Functional Inhibition of Aquaporin-3 With a Gold-Based Compound Induces Blockage of Cell Proliferation

Abstract

AQP3 has been correlated with higher transport of glycerol, increment of ATP content, and larger proliferation capacity. Recently, we described the gold(III) complex Auphen as a very selective and potent inhibitor of AQP3's glycerol permeability (Pgly ). Here we evaluated Auphen effect on the proliferation of various mammalian cell lines differing in AQP3 expression level: no expression (PC12), moderate (NIH/3T3) or high (A431) endogenous expression, cells stably expressing AQP3 (PC12-AQP3), and human HEK293T cells transiently transfected (HEK-AQP3) for AQP3 expression. Proliferation was evaluated in the absence or presence of Auphen (5 μM) by counting number of viable cells and analyzing 5-bromo-2'-deoxyuridine (BrdU) incorporation. Auphen reduced ≈50% the proliferation in A431 and PC12-AQP3, ≈15% in HEK-AQP3 and had no effect in PC12-wt and NIH/3T3. Strong arrest in the S-G2/M phases of the cell cycle, supported by analysis of cyclins (A, B1, D1, E) levels, was observed in AQP3-expressing cells treated with Auphen. Flow-cytometry of propidium iodide incorporation and measurements of mitochondrial dehydrogenases activity confirmed absence of cytotoxic effect of the drug. Functional studies evidenced ≈50% inhibition of A431 Pgly by Auphen, showing that the compound's antiproliferative effect correlates with its ability to inhibit AQP3 Pgly . Role of Cys-40 on AQP3 permeability blockage by Auphen was confirmed by analyzing the mutated protein (AQP3-Ser-40). Accordingly, cells transfected with mutated AQP3 gained resistance to the antiproliferative effect of Auphen. These results highlight an Auphen inhibitory effect on proliferation of cells expressing AQP3 and suggest a targeted therapeutic effect on carcinomas with large AQP3 expression.

Role of aquaporins in cell proliferation: What else beyond water permeability?

“…For instance, we reported that cells with endogenous or exogenous, stable or transient, expression of AQP3 treated with Auphen, a potent inhibitor of the glycerol permeability of AQP3, 59 were arrested in the S-G2/M phases of the cell cycle, suggesting the possibility that the inhibition of AQP3 permeability somehow detains the progression of the cell cycle, thereby slowing cell proliferation. 18 In agreement with our results others have found that cell cycle of neurospheres derived from adult neuron stem cells of the subventricular zone (SVZ) in AQP4 knockout mice get arrested at the G2-M stage, 87 functionally implicating AQP4 in the activation and differentiation of the mice SVZ neurogenic niche.…”

“…18 Since the transport of glycerol by AQP3 is bidirectional, 59 proliferation could be related to the entry or exit of glycerol in cells; and unlike other authors, [60][61][62] we did not rule out the effect of Auphen on cell proliferation being caused by glycerol accumulation inside the cell due to blocking of the exit of glycerol. 18 AQP7 is the major pathway for glycerol efflux from adipocytes after lipolysis, 63,64 its absence resulting in increases in adipocyte cell volume and adipose tissue mass. 65,66 In the renal proximal tubule, AQP7 is a reabsorption pathway that may be important for preventing glycerol from being excreted into urine.…”

“…Reviewing the literature, we find that this association has been suggested mainly in relation to a role for these proteins in cell volume changes, 3,5,8 or in the case of a subgroup of AQPs, called aquaglyceroporins, 11,12 to their permeability to solutes such as glycerol and hydrogen peroxide. [13][14][15][16][17][18] Additionally, various studies have demonstrated that AQP expression is modified over the course of the cell cycle [19][20][21][22][23] and, more recently, we have shown that overexpression of AQP1 and AQP3 alters the levels of essential checkpoint proteins (cyclins) relevant for cell cycle progression, in addition to modifying the levels of transcription factors, and cytokines. 13,24 In this review, we have sought to gather the most relevant evidence associating AQPs with cell proliferation and we will discuss whether the canonical function of water permeation of these proteins would be always essential for this process or the role in proliferation might be independent of this function.…”

Nanoformulations of a Potent copper-based Aquaporin Inhibitor With Cytotoxic Effect Against Cancer Cells

“…In a previous work, the antiproliferative effect of the gold(III) complex Auphen, with human AQP3 inhibition properties, was demonstrated against a human skin carcinoma cell line A432 [18]. In an attempt to select other potential cytotoxic metallodrugs, also acting as AQP3 inhibitors, an in vitro screening was performed where the same epidermoid carcinoma A431 cells were incubated for 72 h with Auphen, Cuphen, AubipyMe and Aubipy, with concentrations ranging from 0.250 up to 60 μM, and cell viability determined Research Article Nave, Castro, Rodrigues, Casini, Soveral & Gaspar by the MTS assay as described in the Experimental section.…”

“…Interestingly, all these gold complexes contain N-donor ligands and are also described as antiproliferative agents against cancer cells in vitro [15 -17] . Notably, no cytotoxic effect of the lead compound [Au(phen)Cl 2 ] (phen=1,10-phenanthroline, Auphen, Figure 1) was observed for tumor cells that do not express AQP3 [18]. Furthermore, other gold complexes were also tested as potent inhibitors of cancer cells growth and they appeared as promising drug candidates [15,19].…”