(P03-062-23) Dark Sweet Cherry Extract Enriched in Anthocyanins (ACN) Synergizes With Drug Doxorubicin (DOX) To Inhibit 4T1 Breast Cancer Growth Drug Resistance and Metastasis

Objectives: To assess the effects of ACN, DOX, and combinations in 4T1 cell drug resistance modulation in vitro and to test methods and procedures needed to assess ACN/DOX synergism for metastasis in vivo.

Methods: The resazurin toxicology kit was used to determine IC₅₀ for ACN and DOX, as well as their synergistic combination in 4T1 BC cells. Biomarkers linked to drug resistance, modification of drugs, and detoxification mechanisms were assessed by gene expression by RT-PCR in treated cells. A pilot in vivo study was performed by injecting 4t1-luc cells into mammary fat pads of BALB/C mice. Treatments with ACN (150mg/kg body weight, BW) by gavage every other day, or DOX (2mg/kg BW) twice a week for 6 weeks were performed while tumor growth and metastasis were assessed using the IVIS Lumina XR Real-Time Bioluminescence system to determine luminescence of Region of Interest (ROI). Tumor weights and degree of metastasis were determined upon animal termination.

Results: ACN and DOX downregulated Cyp-3 mRNA levels, which plays a role in degradation of drugs, down to 0.57- and 0.54-fold of control. P-glycoprotein mRNA, linked to drug resistance, was downregulated by ACN and ACN/DOX (down to 0.56- and 0.61-fold). Cell antioxidant defenses enable cancer cells the development of drug resistance. ACN/DOX combination downregulated GPx mRNA (down to 0.61-fold). Preliminary results from the pilot in vivo study indicated the DOX and ACN protection against tumor growth and lung metastasis. Tumor volumes in ACN- and DOX-treated mice were 0.65- and 0.5-fold of control, while tumor weights were 0.65- and 0.3-fold of control. However, DOX-treated mice BW was 13.5% lower than controls. ACN and DOX treatments protected against lung metastasis after tumor surgical removal at similar levels compared to control (1.25, 1.28, an 11.8 ROI increase per week, respectively).

Conclusions: This study showed the potential of ACN against BC growth, drug resistance, and metastasis in stage IV human BC mimicked by highly tumorigenic and invasive 4T1 cells. A full in vivo study is guaranteed to confirm the ACN/DOX anticancer synergistic implications for drug resistance.

Funding Sources: Northwest Cherry Growers, Washington State Fruit Commission