1. Efficacy of Tabebuia avellandae Extract on a Cell Culture Model for Triple Negative Breast Cancer
Nitin T. Telang1, Hareesh B. Nair2 and George Y.C. Wong3, 4
1Cancer Prevention Research Program,
Palindrome Liaisons Consultants, Montvale, NJ
2Texas Biomedical Research Institute, San Antonio, TX
3American Foundation for Chinese Medicine, New York, NY
4Department of Integrative Medicine,
Mount Sinai Beth Israel Medical Center, New York, NY
San Antonio Breast Cancer Symposium (SABCS-2014)

2. Abstract
Background: The triple negative breast cancer (TNBC), a molecular subtype of clinical breast cancer consisting of epithelial cells that lack estrogen receptor-α (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER-2) expression, is non-responsive to either endocrine therapy or HER-2 targeted therapy. Chemotherapy is frequently associated with long-term systemic toxicity, acquired tumor resistance and resultant compromised treatment efficacy. These aspects emphasize a need to identify efficacious non-toxic agents for secondary prevention/therapy of TNBC. Non-fractionated aqueous extract from the inner bark of the Tabebuia avellandae (TA) tree found in the Amazon rainforest, available as a dietary supplement under the name of Taheebo or Pau d’arco, has documented efficacy in a cell culture model for the Luminal A breast cancer subtype, as well as for several other cancers. Present study examines the inhibitory effects of the TA extract, and identifies possible mechanistic targets for its efficacy in a cell culture model for TNBC. Nutritional Supplement, Experimental Model and Biomarkers: Lyophilized powder of non-fractionated TA extract from Taheebo Japan, Osaka, Japan, provides the source material for the study. The ER-/PR-/HER-2- MDA-MB-231 cell line represents the cell culture model for TNBC. Anchorage dependent growth, cell cycle progression, status of cell cycle regulatory proteins and anchorage independent colony formation, represent the quantitative biomarkers for efficacy. Results: Relative to the non-tumorigenic 184-B5 human mammary epithelial cells, the tumor derived MDA-MB-231 cells exhibited decreased population doubling time, increased saturation density, decreased G1: S+G2/M ratio and increased S+G2/M: Sub G0 ratio, indicating loss of homeostatic growth control. Additionally, unlike 184-B5 cells, MDA-MB-231 cells exhibited increased anchorage independent growth in vitro and tumor development in vivo, indicating enhanced cancer risk. Treatment of MDA-MB-231 cells with TA resulted in a substantial dose dependent cytostatic growth arrest (IC50:1.0%; IC90: 2.5%). Cell cycle analysis of TA treated cells revealed G1 arrest, leading to a progressive dose dependent increase in the G1: S+G2/M ratio. Mechanistically, TA decreased Cyclin D1 expression and attenuated RB phosphorylation, predicting Cyclin D-CDK4-pRB pathway as a molecular target for efficacy. Furthermore, TA effectively inhibited anchorage independent growth in a dose dependent manner. Conclusions: Present data demonstrated pronounced efficacy of TA as a naturally occurring nutritional substance in a cell culture model for TNBC, and validated TA as a promising non-toxic natural agent for secondary prevention/therapy of clinical TNBC.

3. Study Rationale - I
The triple negative molecular subtype of clinical breast cancer (TNBC) lacks the expressions of ER-α, PR and HER-2 (1).
This molecular subtype is resistant to conventional endocrine and HER-2 targeted therapy (2-4).
Current treatment options for TNBC include conventional chemotherapy and Poly (ADP-ribose) polymerase (PARP) inhibitors (5, 6).
These treatment options are associated with long-term systemic toxicity and acquired resistance impacting therapeutic efficacy (3, 4, 7).

4. Study Rationale - II
Natural products with minimal systemic toxicity may provide a testable alternative and/or adjuvant to conventional chemotherapy (8).
Non-fractionated extract of Tabebuia avellandae (TA) has documented anti-bacterial, anti-angiogenic and anti-cancer activities (9,10-12).
The bioactive agents in TA extract include naphthoquinone derivatives and flavonoids (12,13).
Non-fractionated aqueous extracts from several mechanistically distinct Chinese nutritional herbs and from TA, have documented growth inhibitory efficacy in a cell culture model for the Luminal A molecular subtype of clinical breast cancer (14-17).

5. Experimental Model and Test Compound
The human mammary carcinoma derived MDA-MB-231 cell line lacks the expressions of ER-α, PR and HER-2 and therefore, represents a preclinical model for the triple negative molecular subtype of clinical breast cancer (18,19).
Tumor suppressive function of Retinoblastoma (RB) gene via the Cyclin D1 - CDK 4/6 - pRB pathway is compromised in therapy resistant basal-like and triple negative molecular subtypes of clinical breast cancer (20-22).
Lyophilized powder of non-fractionated Tabebuia avelledae (TA) extract was provided by Taheebo Japan, Osaka, Japan.

6. Mechanistic End Point Biomarkers
Anchorage dependent growth (viable cell number)
Anchorage independent growth (colony number)
Cell cycle progression (G1: S+G2/M ratio)
Cell cycle regulatory proteins (RB pathway)
Cellular Apoptosis (Caspase activity)

7. Status of Homeostatic Growth Control and Cancer Risk in MDA-MB-231 Cells
__________________________________________________________________________
End Point Cell Line
Biomarker __________________________________________
184-B MDA-MB-231
Population Doubling (hr.)
Saturation density (x105) ± ±2.3
G1: S+G2/M ± ±0.3
S+G2/M: Sub G ± ±3.2
Anchorage independent
Colonies / /24
Tumor Development / /10
Tumor Latency weeks weeks
______________________________________________________________________

8. Dose Response of Tabebuia avellandae (TA) Extract on MDA-MB-231 Cells
Viable Cell Number (x105)
Initial Control TA (%)
Seeding
Control: 40.7 fold increase
IC50: 1.0%; IC90: 2.5%

9. Effect of Tabebuia avellandae (TA) Extract on Anchorage Independent Growth
Number of Anchorage Independent Colonies
Control TA (%)

10. Effect of Tabebuia avellandae (TA) Extract on Cell cycle Progression
G1: S+G2/M Ratio Control TA (%)

11. Effect of Tabebuia avellandae (TA) Extract on the Retinoblastoma (RB) Pathway
Control TA (%)
Actin
Cyclin D1
CDK4
Total RB
pRB

12. Effect of Tabebuia avellandae (TA) Extract on Cellular Apoptosis
Caspase Activity (Caspase 3/7 Relative Luminescent Units, RLU)
Control TA (%)

13. Summary of Results TNBC Model:
Loss of homeostatic growth control (hyper-proliferation, aberrant cell cycle progression and down-regulated cellular apoptosis).
Increased cancer risk (Increased anchorage independent growth in vitro and tumor development in vivo).
Effects of Tabebuia avellandae (TA) Extract:
Reduction in the number of anchorage independent colonies.
Increase in G1: S+G2/M ratio.
Modulated expressions of Cyclin D1, CDK4 and pRB.
Increase in cellular apoptosis associated Caspase 3/7 activity.

14. Conclusions
The present data demonstrate growth inhibitory effects of non-fractionated aqueous extract from Tababuia avellandae (TA) on a model for triple negative breast cancer (TNBC), and identifies possible mechanistic leads for its efficacy.
These data validate a clinically relevant human mammary carcinoma derived cell culture model for TNBC to prioritize efficacious natural products for prevention/therapy.

15. Acknowledgements
Major funding for this research is provided by the following philanthropic contributions to the American Foundation for Chinese Medicine.
The Saint Agatha Foundation
The Sophie Stenbeck Family Foundation

16. Dedication
This study is dedicated to the memory of Laurie Mezzalingua ( ). During this period Laurie selflessly and generously devoted herself to helping many others suffering from breast cancer.

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