPB-22 did not cause substantial apoptosis in either cell line, but resulted in cytosolic vacuoles and increased LC3-II formation (suggestive of autophagy) and S and G2/M cell cycle arrests. Combining each fluoro compound with an autophagy inhibitor, hydroxychloroquine, enhanced the apoptosis. Conclusions: 5-Fluoro NPB-22, FUB-NPB-22, and LY2183240 represent new leads against prostate and pancreatic cancer cells in addition to the previously reported compounds, HU-331, (±)5-epi-CP55,940, and PTI-2. Mechanistically, the two fluoro compounds and (±)5-epi-CP55,940 differed regarding their structures, CB receptor involvement, and death/fate responses and signaling. Safety and antitumor efficacy studies in animal models are warranted to guide further R&D.

Enhanced apoptotic effects by the combination of curcumin and methylseleninic acid: potential role of Mcl-1 and FAK.

Curcumin and methylseleninic acid (MSeA) are well-documented dietary chemopreventive agents. Apoptosis appears to be a major mechanism for both agents to exert anti-cancer activity. The purpose of the present study was designed to determine whether the apoptotic effect on human cancer cells can be enhanced by combining curcumin with MSeA. Apoptosis was evaluated by Annexin V staining of externalized phosphatidylserine by flow cytometry. Expression of protein was analyzed by Western blotting. Localization of apoptosis-inducing factor (AIF) was detected by immunocytochemistry. RNA interference was employed to inhibit expression of specific protein. We found here that combining curcumin with MSeA led to a significantly enhanced apoptosis in both MDA-MB-231 breast cancer cells and DU145 prostate cancer cells. Further mechanistic investigations revealed that curcumin treatment alone caused a concentration dependent upregulation of Mcl-1, which can be overcome by combining it with MSeA. In line with the Mcl-1 reduction, an enhanced mitochondrial permeability transition and AIF nuclear translocation by the combination were achieved. In addition, an increased suppression of focal adhesion kinase activity was observed in the combination-treated cells which were associated with cell detachment-induced apoptosis by the combination. Our findings suggest that curcumin/MSeA combination holds excellent potential for improving their efficacy against human breast and prostate cancer through enhanced apoptosis induction.

Single oral dose pharmacokinetics of decursin, decursinol angelate, and decursinol in rats.

Decursin and decursinol angelate are the major components in the alcoholic extract of the root of Angelica gigas Nakai. Our previous work convincingly demonstrated that both decursin and decursinol angelate were rapidly converted to decursinol in mice after administration by either oral gavage or i. p. injection. In the current study, we compared for the first time the plasma profiles of decursinol, when equal moles of decursin/decursinol angelate or decursinol were given to rats by oral gavage, and investigated the effect of different formulas and other chemicals in Angelica gigas extract on the bioavailability of decursinol. Our results show that gavage of decursinol led to a faster attainment of plasma decursinol peak (Tmax ~ 0.7 h) and much higher peak levels than an equal molar amount administered as decursin/decursinol angelate mixture or as Angelica gigas ethanol extract, resulting in 2-3 fold higher bioavailability as estimated by the area under the curve of the respective regimens (65 012 vs. 27 033 h · ng/mL for decursinol and decursin/decursinol angelate treatment groups, respectively). Compared to a formula based on ethanol-PEG400-Tween80, carboxyl methyl cellulose was a less optimized vehicle. In addition, we detected peak levels of decursin and decursinol angelate in the plasma of rats administered with decursin/decursinol angelate or Angelica gigas extract in the nM range (Tmax ~ 0.5 h) with a newly established sensitive UHPLC-MS/MS method. Furthermore, our data support the liver, instead of intestine, as a major organ site where decursin and decursinol angelate were hydrolyzed to decursinol with a S9 microsomal in vitro metabolism assay. Taken together, our study provided important PK, LC-MS/MS methodology, formulation and metabolism insights in a rodent model for the rational design of in vivo efficacy studies of the corresponding chemicals in the future.