Mandy Liu

Developing Phytoestrogens for Breast Cancer Prevention

Breast cancer is one of the most common types of cancer in women, and is the second leading cause of cancer-related deaths in the United States. Chemoprevention using phytoestrogens (PEs) for breast cancer may be a valid strategy. PEs are phytochemicals with estrogen-like structures and can be classified into four types: isoflavones, lignans, stilbenes and coumestans. They are widely distributed in diet and herbs and have shown anti-cancer activity via mechanisms including estrogen receptor modulation, aromatase inhibition, and anti-angiogenesis. Genistein, daidzein and resveratrol are some of the most studied PE examples. Quality control in product manufacturing and clinical study design is a critical issue in developing them as clinically effective chemopreventive agents for breast cancer.

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.

Transcriptional profiling of Chinese medicinal formula Si-Wu-Tang on breast cancer cells reveals phytoestrogenic activity

BackgroundSi-Wu-Tang (SWT), comprising the combination of four herbs, Paeoniae, Angelicae, Chuanxiong and Rehmanniae, is one of the most popular traditional oriental medicines for women’s diseases. In our previous study, the microarray gene expression profiles of SWT on breast cancer cell line MCF-7 were found similar to the effect of β-estradiol (E2) on MCF-7 cells in the Connectivity Map database.MethodsFurther data analysis was conducted to find the main similarities and differences between the effects of SWT and E2 on MCF-7 gene expression. The cell proliferation assay on MCF-7 (ER-positive) and MDA-MB-231 (ER-negative) cells were used to examine such estrogenic activity. The estrogenic potency of SWT was further confirmed by estrogen-responsive element (ERE) luciferase reporter assay in MCF-7 cells.ResultsMany estrogen regulated genes strongly up-regulated by E2 were similarly up-regulated by SWT, e.g., GREB1, PGR and EGR3. Of interest with regard to safety of SWT, the oncogenes MYBL1 and RET were strongly induced by E2 but not by SWT. Quantitative RT-PCR analysis revealed a highly concordant expression change in selected genes with data obtained by microarrays. Further supporting SWT’s estrogenic activity, in MCF-7 but not in MDA-MB-231 cells, SWT stimulated cell growth at lower concentrations (< 3.0 mg/ml), while at high concentrations, it inhibits the growth of both cell lines. The growth inhibitory potency of SWT was significantly higher in MDA-MB-231 than in MCF-7 cells. The SWT-induced cell growth of MCF-7 could be blocked by addition of the estrogen receptor antagonist tamoxifen. In addition, SWT was able to activate the ERE activity at lower concentrations. The herbal components Angelicae, Chuanxiong and Rehmanniae at lower concentrations (< 3.0 mg/ml) also showed growth-inducing and ERE-activating activity in MCF-7 cells.ConclusionsThese results revealed a new mechanism to support the clinical use of SWT for estrogen related diseases and possibly for cancer prevention. This study also demonstrated the feasibility of using microarray transcriptional profiling to discover phytoestrogenic components that are present in natural products.

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.

Abstract 2575: DNA microarray and connectivity map analysis reveals estrogen-like activity of Chinese medicinal formula Si-Wu-Tang

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL To pursue a systematic approach for discovery of potential mechanisms of action and new therapeutic use of traditional herbal medicines, we used DNA microarray, bioinformatics and the “Connectivity Map” (CMAP) analysis to investigate the gene expression profiling of Chinese herbal medicines. We demonstrated that this approach can be used to elucidate novel molecular mechanisms for the Chinese medicinal formula Si-Wu-Tang (SWT) which is widely used for womens health in Asian countries. The human breast cancer MCF-7 cells treated with beta-estradiol (E2, 0.1 µM) or SWT extract (0.0256, 0.256, and 2.56 mg/ml) for six hours showed significant gene expression changes. The differentially expressed genes related to SWT treatment were used to compare with the gene expression profiles of 1,309 compounds in the CMAP database. Such comparison revealed that the CMAP profile of E2-treated MCF-7 cells showed an excellent match with SWT treatment (permutation p 6.0 mg/ml), it inhibited the growth of MCF-7. The combination of SWT with tamoxifen, a selective estrogen receptor modulator, demonstrated enhanced growth inhibitory effects on MCF-7 cells. The results our study suggest that SWT may possess a non-toxic chemopreventive effect for human breast cancer. This study also demonstrated the feasibility of using microarray gene expression profiling in combination with the CMAP data mining to discover new molecular signature that are present in natural products. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2575. doi:1538-7445.AM2012-2575

Impact of Genomics on Personalization of Breast Cancer Care

Over the past 50 years, progress on multiple fronts has dramatically altered the nature of the disease known as breast cancer. The initiation of randomized prospective clinical trials in 1959, a novel concept at the time, by the National Surgical Adjuvant Breast and Bowel Project (NSABP) under the guidance of Bernard Fisher established a scientific philosophy as the guiding force in breast cancer treatment. Since 1975, multiple innovations have increased the therapeutic options and improved the outcomes available to women with breast cancer. Increased awareness of breast cancer, improvements in breast imaging, and the development of screening programs have made early diagnosis commonplace. The de-radicalization of surgical techniques used to obtain local control and the application of plastic surgical techniques for breast reconstruction have dramatically reduced the morbidity associated with mastectomy and axillary dissection. The development of pharmacologic hormonal therapy, more effective cytotoxic chemotherapy, and targeted HER2 therapy has improved survival for women with the most common types of breast cancer as well as less common but highly aggressive cancers. The development of predictive assays for response to chemotherapy has spared many patients from unnecessary toxicity and improved their quality of life. Critical to these advances has been the recognition that all breast cancers are not the same and the belief that treatment should be tailored so that every patient receives the best chance of survival with the least morbidity. New insights into the genomic heterogeneity of breast cancer offer the prospect for improved outcomes for patients with breast cancer by further personalization of breast cancer care.

Effects of bioactive constituents in the Traditional Chinese Medicinal formula Si–Wu–Tang on Nrf2 signaling and neoplastic cellular transformation

BACKGROUND The nuclear factor erythroid 2-related factor 2 (Nrf2) is a potential molecular target for cancer chemoprevention. Si-Wu-Tang (SWT), a popular traditional Chinese medicine for womens health, was reported with a novel activity of cancer prevention. PURPOSE The present study was aimed to identify the bioactive constituents in SWT responsible for the Nrf2 activating and cancer preventive activity and explore the pharmacological mechanisms. METHODS Nine compounds detectable from various batches of SWT were ranked using in silico molecular docking based on their ability to interfere the forming of Nrf2-Keap1 complex. The predicted Nrf2 activating effect was validated using the antioxidant response element (ARE) luciferase reporter assay and quantitative RT-PCR analysis for select Nrf2 regulated genes Hmox1, Nqo1 and Slc7a11. The antimutagenic activity of the compounds were determined by the Ames test. The chemopreventive activity of these compounds were assessed on EGF-induced neoplastic transformation of JB6 P+ cells, an established non-cancerous murine epidermal model for studying tumor promotion and identifying cancer preventive agents. These compounds were further characterized using luciferase reporter assay on EGF-induced activation of AP-1, a known transcription factor mediating carcinogenesis. RESULTS Three of the nine compounds predicted as Nrf2 activators by molecular docking, gallic acid (GA), Z-liguistilide (LIG), and senkyunolide A (SA), were confirmed with highest potency of increasing the Nrf2/ARE promoter activity and upregulating the expression of Hmox1, Nqo1 and Slc7a11. In addition, GA, LIG and SA exhibited an antimutagenic activity against the direct mutagen 2-nitrofluorene while no mutagenic effects were observed at the same time in Ames test. At nontoxic concentrations, GA, LIG, and SA inhibited EGF-induced neoplastic transformation of JB6 P+ cells. Combined treatment of GA, LIG and SA, in the same ratio as detected in SWT, showed enhanced effect against JB6 transformation compared with that of the single compound alone. GA, LIG and SA, alone or in combination, suppressed EGF-induced activation of AP-1. CONCLUSION We identified three bioactive constituents in SWT responsible for the Nrf2 activating and cancer preventive activity. This study provides evidence supporting novel molecular basis of SWT in cancer prevention.

Abstract 5252: Skin cancer prevention by traditional Chinese medicinal formula Si-Wu-Tang and its constituents

Si-Wu-Tang (SWT), comprising the combination of four herbs, Rehmanniae, Angelica, Chuanxiong and Paeoniae, is one of the most popular Chinese medicines for women9s diseases. Previously we showed that SWT was able to upregulate genes in the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, suggesting a potential application for cancer chemoprevention. The present study examined the chemopreventive activity of SWT using models of skin carcinogenesis. In JB6 P+ cells, a non-cancerous murine epidermal cell line for studying skin tumor promotion, SWT inhibited epidermal growth factor (EGF)-induced neoplastic transformation. In a 7,12-dimethylbenz(a)anthracene (DMBA)-induced murine skin tumorigenesis model, both topical and oral treatment of SWT inhibited epidermal hyperplasia, proliferating cell nuclear antigen expression, and H-ras mutations induced by DMBA treatment. In addition, SWT exhibited a significant antimutagenic activity against DMBA-induced mutagenicity, determined by the Ames Test using Salmonella typhimurium TA100 in the presence of metabolic activator S9 system. To identify active components in SWT, among nine compounds previously reported in commercial SWT products, in silico molecular docking analysis predicted some as potential Nrf2 activators due to an ability of interfering the forming of Nrf2-Keap1 complex. Three of these compounds, gallic acid, Z-liguistilide and senkyunolide A, were confirmed with highest potency of increasing the antioxidant response element luciferase reporter activity, inducing Nrf2-regulated genes Hmox1, Slc7A11 and Nqo1, and inhibiting EGF-induced JB6 P+ transformation. Further mechanistic studies showed that SWT and the three compounds suppressed EGF-induced activation of the activator protein 1 (AP-1), an essential transcription factor involved in skin carcinogenesis. The antimutagenic activity for the three compounds was also confirmed with the Ames Test. In conclusion, these results provide evidence that SWT and its constituents are able to prevent skin cancer, at least partly, by activating the Nrf2 pathway and blocking the activation of AP-1. Thus, this widely used Chinese medicinal formula may provide a promising option toward preventing skin cancer or may be other types of cancer. Citation Format: Kevin Huang, Mandy Liu, Suhui Zhang, Steven Yeung, Andy Chang, Li Qian, Payal Chatterjee, Rui Li, Su Zhou, Nan Mei, Zhijun Wang, Cyrus Parsa, Robert Orlando, Yun Luo, Ying Huang. Skin cancer prevention by traditional Chinese medicinal formula Si-Wu-Tang and its constituents. [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 5252.