Activities toward Prostate Carcinoma baicalensis with Antiandrogenic and Growth-Inhibitory Characterization of Chemical Constituents in Scutellaria
semanticscholar(2005)
Abstract
Purpose:Botanicalpreparations arewidelyusedbypatientswithprostate cancer.Scutellariabaicalensis, a botanical with a longhistory of medicinal use in China, was a constituent of the herbal mixture PC-SPES, a product that inhibited prostate cancer growth in both laboratory and clinical studies. Due to the difficulties encounteredwhen evaluating the efficacy of complex natural products, we sought to identify active chemical constituents within Scutellaria and determine their mechanismsofaction. Experimental Design and Results:Weusedhigh-performance liquid chromatography to fractionateS. baicalensis and identified four compounds capable of inhibitingprostate cancer cellproliferation; baicalein, wogonin, neobaicalein, and skullcapflavone. Comparisons of the cellular effects inducedby theentireextract versus the four-compoundcombinationproducedcomparable cell cycle changes, levels of growth inhibition, andglobal gene expressionprofiles (r = 0.79). Individual compounds exhibited antiandrogenic activities with reduced expression of the androgen receptor and androgen-regulated genes. In vivo, baicalein (20mg/kg/d p.o.) reduced the growth ofprostate cancer xenografts innudemiceby55%at2weekscomparedwithplaceboanddelayed the average time for tumors to achieve a volume off1,000mm from16 to 47 days (P < 0.001). Conclusions:Most of the anticancer activities of S. baicalensis can be recapitulated with four purified constituents that function in part through inhibition of the androgen receptor signaling pathway.We conclude that clinical studies evaluating the efficacy of these agents in the context of chemoprevention or the treatment of prostate cancer are warranted. Prostate adenocarcinoma represents a major cause of cancerrelated morbidity and mortality. One in six American men will develop prostate cancer and the disease claims >30,000 lives yearly (1). To reduce this tremendous health burden, new approaches have been directed toward extremes of the disease spectrum centering on strategies for prostate cancer prevention and for treating advanced androgen-independent cancers. In this context, environmental and lifestyle factors have been identified that influence prostate carcinogenesis. Nutritional studies indicate that diets rich in soy isoflavones, green tea, lycopene, vitamin E, and selenium are associated with reduced prostate cancer incidence (2–6). Phytoestrogens are thought to contribute to the lower frequency of prostate cancer found in countries with high soy consumption, such as China and Japan (7). In support of these observations, studies in rodent models of prostate cancer show that ingestion of soy isoflavones can delay prostate tumor growth, lower tumor incidence, and decrease the expression of the androgen receptor (AR; refs. 3, 4, 8). Activation of the AR signaling pathway by androgenic ligands plays a permissive and potentially a promoting role in the development and progression of prostate cancer. Antiandrogen therapy, the initial treatment for advanced prostate cancer, is achieved through reductions in circulating androgen levels or inhibition of ligand binding to the AR. Although this approach is initially successful in slowing tumor growth, it is rarely curative due to the emergence of neoplastic cells capable of proliferating in a low-androgen environment (9). Importantly, the AR signaling pathway seems to be active in the vast majority of tumors described as ‘‘androgen independent’’ as shown by the expression of androgenregulated genes (ARG), such as prostate-specific antigen (PSA). Several distinct mechanisms have been identified that seem capable of promoting AR signaling (10). These include overexpression of the AR by amplification, the selection of AR mutations conferring receptor activation through promiscuous ligand binding, and the cross-talk mediated by other growth factor signal transduction pathways. These observations affirm the critical importance of the AR pathway in sustaining prostate www.aacrjournals.org Clin Cancer Res 2005;11(10)May15, 2005 3905 Authors’Affiliations:Divisions of Human Biology and Clinical Research, Fred HutchinsonCancer ResearchCenter and Department of Medicine and Oncology, Veterans Affairs Puget Sound Health Care System, University of Washington, Seattle, Washington Received 9/27/04; revised12/22/04; accepted1/18/05. Grant support: Prostate Cancer Foundation, Damon Runyon Cancer Research Foundation, NIH grant R01DK65204 (P.S. Nelson), MolecularTraining Program in Cancer Research Fellowship T32 CA09437 (M. Bonham), and Chromosome Metabolism and Cancer training grantT32 CA09657 (J. Posakony). The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section1734 solely to indicate this fact. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Requests for reprints: Peter S. Nelson, Division of Human Biology, Fred Hutchinson Cancer Research Center, Mailstop D4-100, 1100 Fairview Avenue North, Seattle, WA 98109-1024. Phone: 206-667-3506; Fax: 206-685-7344; E-mail: pnelson@fhcrc.org. F2005 American Association for Cancer Research. CancerTherapy: Preclinical Research. on May 27, 2013. © 2005 American Association for Cancer clincancerres.aacrjournals.org Downloaded from cancer cell viability and support efforts designed to target the AR for therapeutic gain. Numerous pharmacologic interventions have been developed in attempts to retard prostate tumor growth after the emergence of androgen-independent disease. Several cytotoxic chemotherapeutics have shown substantial palliative benefits but little improvement delaying disease progression or mortality (11). The inability of conventional approaches to reverse the progression of advanced disease coupled with a desire for therapies with fewer perceived toxicities has prompted patients and clinicians to consider unconventional or complementary alternatives. One such complementary therapy that garnered significant interest due to clinical studies reporting measurable responses in advanced prostate cancer consisted of a mixture of herbal extracts marketed under the name PC-SPES (12). Importantly, laboratory assays indicated that one mechanism of growth-inhibitory activity was through modulation of the AR pathway (13). Although PC-SPES administration was generally well tolerated and early-phase clinical trials suggested therapeutic benefits, the difficulties associated with the analyses of poorly standardized and regulated compounds was highlighted through studies demonstrating variable quantities of synthetic drugs in lots of the dispensed PC-SPES capsules (14, 15). Although present in small quantities, several of the identified drugs could have contributed to both beneficial and adverse clinical effects seen with this therapy. However, the possibility that one or more natural constituents of the botanical extracts could exhibit anticancer activities has not been excluded. The objective of this study was to identify and characterize individual chemical compounds derived from specific botanical extracts reportedly used in the PC-SPES formulation that exhibit antiandrogenic and/or growth suppressive effects toward prostate carcinoma. Materials andMethods High-performance liquid chromatography fractionation of Scutellaria baicalensis. High-performance liquid chromatography (HPLC) analyses were done on HP1050 and HP1100 HPLC systems using a Discovery C18 (25 cm 10 mm, 5 Am, Supelco, Bellefonte, PA) semipreparative column or a Supelcosil LC-18 column (25 cm 2.1 mm, 3 Am, Supelco) analytic column. Ethanolic extract (1 mL) of S. baicalensis was injected in aliquots (100 AL each, 10 separate injections) onto the semipreparative column, which was eluted using gradient elution method A (Supplementary Material 1) and the absorbance at 270 nm was monitored. The eluate was collected in 30-second fractions and the solvent was removed from these fractions by evaporation. The dried fractions were resuspended in 1 mL DMSO and then were screened for growth inhibition of LNCaP cells by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. To assess the purity of the active fractions, aliquots (5 AL) were injected onto the analytic column, which was eluted using isocratic elution method B with monitoring at 270 nm. Methods for purifying larger amounts of compounds in the active fractions of Scutellaria are detailed in Supplementary Material 1. General compound identification and purification methods. Lowresolution mass spectrometry (MS; electrospray ionization) was done on a Bruker Esquire ion trap mass spectrometer or a HP Series 1100 MSD. High-resolution MS was done using a Bruker APEX III 47e Fourier transform (ion cyclotron resonance) mass spectrometer. H nuclear magnetic resonance spectra were obtained on Tecmag or Bruker Avance 300 MHz spectrometer and H chemical shifts are reported in ppm (y). Flash column chromatography was done using silica gel (grade 9385, 230-400 mesh, Merck, Whitehouse Station, NJ). Analytic TLC was done using silica gel Analtech GF plates (0.25 mm) and products were visualized using UV light. Unless otherwise noted, reagents were purchased from Sigma-Aldrich Corp. (St. Louis, MO) or Lancaster Synthesis (Windham, NH). Solvents were ACS reagent grade or better, and anhydrous solvents were used as received unless otherwise indicated. Baicalin and baicalein were obtained from Sigma-Aldrich. Wogonin was obtained from Wako Pure Chemicals Industries Ltd. (Richmond, VA) Detailed methods for compound characterization using HPLC-MS are described in Supplementary Material 1. Cell lines and tissue culture. LNCaP and PC-3 cell lines were obtained from the American Type Culture Collection (Manassas, VA) and propagated according to the instructions of the supplier. Cells were grown in 10% fetal bovine serum for a
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