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.

Angelica gigas: Signature Compounds, In Vivo Anticancer, Analgesic, Neuroprotective and Other Activities, and the Clinical Translation Challenges.

Angelica gigas Nakai (AGN) root is a medicinal herbal widely used in traditional medicine in Korea. AGN root ethanolic extract dietary supplements are marketed in the United States for memory health and pain management. We comprehensively reviewed the anticancer, analgesic, pro-memory and other bio-activities of AGN extract and its signature phytochemicals decursin, decursinol angelate, and decursinol a decade ago in 2012 and updated their anticancer activities in 2015. In the last decade, significant progress has been made for understanding the pharmacokinetics (PK) and metabolism of these compounds in animal models and single dose human PK studies have been published by us and others. In addition to increased knowledge of the known bioactivities, new bioactivities with potential novel health benefits have been reported in animal models of cerebral ischemia/stroke, anxiety, sleep disorder, epilepsy, inflammatory bowel disease, sepsis, metabolic disorders, osteoporosis, osteoarthritis, and even male infertility. Herein, we will update PK and metabolism of pyranocoumarins, review in vivo bioactivities from animal models and human studies, and critically appraise the relevant active compounds, the cellular and molecular pharmacodynamic targets, and pertinent mechanisms of action. Knowledge gaps include whether human pyranocoumarin PK metrics are AGN dose dependent and subjected to metabolic ceiling, or metabolic adaptation after repeated use. Critical clinical translation challenges include sourcing of AGN extracts, product consistency and quality control, and AGN dose optimization for different health conditions and disease indications. Future research directions are articulated to fill knowledge gaps and address these challenges.

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.

Interception Targets of Angelica Gigas Nakai Root Extract versus Pyranocoumarins in Prostate Early Lesions and Neuroendocrine Carcinomas in TRAMP Mice.

We reported efficacy of Angelica gigas Nakai (AGN) root ethanol extract and equimolar decursin (D)/decursinol angelate (DA) through daily gavage starting at 8 weeks of age (WOA) to male transgenic adenocarcinoma of mouse prostate (TRAMP) mice such that these modalities suppressed precancerous epithelial lesions in their dorsolateral prostate (DLP) to similar extent, but AGN extract was better than the D/DA mixture at promoting the survival of mice bearing prostate neuroendocrine carcinomas to 28 WOA. Here, we compared by microarray hybridization the mRNA levels in pooled DLP tissues and individual neuroendocrine carcinomas to characterize potential molecular targets of AGN extract and D/DA. Clustering and principal component analyses supported distinct gene expression profiles of TRAMP DLP versus neuroendocrine carcinomas. Pathway Enrichment, Gene Ontology, and Ingenuity Pathway Analyses of differential genes indicated that AGN and D/DA affected chiefly processes of lipid and mitochondrial energy metabolism and oxidation-reduction in TRAMP DLP, while AGN affected neuronal signaling, immune systems and cell cycling in neuroendocrine carcinomas. Protein-Protein Interaction Network analysis predicted and reverse transcription-PCR verified multiple hub genes common in the DLP of AGN- and D/DA-treated TRAMP mice at 28 WOA and select hub genes attributable to the non-D/DA AGN components. The vast majority of hub genes in the AGN-treated neuroendocrine carcinomas differed from those in TRAMP DLP. In summary, the transcriptomic approach illuminated vastly different signaling pathways and networks, cellular processes, and hub genes of two TRAMP prostate malignancy lineages and their associations with the interception efficacy of AGN and D/DA. PREVENTION RELEVANCE: This study explores potential molecular targets associated with in vivo activity of AGN root alcoholic extract and its major pyranocoumarins to intercept precancerous epithelial lesions and early malignancies of the prostate. Without an ethically-acceptable, clearly defined cancer initiation risk reduction strategy available for the prostate, using natural products like AGN to delay formation of malignant tumors could be a plausible approach for prostate cancer prevention.

Kava as a Clinical Nutrient: Promises and Challenges.

Kava beverages are typically prepared from the root of Piper methysticum. They have been consumed among Pacific Islanders for centuries. Kava extract preparations were once used as herbal drugs to treat anxiety in Europe. Kava is also marketed as a dietary supplement in the U.S. and is gaining popularity as a recreational drink in Western countries. Recent studies suggest that kava and its key phytochemicals have anti-inflammatory and anticancer effects, in addition to the well-documented neurological benefits. While its beneficial effects are widely recognized, rare hepatotoxicity had been associated with use of certain kava preparations, but there are no validations nor consistent mechanisms. Major challenges lie in the diversity of kava products and the lack of standardization, which has produced an unmet need for quality initiatives. This review aims to provide the scientific community and consumers, as well as regulatory agencies, with a broad overview on kava use and its related research. We first provide a historical background for its different uses and then discuss the current state of the research, including its chemical composition, possible mechanisms of action, and its therapeutic potential in treating inflammatory and neurological conditions, as well as cancer. We then discuss the challenges associated with kava use and research, focusing on the need for the detailed characterization of kava components and associated risks such as its reported hepatotoxicity. Lastly, given its growing popularity in clinical and recreational use, we emphasize the urgent need for quality control and quality assurance of kava products, pharmacokinetics, absorption, distribution, metabolism, excretion, and foundational pharmacology. These are essential in order to inform research into the molecular targets, cellular mechanisms, and creative use of early stage human clinical trials for designer kava modalities to inform and guide the design and execution of future randomized placebo controlled trials to maximize kava's clinical efficacy and to minimize its risks.

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.

The Impact of One-week Dietary Supplementation with Kava on Biomarkers of Tobacco Use and Nitrosamine-based Carcinogenesis Risk among Active Smokers.

Tobacco smoking is the primary risk factor for lung cancer, driven by the addictive nature of nicotine and the indisputable carcinogenicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) as well as other compounds. The integration of lung cancer chemoprevention with smoking cessation is one potential approach to reduce this risk and mitigate lung cancer mortality. Experimental data from our group suggest that kava, commonly consumed in the South Pacific Islands as a beverage to promote relaxation, may reduce lung cancer risk by enhancing NNK detoxification and reducing NNK-derived DNA damage. Building upon these observations, we conducted a pilot clinical trial to evaluate the effects of a 7-day course of kava on NNK metabolism in active smokers. The primary objective was to compare urinary total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL plus its glucuronides, major metabolites of NNK) before and after kava administration as an indicator of NNK detoxification. Secondary objectives included determining kava's safety, its effects on DNA damage, tobacco use, and cortisol (a biomarker of stress). Kava increased urinary excretion of total NNAL and reduced urinary 3-methyladenine in participants, suggestive of its ability to reduce the carcinogenicity of NNK. Kava also reduced urinary total nicotine equivalents, indicative of its potential to facilitate tobacco cessation. Plasma cortisol and urinary total cortisol equivalents were reduced upon kava use, which may contribute to reductions in tobacco use. These results demonstrate the potential of kava intake to reduce lung cancer risk among smokers.

Role of P53-Senescence Induction in Suppression of LNCaP Prostate Cancer Growth by Cardiotonic Compound Bufalin.

Bufalin is a major cardiotonic compound in the traditional Chinese medicine, Chansu, prepared from toad skin secretions. Cell culture studies have suggested an anticancer potential involving multiple cellular processes, including differentiation, apoptosis, senescence, and angiogenesis. In prostate cancer cell models, P53-dependent and independent caspase-mediated apoptosis and androgen receptor (AR) antagonism have been described for bufalin at micromolar concentrations. Because a human pharmacokinetic study indicated that single nanomolar bufalin was safely achievable in the peripheral circulation, we evaluated its cellular activity within range with the AR-positive and P53 wild-type human LNCaP prostate cancer cells in vitro Our data show that bufalin induced caspase-mediated apoptosis at 20 nmol/L or higher concentration with concomitant suppression of AR protein and its best-known target, PSA and steroid receptor coactivator 1 and 3 (SRC-1, SRC-3). Bufalin exposure induced protein abundance of P53 (not mRNA) and P21CIP1 (CDKN1A), G2 arrest, and increased senescence-like phenotype (SA-galactosidase). Small RNAi knocking down of P53 attenuated bufalin-induced senescence, whereas knocking down of P21CIP1 exacerbated bufalin-induced caspase-mediated apoptosis. In vivo, daily intraperitoneal injection of bufalin (1.5 mg/kg body weight) for 9 weeks delayed LNCaP subcutaneous xenograft tumor growth in NSG SCID mice with a 67% decrease of final weight without affecting body weight. Tumors from bufalin-treated mice exhibited increased phospho-P53 and SA-galactosidase without detectable caspase-mediated apoptosis or suppression of AR and PSA. Our data suggest potential applications of bufalin in therapy of prostate cancer in patients or chemo-interception of prostate precancerous lesions, engaging a selective activation of P53 senescence. Mol Cancer Ther; 17(11); 2341-52. ©2018 AACR.

Prostate Cancer Xenograft Inhibitory Activity and Pharmacokinetics of Decursinol, a Metabolite of Angelica gigas Pyranocoumarins, in Mouse Models.

We have previously shown that the ethanol extract of dried Angelica gigas Nakai (AGN) root exerts anticancer activity against androgen receptor (AR)-negative human DU145 and PC-3 prostate cancer xenografts and primary carcinogenesis in the transgenic adenocarcinoma of mouse prostate (TRAMP) model. The major pyranocoumarin isomers decursin (D) and decursinol angelate (DA), when provided at equi-molar intake to that provided by AGN extract, accounted for the inhibitory efficacy against precancerous epithelial lesions in TRAMP mice. Since we and others have shown in rodents and humans that D and DA rapidly and extensively convert to decursinol, here we tested whether decursinol might be an in vivo active compound for suppressing xenograft growth of human prostate cancer cells expressing AR. In SCID-NSG mice carrying subcutaneously inoculated human LNCaP/AR-Luc cells overexpressing the wild type AR, we compared the efficacy of 4.5[Formula: see text]mg decursinol per mouse with equi-molar dose of 6[Formula: see text]mg D/DA per mouse. The result showed that decursinol decreased xenograft tumor growth by 75% and the lung metastasis, whereas D/DA exerted a much less effect. Measurement of plasma decursinol concentration, at 3[Formula: see text]h after the last dose of respective dosing regimen, showed higher circulating level in the decursinol-treated NSG mice than in the D/DA-treated mice. In a subsequent single-dose pharmacokinetic experiment, decursinol dosing led to 3.7-fold area under curve (AUC) of plasma decursinol over that achieved by equi-molar D/DA dosing. PK advantage notwithstanding, decursinol represents an active compound to exert in vivo prostate cancer growth and metastasis inhibitory activity in the preclinical model.

Pyranocoumarin Tissue Distribution, Plasma Metabolome and Prostate Transcriptome Impacts of Sub-Chronic Exposure to Korean Angelica Supplement in Mice.

Herbal products containing Korean Angelica gigas Nakai (AGN) root extract are marketed as dietary supplements for memory enhancement, pain killing, and female menopausal symptom relief. We have shown the anticancer activities of AGN supplements in mouse models. To facilitate human anticancer translational research, we characterized the tissue distribution of AGN marker pyranocoumarin compounds decursin (D) and decursinol angelate (DA) ([Formula: see text]% in AGN) and their metabolite decursinol (DOH), assessed the safety of sub-chronic AGN dietary exposure in mice, and explored its impact on plasma aqueous metabolites and the prostate transcriptome. The data show that after a gavage dose, plasma contained readily detectable DOH, but little D and DA, mirroring patterns in the liver. Extra-hepatic tissues retained greater levels of DA and D than the liver did. For sub-chronic exposures, male mice were provided ad libitum AIN93M-pellet diets with 0.5 and 1% AGN for six weeks. No adverse effects were observed on the plasma biochemistry markers of liver and kidney integrity in spite of their enlargement. Histopathological examinations of the liver, kidney and other visceral organs did not reveal tissue abnormalities. Metabolomic assessment of plasma from mice fed the 1%-AGN diet suggested metabolic shifts of key amino acids especially in the methionine-cysteine cycle, purine cycle, and glycolysis-citrate cycle. Prostate transcriptomic profiling identified gene signature changes in the metabolisms of drugs, lipids and cellular energetics, neuro-muscular features, immunity and inflammation, and tumor suppressor/oncogene balance. The safety profile was corroborated with a daily [Formula: see text] injection of AGN extract (100-300[Formula: see text]mg/kg) for four weeks, which resulted in much greater systemic pyranocoumarin exposure than the dietary route did.

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 with Korean Angelica: Active Compounds, Pharmacokinetics, and Human Translational Considerations.

Angelica gigas Nakai (AGN) is a major medicinal herb used in Korea and several other Asian countries. Traditionally, its dried root has been used to treat anemia, pain, infection and articular rheumatism, most often through boiling in water to prepare the dosage forms. AGN extract or AGN-containing herbal mixtures are sold in the US and globally as dietary supplements for pain killing, memory enhancement and post-menopausal symptom relief. Decursin (D) and its isomer decursinol angelate (DA) are the major chemicals in the alcoholic extracts of the root of AGN. The anti-cancer activity of AGN alcoholic extract has been established in a number of animal cancer models, including a transgenic model of prostate carcinogenesis. Cell culture structure-activity studies have uncovered distinct cellular and molecular effects of D and DA vs. their pyranocoumarin core decursinol (DOH) with respect to cancer cells and those associated with their microenvironment. Pharmacokinetic (PK) study by us and others in rodent models indicated that DOH is the major and rapid in vivo first-pass liver metabolite of D and DA. Cognizant of metabolic differences among rodents and humans, we carried out a first-in-human PK study of D/DA to inform the translational relevance of efficacy and mechanism studies with rodent models. The combined use of vigorous animal tests and human PK studies can provide stronger scientific rationale to inform design and execution of translational studies to move AGN toward evidence-based herbal medicine.

Cytochrome P450 Isoforms in the Metabolism of Decursin and Decursinol Angelate from Korean Angelica.

We have shown that the in vitro hepatic microsomal metabolism of pyranocoumarin compound decursinol angelate (DA) to decursinol (DOH) exclusively requires cytochrome P450 (CYP) enzymes, whereas the conversion of its isomer decursin (D) to DOH can be mediated by CYP and esterase(s). To provide insight into specific isoforms involved, here we show with recombinant human CYP that 2C19 was the most active at metabolizing D and DA in vitro followed by 3A4. With carboxylesterases (CES), D was hydrolyzed by CES2 but not CES1, and DA was resistant to both CES1 and CES2. In human liver microsomal (HLM) preparation, the general CYP inhibitor 1-aminobenzotriazole (ABT) and respective competitive inhibitors for 2C19 and 3A4, (+)-N-3-benzylnirvanol (NBN) and ketoconazole substantially retarded the metabolism of DA and, to a lesser extent, of D. In healthy human subjects from a single-dose pharmacokinetic (PK) study, 2C19 extensive metabolizer genotype (2C19*17 allele) tended to have less plasma DA AUC0-48h and poor metabolizer genotype (2C19*2 allele) tended to have greater DA AUC0-48h. In mice given a single dose of D/DA, pretreatment with ABT boosted the plasma and prostate levels of D and DA by more than an order of magnitude. Taken together, our findings suggest that CYP isoforms 2C19 and 3A4 may play a crucial role in the first pass liver metabolism of DA and, to a lesser extent, that of D in humans. Pharmacogenetics with respect to CYP genotypes and interactions among CYP inhibitor drugs and D/DA should therefore be considered in designing future translation studies of DA and/or D.

Chemopreventive Effects of Korean Angelica versus Its Major Pyranocoumarins on Two Lineages of Transgenic Adenocarcinoma of Mouse Prostate Carcinogenesis.

We showed previously that daily gavage of Angelica gigas Nakai (AGN) root ethanolic extract starting 8 weeks of age inhibited growth of prostate epithelium and neuroendocrine carcinomas (NE-Ca) in the transgenic adenocarcinoma of mouse prostate (TRAMP) model. Because decursin (D) and its isomer decursinol angelate (DA) are major pyranocoumarins in AGN extract, we tested the hypothesis that D/DA represented active/prodrug compounds against TRAMP carcinogenesis. Three groups of male C57BL/6 TRAMP mice were gavage treated daily with excipient vehicle, AGN (5 mg per mouse), or equimolar D/DA (3 mg per mouse) from 8 weeks to 16 or 28 weeks of age. Measurement of plasma and NE-Ca D, DA, and their common metabolite decursinol indicated similar retention from AGN versus D/DA dosing. The growth of TRAMP dorsolateral prostate (DLP) in AGN- and D/DA-treated mice was inhibited by 66% and 61% at 16 weeks and by 67% and 72% at 28 weeks, respectively. Survival of mice bearing NE-Ca to 28 weeks was improved by AGN, but not by D/DA. Nevertheless, AGN- and D/DA-treated mice had lower NE-Ca burden. Immunohistochemical and mRNA analyses of DLP showed that AGN and D/DA exerted similar inhibition of TRAMP epithelial lesion progression and key cell-cycle genes. Profiling of NE-Ca mRNA showed a greater scope of modulating angiogenesis, epithelial-mesenchymal transition, invasion-metastasis, and inflammation genes by AGN than D/DA. The data therefore support D/DA as probable active/prodrug compounds against TRAMP epithelial lesions, and they cooperate with non-pyranocoumarin compounds to fully express AGN efficacy against NE-Ca.

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.

Kava blocks 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumorigenesis in association with reducing O6-methylguanine DNA adduct in A/J mice.

We previously reported the chemopreventive potential of kava against 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)- and benzo(a)pyrene (BaP)-induced lung tumorigenesis in A/J mice during the initiation and postinitiation stages. In this study, we investigated the tumorigenesis-stage specificity of kava, the potential active compounds, and the underlying mechanisms in NNK-induced lung tumorigenesis in A/J mice. In the first experiment, NNK-treated mice were given diets containing kava at a dose of 5 mg/g of diet during different periods. Kava treatments covering the initiation stage reduced the multiplicity of lung adenomas by approximately 99%. A minimum effective dose is yet to be defined because kava at two lower dosages (2.5 and 1.25 mg/g of diet) were equally effective as 5 mg/g of diet in completely inhibiting lung adenoma formation. Daily gavage of kava (one before, during, and after NNK treatment) completely blocked lung adenoma formation as well. Kavalactone-enriched fraction B fully recapitulated kava's chemopreventive efficacy, whereas kavalactone-free fractions A and C were much less effective. Mechanistically, kava and fraction B reduced NNK-induced DNA damage in lung tissues with a unique and preferential reduction in O(6)-methylguanine (O(6)-mG), the highly tumorigenic DNA damage by NNK, correlating and predictive of efficacy on blocking lung adenoma formation. Taken together, these results demonstrate the outstanding efficacy of kava in preventing NNK-induced lung tumorigenesis in A/J mice with high selectivity for the initiation stage in association with the reduction of O(6)-mG adduct in DNA. They also establish the knowledge basis for the identification of the active compound(s) in kava.

In vitro metabolism of pyranocoumarin isomers decursin and decursinol angelate by liver microsomes from man and rodents.

The aim of this study is to investigate and compare the metabolic rate and profiles of pyranocoumarin isomers decursin and decursinol angelate using liver microsomes from humans and rodents, and to characterize the major metabolites of decursin and decursinol angelate in human liver microsomal incubations using LC-MS/MS. First, we conducted liver microsomal incubations of decursin and decursinol angelate in the presence or absence of NADPH. We found that in the absence of NADPH, decursin was efficiently hydrolyzed to decursinol by hepatic esterase(s), but decursinol angelate was not. In contrast, formation of decursinol from decursinol angelate was mediated mainly by cytochrome P450(s). Second, we measured the metabolic rate of decursin and decursinol angelate in liver S9 fractions from mice and humans. We found that human liver S9 fractions metabolized both decursin and decursinol angelate more slowly than those of the mouse. Third, we characterized the major metabolites of decursin and decursinol angelate from human liver microsomes incubations using HPLC-UV and LC-MS/MS methods and assessed the in vivo metabolites in mouse plasma from a one-dose PK study. Decursin and decursinol angelate have different metabolite profiles. Nine metabolites of decursin and nine metabolites of decursinol angelate were identified in human liver microsome incubations besides decursinol using a hybrid triple quadruple linear ion trap LC-MS/MS system, and many of them were later verified to be also present in plasma samples from rodent PK studies.

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.

Tanshinones: sources, pharmacokinetics and anti-cancer activities.

Tanshinones are a class of abietane diterpene compound isolated from Salvia miltiorrhiza (Danshen or Tanshen in Chinese), a well-known herb in Traditional Chinese Medicine (TCM). Since they were first identified in the 1930s, more than 40 lipophilic tanshinones and structurally related compounds have been isolated from Danshen. In recent decades, numerous studies have been conducted to investigate the isolation, identification, synthesis and pharmacology of tanshinones. In addition to the well-studied cardiovascular activities, tanshinones have been investigated more recently for their anti-cancer activities in vitro and in vivo. In this review, we update the herbal and alternative sources of tanshinones, and the pharmacokinetics of selected tanshinones. We discuss anti-cancer properties and identify critical issues for future research. Whereas previous studies have suggested anti-cancer potential of tanshinones affecting multiple cellular processes and molecular targets in cell culture models, data from in vivo potency assessment experiments in preclinical models vary greatly due to lack of uniformity of solvent vehicles and routes of administration. Chemical modifications and novel formulations had been made to address the poor oral bioavailability of tanshinones. So far, human clinical trials have been far from ideal in their design and execution for the purpose of supporting an anti-cancer indication of tanshinones.