SABCS Abstract Number 850753

Chinese Herb Fructus Cornii Exhibits Efficacy in a Cell CultureModel for Hormone Responsive Breast Cancer

Nitin Telang1, Daniel Sepkovic2, H. Leon Bradlow2George Y. C. Wong3, 4

1Cancer Prevention Research Program,

Palindrome Liaisons, Montvale, NJ.2Hackensack University Medical Center, Hackensack, NJ,

3American Foundation for Chinese Medicine, NY4Department of Integrative Medicine, Beth Israel Medical Center, NY

Study Rationale – I

• Selective estrogen receptor modulators (SERM), independently or incombination with chemotherapeutic drugs represent conventional strategyfor management of ER+ clinical breast cancer [Lippman Breast CancerRes. Treat. 3: 117-127, 1983].

• Long-term use of chemo-endocrine therapy is associated with systemic

toxicity, acquired tumor resistance and resultant compromised patientcompliance [Early Breast Cancer Trialist’s Group Lancet 365: 1687-1717, 2005].

• Toxicity issues with chemo-endocrine therapy has rationalized the use of

herbal medicines independently or in combination with clinical therapy toenhance efficacy and reduce toxicity [Tindle et al Alt. Ther. Health Med.11: 42-49, 2005; Molassiotis et al Support Care Cancer 14: 260-267,2006].

Study Rationale - II

• Preclinical investigations on human breast carcinoma cell lines may facilitateprioritization of efficacious lead compounds and identify clinicallytranslatable mechanistic targets.

• Non-fractionated extracts from medicinal herbs have documented growth

inhibitory activity via distinct mechanisms in ER+ MCF-7 cells [Li et al Nutr.& Cancer 61: 408-414, 2009; Mukherjee et al Int. J. Mol. Med. 24: 253-260, 2009].

• Chinese nutritional herb Fructus Cornii represents a major component in

herbal mixtures for prevention of chemo-radiotherapy associated toxicity inbreast cancer [Zhou et al Aust. J. Acupunc. Chinese Med. 2: 30-32,2007].

• Combinatorial efficacy of herbal lead compounds with conventional chemo-

endocrine therapeutics should be confirmed in preclinical animal modelsand clinical trials for ER+ breast cancer.

Study Objectives

• To characterize the growth properties of ER+ human mammary carcinomaMCF-7 cells adapted to grow in chemically defined serum depleted medium.

• To determine the growth modulating effects of the non-fractionated aqueous

extract from the Chinese herb Fructus Cornii (FC) on ER+ human mammarycarcinoma MCF-7 cells.

• To identify the possible mechanistic leads for the efficacy of FC.

Mechanistic Biomarkers

• Population doubling time • Saturation density • Anchorage dependent growth • Anchorage independent growth • 2-hydroxyestrone : 16α-hydroxyestrone ratio (Endocrine biomarker)

Experimental Methods - I

Aqueous Extract of Fructus Cornii • 20g of FC were boiled in 200 ml of distilled water till the volume was

reduced to 100 ml (aqueous extract # 1).• Residual FC was boiled in 100 ml of distilled water till the volume was

reduced to 50 ml (aqueous extract # 2).• Extract # 1(100 ml) and extract # 2 (50 ml) were combined and boiled till the

volume was reduced to 25 ml.• Combined extract was centrifuged at 5000 rpm at room temperature for 10

min.• The supernatant (20ml) was collected and stored at -20 o C in 5ml aliquots.

Experimental Methods - II

Estradiol Metabolism • MCF-7 cell cultures were treated with the maximum cytostatic dose of FC in

the presence of 20 nM 17ß-estradiol (E2) for 48 hrs, and the mediumcollected for metabolite analysis.

• The samples were incubated in sodium acetate buffer containing ß-

glucuronidase at 370 C for 24 hours to de-conjugate the steroids.Deuterated E2 was added as internal standard and the media wereextracted with chloroform.

• Extracted samples were derivatized in the presence of 50 µl dry pyridine

and 10µl bis (trimethylsilyl) trifluoroacetamide (BSTFA).

Experimental Methods - III

GC-MS Assay

• Select estradiol metabolites were analyzed on an Agilent 6980N gaschromatograph equipped with an Agilent 5973 mass selective detector andan HP G2589 MSD chemstation, and separated on an HP Ultra- 2 capillarycolumn.

• Individual steroids were identified using selected mass ion monitoring and

GC elution times, and were quantified using a five-point calibration curve(Range: 0 to 50 ng).

• The data were expressed as ng metabolite/ 106 cells.

Endocrine Responsiveness of ER+ Human Mammary CarcinomaMCF-7 Cells ________________________________________________________

Treatment Concentration Biomarker ___________________________________ Population Saturation AI Doubling (hr) a Density (x105) b Colonies c________________________________________________________Serum 0.7% 33.0 11.8±0.9 16.7±3.4E2 20 nM 28.5 17.9±1.6 37.2±2.1TAM 20 nM 35.9 3.9±0.6 7.7±0.4________________________________________________________a determined from exponential growth phase. b viable cell number at day 7 post-seeding of 1x105 cells. Mean ± SD,

n=3 per treatment group.

Serum versus E2 p <0.05.

Serum versus TAM p <0.05.

c Anchorage Independent (AI) colonies at day 21 post-seeding of 1,000 cells. Mean ± SD, n=6 per treatment group.

Serum versus E2 p<0.05.

Serum versus TAM p<0.05.

Cytostatic Growth Arrest of ER+ Human Mammary Carcinoma MCF-7 Cellsby Fructus Cornii (FC)

Viable cell number (x105) ISD Serum E2 E2 + FC 0.7% 20 nM 0.01% 0.05% 0.1% 0.5%

Viable cell number at day 7 post-seeding.

E2 versus E2+FC 0.05% p <0.05. E2 versus E2+FC 0.1% p <0.05.

Inhibition of Anchorage independent Growth in ER+ HumanMammary Carcinoma MCF-7 Cells by Fructus Cornii (FC)

Number of AI colonies Serum E2 E2 + FC 0.7% 20 nM 0.01% 0.05% 0.1%

AI colonies at day 21 post-seeding.

E2 versus E2+FC p <0.05.

Growth Modulation in ER+ Human Mammary Carcinoma MCF-7Cells by Estradiol Metabolites

Viable cell number (x105) ISD E2 16α-OHE1 2-OHE1 20 nM 20 nM 20 nM

Viable cell number at day 7 post-seeding. E2 versus 16α-OHE1 p=0.01. E2 versus 2-OHE1 p=0.04.

Effect of Fructus Cornii (FC) on Estradiol Metabolism in ER+ HumanMammary Carcinoma MCF-7 Cells

________________________________________________________Treatment concentration E2 metabolite (ng/106 cells) __________________________________ E1 2-OHE1 16α-OHE1 E3_______________________________________________________________E2 20 nM 7.8±0.6 0.9±0.2 2.2±0.1 0.22±0.03FC 0.1% 14.4±1.9 5.5±1.3 0.8±0.2 0.20±0.04________________________________________________________Mean ± SD, n=3 per treatment group.E1: E2 versus FC p=0.03. 2-OHE1: E2 versus FC p=0.01. 16α-OHE1: E2 versus FC p=0.04.

Up-regulation of Endocrine Biomarker in ER+ Human MammaryCarcinoma MCF-7 Cells by Fructus Cornii (FC)

2-OHE1: 16α-OHE1 Ratio E2 FC

20 nM 0.1% E2 versus FC p <0.001

Study Conclusions

• ER+ human mammary carcinoma MCF-7 cells adapted for growth in achemically defined serum depleted culture medium retain theirresponsiveness to physiologically relevant levels of E2.

• FC produces dose dependent cytostatic growth arrest and reduction in

anchorage independent colonies in E2 stimulated MCF-7 cells. • FC at the maximum cytostatic concentration induces increased 2-OHE1 and

decreased 16α-OHE1 formation, resulting in about a 13 fold increase in the2-OHE1: 16α-OHE1 ratio over the control.

• These data identify a mechanistic lead for the efficacy of FC and thereby,

validate a human cell culture based approach to prioritize herbal medicinalproducts for prevention/therapy of ER+ clinical breast cancer.

Acknowledgements

Major funding for this research was provided by philanthropic contributionsto American Foundation for Chinese Medicine from the family of Daniel andKathleen Mezzalingua, the family of Hakan and Marie Ledin and Issac andLaura Perlmutter Fund.

Dedication

This research 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 time period, she also selflessly and generously devotedherself to helping many others suffering from breast cancer.