Steven Yeung

Nutraceuticals for prostate cancer chemoprevention: from molecular mechanisms to clinical application

Introduction: Nutraceutical is a food, or part of a food, used for the prevention and/or treatment of diseases. A number of nutraceuticals serve as candidates for development of prostate cancer chemopreventive agents because of promising epidemiological, preclinical and pilot clinical findings. Their mechanisms of action may involve an ability to target multiple molecular pathways in carcinogenesis without eliciting toxic side effects. Areas covered: This review provides an overview of several nutraceuticals, including green tea polyphenol, omega-3 fatty acids, vitamin D, lycopene, genistein, quercetin, resveratrol and sulforaphane, for the clinical relevance to chemoprevention of prostate cancer. Their mechanisms of action on regulating key processes of carcinogenesis are also discussed. For each of these agents, a brief summary of completed or currently ongoing clinical trials related to the chemopreventive efficacy on prostate cancer is given. Expert opinion: Even though a few clinical trials have been conducted, review of these results indicate that further studies are required to confirm the clinical efficacy and safety, and to provide a guidance on how to use nutraceuticals for optimal effect. Future cancer prevention clinical trials for the nutraceuticals should recruit men with an increased risk of prostate cancer.

Ginseng and Anticancer Drug Combination to Improve Cancer Chemotherapy: A Critical Review

Ginseng, a well-known herb, is often used in combination with anticancer drugs to enhance chemotherapy. Its wide usage as well as many documentations are often cited to support its clinical benefit of such combination therapy. However the literature based on objective evidence to make such recommendation is still lacking. The present review critically evaluated relevant studies reported in English and Chinese literature on such combination. Based on our review, we found good evidence from in vitro and in vivo animal studies showing enhanced antitumor effect when ginseng is used in combination with some anticancer drugs. However, there is insufficient clinical evidence of such benefit as very few clinical studies are available. Future research should focus on clinically relevant studies of such combination to validate the utility of ginseng in cancer.

Overcoming chemoresistance in prostate cancer with Chinese medicine Tripterygium wilfordii via multiple mechanisms

A leading cause of cancer chemotherapy failure is chemoresistance, which often involves multiple mechanisms. Chinese medicines (CM) usually contain multiple components which could potentially target many mechanisms simultaneously and may offer an advantage over single compounds that target one mechanism at a time. The purpose of this study was to investigate the chemosensitizing effect (CE) of a specific CM, Tripterygium wilfordii (TW), on prostate cancer cells resistant to docetaxel (Dtx) and identify the potential mechanisms. The CE of TW (in combination with Dtx) was evaluated in two Dtx resistant prostate cancer cell lines (PC3-TxR and DU145-TxR) and the efficacy of the combination for resistant PC3-TxR tumor was investigated using a xenograft mouse model. For mechanistic study, the inhibitory effect of TW on P-glycoprotein activity was assessed. In addition, novel gene targets of TW were identified using DNA microarray and quantitative PCR. Results showed that TW induced a CE of 8 and >38 folds in PC3-TxR and DU145-TxR cells, respectively with Dtx IC50 reversed back to that of the sensitive parent cells. An optimum dose of TW+Dtx significantly retarded tumor growth in mice compared to TW or Dtx alone. TW inhibited P-glycoprotein activity and induced a significant gene expression changes in genes related to angiogenesis, cell cycle regulation and differentiation. Our in vitro and in vivo studies demonstrate that TW in combination with Dtx was able to overcome the chemoresistance and suppress resistant prostate tumor growth via multi-mechanisms.

Prevention of skin carcinogenesis by the β-blocker carvedilol.

The stress-related catecholamine hormones and the α- and β-adrenergic receptors (α- and β-AR) may affect carcinogenesis. The β-AR GRK/β-arrestin biased agonist carvedilol can induce β-AR–mediated transactivation of the EGFR. The initial purpose of this study was to determine whether carvedilol, through activation of EGFR, can promote cancer. Carvedilol failed to promote anchorage-independent growth of JB6 P+ cells, a skin cell model used to study tumor promotion. However, at nontoxic concentrations, carvedilol dose dependently inhibited EGF-induced malignant transformation of JB6 P+ cells, suggesting that carvedilol has chemopreventive activity against skin cancer. Such effect was not observed for the β-AR agonist isoproterenol and the β-AR antagonist atenolol. Gene expression, receptor binding, and functional studies indicate that JB6 P+ cells only express β2-ARs. Carvedilol, but not atenolol, inhibited EGF-mediated activator protein-1 (AP-1) activation. A topical 7,12-dimethylbenz(α)anthracene (DMBA)-induced skin hyperplasia model in SENCAR mice was utilized to determine the in vivo cancer preventative activity of carvedilol. Both topical and oral carvedilol treatment inhibited DMBA-induced epidermal hyperplasia (P < 0.05) and reduced H-ras mutations; topical treatment being the most potent. However, in models of established cancer, carvedilol had modest to no inhibitory effect on tumor growth of human lung cancer A549 cells in vitro and in vivo. In conclusion, these results suggest that the cardiovascular drug carvedilol may be repurposed for skin cancer chemoprevention, but may not be an effective treatment of established tumors. More broadly, this study suggests that β-ARs may serve as a novel target for cancer prevention. Cancer Prev Res; 8(1); 27–36. ©2014 AACR.

Inhibition of Neoplastic Transformation and Chemically-Induced Skin Hyperplasia in Mice by Traditional Chinese Medicinal Formula Si-Wu-Tang

Exploring traditional medicines may lead to the development of low-cost and non-toxic cancer preventive agents. Si-Wu-Tang (SWT), comprising the combination of four herbs, Rehmanniae, Angelica, Chuanxiong, and Paeoniae, is one of the most popular traditional Chinese medicines for women’s diseases. In our previous studies, the antioxidant Nrf2 pathways were strongly induced by SWT in vitro and in vivo. Since Nrf2 activation has been associated with anticarcinogenic effects, the purpose of this study is to evaluate SWT’s activity of cancer prevention. In the Ames test, SWT demonstrated an antimutagenic activity against mutagenicity induced by the chemical carcinogen 7,12-dimethylbenz(a)anthracene (DMBA). In JB6 P+ cells, a non-cancerous murine epidermal model for studying tumor promotion, SWT inhibited epidermal growth factor (EGF)-induced neoplastic transformation. The luciferase reporter gene assays demonstrated that SWT suppressed EGF-induced AP-1 and TNF-α-induced NF-κB activation, which are essential factors involved in skin carcinogenesis. In a DMBA-induced skin hyperplasia assay in ‘Sensitivity to Carcinogenesis’ (SENCAR) mice, both topical and oral SWT inhibited DMBA-induced epidermal hyperplasia, expression of the proliferation marker Proliferating cell nuclear antigen (PCNA), and H-ras mutations. These findings demonstrate, for the first time, that SWT prevents tumor promoter and chemical-induced carcinogenesis in vitro and in vivo, partly by inhibiting DNA damage and blocking the activation of AP-1 and NF-κB.

Topically Applied Carvedilol Attenuates Solar Ultraviolet Radiation Induced Skin Carcinogenesis

In previous studies, the β-blocker carvedilol inhibited EGF-induced epidermal cell transformation and chemical carcinogen-induced mouse skin hyperplasia. As exposure to ultraviolet (UV) radiation leads to skin cancer, the present study examined whether carvedilol can prevent UV-induced carcinogenesis. Carvedilol absorbs UV like a sunscreen; thus, to separate pharmacological from sunscreen effects, 4-hydroxycarbazole (4-OHC), which absorbs UV to the same degree as carvedilol, served as control. JB6 P+ cells, an established epidermal model for studying tumor promotion, were used for evaluating the effect of carvedilol on UV-induced neoplastic transformation. Both carvedilol and 4-OHC (1 μmol/L) blocked transformation induced by chronic UV (15 mJ/cm2) exposure for 8 weeks. However, EGF-mediated transformation was inhibited by only carvedilol but not by 4-OHC. Carvedilol (1 and 5 μmol/L), but not 4-OHC, attenuated UV-induced AP-1 and NF-κB luciferase reporter activity, suggesting a potential anti-inflammatory activity. In a single-dose UV (200 mJ/cm2)-induced skin inflammation mouse model, carvedilol (10 μmol/L), applied topically after UV exposure, reduced skin hyperplasia and the levels of cyclobutane pyrimidine dimers, IL1β, IL6, and COX-2 in skin. In SKH-1 mice exposed to gradually increasing levels of UV (50–150 mJ/cm2) three times a week for 25 weeks, topical administration of carvedilol (10 μmol/L) after UV exposure increased tumor latency compared with control (week 18 vs. 15), decreased incidence and multiplicity of squamous cell carcinomas, while 4-OHC had no effect. These data suggest that carvedilol has a novel chemopreventive activity and topical carvedilol following UV exposure may be repurposed for preventing skin inflammation and cancer. Cancer Prev Res; 10(10); 598–606. ©2017 AACR.

Phosphoproteome profiling provides insight into the mechanism of action for carvedilol-mediated cancer prevention

Recent studies suggest that the β‐blocker drug carvedilol prevents skin carcinogenesis but the mechanism is unknown. Carvedilol is one of a few β‐blockers identified as biased agonist based on an ability to promote β‐arrestin‐mediated processes such as ERK phosphorylation. To understand the role of phosphoproteomic signaling in carvedilols anticancer activity, the mouse epidermal JB6 P+ cells treated with EGF, carvedilol, or their combination were analyzed using the Phospho Explorer Antibody Array containing 1318 site‐specific and phospho‐specific antibodies of over 30 signaling pathways. The array data indicated that both EGF and carvedilol increased phosphorylation of ERKs cytosolic target P70S6 K while its nuclear target ELK‐1 were activated only by EGF; Furthermore, EGF‐induced phosphorylation of ELK‐1 and c‐Jun was attenuated by carvedilol. Subcellular fractionation analysis indicated that ERK nuclear translocation induced by EGF was blocked by co‐treatment with carvedilol. Western blot and luciferase reporter assays confirmed that the biased β‐blockers carvedilol and alprenolol blocked EGF‐induced phosphorylation and activation of c‐Jun/AP‐1 and ELK‐1. Consistently, both carvedilol and alprenolol strongly prevented EGF‐induced neoplastic transformation of JB6 P+ cells. Remarkably, oral carvedilol treatment significantly inhibited the growth of A375 melanoma xenograft in SCID mice. As nuclear translocation of ERK is a key step in carcinogenesis, inhibition of this event is proposed as a novel anticancer mechanism for biased β‐blockers such as carvedilol.

Bioavailability of wilforlide A in mice and its concentration determination using an HPLC-APCI-MS/MS method

Wilforlide A (WA), an active compound in Tripterygium wilfordii Hook F (TW) which is a traditional Chinese medicine for treatment of autoimmune diseases, is a quality control marker for TW product. At present, the bioavailability/pharmacokinetics of WA is not known. Such information is not only essential to evaluate the relevance of WA as a quality control maker, but also important for future clinical efficacy studies. Therefore, a high-performance liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometric method (HPLC-APCI-MS/MS) was developed and applied to a bioavailability/pharmacokinetic study of WA. WA and celastrol (the internal standard, IS) were extracted by a liquid-liquid extraction method using methyl tert-butyl ether. Multiple reaction monitoring (MRM) scanning in positive ionization mode was used to monitor the transition of m/z 455.1 to 191.3 for WA and 451.3 to 201.2 for IS. This method was validated and applied to a pharmacokinetic study of WA in mice following intravenous administration (IV, 1.2 mg/kg), intraperitoneal injection (IP, 6 mg/kg) and oral administration (PO, 30 mg/kg). The lower limit of quantification (LLOQ) for WA was 10 ng/ml. The intra- and inter-day precision was found to be within 15.4% while the accuracy within 94.1-115.7% for all the quality control and LLOQ samples. The samples were stable under all the usual storage and experimental conditions. The terminal elimination half-lives were 14.7, 9.1 and 22.7 min following IV, IP and PO dosing, while the absolute bioavailability for IP and PO WA were 9.39% and 0.58% respectively. These results indicated that the HPLC-APCI-MS/MS assay was suitable for the pharmacokinetic study of WA. WA was found poorly absorbed when given orally and therefore it may not be a relevant marker for the oral TW products in the market.

Development of a simple and rapid HPLC-MS/MS method for quantification streptomycin in mice and its application to plasma pharmacokinetic studies

Streptomycin was the first discovered aminoglycoside antibiotic. It has been widely applied in veterinary medicine for the prevention and treatment of bacterial infection. However, the current detection methods are not satisfactory in terms of sensitivity and sample process, which makes them unsuitable for a pharmacokinetic study. A high-performance liquid chromatography-mass spectrometric method employing positive electrospray ionization was developed and validated for the determination of streptomycin concentration in mice plasma. A simple protein precipitation method was utilized to extract streptomycin as well as the internal standard (kanamycin) from mouse plasma. This assay method was validated in terms of specificity, sensitivity, precision, accuracy and recovery. This method was applied to a pharmacokinetic study in mice following intramuscular administration of 200 mg/kg streptomycin. The lower limit of quantification of the developed assay method for streptomycin was 10 ng/mL. The intra-day and inter-day precision was evaluated with the coefficient of variations <14.3%, whereas the mean accuracy ranged from 87.0 to 105.0%. The samples were stable under the experimental conditions. The present method provides a robust, fast and sensitive analytical approach for the quantification of streptomycin in mouse plasma and has been successfully applied to a pharmacokinetic study in mice.

Abstract 2617: Protective effect of the β-blocker carvedilol on UVB-induced skin damage

The response to ultraviolet (UV) radiation is the primary etiologic factor leading to carcinogenesis of the skin. The effects of UV radiation on the skin is generally transient, but the long term consequences can be carcinogenic. Despite growing public awareness and the use of sunscreen to prevent skin cancer, the incidence continues to rise, and therefore there is a need to develop innovative chemoprevention strategies. Previously, we showed that the receptor subtype nonselective β-adrenergic receptor antagonist (i.e., β-blocker) carvedilol prevented EGF and UVB-mediated neoplastic transformation of the mouse epidermal cell line JB6 P+, indicating its chemopreventive potential against skin cancer. To demonstrate the in vivo effects of carvedilol, in the present study, hairless SKH-1 mice were irradiated for two weeks with 200 mJ/cm2 of UVB and drugs were topically applied immediately after the radiation. Since carvedilol is able to absorb UV, its analogue 4-hydroxycarbazole, which has the same UVB absorption profiling as carvedilol but does not function as a β-blocker, was included as a sunscreen control. Significant skin thickening measured by bi-fold skin thickness on the dorsal skin was observable within a week and over the course of the two-week period. Treatment with carvedilol, but not the analogue, reduced UVB-induced skin hyperplasia, reddening and inflammation. Furthermore, carvedilol, but not the analogue, reduced H2O2 and UVB- induced cytotoxicity and reactive oxygen species production in JB6 P+ cells. To elucidate the molecular mechanism(s) for carvedilol9s chemopreventive activity, the signaling profile for JB6 P+ cells treated with EGF and/or carvedilol for 15 min was examined using a Phospho Explorer Antibody Microarray containing 1318 site and phosphor-specific antibodies from over 30 signaling pathways. The antibody array data suggested that the phosphorylation of the intracellular signaling pathways involved in skin carcinogenesis, AP-1 and NF-kB, were increased by EGF but attenuated by carvedilol. These results were validated using luciferase reporter assays. The array data also suggested that carvedilol was able to induce the expression of tumor suppressors p53 and PTEN. In addition, the checkpoint kinases CHK1 and CHK2 and other cell-cycle regulators such as cyclins were also upregulated by carvedilol, suggesting a role in carvedilol9s protective effect. Since highly abundant mutations are observable in sun-exposed skin, our current work is to examine the effects of carvedilol on the production of cyclo-pyrimidine dimers (CPDs) induced by UVB. In conclusion, our study has demonstrated novel mechanisms underlying carvedilol9s chemopreventive activity against skin cancer. Since carvedilol and other β-blockers are FDA approved drugs and relatively safe, they may offer a new approach of prevention for UVB-induced skin cancer. Citation Format: Kevin M. Huang, Kristan H. Cleveland, Steven Yeung, Bradley T. Andresen, Ying Huang. Protective effect of the β-blocker carvedilol on UVB-induced skin damage. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2617.