comparative preventive efficacy of select chinese herbs in
TRANSCRIPT
Comparative Preventive Efficacy of Select Chinese Herbsin Breast Carcinoma Derived Isogenic Cells with
Modulated Estrogen Receptor Functions
Telang, N1, Li G2, Katdare, M 3,4, Sepkovic, DW 5, Bradlow, HL 5 and Wong, GYC2, 6
1 Cancer Prevention Research Program, Palindrome Liaisons,New Jersey
2 American Foundation for Chinese Medicine, New York3 Hampton University Skin of Color Research Institute, Virginia4 Department of Dermatology and Leroy T Canoles Jr. Cancer
Research Center, Eastern Virginia Medical School, Virginia5 Hackensack University Medical Center, New Jersey
6 Department of Integrative Medicine, Beth Israel Medical Center,New York
Abstract
Background: Human mammary carcinoma derivedestrogen receptor positive (ER+) MCF-7 cells dependon 17ß-estradiol (E2) for cellular proliferation in vitroand for tumor development in vivo. Traditionally, ERfunctions as a ligand activated nuclear transcriptionfactor with E2 as its physiological ligand, and limitedavailability of E2 modulates ER functions. We haverecently demonstrated preventive efficacy of selectedChinese nutritional herbs in this MCF-7 cell culturemodel. The present study screened additional Chinesenutritional herbs for their preferential preventiveefficacy on MCF-7 cells with modulated ER functions.Methods: MCF-7 cells were maintained in mediumsupplemented with either 0.7% serum, <1 nM E2, or0.7% serum+20 nM E2.This approach, whilepreserving the MCF-7 genotype, provides isogeniccells with either non-functional ER (ER-NF) or
Study Rationale - I
• Estrogen receptor positive (ER+) clinical breast cancer responds toendocrine therapy involving the use of receptor antagonists and selectiveinhibitors of estradiol biosynthesis (1,2).
• Long-term treatment with these agents is associated with acquired tumor
resistance and systemic toxicity, impacting on patient compliance (3). • Chinese nutritional herbs have been widely used in traditional Chinese
medicine for a variety of health conditions, including cancer in women (4,5). • Naturally occurring herbal preparations may exhibit acceptable toxicity
profile and interact with conventional endocrine therapy to reduce toxicityand enhance efficacy of pharmacological therapeutic agents (4-7).
Experimental Model• Estrogen receptor positive (ER+) MCF-7 cell line represents a human tissue
derived preclinical cell culture model for hormone responsive clinical breastcancer (8).
• MCF-7 cells depend on 17ß-estradiol (E2 ) for cell proliferation in vitro and
for tumor development in vivo (1,3,8). • ER functions as a ligand activated nuclear transcription factor with E2 as its
physiological ligand to induce the expression of E2 responsive genes and topromote cellular proliferation and tumorigenic growth via complex signalingcascades (9,10).
• Sub-physiological concentrations of E2, the ER ligand, render ER astransiently non-functional receptor (11-13).
• MCF-7 cells adapted to grow in chemically defined, serum-depleted culture
condition retain their responsiveness to E2, and respond to growth inhibitoryeffects of several mechanistically distinct herbal extracts (11-14).
Cell Culture Model and Herbal Extracts
Model: MCF-7 cells adapted for growth in a chemically defined serum-depletedculture medium to isolate isogenic phenotypes with functional estrogenreceptor (ER-F) and non-functional estrogen receptor (ER-NF). Herbal Extracts: Non-fractionated aqueous extracts from select Chinesenutritional herbs.• 20 g of herbs boiled in 200 ml of distilled water to reduce the volume to 100
ml (Extract # 1). Residue from this extract boiled in 100 ml of distilled waterto reduce the volume to 50 ml (Extract # 2).
• Two extracts (150 ml) combined and boiled to reduce the volume to 25 ml.• The combined extracts centrifuged (5,000 rpm, at room temperature) to
collect the supernatant (20 ml). This aqueous supernatant served as thestock solution (100%). Working solutions diluted with the culture medium to2%, 1% 0.5%, 0.02%, 0.01% and 0.05% used to determine IC50 .
Mechanistic Biomarkers and Quantitative End Points
_______________________________________________________________Biomarker End Point_______________________________________________________________Cytostatic growth arrest Number of viable cellsAnchorage independent growth Number of anchorage independent coloniesCell cycle progression G1: S+G2/M ratioCellular apoptosis % sub G0Cellular metabolism 2-OHE1: 16α-OHE1 ratioof estradiol E3: 16α-OHE1 ratio_______________________________________________________________
Figure 1Effect of Serum Depletion on the Growth of Human Mammary
Carcinoma MCF-7 Cells Viable Cell Number (x105) Initial Seeding 10% 7.5% 5.0% 2.5% 1.0% Density Serum Concentration
Figure 2Dose Response of 17ß-estradiol (E2) on Human Mammary
Carcinoma MCF-7 Cells!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!Viable Cell Number (x105) Initial Seeding 0.7% 1 nM 5 nM 10 nM 20 nM Density Serum 0.7% Serum + E2
Chinese Nutritional Herbs
_______________________________________________________________Botanical Name Abbreviation_______________________________________________________________Aconitum carmichaeli ACAngelica sinensis ASCibotium barometz CBCornus officinalis COCuscuta sinensis CSDipsacus asperoides DAEpimedium grandiflorum EGEucommia ulmoides EULigusticum chuanxiong LCLigustrum lucidum LLLycium barbarum bark LBBLycium barbarum fruit LBFMorinda officinalis MOPsoralia corylifolia PC__________________________________________________________________
Table 1Ranking by IC50 on Estrogen Receptor Functional ( ER-F)
Phenotype_________________________________________________________________________________Chinese Herbal Rank OrderExtract (IC50) % a_________________________________________________________________________________LBB 0.001CO 0.07 EU 0.09 DA 0.13PC 0.26LL 0.28LBF 0.39EG 0.44CB 0.52CS 0.57
Table 2Ranking by IC50 on Estrogen Receptor Non-functional
(ER-NF) Phenotype_______________________________________________________________________Chinese Herbal Rank Order Extract (IC50 ) % a _______________________________________________________________________LBB 0.01CO 0.03DA 0.19EU 0.23LL 0.30PC 0.40EG 0.46CS 0.60LC 0.60AS 0.70LBF 0.90AC 1.78CB 2.72MO 5.13
_______________________________________________________________a determined after a 7 day treatment of cells cultured in a medium supplemented with 0.7% serum.
Figure 3Inhibition of Anchorage Independent Growth in Human Mammary
Carcinoma MCF-7 Cells by Select Chinese Herbal Extracts
AnchorageIndependent (AI) Colony Number
Figure 4Inhibition of Cell Cycle Progression in Human Mammary Carcinoma
MCF-7 Cells by Select Chinese Herbal Extracts
G1:S+G2/M Ratio
20 nM 0.05% 0.1%
Figure 5Induction of Cellular Apoptosis in Human Mammary Carcinoma
MCF-7 Cells by Select Chinese Herbal Extracts
% SubG0
Study Rationale - II
• Oxidative metabolism of 17β-estradiol (E2) impacts on carcinogenesis ofendocrine responsive target organs (15-17).
• E2 metabolites exhibit pleotropic growth modulatory effects on initiated
mammary epithelial cells as well as breast carcinoma derived cells (18-20). • Altered E2 metabolism provides an endocrine biomarker for carcinogenic
risk and cancer prevention/therapy (11,14,18).
Metabolic Pathways of Estrogen Metabolism
Figure 6Modulation of Cellular Metabolism of 17ß-estradiol (E2) in
Human Mammary Carcinoma MCF-7 Cells by Select ChineseHerbal Extracts
2-OHE1: 16α-OHE1 Ratio
20 nM 0.05% 0.1%
Figure 7 Modulation of Cellular Metabolism of 17ß-estradiol (E2) in Human
Mammary Carcinoma MCF-7 Cells by Select Chinese HerbalExtracts
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!E3: 16α-OHE1 Ratio 20 nM 0.05% 0.1% 0.8% E2 E2+LBB E2+CO E2+EG
Study Outcome-I
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!Experimental Model• Sequential decrease of serum concentration in the culture medium
facilitated effective growth adaptation of estrogen receptor positive (ER+ )MCF-7 cells in a medium supplemented with 0.7% serum (E2 <1 nM). • MCF-7 cells maintained in a medium supplemented with 0.7% serum
exhibited progressive dose dependent growth in the presence ofphysiologically relevant concentrations of E2.
• Long-term maintenance of MCF-7 cells in a medium supplemented with
0.7% serum selected a proliferative sub-population with transiently non-functional ER (ER-NF) due to limited availability of E2.
• Maintenance of MCF-7 cells in medium supplemented with 0.7% serum +20
nM E2 and with 0.7% serum < 1 nM E2 selected isogenic cells with ER-F and ER-NF phenotypes, respectively.!"
Study Outcome-II
Efficacy of Chinese Herbal Extracts • Comparative dose response experiments with non-fractionated
aqueous extracts from Chinese nutritional herbs facilitated rankorder for their IC50 concentration on ER functional (ER-F)phenotype and ER non-functional (ER-NF) phenotype.
• Preferential Efficacy: ER-F > ER-NF: AC, CB, DA, EU, LBB, LBF, MO, PC ER-NF > ER-F: AS, CO ER-F = ER-NF: CS, EG, LC, LL • Representative herbal extracts LBB, CO and EG at their
respective maximum cytostatic concentrations, inhibitedanchorage independent growth, down-regulated cell cycleprogression through G2/M arrest and cellular apoptosis or G1arrest, and altered cellular metabolism of estradiol that favoredthe formation of the anti-proliferative 2-OHE1 and E3
Conclusions
• Isogenic phenotypes from MCF-7 cells with modulated ER functionsvalidate a facile cell culture approach for mechanism based evaluation ofChinese nutritional herbs having multiple functions.
• Rank order of herbal extracts preferentially efficacious in cells with ER-Fand ER-NF prioritizes promising herbs that may be effective in ER+ and/orER- clinical breast cancer.
Acknowledgements and Dedication
Major funding for this study was provided by philanthropic contributions to theAmerican Foundation for Chinese Medicine by• Peter Cheney• Suzanne Hoyt • The family of Hakan and Marie Ledin• The family of Daniel and Kathleen Mezzalingua• The Issac and Laura Perlmutter Fund This study is dedicated to the memory of Laurie Mezzalingua (1968-2009).Laurie fought gallantly against her breast cancer from 1993 to July 4, 2009.During that period she also selflessly and generously devoted herself to helpingmany others suffering from breast cancer.
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