Published OnlineFirst August 18, 2009; DOI: 10.1158/1078-0432.CCR-09-0298 Cancer Therapy: Preclinical Herbal Extract of Wedelia chinensis Attenuates Androgen Receptor Activity and Orthotopic Growth of Prostate Cancer in Nude Mice Chin-Hsien Tsai,1,3 Feng-Min Lin,1 Yu-Chih Yang,1 Ming-Ting Lee,2,3 Tai-Lung Cha,4 Guan-James Wu,1 Shih-Chuan Hsieh,1 and Pei-Wen Hsiao1 Abstract Purpose: Wedelia chinensis is a common ingredient of anti-inflammatory herbal med- icines in Taiwan and southern China. Inflammation is involved in promoting tumor growth, invasion, and metastasis. This study aims to test the biological effects in vivo of W. chinensis extract on prostate cancer. Experimental Design: The in vivo efficacy and mechanisms of action of oral adminis- tration of a standardized extract of W. chinensis were analyzed in animals bearing a subcutaneous or orthotopic prostate cancer xenograft. Results: Exposure of prostate cancer cells to W. chinensis extract induced apoptosis selectively in androgen receptor (AR)–positive prostate cancer cells and shifted the pro- portion in each phase of cell cycle toward G2-M phase in AR-negative prostate cancer cells. Oral herbal extract (4 or 40 mg/kg/d for 24-28 days) attenuated the growth of pros- tate tumors in nude mice implanted at both subcutaneous (31% and 44%, respectively) and orthotopic (49% and 49%, respectively) sites. The tumor suppression effects were associated with increased apoptosis and lower proliferation in tumor cells as well as reduced tumor angiogenesis. The antitumor effect of W. chinensis extract was correlat- ed with accumulation of the principle active compounds wedelolactone, luteolin, and apigenin in vivo. Conclusion: Anticancer action of W. chinensis extract was due to three active com- pounds that inhibit the AR signaling pathway. Oral administration of W. chinensis ex- tract impeded prostate cancer tumorigenesis. Future studies of W. chinensis for chemoprevention or complementary medicine against prostate cancer in humans are thus warranted. (Clin Cancer Res 2009;15(17):5435–44) Carcinoma of the prostate gland is the most common malig- the most effective means of treating metastatic prostate cancer nancy in males in the western world (1). Despite the low inci- tumors (2, 3). This therapy often induces apoptosis in the ma- dence of prostate cancer in oriental countries, statistics from jority of prostate cancer cells by blocking testosterone signaling Taiwan reveal prostate cancer deaths have continued to increase at the androgen receptor (AR) and lowering the expression of in the past two decades.5 Androgen ablation therapy remains AR-regulated genes including prostate-specific antigen (PSA), a serologic biomarker up-regulated by androgens (4, 5). The CWR22 tumor model, based on implantation of a hu- man prostate cancer in an athymic nude mouse, progresses in Authors' Affiliations: 1Agricultural Biotechnology Research Center and the same androgen-dependent manner as observed in clinical 2 3 Institute of Biochemistry, Academia Sinica; Institute of Biochemical prostate cancer tumors. Further development from castration- Sciences, National Taiwan University; 4Division of Urology, Department of Surgery, Tri-Service General Hospital, National Defense Medical relapsed CWR22 tumors created subline tumors and a cell line, Center, Taipei, Taiwan 22Rv1, which retain expression of AR and show androgen sti‐ Received 2/6/09; revised 5/25/09; accepted 5/27/09; published OnlineFirst mulation of neoplasia (6, 7). Earlier, we devised the androgen- 8/18/09. induced PSA-luciferase activity in prostate cancer 22Rv1 Grant support: Academia Sinica, National Science and Technology Pro- gram for Agricultural Biotechnology (P.-W. Hsiao), and Academia Sinica cells and a derived stable clone, 103E, as a cell-based assay to postdoctoral fellow training grant (F.-M. Lin). detect any AR modulator effect of herbal extract or compounds The costs of publication of this article were defrayed in part by the payment of (8–10). To determine whether herbal remedies or compounds page charges. This article must therefore be hereby marked advertisement can inhibit prostate cancer growth in orthotopic sites, we in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. constructed a tumor model by orthotopic implantation of lucif- Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). erase-expressing 103E cells into nude mice to serially image Requests for reprints: Pei-Wen Hsiao, Agricultural Biotechnology Research prostate cancer growth. The present study used this biolumines- Center, Academia Sinica, 128 Academia Road, Section 2, Nankang, Taipei cent tumor model to assess the preventive and therapeutic 115, Taiwan. Phone: 886-2-2651-5747; Fax: 886-2-2785-9360; E-mail: effects of a standardized herbal extract against prostate cancer. [email protected]. F 2009 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-09-0298 5 http://www.doh.gov.tw/ www.aacrjournals.org 5435 Clin Cancer Res 2009;15(17) September 1, 2009 Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2009 American Association for Cancer Research. Published OnlineFirst August 18, 2009; DOI: 10.1158/1078-0432.CCR-09-0298 Cancer Therapy: Preclinical Cell culture. LNCaP, PC-3, and 22Rv1 prostate cancer cell lines Translational Relevance were obtained from the American Type Culture Collection. 103E was derived by stable transfection of p5.7kb-PSA-luciferase and pGK-puro Herbal remedies are the most accepted comple- and selected by puromycin resistance and androgen-dependent lucifer- mentary and alternative medicines used among pa- ase expression as described previously (8). LNCaP, 22Rv1, and 103E tients with prostate cancer. Plant extracts contain cells were cultured in RPMI 1640 (Invitrogen) supplemented with various compounds that have been shown to inhibit 10% fetal bovine serum (Hyclone). PC-3 cells were cultured in DMEM many different process of inflammation. It is intrigu- (Hyclone) supplemented with 10% fetal bovine serum. Cultures were maintained in a humidified incubator at 37°C in 5% CO . ing that Wedelia chinensis, an ingredientof anti- 2 Luciferase, proliferation, and flow cytometry analysis of cell cycle and inflammatory herbal medicines, also contains a apoptosis. Luciferase assay and colony formation analyses were done set of compounds that inhibit androgen receptor sig- as described previously (8). LNCaP, 22Rv1, and PC-3 cells were treated naling and in vitro growth of prostate cancer cell lines. with indicated treatments for 48 h. The cell cycle of treated cells was This study examines the use of a herbal extract to examined by flow cytometry after cellular staining with PI and analyzed treat a clinically advanced prostate cancer in animal as described previously (11). For apoptosis analysis, treated cells were models with castration-resistant tumors. The herbal washed in PBS and resuspended in 500 μL staining solution containing extract restrained the prostate cancer tumor growth FITC-Annexin V and PI in HEPES. After incubation at room temperature and decreased angiogenesis without observable for 5 min, cells were analyzed by flow cytometry. In vivo toxicity. This preclinical study thus provides strong studies. Athymic (nu/nu) nude mice (6-7 weeks old) were evidence that W. chinensis is a good candidate for obtained from the National Laboratory Animal Center and housed as described previously (9). All animal work was done in accordance with chemoprevention or complementary medicine. the protocol approved by the Institutional Animal Care and Use Com- mittee, Academia Sinica, Taiwan. 22Rv1 cells (1 × 106) suspended in PBS were mixed 1:1 with Matrigel (BD Biosciences) and subcutaneously We have shown previously that Wedelia chinensis (Astera- inoculated into the right flank of each mouse and allowed to reach a ceae), an oriental herb, contains four compounds capable of tumor diameter of 0.4 to 0.5 cm. For orthotopic implantation, a mouse suppressing androgen activity: indole-3-carboxylaldehyde, we- prostate was exposed with a surgical incision and 103E cells in suspen- 5 μ delolactone, luteolin, and apigenin (8). In vitro experiments sion (3 × 10 in 20 L PBS) were injected into the left side of prostate. Two weeks after implantation, mice were randomly assigned to three showed that a combination of these bioactive compounds in groups (n = 7) that received vehicle control or W. chinensis extract at the ratio present in the herbal extract synergistically inhibited different dosages (4 or 40 mg/kg/d) by gavage in 200 μL PBS containing the clonogenic growth of AR-positive prostate cancer cells. 10% DMSO. Subcutaneous tumors were measured twice per week using Luteolin and apigenin also had additive effects on AR-negative calipers and their volumes were calculated using a standard formula prostate cancer. In continued evaluation of the potential of (width2 × length × 0.5). Bioluminescence intensity of implanted tumors W. chinensis extracts for treating prostate cancer, we prepared was monitored in living mice once a week. Body weight was measured a standardized herbal extract of W. chinensis based on the po- weekly. Mice received 24 or 28 doses, and 24 h after the last dose, they tential anti–prostate cancer compounds, examined its action were sacrificed to harvest plasma and tumors. A portion of each tumor in vivo against prostate cancer models, and determined the was snap-frozen in liquid nitrogen and stored at -80°C until needed for in vivo bioavailability of the hypothesized active compounds. analysis of attained compounds in the tumors, and the remainder was fixed in 10% formalin overnight. Evidence of the antitumor effect of the extract in cultured cell Immunohistochemistry. The paraffin-embedded tumor sections lines and the tumor specimens is also provided. (4 μm thickness) were heat immobilized, deparaffinized using xylene, and rehydrated in a graded series of ethanol with a final wash in dis- Materials and Methods tilled water. Antigen retrieval in Target Retrieval Solution (DAKOCy- tomation) in a Decloaking Chamber (Biocare Medical) was followed Plant material and extract preparation.