Combined pharmacological administration of AQP1 ion channel blocker AqB011 and water channel blocker Bacopaside II amplifies inhibition of colon cancer cell migration

Abstract

Aquaporin-1 (AQP1) has been proposed as a dual water and cation channel that when upregulated in cancers enhances cell migration rates; however, the mechanism remains unknown. Previous work identified AqB011 as an inhibitor of the gated human AQP1 cation conductance, and bacopaside II as a blocker of AQP1 water pores. In two colorectal adenocarcinoma cell lines, high levels of AQP1 transcript were confirmed in HT29, and low levels in SW480 cells, by quantitative PCR (polymerase chain reaction). Comparable differences in membrane AQP1 protein levels were demonstrated by immunofluorescence imaging. Migration rates were quantified using circular wound closure assays and live-cell tracking. AqB011 and bacopaside II, applied in combination, produced greater inhibitory effects on cell migration than did either agent alone. The high efficacy of AqB011 alone and in combination with bacopaside II in slowing HT29 cell motility correlated with abundant membrane localization of AQP1 protein. In SW480, neither agent alone was effective in blocking cell motility; however, combined application did cause inhibition of motility, consistent with low levels of membrane AQP1 expression. Bacopaside alone or combined with AqB011 also significantly impaired lamellipodial formation in both cell lines. Knockdown of AQP1 with siRNA (confirmed by quantitative PCR) reduced the effectiveness of the combined inhibitors, confirming AQP1 as a target of action. Invasiveness measured using transwell filters layered with extracellular matrix in both cell lines was inhibited by AqB011, with a greater potency in HT29 than SW480. A side effect of bacopaside II at high doses was a potentiation of invasiveness, that was reversed by AqB011. Results here are the first to demonstrate that combined block of the AQP1 ion channel and water pores is more potent in impairing motility across diverse classes of colon cancer cells than single agents alone.

Important players in carcinogenesis as potential targets in cancer therapy: an update

“…The attenuation of HT-29 cell migration in monotherapy has been demonstrated as well as a significant improvement of this process in combined therapy. Moreover, a decreased invasiveness of cells was observed after bacopaside II treatment [191]. A similar effect was demonstrated in colon cancer cells (HCT116, SW480) after AQP-5 knock-down with specific shRNA [192] .…”

Combined Systematic Review and Transcriptomic Analyses of Mammalian Aquaporin Classes 1 to 10 as Biomarkers and Prognostic Indicators in Diverse Cancers

“…Effects on motility could arise from AQP1-associated effects on actin organization via RhoA and Rac signaling pathways [ 123 ]. Alternatively, the role of AQP1 in colon cancer might depend on a dual water and ion channel function suggested to promote lamellipodial extension and cell migration [ 5 , 12 , 123 ]. AQP5 similarly has been proposed as a prognostic biomarker for colorectal cancer, with AQP5 levels found to be proportional to numbers of circulating tumors cells [ 124 , 125 , 126 ] and risk of liver metastases [ 126 ].…”

“…This reliance on an AQP subtype indicates that the tumorigenic roles are unlikely to stem simply from increasing water channel activity and osmotic water flux, but must be exploiting features such as substrate permeability, mechanisms of regulation, subcellular localization or other properties that differ between subtypes. AQPs have been proposed to promote cancer metastasis by facilitating tumor cell migration [ 6 , 7 , 12 ], but a broader range of possible functions is likely, given the expanding understanding of their diverse roles in normal cell physiology.…”

“…AQPs 11 and 12 are more distantly related to other mammalian AQPs, based on amino acid sequence; functions and regulation remain to be fully defined [ 19 ]. Diverse contributions of AQP classes could augment pro-cancer conditions by enhancing signaling via nitric oxides or hydrogen peroxide [ 20 , 21 , 22 , 23 ], facilitating gas exchange of O 2 and CO 2 [ 24 , 25 ], promoting cell cycle progression [ 26 ], providing metabolic support [ 27 , 28 ], mediating dual ion and water fluxes for localized control of process extension [ 12 ], driving angiogenesis [ 29 ], boosting mechanical compliance needed for rapid volume changes [ 6 , 30 ] and other processes. Dynamic translocation between membrane and intracellular vesicle pools is controlled by signaling; for example, AQP2 localization depends on cyclic AMP-dependent kinase activity [ 31 ], and AQPs are associated via protein–protein interactions into signaling complexes [ 32 , 33 , 34 ].…”

Natural Health Products (NHP’s) and Natural Compounds as Therapeutic Agents for the Treatment of Cancer; Mechanisms of Anti-Cancer Activity of Natural Compounds and Overall Trends

“…Additionally, an interesting finding has been reported recently by De Leso et al where it was observed that the blockers AqB011 and bacopaside II exhibit synergistic action, amplifying the inhibition of 2D cell migration in two colon cancer cell lines [ 132 ].…”