IN VITRO AND PRE-CLINICAL EVALUATION OF ANTI-METASTATIC ACTIVITY OF PHLORIDZIN DOCOSAHEXAENOATE (PZ-DHA) VERSUS TRIPLE-NEGATIVE BREAST CANCER

Abstract

The World Health Organization identifies breast cancer as the most common cancer among women worldwide. Triple-negative breast cancer (TNBC) lacks receptors for estrogen (ER), progesterone (PR), and human epidermal growth factor 2 (HER2); therefore, TNBC does not respond to hormone- or HER2-targeted therapy. Even though TNBC responds initially to chemotherapy, these cells eventually develop resistance to chemotherapy and metastasize to distant organs. Therefore, research is continued to identify novel remedies to treat TNBC more effectively. Phloridzin docosahexaenoate (PZ-DHA) is a novel polyphenol fatty acid ester derivative that combines a flavonoid precursor compound known as phloridzin (PZ) with an omega-3 fatty acid known as docosahexaenoic acid (DHA) through an acylation reaction catalyzed by lipase B enzyme extracted from Candida antarctica. The current project was designed to investigate the pharmacokinetic parameters, anti-proliferative, anti-metastatic, and anti-angiogenic activities of PZ-DHA using in vitro, ex-vivo and in vivo models of breast cancer. PZ-DHA improved the cellular uptake of PZ and intracellular stability of DHA. PZ-DHA was absorbed following intraperitoneal administration into Balb/c female mice and underwent phase I and II metabolism. PZ-DHA and its metabolites were readily distributed throughout the body. PZ-DHA was selectively cytotoxic to mammary carcinoma cells, including TNBC cells (MDA-MB-231, MDA-MB-468, 4T1), and showed minimum cytotoxic activity against non-malignant cells (MCF-10A mammary epithelial cells and human dermal fibroblasts). Sub-cytotoxic concentrations of PZ-DHA attenuated the proliferation of MDA-MB-231 cells by arresting the cell cycle at G2/M, and inhibiting protein kinase B (Akt) and mitogen activated protein kinase signaling. PZ-DHA inhibited the migration, invasion, TGF-β-induced small molecular Rho GTPase signaling, and expression of transcription factors (β-catenin, Slug and ZEB1) involved in epithelial-to-mesenchymal transition. Furthermore, PZ-DHA inhibited the expression of MMP2 and increased E-cadherin levels in MDA-MB-231 cells. Intraperitoneal administration of PZ-DHA inhibited the growth and metastasis of orthotopically implanted 4T1 and MDA-MB-231 cells to the lungs of Balb/c and NOD-SCID female mice, respectively, as well as suppressing the expression of Ki67, MMP2 and CD31in primary tumors. Sub-cytotoxic concentrations of PZ-DHA suppressed the proliferation of human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMVEC), and arrested HUVECs at G0/G1. Furthermore, PZ-DHA blocked tube formation by HUVECs and HMVECs in vitro, and vessel sprouting by thoracic aortic sections harvested from Wistar rats. PZ-DHA-induced inhibitory effects on Akt and vascular endothelial growth factor (VEGF165)-stimulated Rho GTPase signaling in HUVECs was also documented. Intraperitoneal administration of PZ-DHA inhibited angiogenesis within VEGF165- and basic fibroblast growth factor (bFGF)-containing Matrigel plugs implanted into Balb/c female mice, confirming anti-angiogenic activity of PZ-DHA. These findings provide strong evidence for PZ-DHA-mediated inhibition of TNBC cell metastasis by inhibiting multiple aspect of the process; therefore, PZ-DHA shows promise as a therapeutic agent to inhibit the progression of TNBC in patients.