Abstract 3638: Combination of metformin and metronomic liposomal doxorubicin exerts a robust anticancer effect in triple negative breast cancer by inhibiting breast cancer stem cells & the Wnt/beta-catenin pathway
Authors: Banerjee Indranil , Das Subhayan , Das Chandan Kanta , Jena Bikash Ch. , Bharadwaj Deblina , Pradhan Anjan K. , Das Swadesh K. , Fisher Paul B. , Mandal Mahitosh 
Published: August 2019

Abstract
Introduction: The high death rate due to breast cancer is often attributed to aggressive triple negative breast cancer (TNBC). We used the combination of metronomic pegylated liposomal doxorubicin (PLD), and metformin {selectively inhibits cancer stem cells (CSCs)} for effective management of TNBC xenografts generated in mice. Down-regulation of β-catenin is responsible for the efficacy of the combination of metronomic PLD and metformin in treating aggressive TNBC.
Methodology: PLD was prepared using the ammonium sulfate gradient method. Morphology, particle size, zeta potential measurement, drug entrapment efficiency, and drug release studies were performed to characterize the prepared liposomal formulation. Cell culture studies were performed on MDA-MB-231 and MDA-MB-468 cell lines (i.e., TNBC cells). MTT assays, mammosphere assays, apoptosis assays, quantitative real-time polymerase chain reactions (qPCR), western blotting experiments were performed. In vivo anticancer efficacy of the combination therapy {antidiabetic dosing of metformin: 150 mg/kg body weight every day, and metronomic dosing of PLD: 1 mg/kg body weight with respect to doxorubicin (Dox) every other day for 3 weeks} was verified in an MDA-MB-231 xenograft model.
Results: Monodisperse PLD having dimensions less than 100 nm was successfully prepared (confirmed by AFM, TEM, and DLS). Zeta potential measurement showed that PLD was negatively charged (- 12.1 ± 2.3 mV, n = 3). Encapsulation efficiency and drug release were found to be satisfactory. IC50 (i.e., 50 % inhibitory concentration) of PLD was found to be in nanomolar range with respect to Dox in TNBC cell lines. However, a combination of metformin (500 µM) and PLD resulted in a leftward shift of the concentration-response curve such that the IC50 value of PLD was further reduced in the nanomolar range with respect to Dox. A similar trend was seen in the apoptosis assay. Metformin works together with PLD to reduce non-stem cancer cells, CSCs, and stem cell markers as indicated by the reduction in the number of mammospheres, qPCR results, and western blotting experiments. We also found attenuation of β-catenin in TNBC cells by the combination treatment regimen, as suggested by qPCR and western blotting. Some of the in vitro observations were also confirmed in the MDA-MB-231 xenograft model.
Conclusion: Our experiments demonstrate that down-regulation of β-catenin is responsible for the strong anticancer effects of the combination of metformin and PLD in vitro and in vivo. Further studies are warranted with this combination regimen in different cancer types as metformin is an approved anti-diabetic drug with an acceptable safety profile, and metronomic chemotherapy is gaining popularity in treating different cancer types.
Citation Format: Indranil Banerjee, Subhayan Das, Chandan Kanta Das, Bikash Ch. Jena, Deblina Bharadwaj, Anjan K. Pradhan, Swadesh K. Das, Paul B. Fisher, Mahitosh Mandal. Combination of metformin and metronomic liposomal doxorubicin exerts a robust anticancer effect in triple negative breast cancer by inhibiting breast cancer stem cells & the Wnt/beta-catenin pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3638.