Evaluation of Anti-Cancer Effect of Synthetic and Plant-based Inhibitors of Aquaporin 1 in Colon Cancer

Abstract

Efficacy of chemotherapy and biological therapy has been established in the management of colorectal cancer (CRC), however, toxicity and development of resistance remain problematic, highlighting the need for more effective and less toxic therapeutics for this disease. Aquaporin (AQP) 1 is a dual water and ion channel, expression of which is induced during early colon cancer tumorigenesis and correlates with clinical features. AqB013 and AqB050 are bumetanide derived synthetic small molecules, while bacopaside (bac) I and II are triterpene saponins isolated from a medicinal plant Bacopa monnieri. These compounds all exhibit anti-AQP1 activity. This thesis describes the research evaluating AQP1 as a prognostic biomarker in CRC patients, down-streaming signalling molecules for AQP1-mediated migration and invasion, and anti-cancer effect of four compounds with anti-AQP1 activity: AqB013, AqB050, bac I and bac II. In vitro cytotoxic, anti-proliferative, anti-migratory, anti-tubulogenic and pro-apoptotic effects of these compounds were examined on HT-29 colon cancer, 2H-11 and 3B-11 murine, and human umbilical vascular endothelial cell (HUVEC) lines, using MTS viability, crystal violet, annexin V and propidium iodide (PI) staining, circular scratch wound, tube formation and caspase 3/7 apoptosis assays. For bac I and II specifically, synergy of the two compounds were assessed using isobologram method and mechanisms underlying their cytotoxicity was explored by examining their effect on Ca2+ flux and the plasma membrane integrity with Fluo-8 calcium flux and PI assays, respectively. In vivo, efficacy of AqB013 and AqB050 was tested with Matrigel assay and in a murine subcutaneous model of colon cancer. In the small cohort of CRC patients from a single institution, no correlation was found between AQP1 transcript expression and overall survival outcome. Expression level of seven EMT associated genes (IL1RN, BMP7, CALD1, CAV2, ITGA5, MMP9 and VCAN) markedly differed between the two colon cancer cell lines with dissimilar AQP1 expression, however, if and how their gene products are involved in AQP1-mediated tumour cell migration and invasion remain unknown. Anti-proliferative, anti-migratory and anti-tubulogenic effect of AqB013 and/or AqB050 was demonstrated in colon cancer and endothelial cells in vitro. Anti-tumour efficacy of AqB050 partly through inhibition of tumour angiogenesis was confirmed in the animal model, raising the potential therapeutic utility of this compound. For bac I and II, their synergism in inhibiting viability of colon cancer and endothelial cells was shown and the combination treatment impaired proliferation, migration and tube formation of colon cancer and endothelial cells in vitro. Treatment-induced induction of apoptosis and disruption of the plasma membrane integrity were cell line-dependent, while cytosolic Ca2+ flux occurred in all three cell lines examined. Findings suggest both apoptotic and non-apoptotic cell death is involved in combined bac I and II-mediated cytotoxicity and support the idea that transmembrane proteins such as AQP1 is a site of their action. Further research is required to better understand their anti-cancer mechanisms and the in vivo efficacy.