GSD-Ib patients may also experience oral symptoms, consisting of dental caries, periodontal diseases, gingivitis, delayed dental maturation and eruption, oral bleeding diathesis and ulcers (Mortellaro et al., 2005). Remarkably, not all GSD-Ib patients manifest neutropenia or frequent infections (Kure et al., 2000; Melis et al., 2005; Angaroni et al., 2006; Martens et al., 2006). In this regard, in a multicentre study investigating the genotype/phenotype correlation on a cohort of 25 GSD-Ib patients, no correlation was found between individual mutations and the presence of neutropenia, bacterial infections or systemic complications. This evidence might suggest the existence of unknown factors able to influence immune phenotype, such as polymorphisms, proteins or genes, capable of modulating neutrophil differentiation, maturation, and apoptosis (Melis et al., 2005). Considering that neutrophils of GSD-Ib patients exhibited enhanced apoptosis, a causal relationship between apoptosis and neutropenia was hypothesized (Kuijpers et al., 2003; Jun et al., 2014). This theory was supported by further studies conducted on animal models, demonstrating that either neutrophils from G6Pase-β −/− mice or those from G6PT −/− mice exhibited enhanced ER stress and apoptosis (Cheung et al., 2007; Kim et al., 2008). So, neutrophil ER stress, higher oxidative stress and apoptosis might be underlying causes of neutropenia in GSD-Ib (Jun et al., 2010). In addition, neutrophil apoptosis in both G6Pase-β −/− (Jun et al., 2011) and G6PT −/− (Kim et al., 2008) mice was mediated by the intrinsic apoptosis pathway. In GSD-Ib, a maturation arrest seems not to be responsible for neutrophil dysfunction (Jun et al., 2014). Since neutrophils, as well as macrophages, have to import glucose from the blood by the glucose transporters (Pessin and Bell, 1992), these cells critically depend on the G6PT/G6Pase-β complex activity, especially when the need for glucose rises (Jun et al., 2014). This might provide a rationale for neutropenia caused by enhanced neutrophil ER stress, oxidative stress and apoptosis, when G6PT and/or G6Pase-β are defective (Kuijpers et al., 2003; Kim et al., 2008; Jun et al., 2010, 2014).

### AUTOIMMUNITY RISK OF SLC37A4 DEFECT

A strong association was found between GSD-Ib and inflammatory bowel disease (IBD), because many GSD-Ib patients with chronic gastrointestinal inflammation were diagnosed with IBD, which was clinically indistinguishable from idiopathic Crohn disease, assuming the involvement of an impaired mucosal innate immunity in the pathogenesis of IBD (Dieckgraefe et al., 2002). The association between neutropenia and IBD was supported by Visser et al., in a retrospective European study, showing that up to 77% of patients with GSD-Ib presented also neutropenia, as well as many GSD-Ib patients suffering from perioral or perianal infections (Visser et al., 2000).

In patients with GSD-Ib an increased risk for Crohnlike disease was reported (Melis et al., 2003), along with a severe deficit of hypothalamus-pituitary-thyroid axis, causing an increased prevalence of thyroid autoimmunity and hypothyroidism (Melis et al., 2007). In one patient, the occurrence of myasthenia gravis was also described (Melis et al., 2008). These observations paved the way to the hypothesis that GSD-Ib patients, but not those affected by GSD-Ia, were at increased risk for autoimmune disorders. The different risk degree between GSD-Ia and GSD-Ib patients might be related to the presence of neutropenia and neutrophil dysfunction only in GSD-Ib (Melis et al., 2007). Some studies have also suggested an association between lymphopenia and autoimmunity (Merayo-Chalico et al., 2016). Chronic lymphopenia might promote autoimmunity inducing the homeostatic expansion of T cell, which represents a normal compensatory reaction during lymphopenic conditions (King et al., 2004).

Recent studies have highlighted the molecular mechanisms underlying the increased frequency of autoimmunity disorders shown in GSD-Ib patients (Melis et al., 2017).

Regulatory T cells (Tregs) are a subset of CD4+ T cells involved in maintaining tolerance to self-antigens, and they play a critical role in human autoimmune diseases, because their imbalance seems to be responsible for the failure of local regulatory mechanisms (Dejaco et al., 2006). Those cells mediate their suppressive function by acting directly on conventional T cells (Tconvs) and antigen-presenting cells, such as dendritic cells (Rueda et al., 2016). Tregs express the transcription factor forkhead box P3 (FOXP3), which is very important for Tregs development and function, since defects in the FOXP3 gene cause a human lethal autoimmune disease (Bennett et al., 2001).

Treg responses can be modulated by several metabolic pathways (Buck et al., 2015), particularly by glycolysis, able to affect human Treg induction and function (De Rosa et al., 2015). In this regard, a defective engagement of glycolysis upon suboptimal T cell receptor (TCR) stimulation of conventional T cells (Tconvs) has been observed in several human autoimmune diseases, and it has been associated with a decreased suppressive function of Tregs (De Rosa et al., 2015).

In GSD-Ib, a defective G6PT causes a decreased capacity to mobilize glucose, leading to diminished glucose utilization. Consequently, a reduced engagement of glycolysis in T cells occurs upon suboptimal TCR stimulation; additionally, qualitative and quantitative alterations of peripheral regulatory T cells (pTregs) have been observed, along with impaired FOXP3 expression by Tconvs during low TCR activation (Melis et al., 2017).

### THERAPEUTIC APPROACHES FOR SLC37A4 DEFECT

Clinical therapies consist of dietary treatment, granulocyte colony-stimulating factor (G-CSF) therapy and transplantations.

Since recurrent hypoglycemia is responsible for lactic acidosis, hyperuricemia and hypertriglyceridemia, the primary goal of nutritional therapy is to achieve a stable normoglycemia. In addition, dietary treatment aims to reduce metabolic impairment, delaying some of the long-term GSD-Ib consequences. Unfortunately, elevated triglycerides and cholesterol blood levels may persist regardless of diet, along with their consequences (Cappello et al., 2016); moreover, even if nutritional therapy can improve long-term outcomes for many patients, several long-term complications remain. Recommended diet composition is 60–70% calories from carbohydrates, 10–15% from proteins and the remaining from fats (Goldberg and Slonim, 1993). Small frequent feedings rich in complex carbohydrates (particularly those higher in fiber) have to be regularly ingested over 24 h to achieve a good metabolic control (Wolfsdorf and Weinstein, 2003; Kishnani et al., 2014). GSD-Ib, as well as GSD-Ia patients, can use the same nutritional therapy to avoid hypoglycemia, especially nighttime (Chou et al., 2010b).

Cornstarch therapy in GSD-I is used since the early 1980s (Chen et al., 1984). Raw uncooked cornstarch is a slowly digested carbohydrate, which allows a slow release of glucose able to prolong the length of euglycemia between meals (Sidbury et al., 1986).

Glucose requirement normally decreases with age, so adults have a longer fasting tolerance with respect to children. Traditionally, in children older than 3 years, as well as in adults, normoglycemia during nighttime is maintained by the ingestion at bedtime of uncooked corn starch (Wolfsdorf and Crigler, 1997; Weinstein and Wolfsdorf, 2002; Shah and O'Dell, 2013) or of modified forms of corn starch (Bhattacharya et al., 2007; Correia et al., 2008). In children <3 years continuous tube feeding is usually needed (Greene et al., 1976), because the level of pancreatic amylase able to hydrolyze raw starch is low before 3 years of age (Chou et al., 2010b). A recent controlled crossover study enrolling adult GSD-Ia and GSD-Ib patients, highlighted that nighttime glucose control can be achieved also by the ingestion of a cooked pasta meal at bedtime, allowing a more palatable alternative to corn starch use (Hochuli et al., 2015).

G-CSF administration promotes the production of granulocytes (Mehta et al., 2015), increasing neutrophile numbers and reducing the frequency and severity of infections in GSD-Ib patients, but it can not improve neutrophile dysfunction (Visser et al., 2002). G-CSF also appears to increase neutrophil survival (Jun et al., 2011). In this regard, studies led in vivo, on murine G6Pase-β-deficient models, highlighted that G-CSF therapy was able to normalize neutrophil energy homeostasis and to improve functionality, as supported by improved neutrophil glucose uptake and increased G6P, ATP, and lactate intracellular concentrations (Jun et al., 2011).

A recent study has evidenced a partial improvement of neutrophil respiratory burst activity in a GSD-Ib patient by oral administration of galactose, even though the role of galactose in sugar metabolism of GSD-Ib neutrophils remains to be clarified (Letkemann et al., 2017).

Neutropenia associated with IBD can be treated by using a combination of G-CSF and 5-aminosalicylic acid (Visser et al., 2002; Koeberl et al., 2009; Chou et al., 2010b; Shah and O'Dell, 2013).

In some GSD-Ib patients, metabolic abnormalities can be corrected by liver or combined liver/kidney transplantation, since such operations have improved metabolic control, restoring normal fasting tolerance (Boers et al., 2014). Patients with impending renal failure are suggested to consider combined liver/kidney transplantation (Labrune, 2002). In GSD-Ib patients, myeloid dysfunctions can be treated by bone marrow transplantation, which appears to be a promising approach, even if it requires further validation (Pierre et al., 2008). According to the guidelines from the European study on GSD-I, liver transplantation is suggested in GSD-I patients with unresectable hepatocellular adenomas unresponsive to nutritional therapy, mainly if adenomas are associated with serious compression or hemorrhage, or in the case of enhanced risk of transformation into hepatocellular carcinomas (Rake et al., 2002). Although liver transplantation improves metabolic impairment (Reddy et al., 2009), its usefulness on renal disease and neutropenia or neutrophil dysfunction is yet to be settled.

### GENE THERAPY FOR SLC37A4 DEFECT

Gene therapy represents a promising strategy to correct metabolic abnormalities in all GSD-I patients (Chou and Mansfield, 2011), although currently it seems not to compensate for myeloid and renal dysfunction in GSD-Ib patients (Kwon et al., 2017).

Several gene transfer vectors, including recombinant adenoassociated virus (rAAV) vectors, have been developed and tested, either on GSD-Ia or on GSD-Ib animal models (Chou et al., 2015; Kwon et al., 2017).

G6PT knockout (G6PT−/−) mice are excellent G6PTdeficient murine models, since they exhibit all the metabolic and myeloid dysfunctions typical of human GSD-Ib (Chen et al., 2003). Some studies led on those mice showed that recombinant adenovirus-mediated G6PT gene transfer was able to deliver the transgene to the liver and bone marrow, improving metabolic and myeloid defects; however, this therapy was short-lived, owing to the fast loss of vector-mediated gene expression (Yiu et al., 2007).

Further studies led on such mice showed that a human G6PT expressing recombinant AAV serotypes 2/8 vector (rAAV8) directed by a hybrid chicken β-actin (CBA) promoter and a cytomegalovirus (CMV) enhancer, was able to deliver the transgene primarily to the liver, thus normalizing metabolic abnormalities. Nevertheless, two of the five treated mice survived for 51–72 weeks, developed multiple hepatocellular adenomas, and one experienced cancer transformation (Yiu et al., 2009). So, this therapeutic approach, while allowing normoglicemia, could not prevent long term complications of hepatocellular adenoma.

A further work, on G6Pase-α-deficient murine models (manifesting all the symptoms of human GSD-Ia), was led in order to compare two different gene transfer methods. In one case, murine G6Pase-α gene transfer in vivo was mediated by recombinant AAV serotypes 2/1 (rAAV1) or rAAV8 vectors, and expression was directed by hybrid CBA promoter/CMV enhancer. In the other case, a rAAV8 vector expressing human G6Pase-α directed by its promoter/enhancer (GPE) was employed (Yiu et al., 2010).

This work highlighted that the gluconeogenic tissuespecific human GPE was more effective in directing persistent in vivo hepatic transgene expression with respect to the CBA promoter/CMV enhancer, because human GPE utilization triggered a lesser humoral immune response, allowing complete normalization of hepatic G6Pase-α deficiency (Yiu et al., 2010). In addition, in murine GSD-Ia models, the use of a G6Pase-α-expressing rAAV vector under the GPE control was able to correct metabolic defects with no hepatocellular adenoma development (Lee Y. M. et al., 2012).

On this basis, recently two different liver directed gene therapy approaches were tested on G6PT−/− mice, using rAAV-GPE-G6PT and rAAV-miGT-G6PT, this latter consisted of a human G6PT expressing rAAV directed by human minimal G6PT promoter/enhancer (miGT) (Hiraiwa and Chou, 2001; Kwon et al., 2017).

Both vectors could deliver the hG6PT transgene to the liver, correcting metabolic anomalies, but rAAV-GPE-G6PT vector directed by the hG6Pase-α promoter/enhancer had greater efficacy. A full restoration of normal G6PT activity was not

### REFERENCES

necessary to obtain significant therapeutic benefits, since a restoration of 3-62% seemed to confer protection against agerelated obesity and insulin resistance, while restoration <6% exposed to hepatic cancer risk (Kwon et al., 2017).

### AUTHOR CONTRIBUTIONS

AC, RC and RL conducted literary review, wrote the article and prepared publication-ready figures, MM and VD designed and edited the article.

### ACKNOWLEDGMENTS

This work was supported by Associazione Italiana per la Ricerca sul Cancro (MM grant no. 16719/2015).

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Cappello, Curcio, Lappano, Maggiolini and Dolce. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Aquaporins as Targets of Dietary Bioactive Phytocompounds

Angela Tesse1†, Elena Grossini 2†, Grazia Tamma3 , Catherine Brenner 4 , Piero Portincasa5 , Raul A. Marinelli 6 and Giuseppe Calamita3 \*

1 Centre National de La Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, l'Institut du Thorax, Universitè de Nantes, Nantes, France, 2 Laboratory of Physiology, Department of Translational Medicine, University East Piedmont, Novara, Italy, 3 Department of Biosciences, Biotecnhologies and Biopharmaceutics, University of Bari "Aldo Moro", Bari, Italy, 4 Institut National de la Santé et de la Recherche Médicale UMR-S 1180-LabEx LERMIT, Université Paris-Sud, Université Paris-Saclay, Châtenay Malabry, France, 5 Clinica Medica "A. Murri", Department of Biomedical Sciences and Human Oncology, Medical School, University of Bari "Aldo Moro", Bari, Italy, 6 Instituto de Fisiología Experimental, CONICET, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina

### Plant-derived bioactive compounds have protective role for plants but may also modulate several physiological processes of plant consumers. In the last years, a wide spectrum of phytochemicals have been found to be beneficial to health interacting with molecular signaling pathways underlying critical functions such as cell growth and differentiation, apoptosis, autophagy, inflammation, redox balance, cell volume regulation, metabolic homeostasis, and energy balance. Hence, a large number of biologically active phytocompounds of foods have been isolated, characterized, and eventually modified representing a natural source of novel molecules to prevent, delay or cure several human diseases. Aquaporins (AQPs), a family of membrane channel proteins involved in many body functions, are emerging among the targets of bioactive phytochemicals in imparting their beneficial actions. Here, we provide a comprehensive review of this fast growing topic focusing especially on what it is known on the modulatory effects played by several edible plant and herbal compounds on AQPs, both in health and disease. Phytochemical modulation of AQP expression may provide new medical treatment options to improve the prognosis of several diseases.

Keywords: aquaporin membrane channels, functional foods, nutraceutics, epigenetics, gut microbiota, antioxidants, anti-inflammatory, chronic diseases

## INTRODUCTION

Growing evidence from epidemiological, in vivo, in vitro, and clinical trial results indicate that the plant-based food can reduce or prevent the risk of chronic diseases such as cardiovascular disease, arterial hypertension, diabetes mellitus, and cancer due to presence of biologically active plant compounds or phytochemicals. Several classes of phytochemicals from edible plants and herbs exist (Steinmetz and Potter, 1991) and exert beneficial effects in disease prevention and in reducing the incidence of certain chronic diseases. The mechanisms modulate the cell signaling pathways underlying inflammation, oxidative stress, metabolic disorder, apoptosis, and so forth (Maraldi et al., 2014).

This review provides an update on the involvement of Aquaporins (AQPs), a family of membrane channel proteins with important role in many body functions, in the beneficial effects imparted by food polyphenols and herbal phytocompounds, both in health and disease.

### Edited by:

Graça Soveral, Universidade de Lisboa, Portugal

#### Reviewed by:

Umberto Laforenza, University of Pavia, Italy Lidija Milkovic, Rudjer Boskovic Institute, Croatia

> \*Correspondence: Giuseppe Calamita<EMAIL_ADDRESS>
†These authors have contributed equally to this work.

#### Specialty section:

This article was submitted to Cellular Biochemistry, a section of the journal Frontiers in Molecular Biosciences

> Received: 10 February 2018 Accepted: 21 March 2018 Published: 18 April 2018

#### Citation:

Tesse A, Grossini E, Tamma G, Brenner C, Portincasa P, Marinelli RA and Calamita G (2018) Aquaporins as Targets of Dietary Bioactive Phytocompounds. Front. Mol. Biosci. 5:30. doi: 10.3389/fmolb.2018.00030

### AQUAPORINS, A FAMILY OF MEMBRANE CHANNELS WIDELY DISTRIBUTED IN HUMAN TISSUES

Aquaporins (AQPs) are channel proteins largely expressed in living organisms mediating the transport of water and some anaelectrolytes across biological membranes (Agre, 2004). The 13 AQPs (AQP0-12) expressed in mammals are summarily grouped into orthodox aquaporins (AQP0, AQP1, AQP2, AQP4, AQP5, AQP6, and AQP8) and aquaglyceroporins (AQP3, AQP7, AQP9, AQP10), depending on their ability to conduct only water or water and neutral solutes, particularly glycerol, respectively (**Figure 1**). AQP11 and AQP12 are often grouped as unorthodox aquaporins due to their distinct evolutionary pathway and transport properties (Ishibashi et al., 2009). Some AQPs are also able to conduct ammonia (AQP3, AQP4, AQP6, AQP8, and AQP9) and/or hydrogen peroxide (AQP1, AQP3, AQP5, AQP8, and AQP9) and, for these biophysical properties, are also denoted as ammoniaporins (or aquaammoniaporins) (Jahn et al., 2004) and/or peroxiporins (Geyer et al., 2013; Almasalmeh et al., 2014; Rodrigues et al., 2016; Watanabe et al., 2016) (**Figure 2**). Moreover, some AQPs also allow permeation of gases of physiological importance such as CO2, NO or O2 (Nakhoul et al., 1998; Herrera et al., 2006; Wang et al., 2007). Expression, transport properties (Agre, 2004), and pharmacological gating (Soveral and Casini, 2017) of AQPs are object of strong interest and intense investigation in all body districts and a number of important roles have been already described, both in health and clinical disorders.

aquaglyceroporins (AQP3, AQP7, AQP9, and AQP10) depending on their ability to conduct only water or glycerol and some other small neutral solutes, in addition to water, respectively. Two of the 13 AQPs found in mammals, AQP11 and AQP12, are called unorthodox aquaporins as they show marked distinctions in terms of evolutionary pathway. Some AQPs also express conductance to gases of physiological relevance.

## DIETARY POLYPHENOLS AND AQUAPORINS

The class of polyphenols is characterized by the presence of phenol units in their chemical structure. Polyphenols are the largest group of phytochemicals, and many of them exist in edible plants (Maraldi et al., 2014). Foods enriched in polyphenols were found to exert a wide spectrum of protective effects (i.e., hypolipidemic, anti-oxidative, anti-proliferative, antiapoptotic, and anti-inflammatory) with the benefit of reducing the prognosis and onset of disease progression (for review see Upadhyay and Dixit, 2015). So far, more than 8,000 phenolic structures have been identified in vegetables, fruits, olive oil, and wine. Due to their diversity and food distribution the latest classification subdivides polyphenols in phenolic acids, curcuminoids, flavonoids, chalcones, stilbenes, lignans, and isoflavonoids (González-Castejón and Rodriguez-Casado, 2011; Upadhyay and Dixit, 2015). Bioactive polyphenols also influence the expression and biophysical properties of mammalian AQPs (Zhang et al., 2014; Fiorentini et al., 2015; Cataldo et al., 2017). The AQPs modulated by polyphenols and related health benefits are summarized in **Table 1**.

### Curcuminoids

Curcuminoids (or curcumins) are characterized by a pronounced yellow color composed of linear diarylheptanoids. They are represented by curcumin and its derivatives (i.e., demethoxycurcumin and bisdemethoxycurcumin). Curcuminoids have been tested in particular for their anti-oxidant activity.

Curcumin is a non-flavonoid polyphenol isolated from spice turmeric, and known for playing anti-inflammatory, antioxidant, anti-proliferative, and anti-angiogenic activities (Tsao, 2010). The beneficial effects of curcumin on human health, however, are

AQP6, AQP7, AQP8, and AQP9), particularly AQP8, and/or hydrogen peroxide (AQP1, AQP3, AQP5, AQP8, and AQP9) and are called ammoniaporins (or aquaammoniaporins) and peroxiporins, respectively.

TABLE 1 | Polyphenolic modulation of AQPs and related beneficial effects.

Arrows: ↑, upregulation; ↓, downregulation; SCI, spinal cord injury; ND, specific AQP homolog to be defined.

downplayed by its poor absorption and bioavailability (Anand et al., 2007). Liposomal curcumin or curcumin nanoparticles have increased bioavailability, while structural analogs of curcumin such as EF-24 feature higher stability and faster absorption (Santiago-Vázquez et al., 2016). EF-24 has been demonstrated effective against cancer (Yang et al., 2014; Santiago-Vázquez et al., 2016), Parkinson's (PD), and Alzheimer's (AD) diseases (Pal et al., 2011).

The central nervous system expresses various AQPs (Badaut et al., 2014) and studies using rodent brain have suggested important roles for AQP1, AQP4, and AQP9 in controlling water transport and volume homeostasis (Badaut et al., 2014). AQP1 is highly expressed in choroid plexus, a secretory epithelium which plays a role in cerebrospinal fluid (CSF) formation and secretion (Nabiuni et al., 2013). After brain injury, mice deficient for AQP1 displayed a decreased intracranial pressure and improved survival compared to wild type mice (Oshio et al., 2005). This strongly suggests that AQP1 downregulation might be protective against several neurological disorders characterized by increased intracranial pressure. Notably, curcumin decreases, in a dosedependent manner, the AQP1 level in choroidal epithelial cells isolated from the lateral ventricle of Wistar rats (Nabiuni et al., 2013). Curcumin could also act inhibiting choroid plexus AQP1 since in a study using Hela cells this phytocompound has been recently suggested to have a direct gating action on some AQPs (Pellavio et al., 2017). AQP4, the most characterized brain water channel, is located mainly on astrocytic endfeets that are in strict contact with blood vessels (Badaut et al., 2014). Curcumin counteracts the brain edema and the effect might include the modulation of the expression of various AQPs, especially AQP4. Indeed, current medical and surgical therapies available for the treatment of intracerebral hemorrhage (ICH) do not adequately control brain edema. Interestingly, curcumin dose-dependently reduced both the gene expression and protein abundance of AQP4 and AQP9 but not those of AQP1 in a mouse model of ICH (Wang et al., 2015). Likely, the protective effects of curcumin may also involve the downregulation of specific water channels.

 Similarly, in a rat model of hypoxia-hypercapnia, curcumin injection attenuated brain edema and restored the levels of AQP4 expression (Yu et al., 2016b). In the mice model of traumatic brain injury, pre- or post-treatment with curcumin reduced the cerebral edema, the pericontusional expression of IL-1β, and reversed the induction of AQP4 (Zhang et al., 2014). The beneficial effect of curcumin was also observed in an animal model of SCI (impaired motor function and spinal cord edema); curcumin counteracted the abnormal activation of JAK/STAT signaling pathways and reduced the glial fibrillary acidic protein (GFAP) and AQP4 overexpression (Zu et al., 2014). While most available data highlight the beneficial effects of curcumin in neurologic diseases, curcumin could aggravate some CNS manifestations in experimental lupus erythematous. Indeed, curcumin treatment was associated with increased cerebral water content and AQP4 expression in mice with systemic lupus erythematous, likely depending on worsened cerebral atrophy and astrocytosis (Foxley et al., 2013). All together the above-mentioned results suggest that AQP4 is the main target through which curcumin would exert its action on brain edema and SCI.

In cultured human ovarian CaOV3 cells, stimulation with the endothelial growth factor (EGF) promoted AQP3 expression and CaOV3 cell migration. AQP3 knocking down by siRNA was associated with significant impairment of CaOV3 cell migration. Also in this context, curcumin (or a stable analog) proposed for therapeutic treatment of ovarian cancer (Terlikowska et al., 2014), downregulated AQP3 and reduced cell migration in CaOV3, an effect mediated by inhibition of EGFR signaling (Ji et al., 2008).

As shown for quercetin and naringenin (see below), in HeLa cells curcumin elicited antioxidant effects by reducing the hydrogen peroxide cellular content, probably by decreasing its entry into the cell through AQPs-mediated mechanisms (Pellavio et al., 2017). Modulation of AQPs by curcumin in cancer cells resulting in elimination of hydrogen peroxide may not always result beneficial to health when considering that accumulation of hydrogen peroxide (among other ROS) is a mechanism by which a number of conventional anti-cancer treatments govern cancer cells to death.

Curcumin could also influence various physiological functions through interactions with different ion channels and transporters involving several signaling pathways, from the well-known CFTR to voltage-gated potassium channels, volumeregulated anion channel (VRAC), Ca2+ release-activated Ca2+ channel (CRAC), and glucose transporters (Zhang et al., 2014). More research is needed in this respect, to better highlight the role of curcumin as modulator of various channel functions and pointing to its protective effects in various disease. Moreover, the use of curcumin could be useful also for research purposes since it could help the understanding of the interplay between AQPs system and other membrane transporters (Zhang et al., 2014).

### Flavonoids

Flavonoids are the biggest representative subgroup of polyphenols, with more than 4,000 molecules described (Harborne and Williams, 2000; Cheynier, 2005).

Pinocembrin is a natural flavonoid compound, which has been isolated from several plants, such as ginger roots and wild marjoram, honey, and propolis (Massaro et al., 2014; Lan et al., 2016). Pinocembrin exerts pleiotropic effects: reduces reactive oxygen species (ROS) production, apoptosis, and controls mitochondrial functions (Massaro et al., 2014). In vitro evidence revealed that pinocembrin can cross the blood-brain-barrier passively indicating possible therapeutic use in nervous system diseases (Yang et al., 2012). In an animal model of focal cerebral ischemia induced by middle cerebral artery occlusion (MCAO), several inflammatory cytokines, tumor necrosis factor-α (TNFα), and interleukin-1β (IL-1β), inducible nitric synthase (iNOS) and AQP4 were significantly upregulated in the ischemic brain (Gao et al., 2010). Administration of pinocembrin via tail vein injection ameliorated the neuronal apoptosis and the edema as well as the typical alterations of endothelial cells and capillaries characterizing ischemia. Pinocembrin treatment decreased the production of cytokines and the expression iNOS and AQP4. These findings suggest that the protective response triggered by the flavonoid might be due to reduced inflammatory signals and decreased level of AQP4, which has been associated with edema subsequent to cerebral ischemia (Gao et al., 2010).

Chrysin is a natural flavonoid occurring in honey and propolis, various fruits, vegetables and mushrooms (Nabavi et al., 2015). Chrysin displays fundamental biological anticancer actions reducing cell proliferation and promoting apoptosis, especially in leukemia cells (Monasterio et al., 2004). In animal studies, UVB and UVA radiation induced skin dehydration and this step was associated with decrease of the expression level of AQP3, one of the main skin AQPs. Other changes included ROS release and apoptosis (Wu et al., 2011). Topical application of chrysin improved the UV-induced skin damage, and significantly increased keratinocyte AQP3, suggesting a chrysin-mediated protection of the deleterious effects exerted by UVs on human skin.

Quercetin is one of the most abundant bioflavonoids in the human diet. It is largely present in different vegetables including onions and broccoli, fruits such as apples, berry crops, and grapes. Quercetin is also found in some herbs, tea, and wine. Similarly to other polyphenols, quercetin displays pleiotropic properties as antioxidant and anti-inflammatory compound. Plant extract of quercetin is the principal ingredient of many potential anti-allergic drugs, supplements, and enriched products, being highly competent in inhibiting IL-8 action, suppressing IL-6 and intracellular calcium level increase (Mlcek et al., 2016). Retinas from streptozotocin-induced diabetic rats showed a remarkable increase of pro-inflammatory cytokines such as TNF-α and IL-1β and a considerable augmentation of AQP4 level, which could mediate the movement of water underlying the retinal edema (Kumar et al., 2014). Changes were accompanied with a significant reduction of retinal glutathione (GSH) and antioxidant enzymes [superoxide dismutase (SOD) and catalase (CAT)]. Oral administration of quercetin leads to an important neuroprotective effect which was characterized by impaired inflammatory cytokines production and release, restoration of GSH, SOD, and CAT levels and significant reduction of AQP4 in Müller cell endfeet and perivascular space with consequent decrease of the edema affecting the retina of diabetic rats. The observed amelioration of retinal edema was suggested as due to quercetin-dependent AQP4 downregulation on Müller cell endfeet and perivascular space. Another orthodox AQP water channel, AQP5, was found to be involved in the beneficial action of quercetin in attenuating the damaged salivary secretion induced in a murine model of impaired salivation by radiation exposure (Takahashi et al., 2015). Quercetin upregulated AQP5 expression and calcium uptake and suppressed the oxidative stress and inflammatory responses induced by the radiation exposure. Increased AQP5 levels were also observed in a mouse model of influenza A virus (IAV)-induced lung injury where AQP5 lung increased after treatment with flavonoid extracts from the Lamiaceae plant Mosla scabra (Yu et al., 2016a). The lung AQP5 modulation by the flavonoid was interpreted as a way to restore the normal water permeability alleviating the IAV-induced pulmonary inflammation and apoptosis.

AQP3, 5, 8, and 9 have been reported to facilitate the transmembrane diffusion of hydrogen peroxide in mammalian cells (Almasalmeh et al., 2014; Rodrigues et al., 2016; Watanabe et al., 2016). This is an important aspect since cellular oxidative stress can interfere with water permeability. In HeLa cells, the role of AQPs as target of antioxidant compounds acting on the osmotic water diffusion in the presence of oxidative stress condition has recently been studied (Pellavio et al., 2017). Quercetin appeared to modulate water transport and acted as antioxidant by increasing the expression of AQP3 and AQP8 (together with that of AQP1 and AQP11) at both mRNA and protein level. Particularly, with quercetin, the water permeability decrease caused by oxidative stress was prevented or restored. Furthermore, quercetin significantly reduced the hydrogen peroxide content to levels even lower than those of control cells. Thus, regulation of AQPs gating by antioxidant compounds like quercetin could represent a novel mechanism to modulate exogenously cell signaling and survival during stress, acting on key signaling pathways in cancer and degenerative diseases (see Tamma et al., 2018 for a review).

Hesperetin is a flavanone isolated from several fruits and highly expressed in the Citrusspecies. This flavonoid has different biological properties, including antioxidant, anti-inflammatory, and anticancer effects (Ahmadi et al., 2015; Bodduluru et al., 2015). Its beneficial actions have been showed in different organs including liver, heart, and kidney (Roohbakhsh et al., 2015; Miler et al., 2016). Neuroprotective effects have also been reported, in particular in the treatment of diabetic retinopathy where inflammation, oxidative stress, and neurovascular disorders are involved. Streptozotocin-induced diabetic rats receiving hesperetin for 24 weeks showed restoration of retinal levels of GSH associated to a positive modulation of antioxidant enzyme activities. An inhibition of caspase-3 activity and expression of GFAP was also seen along with reduced inflammatory cytokines (Kumar et al., 2013). Treatment with hesperetin was accompanied by downregulation of AQP4 at Müller cell endfeet and consequent reduction of the edematous state indicating modulation of AQP-associated water permeability.

Alpinetin is a flavanone isolated from the seed of Alpinia katsumadai (Zingiberaceae). The compound is widely used in Korean traditional medicine (Lee et al., 2012). Alpinetin has been found to control cell signaling pathways at the base of cell growth, proliferation, and apoptosis (Wang et al., 2013). Alpinetin also causes vasorelaxation (Wang et al., 2001) and counteracts the hydrogen peroxide-induced vascular smooth muscle cell proliferation and migration (Li and Du, 2004). In lipopolysaccharide (LPS)-induced lung injury, alpinetin prevented the LPS-induced TNF-α, IL-6, and IL-1β release and alleviated the inflammatory associated lung hystopathological alterations (Hou et al., 2009). Considerable AQP1 downregulation, a condition negatively correlated with pulmonary edema, has been described in acute lung injury (ALI) and, associated with severe acute pancreatitis (SAP). Interestingly, alpinetin has been found to inhibit TNF-α expression, promote human pulmonary microvascular endothelial cell proliferation and increase the expression level of AQP1 thereby improving the SAP-induced ALI symptoms (Liang et al., 2016). Overall, these findings propose alpinetin as possible therapeutical tool against lung inflammation diseases.

Naringenin is a natural flavonoid widely found in citrus fruits and tomatoes, that has been reported to act as anti-inflammatory, anti-atherogenic, anti-mutagenic, hepatoprotective, and anticancer agent (Yin et al., 2018). Naringenin relieved the loperamide-induced constipation by targeting AQP3. It is known that AQPs are primarily expressed in the mucosal epithelial cells in the colon, in which AQP3 plays a central role in water reabsorption across colonic surface cells. Yin et al. found that naringenin increased the mRNA and protein expression levels of AQP3 in the colon, both in apical and lateral mucosal epithelial cells. Furthermore, a positive correlation was observed between this increase in the AQP3 level and the increase in fecal water content (Yin et al., 2018). As also shown for quercetin, the antioxidant effects elicited by naringenin in HeLa cells have been recently related to its capacity to facilitate hydrogen peroxide elimination through AQPs. These observations would strengthen the role of AQPs as physiologic modulators of hydrogen peroxide diffusion in mammalian cells (Pellavio et al., 2017).

Liquiritigenin belongs to the chiral flavanone family and is an important compound extracted from Glycyrrhiza uralensis and found in a variety of plants, including Glycyrrhiza glabra (licorice). Liquiritigenin possesses kinds of healthy properties including antioxidation, anti-inflammation, antidiabetes, cardioprotection, and neuroprotection (Hosseinzadeh and Nassiri-Asl, 2015). As regarding the mechanisms, the inhibition of NF-κB and MAPK signaling pathways would be at basis of its antioxidant effects. Liquiritigenin has been shown to exhibit renal protective effects in the animal model of potassium oxonate-induced hyperuricemia, as well, by targeting the AQPs (Hongyan et al., 2016). AQP4 would play an important role in inflammatory responses involving the kidney, also by the regulation of endoplasmic reticulum stress. In the hyperuricemic rat, liquiritigenin has been able to suppress the activation of renal AQP4/NF-κB/IκBα signaling and nod-like receptor protein 3 (NLRP3) inflammasome, resulting in renal inflammation reduction. These findings would suggest that liquiritigenin could act as a potential drug for the treatment of hyperuricemia and renal injury by targeting the AQPs system.

Epigallocatechin gallate (EGCG) is a flavonol esterified with gallic acid mainly found in the green tea Camellia sinensis L. EGCG accounts for more than 50% of total green tea polyphenols. This phenolic compound and/or its metabolites exert cardioprotection, neuroprotection, renal protection, osteoprotection, and anticancer actions. Beneficial effects have also been shown in diseases with metabolic disorders such as obesity, metabolic syndrome and type 2 diabetes (Afzal et al., 2015). At a molecular level, EGCG promotes the expression and the activity of several anti-oxidant and anti-inflammatory enzymes. EGCG is also proved to counteract the activation of Toll-like receptor 4 (TLR4) (Marinovic et al., 2015) and nuclear factor-κB (NF-κB) (Albuquerque et al., 2016), pathways associated with the production of inflammatory cytokines. In a mouse model of Sjögren syndrome, treatment with EGCG increased the abundance of AQP5 at the apical plasma membrane of the acinar cells. AQP5 expression resulted upregulated by mechanisms leading to protein kinase A (PKA) activation and NF-κB inhibition (Saito et al., 2015). Conversely, by means of other pathways, EGCG was found to downregulate the expression of AQP5 in the ovarian cancer cell line SKOV3. The lower abundance of AQP5 was suggested to counteract the tumor growth through NF-κB activation (Yan et al., 2012). Considerable decrease of the spinal cord water content was seen in a work employing a rat model of acute SCI where EGCG was administered immediately following the injury (Ge et al., 2013). The anti-edema action exerted by EGCG was ascribed to the marked reduction of spinal cord AQP4 induced by the EGCG administration.

### Chalcones

Chalcones are a variety of aromatic ketones, precursors of flavonoids, and isoflavonoids. They are abundant in edible plants and their derivatives have been reported to have an extremely wide variety of biological activities (i.e., anti-bacterial, anti-fungal, anti-neoplastic, anti-inflammatory, anti-diabetic, anti-obesity, immunosuppressant actions) depending on the substitution made on them (Mahapatra et al., 2015).

Phloridzin, one of the most characterized bioactive chalcones, is also a competitive inhibitor of the isoforms 1 and 2 of the sodium glucose cotransporter (SGLT1 and SGLT2, respectively) as it competes with D-glucose in binding the carrier. This action leads to a decrease in glucose absorption and reabsorption by the small intestine and renal proximal tubules, respectively, lowering the glucose level in the blood. However, phloridzin is not an effective drug because when orally consumed, it is nearly entirely converted into phloretin and glucose by hydrolytic enzymes in the small intestine.

Phloretin, an abundant chalcone in apples, is a protective agent with anti-oxidative stress and anti-inflammatory actions (Aliomrani et al., 2016). Phloretin is also known for gating the aquaglyceroporins by inhibiting the AQP-mediated transport of glycerol and urea (Tsukaguchi et al., 1999; Calamita et al., 2008, 2012; Rodriguez et al., 2014), a metabolic intermediate substrate of gluconeogenesis and triacylglycerols (TAG) synthesis (Calamita et al., 2012), and a variety of urea transporters and urea-conducting AQPs such as AQP3, AQP7, and AQP9 (Shayakul et al., 2001; Fenton et al., 2004). AQP9-facilitated urea extrusion out of liver was evoked to explain the loss of urea and diuresis that characterizes mice submitted to high protein diet. This function played by liver AQP9 can be blocked by phloretin (Jelen et al., 2012). It is tempting to think that the antiinflammatory action exerted by phloretin may also involve the inhibition of AQP9, an AQP that has been suggested to play a role in inflammation (Matsushima et al., 2014).

### Stilbenes

Stilbenes belong to the family of phenylpropanoids and are better known as stilbenoids, their hydroxylated derivatives. The most studied stilbenoid is resveratrol, a compound having numerous health benefits.

Trans-resveratrol is a natural hydroxystilbene found in a variety of edible plants including grapes, blueberry, raspberry, and senna leaves. Research on the biological actions of resveratrol on human health has focused on cancer, neurodegenerative, and cardiovascular diseases, and metabolic disorders. At molecular level, resveratrol plays pleiotropic effects including inhibition of kinases, anti-inflammatory, analgesic and anticancer activities, and detoxification, by inhibiting the aryl hydrocarbon and dioxin receptor (AhR), (de Medina et al., 2005). AhR stimulation causes oxidative stress, inflammation, apoptosis and immunosuppression, and is associated with an increased

risk of osteoporosis, cancers and metabolic disorders such as diabetes (Zollner et al., 2010). As a polyphenol, resveratrol can also act as tyrosine kinases inhibitor and as a modulator of the mitogen activated protein kinase/extracellular signal-regulated kinase 1/2 (MEK-ERK1/2), mitogen-activated protein kinases (MAPK), activator protein 1 (AP-1), and NF-κB pathways in different tissues (Yu et al., 2001; Gao et al., 2004; Kundu et al., 2004). Resveratrol has been reported to influence the gene expression of various AQPs (see Cataldo et al., 2017 for a review). In human keratinocytes, AQP3 has been found to play a pivotal role in skin hydration, although overexpression of AQP3 is also linked with hyperplastic epidermal disorders (Nakahigashi et al., 2011). In normal human epidermal keratinocytes (NHEKs), treatment with resveratrol reduced cell proliferation and the expression of AQP3, the major skin AQP. Particularly, AQP3 downregulation appeared to be secondary to ERK signaling inhibition via upregulation of both Sirtuin 1 (SIRT1) and aryl hydrocarbon receptor AhR (Wu et al., 2014). These novel findings may be important to drug design and development against hyperproliferative skin disorders.

Cerebral ischemia-reperfusion (I/R) is associated with a strong increase of ROS production and brain edema. Resveratrol exerts a beneficial action by modulating the activity of SOD and reducing the iNOS and AQP4 expression levels (Li et al., 2015). More recently, functionalized AQP4 antibody nanoparticles were synthesized to deliver resveratrol in rat optic nerve transection. These engineering nanoparticles displayed high efficacy in reducing oxidative damage and AQP4 immunoreactivity thus preserving the visual function (Lozic et al., 2016 ´ ).

### Isoflavonoids

The isoflavonoids genistein and daidzein are important components of Leguminosae. Based on their chemical structure they function as phytoestrogens displaying anti-tumor features. These phytocompounds play an important role in modulating the genes involved in controlling cell-cycle progression.

Genistein and daidzein are considered the "sharp edge of balance" between cell survival and cell progression because they control the activation or the inhibition of pivotal signal molecules such as NF-κB. Importantly, they also play a role in reversion of epigenetic events associated with prostate cancer (Adjakly et al., 2013). The bioavailability of these compounds is strongly influenced by gut microbiota, antibiotic administration and individual's age and health status (Franke et al., 2014). A work addressing the effects of lifelong dietary isoflavone on estrogen sensitive tissues was carried out studying the effects of genistein and daidzein on rat uterus (Möller et al., 2010). The effect of genistein either alone or in association with daidzein was compared to that of isoflavone-free diet in rats throughout their whole lifetime. The dietary isoflavone pre-exposure resulted in a much stronger uterine weight increase following external ERαmediated estrogenic stimuli than that seen in the phytoestrogen– free group. Gene expression analysis showed that the uterine levels of AQP1, and, at a lesser extent, those of AQP3, AQP5, and AQP9, were increased by ovary estrogens. This modulation was considerably influenced by the isoflavone-containing diets, likely by an epigenetic mechanism. Lifelong dietary isoflavone ingestion was suggested to increase the uterine responsiveness to ERα-mediated estrogenic stimuli in female rats where the water homeostasis was highly affected whereas the proliferation rate remained unchanged.

Puerarin is a flavonoid glycoside that is extracted from the root of the leguminous plants Pueraria lobata and Thomson Kudzuvine Root. Puerarin displays a series of beneficial activities on hangover, cardiovascular disease, osteoporosis, neurological dysfunction (ischemic stroke, cerebrovascular disease) fever, and liver injury both in clinical treatment and experimental research (Wang et al., 2018). In addition to inhibit inflammation, protective effects elicited by puerarin against cerebral damage would be related to modulation of AQP4 function. Hence, AQP4, which can be mainly found in the primate and rodent perivascular astrocyte end feet, would play a role not only in water movement, but also, as previously reported, in neuroinflammation and brain edema. AQP4 expression would be increased by pro-inflammatory factors like TNF-α. Furthermore, increased phosphorylation of MAPK (p38, ERK1/2, c-Jun Nterminal kinase 1/2) would participate in AQP4 regulation in astrocytes exposed to the inflammatory cytokines released by microglia under the condition of hypoxia, too. Various interventions have been performed to prevent or to treat people rapidly ascending to high altitude. Currently, traditional Chinese medicine has been used to prevent or treat symptoms although the specific mechanisms are still a matter of debate. In rats undergone hypobaric hypoxia, puerarin was able to elicit protective effects against cerebral edema by inhibiting the increase of AQP4 through inhibition of TNF-α release and by counteracting the activation of NF-κB and MAPK pathway (Wang et al., 2018).

## CAPSAICINOIDS

Capsaicin is the most representative compound of a group of phytochemicals called capsaicinoids also including dihydrocapsaicin, nordihydrocapsaicin, and some other compounds such capsinoids. In chili peppers, capsaicin, a phenolic amide, gives the sensation of spiciness by acting through the transient receptor potential vanilloid-1 (TRPV1). TRPV1 is a non-selective permeable cation channel expressed in brain, bladder, kidneys, intestines, keratinocytes of epidermis, glial cells, liver, and polymorphonuclear granulocytes, mast cells, and macrophages (Reyes-Escogido Mde et al., 2011). In tumor cells, capsaicin inhibits cell growth and promotes apoptosis by increasing the intracellular calcium concentration and ROS levels, disrupting mitochondrial membrane transition potential and activating NF-κβ transcription factor (Chapa-Oliver and Mejia-Teniente, 2016). In addition, capsaicin stimulates the phosphorylation of p53 at serine-15 and its acetylation through downregulation of SIRT1. Altogether, these posttranslational modifications lead to apoptosis (Ito et al., 2004). Similarly to anti-obesity, anti-diabetic, and anti-inflammatory compounds, capsaicin plays multiple roles in nociceptive heat sensation. Capsaicin can activate sympathetic system to induce brown adipose tissue thermogenesis (Saito, 2015).

Capsaicin upregulated TRPV1 and AQP5 in rabbit salivary glands (**Table 2**). Specifically, in transplanted rabbit submandibular gland cells, capsaicin upregulated and stimulated the translocation of AQP5 from an intracellular pool to the plasma membrane via TRPV1 signaling and ERK phosphorylation (Ding et al., 2010; Zhang et al., 2010). This finding may provide a new therapeutic tool to stimulate submandibular gland hypofunction.

### TERPENES

Terpenes are a large and diverse class of organic compounds derived biosynthetically from units of isoprene. They are produced by a variety of plants and even some insects. Terpenoids are compounds related to terpenes characterized by an isoprenoid chemical structure as they may include some oxygen functionality or some rearrangement. Terpenoids are present in a great variety of fruits, vegetables and medicinal plants, representing the largest and most diverse class of chemicals among the myriad of compounds produced by plants. Terpenoids are used extensively for their aromatic qualities and play a role in traditional herbal remedies. The terms terpenes and terpenoids are often used interchangeably. As many other bioactive phytocompounds, terpenes, and terpenoids have been reported to influence the expression of AQPs (**Table 2**).

### Monoterpenoids

Carvacrol (or cymophenol) is a monoterpenoid extracted from many plants of the Lamiaceae family. Carvacrol has been reported to exert neuroprotective effects in central nervous system diseases such as AD and cerebral ischemia (Zhong et al., 2013). Modulation of brain AQP4 was reported in a preclinical study employing a bacterial collagenase-induced ICH murine model to address the effect of the monoterpenoid on cerebral edema after ICH. Administration of carvacrol improved the neurological deficits following ICH by significantly reducing cerebral edema and AQP4 in perihematomal area (Zhong et al., 2013). It was suggested that carvacrol exerts its protective effect on ICH injury by ameliorating AQP4-mediated cerebral edema.

Marrubiin (MARR) is a terpenoid abundantly found in many Lamiaceae species (i.e., Marrubium vulgare, Plomis bracteosa, Leonotis nepetifolia). MARR is a compound featuring high stability and limited catabolism, two properties favoring its bioavailability: MARR is reported to exert cardioprotective, vasorelaxant, gastroprotective, and antidiabetic effects (Popoola et al., 2013). Antioxidant action involving AQPs has been reported in a recent work where pre- or post-treatment of heat-stressed HeLa cells with MARR prevented or reversed, respectively, the intracellular H2O2 accumulation induced by the heat (Pellavio et al., 2017).

### Triterpenes

Bacopasides are triterpene saponins isolated from the medicinal plant Bacopa monnieri. Recently, two structurally related bacopaside compounds, bacopaside I and bacopaside II, have been shown to block differentially the transport activity of AQP1, a finding that fitted with predictions made by in silico molecular modeling (Pei et al., 2016). When tested in migration assays using HT29 and SW480 cells, two colon cancer cell lines characterized by high and low expression levels of AQP1, respectively, both bacopasides impaired migration of HT29 cells showing minimal effect on migration of SW480 cells. Based on these results bacopasides were suggested as possible novel lead compounds for pharmaceutical development of selective AQPs blockers in cancer treatment.

The triterpenoid 18β-glycyrrhetinic acid (β-GA) is a natural compound derived from Glycyrrhiza glabra (licorice) root shown to exert antiviral, antitumor and immunosuppressive effects. Studies using rat models of nasal mucosa of allergic rhinitis (AR) showed that intranasal administration of β-GA downregulates AQP1 together with that of eotaxin 1 (CCL11) and eosinophil (EOS) in nasal mucosa of allergic rhinitis rats and cast effects on inhibiting the progress of AR (Li et al., 2015).

Glycyrrhizic acid, a triterpenoid also known as glycyrrhizin, is the main sweet-tasting constituent of licorice root. Based on its inhibiting effect on liver cell injury glycyrrhizin is used for the treatment of chronic viral hepatitis and cirrhosis (van Rossum et al., 1998). Glycyrrhizin is also employed to prevent disease progression in subjects with acute onset autoimmune hepatitis (Yasui et al., 2011). Enoxolone, the aglycone of the glycyrrhizic acid, is used to prevent liver carcinogenesis in patients with chronic hepatitis C. Glycyrrhizin was reported to influence the renal functions by modulating AQP2 and AQP3, two AQPs expressed in the principal cells of the renal distal tubules and collecting ducts where they mediate the antidiuretic hormone (ADH)-induced water reabsorption (Kang et al., 2003). Using a rat model of gentamicin-induced acute renal failure the protective effects of glycyrrhizin downregulated AQP2 in the inner and outer renal medulla, and cortex (Sohn et al., 2003) suggesting that this triterpenoid involves renal AQPs in its beneficial action on renal function.

Ginsenoside Rg1 (Rg1) is a triterpenoid saponin known as one of the main compounds harvested from ginseng with pharmaceutical action and potential neuroprotective properties, empirically used in traditional Chinese medicine to treat stroke (Xie et al., 2015).

Ginsenoside Rg3 (Rg3), a triterpene saponin, is one of the bioactive extracts contained in ginseng root. Rg3 has been shown to have anticancer activity in various cancer models. Thus, in the highly metastatic prostate cancer cell line PC-3M, treatment with Rg3 was found to lead to a remarkable inhibition of cell migration. In particular, exposure of PC-3M cells to Rg3 suppressed the expression of AQP1, an AQP with a role in cell migration. The anti-metastatic effect of Rg3 was found to occur through the p38 MAPK pathway and some transcription factors acting on the Aqp1 gene promoter (Pan et al., 2012).

TABLE 2 | Modulation of AQPs by non-polyphenolic phytocompounds and related beneficial effects.

Arrows: ↑, upregulation; ↓, downregulation; ND, specific AQP homolog to be defined.

### SULPHORAPHANE

Sulphoraphane (SUL) is a chemical belonging to isothiocyanates, a group of organosulfur compounds. SUL was identified in broccoli sprouts, which, of the cruciferous vegetables have the highest concentration of this compound (Zhang et al., 1992). Among the reported beneficial effects sulforaphane appears to be a promising phytochemical with neuroprotective properties (Tarozzi et al., 2013).

Using a rodent controlled cortical impact injury model of traumatic brain edema it was observed that post-injury administration of SUL counteracted AQP4 loss in the contusion core and further increased the protein levels of this water channel in the penumbra region compared with injured animals receiving vehicle. This increase in AQP4 expression was accompanied by a significant amelioration of cerebral edema (Zhao et al., 2005). It was suggested that the reduction of the edema in response to SUL administration could be ascribed to water clearance through AQP4 from the injured brain.

### TETRAHYDROANTHRACENES

Tetrahydroanthracenes are polycyclic aromatic phytocompounds deriving from the phytochemical hydrogenation of anthracene in Liliaceae. This reaction is influenced by light exposition in roots of Aloe plants and tetrahydroanthracenes are markers of subterranean anthranoid metabolism. These molecules are used mainly to treat parasitic infections with potential anti-oxidant properties because of their chemical structure.

(**R**)-Aloesaponol III 8-methyl ether. The (**R**)-Aloesaponol III 8-methyl ether (ASME) was extracted for the first time from Eremurus persicus root but it is contained in other plants such as Aloe saponaria, Kniphonia foliosa, Eremurus chinensis, and others. ASME is a tetrahydroanthracene known for having biological activity against Leishmania infection. Like the terpenoid MARR, ASME improved the HeLa cells membrane permeability by restoring the AQP-mediated cellular extrusion of H2O2 contributing to the reduction in oxidative stress (Pellavio et al., 2017). However, further work is needed to better investigate this preliminary observation since the specific AQP channel responsible for the ASME-induced efflux of H2O2 out of the cells is still elusive.

### BIOACTIVE EXTRACTS

The beneficial effects of a number of plants are likely due to their original composition and the synergistic action played by the different bioactive phytochemicals they contain rather than to a unique active compound. Here, we review bioactive extracts from plants whose healthy effects have been reported to involve modulation of AQPs (**Table 3**).

Heliotropium indicum is a plant belonging to family of Boraginaceae used in folk medicine in many countries. In Ghana, extracts of H. indicum are employed as a remedy against cataract formation without any scientific evidence. The

Arrow: ↑, upregulation.

extract of this plant contains an original composition showing numerous bioactive compounds with antibacterial, antitumor, anti-inflammatory and diuretic activities. In a rat model of selenite-induced cataract development, the aqueous total extract of H. indicum significantly alleviated cataract at all dose levels tested (0.1–3.0 mg/ml) preserving AQP0, an AQP highly expressed in fiber cells of lenses, and other markers of lens transparency proteins (Kyei et al., 2015).

Garlic has shown versatile therapeutic action in the prevention and treatment of pathologies such as chronic obstructive pulmonary disease (COPD). However, the specific garlic bioactive component underlying this medicinal activity remains elusive. Viscous COPD mucus secretions have been explained also due to a down-regulation to which AQP5, an AQP water channel highly expressed in lungs, undergoes. Interestingly, S-allylmercapto-l-cysteine (SAMC), one of the bioactive phytochemicals found in aged garlic, improved the LPS-induced mucus secretion of a COPD cell model, the human airway submucosal gland cell line (SPC-A1), up-regulating AQP5 and mucin 5AC (MUC5AC) via NF-κB signaling pathway (Yang et al., 2016).

Isotetrandrine is a biscoclaurine alkaloid, a bioactive compound isolated from traditional Chinese herbs known for reducing the astrocyte cytotoxicity in the neurological autoimmune disorder such as neuromyelitis optica (NMO).

### REFERENCES

This phytochemical was reported to be a small molecule inhibitor of NMO-IgG binding to AQP4 without impairing the expression and water channel activity of AQP4 (Sun et al., 2016).

### CONCLUSIONS AND FUTURE PERSPECTIVES

Foods are enriched with myriads of bioactive phytocompounds, and research on their effects on human health is a quickly growing field. The recent recognition that AQPs are among the targets of food phytochemicals paves the way to potentially novel therapeutic options dealing with prognosis and cure of highly prevalent diseases (i.e., metabolic disorders, cancer, neurological diseases, and inflammatory chronic diseases). While the molecular mechanisms by which phytocompounds modulate the expression or gating (i.e., by phloretin, curcumin, and bacopaside II) of AQPs remains to be fully ascertained, there are no doubts that the list of functional food ingredients and herbal phytochemicals influencing AQPs is far from complete. Studies are also warranted to confirm in humans the results that have been obtained in vitro or with the use of rodent models also taking into account the gender dimorphism that may exist in terms of AQP expression and localization (Rodríguez et al., 2015). It should also be considered that bioactive phytocompounds are often not active per se, while their metabolites are. Thus, new and potentially important translational acquisitions are anticipated in the near future, in this exciting field.

### AUTHOR CONTRIBUTIONS

AT and EG contributed to article design, bibliographic search, writing, illustrations, and critical discussion. GT contributed to writing and bibliographic enrichment. CB provided critical discussion; PP contributed to writing and provided critical discussion; RM contributed to article design and writing, and provided critical discussion; GC designed and wrote the first draft of the article and contributed to article refinement, illustrations, bibliographic enrichment, and critical discussion. All authors approved the final version for submission.

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treatment for chronic hepatitis C. Aliment. Pharmacol. Ther. 12, 199–205. doi: 10.1046/j.1365-2036.1998.00309.x

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Tesse, Grossini, Tamma, Brenner, Portincasa, Marinelli and Calamita. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Mechanisms of Aquaporin-Facilitated Cancer Invasion and Metastasis

### Michael L. De Ieso and Andrea J. Yool\*

Department of Physiology, Adelaide Medical School, University of Adelaide, Adelaide, SA, Australia

Cancer is a leading cause of death worldwide, and its incidence is rising with numbers expected to increase 70% in the next two decades. The fact that current mainline treatments for cancer patients are accompanied by debilitating side effects prompts a growing demand for new therapies that not only inhibit growth and proliferation of cancer cells, but also control invasion and metastasis. One class of targets gaining international attention is the aquaporins, a family of membrane-spanning water channels with diverse physiological functions and extensive tissue-specific distributions in humans. Aquaporins−1,−2,−3,−4,−5,−8, and−9 have been linked to roles in cancer invasion, and metastasis, but their mechanisms of action remain to be fully defined. Aquaporins are implicated in the metastatic cascade in processes of angiogenesis, cellular dissociation, migration, and invasion. Cancer invasion and metastasis are proposed to be potentiated by aquaporins in boosting tumor angiogenesis, enhancing cell volume regulation, regulating cell-cell and cell-matrix adhesions, interacting with actin cytoskeleton, regulating proteases and extracellular-matrix degrading molecules, contributing to the regulation of epithelial-mesenchymal transitions, and interacting with signaling pathways enabling motility and invasion. Pharmacological modulators of aquaporin channels are being identified and tested for therapeutic potential, including compounds derived from loop diuretics, metal-containing organic compounds, plant natural products, and other small molecules. Further studies on aquaporin-dependent functions in cancer metastasis are needed to define the differential contributions of different classes of aquaporin channels to regulation of fluid balance, cell volume, small solute transport, signal transduction, their possible relevance as rate limiting steps, and potential values as therapeutic targets for invasion and metastasis.

#### Edited by:

Graça Soveral, Universidade de Lisboa, Portugal

#### Reviewed by:

Ali Mobasheri, University of Surrey, United Kingdom Miriam Echevarria Irusta, Universidad de Sevilla, Spain

> \*Correspondence: Andrea J. Yool<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Chemical Biology, a section of the journal Frontiers in Chemistry

Received: 15 February 2018 Accepted: 09 April 2018 Published: 25 April 2018

### Citation:

De Ieso ML and Yool AJ (2018) Mechanisms of Aquaporin-Facilitated Cancer Invasion and Metastasis. Front. Chem. 6:135. doi: 10.3389/fchem.2018.00135 Keywords: aquaporin, cell migration, metastasis, cancer, invasion, pharmacology, drug

### INTRODUCTION

### Aquaporins

Aquaporins (AQPs) are a family of water channels that also include a subset of classes shown to mediate transport of glycerol, ions, and other molecules (Li and Wang, 2017). The first aquaporin to be cloned, aquaporin-1 (AQP1), was identified in red blood cells and renal proximal tubules (Denker et al., 1988; Preston and Agre, 1991). In the Xenopus laevis expression system, introduced AQP1 channels enabled high osmotic water flux across the plasma membrane as compared to non-AQP control oocytes (Preston et al., 1992), explaining the mechanism enabling rapid transmembrane passage of water in certain types of cells. To date, 15 classes of aquaporin genes have been identified in mammals (AQP0–AQP14), with AQPs 13 and 14 found in older lineages of mammals (Metatheria and Prototheria) (Ishibashi et al., 2009; Finn et al., 2014; Finn and Cerda, 2015). The first 13 aquaporins (AQP0–AQP12) have been divided into categories based on functional properties (Li and Wang, 2017).  A second category consists of the aquaglyceroporins (AQP3,−7,−9, and−10), which are permeable to water and glycerol, with some also exhibiting urea, arsenite, and hydrogen peroxide permeability (Ishibashi et al., 1997a, 1998, 2002; Yang and Verkman, 1997; Liu et al., 2002; Lee et al., 2006; Rojek et al., 2008; Miller et al., 2010; Watanabe et al., 2016). A possible third category consists of AQP11 and AQP12, distantly related paralogs with only 20% homology with other mammalian AQPs (Ishibashi, 2009), which appear to carry both water and glycerol (Yakata et al., 2011; Bjørkskov et al., 2017). The permeability of AQP11 to glycerol could be important for its function in human adipocytes, in which it is natively expressed (Madeira et al., 2014). Aquaporins assemble as homo-tetramers, with monomers ranging 26–34 kDa (Verkman and Mitra, 2000). In most AQPs, each monomer is composed of six transmembrane domains and intracellular amino and carboxyl termini, with highly conserved asparagine-proline-alanine (NPA) motifs in cytoplasmic loop B and in extracellular loop E (Jung et al., 1994). The NPA motifs in loops B and E contribute to a monomeric pore structure that mediates selective, bidirectional, single-file transport of water in the classical aquaporins (Sui et al., 2001), and water and glycerol in aquaglyceroporins (Jensen et al., 2001).

Intracellular signaling processes regulate AQP channels by altering functional activity, intracellular localization, and levels of expression in different cells and tissues. For example, the peptide hormone vasopressin regulates excretion of water in the kidney by augmenting water permeability of collecting duct cells. Vasopressin induces phosphorylation of AQP2 (Hoffert et al., 2006), stimulating the reversible translocation of AQP2 from intracellular vesicles to the apical plasma membrane (Nielsen et al., 1995). Guanosine triphosphate (GTP) stimulates AQP1-induced swelling of secretory vesicles in the exocrine pancreas (Cho et al., 2002), with functional implications in pancreatic exocrine secretions. Additionally, AQP1 ion channel activity is activated by intracellular cGMP (Anthony et al., 2000), and phosphorylation of Y253 in the carboxyl terminal domain regulates responsiveness of AQP1 ion channels to cGMP (Campbell et al., 2012). Given the diverse array of functional properties, mechanisms of regulation, and tissuespecific distributions being discovered for aquaporins, it is not surprising that different classes of aquaporins (AQP-1,−2,−3,−4,−5,−8, and−9) have been implicated specifically in the complex steps associated with cancer invasion and metastasis (**Table 1**), suggesting specialized roles for these channels have been arrogated into the pathological processes.

### Cancer Invasion and Metastasis

Cancer is a leading cause of death worldwide, accounting for 8.2 million deaths in 2012 (Ferlay et al., 2015). The incidence of cancer is rising steadily in an aging population, with numbers expected to increase 70% in the next two decades (Ferlay et al., 2015). Current treatments involve chemotherapy, radiation therapy, and surgery (Miller et al., 2016), associated with an array of side effects including nausea (Koeller et al., 2002), impaired fertility and premature menopause (Howard-Anderson et al., 2012; Wasilewski-Masker et al., 2014), painful neuropathy (Gamelin et al., 2002; Rivera and Cianfrocca, 2015), increased risk of cardiovascular disease (Monsuez et al., 2010; Willemse et al., 2013), and loss of bone density (Gralow et al., 2013). Inhibiting proliferation remains the primary focus of cancer treatments, although the predominant cause of death is cancer metastasis (Yamaguchi et al., 2005; Spano et al., 2012). Less devastating cancer therapies might be achievable via a combination of strategies that not only inhibit proliferation, but also control metastasis of tumor cells from their primary site to distant organs (Friedl and Wolf, 2003). Cancer cell migration through the body exploits pathways including blood stream, lymphatic system, and transcoelomic movement across body cavities (Wyckoff et al., 2000; Pepper et al., 2003; Tan et al., 2006). The hierarchical nature of the metastatic cascade suggests it should be vulnerable to intervention at multiple levels including angiogenesis, detachment of cells from the primary tumor, and infiltration of dissociated tumor cells into and out of circulatory pathways via intravasation and extravasation, respectively (**Figure 1**). AQPs that serve as rate-limiting steps in the metastatic cascade should have substantial value as prognostic markers and pharmacological targets for treatments.

### ANGIOGENESIS

Both cancer invasion and metastasis are enhanced by angiogenesis. Angiogenesis, activated in response to inadequate oxygen perfusion, triggers extracellular matrix breakdown; endothelial cell proliferation, differentiation, and migration; and recruitment of periendothelial cells (Clapp and de la Escalera, 2006) which form discontinuous layers around vessels and exert developmental and homeostatic control (Njauw et al., 2008). Under physiological conditions, angiogenesis is seen in the proliferative phase of the menstrual cycle (Demir et al., 2010), development of fetal and placental vasculature (Demir et al., 2007), and skeletal muscle following physical activity (Egginton, 2009). In pathological scenarios such as tumorigenesis, tissue hypoxia stimulates the formation of new vasculature, enabling tumors to better obtain nutrients, exchange gases, and excrete waste (Nishida et al., 2006). Folkman et al. (1966) showed that tumors up to 2 mm in diameter could survive via passive diffusion from surrounding tissue; but angiogenesis was essential for support of larger tumors.

#### TABLE 1 | Key roles of AQPs involved in cancer invasion and metastasis.

(Continued)

#### TABLE 1 | Continued

tumors with tissue hypoxia rely on angiogenesis for vascular exchange of nutrients and waste. Primary tumor cells undergo phenotypic changes including loss of cell-cell adhesions which enables cells to dissociate from primary tumor, invade the adjacent extracellular matrix (ECM), and intravasate into the blood or lymph systems. Circulating tumor cells extravasate to seed secondary sites at which the process can reoccur.

AQP1, expressed in peripheral vascular endothelial cells, is involved in tumor angiogenesis (Nielsen et al., 1993; Endo et al., 1999; Saadoun et al., 2002a; El Hindy et al., 2013; Verkman et al., 2014). AQP1 knock-down in chick embryo chorioallantoic membrane resulted in a dramatic inhibition of angiogenesis (Camerino et al., 2006). Saadoun et al. (2005a) found AQP1-deficient mice exhibited reduced tumor growth and angiogenesis as compared to wild type, following subcutaneous or intracranial B16F10 melanoma cell implantation. Their work showed AQP1-null endothelial cells from mouse aorta had reduced motility as compared to wild-type, suggesting AQP1 was needed to facilitate cell migration for angiogenesis. Monzani et al. (2009) confirmed a reduced migration capacity in human microvascular endothelial cells (HMEC-1) after AQP1 knockdown by siRNA. AQP1 mRNA and protein levels are increased in response to tissue hypoxia (Kaneko et al., 2008; Abreu-Rodríguez et al., 2011). AQP1 facilitates hypoxia-induced angiogenesis by enhancing endothelial cell migration.

Angiogenesis is regulated by growth factors such as vascular endothelial growth factor (VEGF), which stimulates endothelial cell proliferation and angiogenesis in response to hypoxia (Suzuki et al., 2006), through processes that could augment AQP1 activity indirectly. Pan et al. (2008) found a positive correlation between levels of AQP1 expression, intratumoral microvascular density, and VEGF in endometrial adenocarcinoma. Similarly, AQP1 gene deletion correlated with reduced VEGF receptor expression in mouse primary breast tumor cells (Esteva-Font et al., 2014), and knockdown of AQP1 in human retinal vascular endothelial cells with concurrent inhibition of VEGF caused an additive inhibition of hypoxia-induced angiogenesis (Kaneko et al., 2008). However, application of VEGF-neutralizing antibodies did not alter AQP1 expression (Kaneko et al., 2008), and levels of VEGF in primary breast tumors were not different between AQP1-null and wild-type mice (Esteva-Font et al., 2014), supporting the idea that VEGF is regulated independently of AQP1 expression or activity.

Other angiogenic factors, such as hypoxia-inducible factor 1-alpha (HIF-1α), induce AQP1 expression in low oxygen conditions (Abreu-Rodríguez et al., 2011). The AQP1 gene promoter carries a HIF-1α binding site which drives AQP1 expression in response to hypoxia in cultured human retinal vascular endothelial cells (HRVECs) (Tanaka et al., 2011), and involves phosphorylation of p38 mitogen-activated protein kinase (MAPK) (Tie et al., 2012). Estrogen signaling also targets the promoter region of the AQP1 gene to increase transcription, inducing enhanced tubulogenesis of vascular endothelial cells as a model for angiogenesis (Zou et al., 2013). In summary, AQP1 is upregulated by angiogenic factors in response to hypoxia, and necessary for endothelial cell migration and angiogenesis. Therapies aimed at blocking transcriptional activation of AQP1 could impede cancer angiogenesis, if the treatment could be spatially limited to the tumor site without impacting normal cell functions.

### CELLULAR DISSOCIATION AND EPITHELIAL-MESENCHYMAL TRANSITION

Epithelial-mesenchymal transition (EMT) occurs in normal physiological conditions such as implantation, embryogenesis, and organ development, as well as pathological processes such as cancer invasion and metastasis (Vicovac and Aplin, ´ 1996; Thiery, 2002). During EMT, polarized epithelial cells undergo biochemical changes to adopt a mesenchymal phenotype, characterized by a loss of cell polarity, reduced cell-cell adhesiveness, and enhanced invasive capacity (Thiery, 2002, 2003; Cavallaro and Christofori, 2004; Kalluri and Weinberg, 2009; van Zijl et al., 2011). Epithelial cadherin (E-cadherin), a transmembrane glycoprotein, enables calciumdependent tight adhesions between epithelial cells and links to cytoskeletal elements (Angst et al., 2001; Alizadeh et al., 2014). Downregulation of E-cadherin is a hallmark feature of EMT (Cano et al., 2000; Chua et al., 2007; Korpal et al., 2008). EMT in cancer is induced by signals from the tumorassociated stroma, including epidermal growth factor (EGF), platelet-derived growth factor (PDGF), hepatocyte-derived growth factor (HGF), and transforming growth factor beta (TGF-β) (Miettinen et al., 1994; Pagan et al., 1999; Lo et al., 2007; Kong et al., 2009; Xu et al., 2009). These signals stimulate transcription factors such as SNAI1 (SNAIL), SNAI2 (SLUG), zinc finger E-box binding homeobox 1 (ZEB1), Mothers against decapentaplegic homolog 2 (SMAD-2) and Twist, which are all E-cadherin transcription repressors (Yang et al., 2004; Medici et al., 2008).

Classes of aquaporins such as AQP3 have been implicated in the EMT process. AQP3 up-regulation in response to EGF in colorectal, gastric, and pancreatic cancers, is associated with augmented cell migration, invasion, and metastasis (Huang et al., 2010; Liu et al., 2012; Li et al., 2013). In gastric cancer, EGF-induced AQP3 upregulation enhances the mesenchymal transformation (Chen et al., 2014). Chen et al. (2014) determined that mRNA and protein levels of vimentin and fibronectin (proteins associated with mesenchymal phenotype) were significantly increased in cells with high levels of AQP3 expression but decreased in AQP3-deficient cells. Conversely, E-cadherin expression was significantly lower in cells with high AQP3 and increased in AQP3-knockdown cells. The mechanisms for AQP3 facilitated pancreatic and colorectal cancer cell migration have not yet been determined. It will be interesting to investigate whether AQP3 promotes EMT in these cancers.

In addition to AQP3, AQPs 1, 4, 5, and 9 also have been linked to EMT in different types of cancer cells. In lung adenocarcinoma cells, AQP1 overexpression correlated with the down-regulation of E-cadherin, and up-regulation of vimentin (Yun et al., 2016). AQP4 knockdown in human breast cancer was associated with increased levels of E-cadherin, and in glioma cells with increased β-catenin (involved in actin reorganization and cell-cell adhesion) and connexin-43 (a gap junction protein that contributes to cell-cell signaling and adhesion) (Ding et al., 2011; Li Y. et al., 2016), suggesting AQP4 might enhance cell detachment from primary tumors. However, opposing evidence showed knockdown of AQP4 in primary human astrocytes correlated with down-regulation of connexin-43 (Nicchia et al., 2005); and transfection of wild type AQP4 into glioma cell lines caused enhanced adhesion (McCoy and Sontheimer, 2007). In primary glial cells, AQP4 expression levels had no appreciable effect on cell-cell adhesion under the conditions tested (Zhang and Verkman, 2008). In human non-small cell lung cancer cells (NSCLCs), AQP5 increased invasiveness; conversely, expression of AQP5 mutant channels lacking membrane targeting signals or the S156 phosphorylation site did not augment invasiveness (Chae et al., 2008b). Overexpression of AQP5 in NSCLCs was associated with a reduction in epithelial cell markers such as E-cadherin, αcatenin, and γ-catenin, and an increase in mesenchymal cell markers such as fibronectin and vimentin, concomitant with a mesenchymal change in morphology. Similarly, AQP3 and AQP5 overexpression in pancreatic ductal adenocarcinoma is accompanied by downregulation of E-cadherin and upregulation of vimentin (Direito et al., 2017). The invasion-promoting properties of AQP5 expression appear to depend on the c-Src signaling pathway, a potent trigger of EMT (Guarino et al., 2007; Chae et al., 2008b). High AQP5 expression correlated with an increase in phosphorylated SMAD2, promoting EMT in colorectal cancer, whereas AQP5 silencing was associated with a down-regulation of phosphorylated SMAD2, and a repressed EMT response (Chen et al., 2017). AQP9 is downregulated in hepatocellular carcinoma; overexpression corresponds to reduced growth and EMT, thus reducing cancer invasion and metastasis (Li C. F et al., 2016; Zhang et al., 2016). Evidence suggests that AQPs have different effects depending on the type of cancer. Moreover, the state of cancer progression, environmental factors, and the types of assays used will be complicating factors; nevertheless, AQPs have clear potential as diagnostic and prognostic

biomarkers, and as therapeutic targets for modulation of EMT, cell-cell adhesion, and dissociation phases of cancer progression.

## INVASION AND CELL MIGRATION

Cell migration involves the translocation of individual and collective groups of cells through fluid or tissues, relevant for survival in multicellular and single-celled organisms (Klausen et al., 2003; Friedl et al., 2004). Migration enables physiological morphogenesis, immunity, and tissue repair (Friedl et al., 2004; Friedl and Weigelin, 2008). In most mammalian cells, migration is highest during development and morphogenesis and decreases after terminal differentiation. In pathological circumstances such as cancer, migration machinery can be reactivated. AQPs−1,−3,−4, and−5,−8, and−9 are known to contribute to cancer cell migration and invasion. Translocation of cancer cells can be initiated by chemokines released from host tissues, and growth factors such as EGF secreted by stromal cells (Dittmar et al., 2008; Roussos et al., 2011).

AQP3 has been suggested to increase EGF-induced cancer growth and migration by mediating H2O2 flux (Miller et al., 2010; Hara-Chikuma et al., 2016). H2O2 is known as an oxidative stressor, but is also a second messenger in cell proliferation, differentiation and migration (Thannickal and Fanburg, 2000; Rhee, 2006). AQP3 knockdown in skin and lung cancer cell lines reduced EGF-induced H2O2 influx, and attenuated EGF signaling cascades (Hara-Chikuma et al., 2016), reducing migration and growth. H2O2 also influenced chemokine-dependent migration of T-cells and breast cancer cells (Hara-Chikuma et al., 2012; Satooka and Hara-Chikuma, 2016). AQP1,−3,−5,−8, and−9 have all been suggested to transport H2O2 (Bienert et al., 2007; Miller et al., 2010; Almasalmeh et al., 2014; Rodrigues et al., 2016; Watanabe et al., 2016). All of these classes also have been linked with cancer cell migration (Hu and Verkman, 2006; Shi et al., 2013; Li et al., 2014; Chen et al., 2015; Zhang et al., 2016); however, H2O2 transport has thus far been linked only to AQP3 as a control mechanism in cancer cell migration. Further work might show H2O2 transport in other classes of AQPs regulates cell motility and invasion.

### Polarization

Key molecular and cellular events involved in cell migration can be classified into five inter-dependent stages, which are polarization, protrusion, cell-matrix adhesion, extracellular matrix (ECM) degradation and retraction (**Figure 2**). Cell polarization provides functionally specialized domains in the membrane and cytoplasm (Drubin and Nelson, 1996), typified by asymmetric distributions of organelles, signaling mechanisms, and membrane channels, transporters and receptors (Swaney et al., 2010). In movement, changes in cell polarization generate leading and trailing edges, predominantly regulated by small GTPases such as CDC42 (Johnson and Pringle, 1990; Allen et al., 1998), which controls the recruitment of partitioningdefective (PAR) proteins, atypical protein kinase C (aPKC), and actin polymerization machinery (Etienne-Manneville and Hall, 2003; Goldstein and Macara, 2007). AQPs−1,−4,−5, and−9 have been shown to show polarized localization at the leading edges of migrating cells. Specific co-distributions with ion transporters such as the Na+/H+ exchanger, the Cl−/HCO− 3 exchanger, and the Na+/-HCO3 co-transporter, suggest sophisticated mechanisms for regulation of fluid influx and efflux (Loitto et al., 2002; Verkman, 2005; Hara-Chikuma and Verkman, 2006; Papadopoulos et al., 2008; Stroka et al., 2014), potentially driving membrane protrusions for cell locomotion (Schwab et al., 2007).

### Protrusion

A migrating cell extends its leading edge into the ECM by assembling a branched network of intracellular actin filaments, predicted to yield a physical force that dynamically pushes the membrane out, alternating with relaxation and actin depolymerization (Wang, 1985; Theriot and Mitchison, 1991; Pollard and Borisy, 2003). Membrane expansion requires the vesicle fusion to support the increase in surface area (Bretscher and Aguado-Velasco, 1998; Pierini et al., 2000; Fletcher and Rappoport, 2010). Three types of protrusions found in motile cells are lamellipodia, filopodia, and invadopodia. Lamellipodia are broad, flat, actin-rich protrusions that extend in the direction of locomotion and provide a foundation on which the cell moves forward (Cramer et al., 1997). Filopodia are long, thin protrusions of the membrane thought to be exploratory, "sensing" the local environment (Mattila and Lappalainen, 2008). Lamellipodial and filopodial formations are modulated by small GTPases in the Rho family, such as Rac1 and CDC42 (Ridley et al., 1992; Allen et al., 1997; Hall, 1998; Machesky, 2008), which stimulate actin polymerization in response to growth factor (Hall, 1998) and integrin receptor activations (Price et al., 1998). Interestingly, AQP9-facilitated water flux appears to critical for filopodial protrusion formation in fibroblasts, via the CDC42 pathway (Loitto et al., 2007). The Arp2/3 (actin-related protein 2/3) complex regulates the formation of new actin filaments in migrating cancer cells, and is regulated by Scar/WAVE complex (otherwise known as WANP), which interacts with the small GTPase Rac1 for lamellipodial assembly (Ibarra et al., 2005). Invadopodia are actin-rich, matrix-degrading protrusions that appear when ECM degradation and cell adhesion are needed to create space for movement, involving proteases such as MMP2, MMP9, and MT1-MMP and src tyrosine kinase (Weaver, 2006). Changes in cell volume during protrusion are assumed to require rapid water flow (Condeelis, 1993), and could occur in part in response to osmotic gradients governed by ion transport and actin polymerization state (Diez et al., 2005; Disanza et al., 2005; Schwab et al., 2007).

AQPs at the leading edges of migrating cells are well positioned to facilitate cell volume changes and cytoskeletal modifications during protrusion formation (Monzani et al., 2009; Jiang and Jiang, 2010; Klebe et al., 2015; Wei and Dong, 2015; Pelagalli et al., 2016). AQP1 overexpression in B16F10 melanoma cells and 4T1 mammary gland tumor cells enhanced cell migration and lamellipodial width in vitro, and augmented metastasis in a mouse model (Hu and Verkman, 2006). AQP1 is proposed to enhance lamellipodial formation by increasing membrane osmotic water permeability (Verkman, 2005; Hu

FIGURE 2 | Key contributions of aquaporins in cell migration. (A) Forward movement is preceded by establishing specialized loci within the cell, with redistribution of aquaporins, ion transporters/exchangers, and actin polymerization machinery to the leading edge. AQP-1,−4,−5, or−9 can be found on leading edges of migrating cancer cells. (B) Protrusions of the membrane might use water influx (down an osmotic gradient established by ion transporters/exchangers) and actin polymerization beneath the plasma membrane to dynamically push the membrane forward. AQP-1,−4, and−5 are implicated in water influx for protrusion extension in cancer cells; AQPs-1 and−4 also appear to interact with actin cytoskeleton. (C) Protrusions adhere to the ECM using integrin to generate "traction" for cellular movement. AQP2 might modulate turnover of integrin at adhesion sites, enabling forward cellular movement. (D) ECM degradation by enzymes can widen gaps through which the cell body can penetrate. AQP-1,−3,−4 and−9 are suggested to interact with ECM-degrading enzymes. (E) The final step is retraction of the cell trailing edge, thought to use aquaporins for water efflux following by K+ export.

and Verkman, 2006; Jiang, 2009), allowing water entry at the leading edge to impose hydrostatic pressure, drive membrane extension, and create space for actin polymerization. In addition to water channel activity, AQP1 is also thought to be an ion channel, proposed to allow gated conduction of monovalent cations through the central tetrameric pore (Anthony et al., 2000; Yu et al., 2006). The dual water and ion conductance of AQP1 is essential for colon cancer cell migration in vitro (Kourghi et al., 2015). Conversely, in clinical cases of cholangiocarcinoma, high AQP1 expression has been correlated with low metastasis (Aishima et al., 2007; Sekine et al., 2016), suggesting that AQP1 might play different roles in different types of cancers.

Other classes of AQP water channels are not necessarily interchangeable with AQP1 in facilitating cell migration (McCoy and Sontheimer, 2007), suggesting features of AQP1 other than simple osmotic water permeability are involved. AQP1-enhanced cell migration might also be due to interactions with cytoskeletal proteins. For example, Monzani et al. (2009) demonstrated that AQP1 knockdown dramatically impeded actin cytoskeletal organization in migrating human melanoma and endothelial cell lines via interaction with Lin-7/β-catenin. The Lin-7/β-catenin complex enables asymmetrical organization of filamentous actin (F-actin). AQP1 might act as a scaffolding protein at the leading edges. Jiang (2009) found that knocking down AQP1 was associated with re-localization of actin in migrating HT20 colon cancer cells, and a reduction in the activity of actin regulatory factors RhoA and Rac. A PDZ domain in Lin-7 could mediate interaction with rhotekin protein, which inhibits Rho GTPase signaling that is involved in cell migration, invasion, and cytoskeletal reorganization (Sudo et al., 2006). Rhotekin merits further evaluation in models of AQP1-dependent cytoskeletal organization.

A role for AQP4 in glioma cell migration has similarly been proposed to occur through regulation of cell volume and cytoskeletal interactions. Protein kinase C (PKC)-mediated phosphorylation of AQP4 at serine 180 correlated with a decreased glioma cell invasion (McCoy et al., 2010). AQP4 facilitated glioma invasion is dependent on co-expression of chloride channels (ClC2) and the potassium-chloride cotransporter 1 (KCC1) in invadopodia, which could provide the ionic driving force for water efflux leading to cell shrinkage that could augment invasiveness through ECM (Mcferrin and Sontheimer, 2006; McCoy et al., 2010). AQP4 effects on actin cytoskeleton suggest a role for α-syntrophin, interacting with the C-terminal domain of AQP4 at a PDZ-binding site (Neely et al., 2001). In human glioma and primary astrocytes, reduced AQP4 expression correlated with dramatic morphological elongation, reduced invasiveness, and impaired F-actin polymerization (Nicchia et al., 2005; Ding et al., 2011).

AQP5 facilitates protrusion formation, volume regulation, cell migration, and metastasis.  Moreover, Jung et al. (2011) showed that a shRNA-induced reduction in AQP5 expression in MCF7 breast cancer cells was associated with significantly reduced cell proliferation and migration. The mechanism of AQP5-facilitated cancer cell invasion and metastasis might be due to its direct or indirect interaction with the epidermal growth factor receptor/extracellular signal-regulated kinase (ERK1/2) pathway (Kang et al., 2008; Zhang et al., 2010), known to be important in cancer metastasis and aggressiveness (Vicent et al., 2004). Additionally, AQP5 mediates lung cancer cell membrane osmotic water permeability, and has been suggested to contribute to cancer cell migration and invasion by enabling rapid cell volume regulation and subsequent protrusion formation (Chen et al., 2011). The complementary role of ion transport for migration in AQP5-expressing cells was supported by Stroka et al. (2014), who found that cell migration through physically confined spaces occurred despite block of actin polymerization and myosin contraction, but relied on co-expression of the Na+/H+ exchanger with AQP5, supporting AQP5-induced cell volume regulation and its importance in cell motility.

 Further work is needed to investigate whether mechanisms of AQP3- and AQP8 facilitated cancer cell migration and invasion involve cell volume regulation, protrusion formation, cytoskeletal interaction, or other functional properties of the AQP channels that remain to be defined.

### Cell-Matrix Adhesion

Cell-matrix adhesions, first observed in cultured fibroblasts, connect the extracellular matrix to the actin cytoskeleton (Curtis, 1964). During migration, contacts with substratum must form to facilitate extension, and must detach to allow forward displacement of the cell. Insufficient anchoring causes protrusions to collapse, leading to a "membrane ruffling" phenomenon (Vicente-Manzanares and Horwitz, 2011). Protrusions adhere to ECM via integrin receptors, in turn linked to intracellular actin filaments (Ridley et al., 2003). The extracellular binding of integrin receptors to ECM ligands initiates integrin clustering, and activates protein tyrosine kinases and small GTPases. The organization of actin cytoskeleton and cell polarity controls the positions of focal adhesions for cell locomotion (Geiger et al., 2001; Martin et al., 2002). Cell-matrix adhesions create the focal points for generation of traction to pull the cell forward over the substratum.

Classes of aquaporins (AQP1-4) have been shown to interact with adhesion molecules and to influence adhesive properties of migrating cells. Increased AQP1 in mesenchymal stem cells enhances migration by a mechanism involving β-catenin and the focal adhesion kinase (FAK) (Meng et al., 2014), which regulates integrin signaling at focal adhesion sites (Schaller et al., 1992; McLean et al., 2005; Zhao and Guan, 2011). Whether AQP1 and FAK also interact in cancer cell migration remains to be tested. AQP2 appears to promote cell migration by modulating integrin β1 at focal adhesion sites, by a mechanism thought to involve an arginine-glycine-aspartate (RGD) motif in the second extracellular loop of AQP2 (Chen et al., 2012). When AQP2 is absent, integrin β1 is retained at focal adhesion sites, delaying recycling of focal adhesions, thus reducing migration rate. AQP2 also enables estradiol-induced migration and adhesion of endometrial carcinoma cells by mechanisms involving annexin-2 and reorganization of F-actin (Zou et al., 2011). Knockdown of AQP3 in human esophageal and oral squamous cell carcinoma with siRNA correlated with reduced phosphorylation of FAK, impaired cell adhesion and cell death (Kusayama et al., 2011); these effects would be predicted to impair cancer cell migration. AQP4 expression has been suggested to enhance cell-matrix adhesion in cancer cells (McCoy and Sontheimer, 2007). More research is needed to identify the intracellular signaling mechanisms and to determine whether other AQP classes alter cell migration via modulation of cell adhesion.

### ECM Degradation

Extracellular matrix degradation widens pathways through which cells can penetrate tissues, and reduces the distortion of the rounded cell body needed for physical progress (Brinckerhoff and Matrisian, 2002; Mott and Werb, 2004). Invadopodia sprout from leading edge filopodia, extending through tiny channels in the ECM, and adhere to ECM collagen fibers (Weaver, 2006; Friedl and Wolf, 2009). To accommodate displacement of the cell body, constraining ECM fibers are cleared by local proteolysis, using surface proteases such as zinc-dependent matrix metalloproteinases (MMP) and serine proteases (Nagase and Woessner, 1999; Netzel-Arnett et al., 2003; Wolf et al., 2007). AQPs−1,−3,−4, and−9 have been shown to interact with specific MMPs to facilitate ECM degradation and invasion.

In lung cancer cells, migration was facilitated by AQP1 expression, linked to expression of MMP2 and MMP9 (Wei and Dong, 2015). In gastric cancer cells (SGC7901), AQP3 levels were correlated with MMP2, MMP9, and MT1-MMP levels, and enhanced invasiveness via phosphoinositide 3-kinase signaling (Xu et al., 2011). Positive correlations between AQP3, MMP2, and MMP9 and cancer invasiveness also occur in lung cancer (Xia et al., 2014; Xiong et al., 2017). In prostate cancer, AQP3 expression is correlated with up-regulation of MMP3 via ERK1/2 signaling, with increased cell motility and invasion (Chen et al., 2015). In glioma, AQP4 levels correlated with migration and invasiveness in vitro and in vivo through a mechanism involving MMP2 (Ding et al., 2011). AQP9 upregulation in prostate cancer could enhance growth, migration, and invasion involving ERK1/2 signaling; reduced levels of phosphorylated ERK1/2 and MMP9 were observed in AQP9-deficient cell lines (Chen et al., 2016). These studies suggest one of the key components of AQPmediated facilitation of cancer cell invasion is the regulation of MMP proteases needed for degradation of ECM.

### Retraction

Following integrin-ligand binding, cross-linking proteins such as myosin II contract the actin filament strands (Vicente-Manzanares et al., 2009), developing tension against the intact adhesion points (Chrzanowska-Wodnicka and Burridge, 1996). The final step in the cycle of cell movement is retraction of the trailing edge. A working model is that membrane tension opens stretch-activated Ca2+ channels, activating calpain and triggering disassembly of focal adhesion proteins on the trailing edge, while concurrent K+ efflux drives volume loss at the cell rear, resulting in detachment and net translocation along the substrate. In this model, the role of AQP channels is to facilitate osmotic water efflux in response to K+ efflux (Huttenlocher et al., 1997; Palecek et al., 1998; Schwab et al., 2007) presumably in parallel with electroneutral efflux of chloride ions.

### AQP PHARMACOLOGY AND THERAPEUTIC IMPLICATIONS IN CANCER INVASION AND METASTASIS

Aquaporin pharmacological agents have attracted keen interest for their potential therapeutic uses in diseases involving impaired fluid homeostasis. Aquaporins in cancer metastasis are new translational targets for AQP modulators. Known and proposed inhibitors of AQPs include cysteine-reactive metals such as mercury (II) chloride (HgCl2) (Preston et al., 1993), goldbased compounds (Martins et al., 2013), carbonic anhydrase inhibitor acetazolamide (Ma et al., 2004a; Gao et al., 2006), and small molecule inhibitors such as tetraethylammonium (TEA+) (Brooks et al., 2000), although the small molecule blockers vary in efficacy between preparations. The pharmacological panel for AQPs has been expanding steadily, with new compounds being discovered around the world, including for example the University of Niigata, Japan (Huber et al., 2009), Radboud University, Netherlands (Detmers et al., 2006), the Faculty of Pharmacy, University of Lisbon, Portugal (Martins et al., 2012), the Institute of Food and Agricultural Research and Technology, Barcelona, Spain (Seeliger et al., 2012), the University of Adelaide, Australia (Niemietz and Tyerman, 2002; Yool, 2007), the University of Groningen, Netherlands (Martins et al., 2013), the University of Kiel, Germany (Wu et al., 2008), and others. This review focuses specifically on selected AQP pharmacological agents that to date have been tested in models of cancer cell migration and metastasis (**Table 2**).

### Acetazolamide and Topiramate

Acetazolamide and topiramate are FDA-approved drugs that inhibit carbonic anhydrase. Acetazolamide at 100 µM was reported to inhibit water channel activity by 39% for AQP1 expressed in human embryonic kidney (HEK293) cells (Gao et al., 2006), and by 81% at 10µM in the Xenopus oocyte expression system (Ma et al., 2004a). AQP4 activity was inhibited by 47% at 1,250µM in proteoliposomes (Tanimura et al., 2009). However, acetazolamide (at doses up to 10,000 µM) did not block water flux in erythrocytes with native AQP1 expression, or epithelial cells transfected with AQP1 (Yang et al., 2006; Søgaard and Zeuthen, 2008). Acetazolamide inhibited angiogenesis in a chick chorioallantoic membrane assay, and tumor growth and metastasis in mice with Lewis lung carcinoma (Xiang et al., 2002, 2004), perhaps as a result of reduced AQP1 expression (Bin and Shi-Peng, 2011). Topiramate reduces Lewis lung carcinoma growth and metastasis, with effects similarly attributed to suppression of AQP1 expression (Ma et al., 2004b). It will be of interest to compare the effects of acetazolamide and topiramate on angiogenesis, tumor growth, and metastasis with those of AQP1 channel inhibitors.

### Tetraethylammonium

TEA+ is an inhibitor of voltage-gated potassium channels, calcium-dependent potassium channels, the nicotinic acetylcholine receptor, and it has also been shown to block AQP-1,−2, and−4 water permeability in Xenopus laevis oocytes and kidney derived cell lines (Brooks et al., 2000; Yool et al., 2002; Detmers et al., 2006). However, inhibition of AQP1 water permeability by TEA+ is variable, having been confirmed by some groups (Detmers et al., 2006), and challenged by others (Søgaard and Zeuthen, 2008). Yang et al. (2006) reported no block of water flux by TEA+ in erythrocytes with native AQP1, or in epithelial cells transfected with AQP1, and suggested previous positive results might have been due to inhibition of K+ channels and altered baseline cell volume; however, the observation that site-directed mutation of AQP1 altered TEA sensitivity (Brooks et al., 2000) ruled out this alternative explanation. TEA+ block of AQP1 water permeability reduced cell migration and invasion in in vitro models of osteosarcoma and hepatocellular carcinoma (Pelagalli et al., 2016), with outcomes interpreted as consistent with action of TEA+ as a possible AQP1 inhibitor. However, given the variability in efficacy and cross-talk with other channels, TEA+ is not an ideal candidate for clinical development, although the targets causing the observed block of cancer cell migration and invasion might merit further investigation.

### Bumetanide Derivatives

Bumetanide is a sulfamoylanthranilic acid derivative used clinically to increase diuresis by blocking sodium cotransporter activity at the loop of Henle in the nephron. Molecular derivatives of bumetanide have been synthesized and found to exhibit inhibitory effects on classes of AQP channels. For example, the bumetanide derivative AqB013 blocks osmotic water fluxes mediated by mammalian AQP1 and AQP4 channels expressed in Xenopus laevis oocytes (Migliati et al., 2009). The water channel blocker AqB013 was shown to inhibit endothelial tube formation and colon cancer cell migration and invasion in vitro (Dorward et al., 2016). Other bumetanide derivatives, AqB011 and AqB007, block the AQP1 ion conductance, but not water flux (Kourghi et al., 2015). In AQP1, the central tetrameric pore is thought to be permeable to monovalent cations, CO2, and NO (Nakhoul et al., 1998; Herrera et al., 2006; Yu et al., 2006; Musa-Aziz et al., 2009), although some work questioned AQP1-mediated CO2 and cation transport properties (Yang et al., 2000; Fang et al., 2002; Tsunoda et al., 2004). An ionic conductance in AQP1-expressing Xenopus oocytes stimulated with forskolin was first reported in 1996 (Yool et al., 1996); however, the forskolin response proved to be inconsistent when repeated by other groups (Agre et al., 1997). Further work showed the forskolin effect was indirect; the direct regulation of the AQP1 cation conductance depended on cGMP binding (Anthony et al., 2000). The reason that AQP1 cation channels have low opening probability (Saparov et al., 2001) or are not detectable (Tsunoda et al., 2004) reflects the availability of AQP1 to be gated by cGMP, which depends on tyrosine phosphorylation status of the carboxyl terminal domain, suggesting the AQP1 ion channel function is highly regulated (Campbell et al., 2012). With the discovery of AQP1 TABLE 2 | Summary of AQP pharmacology used in cancer invasion and metastasis.

(Continued)

#### TABLE 1 | Continued

ion blocking agents, AqB011 and AqB007, the physiological function of the ion channel activity could finally be addressed. When applied to AQP1-expressing HT29 colon cancer cells, these inhibitory compounds significantly reduced cancer cell motility (Kourghi et al., 2015), suggesting a physiological role of AQP1 ion conductance in cell migration. Mutation of the candidate binding site in the AQP1 intracellular loop D domain removed sensitivity to AqB011, showing that the inhibitory mechanism directly involved the AQP1 channel and could not readily be attributed to off-target actions on other channels or transporters (Kourghi et al., 2018). Another bumetanide derivative AqB050 was shown to inhibit mesothelioma cell motility and metastatic potential in vitro, but not in vivo (Klebe et al., 2015). The mechanism of action of AqB050 in blocking mesothelioma cell motility in vitro remains to be determined.

### Plant-Based Derivatives

Plant-based derivatives that reduce cancer cell migration and invasion include agents that have also have been found to inhibit AQPs. Bacopa monnieri is a perennial herb native to the wetlands of India that is used in alternative medicinal therapies. Chemical constituents bacopaside-I and bacopaside-II, were shown to block AQP1 but not AQP4 water channels (Pei et al., 2016). Pei and colleagues also found that bacopaside-I and bacopaside-II attenuated migration of colon cancer cell lines expressing high levels of AQP1, but had no effect on lines with low AQP1, suggesting the inhibitory effects were AQP1 specific. Ginsenoside Rg3 from a traditional Asian medicinal plant Panax ginseng is an intriguing candidate for possible antimetastatic therapies. Ginsenoside Rg3 inhibited prostate cancer cell migration and was associated with downregulation of AQP1 expression via the p38 MAPK pathway and transcription factors (Pan et al., 2012). Effects of Ginsenoside Rg3 directly on water channel activity, or on expression levels of other aquaporins, remain unknown. Curcumin is a naturally occurring ingredient in turmeric, used as therapeutic tool for pathologies including cancer (Gupta et al., 2013). Curcumin was found to inhibit EGF-induced upregulation of AQP3 and migration in human ovarian cancer cells, via inhibition of AKT/ERK and PI3K pathways (Ji et al., 2008); however, curcumin affects a number of biochemical pathways and might not be suited when AQPspecific modulation is required (Aggarwal et al., 2003). Research on the effects of curcumin in other cancers such as gastric cancer, in which EGF-induced AQP3 up-regulation occurs, might further understanding of the role of AQP3 in cell migration and invasion (Huang et al., 2010).

### Metal-Based Inhibitors

Mercury has classically been used as an AQP1 inhibitor. In the human AQP1 monomer, the NPA motif in loop E is near cysteine 189, which is the site at which mercury inhibits osmotic water permeability (Preston et al., 1993). Lack of a cysteine in the corresponding position is consistent with mercury insensitivity in mammalian AQP4 (Preston et al., 1993). However, mercury is not a promising candidate for AQP-specific modulation or therapeutic application due to its toxicity and non-specific sideeffects. Metal-based inhibitors that have been tested in models of cancer include AQP3 inhibitors such as NiCl2 (Zelenina et al., 2003) and CuSO4 (Zelenina et al., 2004), which inhibited EGF-induced cell migration in human ovarian cancer cells. Auphen is a gold-based compound which, when administered at concentrations of 100µM, blocks AQP3 glycerol transport by 90%, and water transport by 20% in human red blood cells (Martins et al., 2012). Auphen also blocks proliferation in various mammalian cell lines, including human epidermoid carcinoma, by inhibiting AQP3 glycerol transport (Serna et al., 2014). This merits more research into the importance of AQP3-facilitated glycerol transport in cancer invasiveness, and whether gold-based compounds such as auphen can also be used to suppress cancer invasion and metastasis.

## CONCLUSION

Aquaporin-dependent mechanisms serve as key steps throughout the process of metastasis, in angiogenesis, cellular dissociation, cell migration and invasion. AQPs−1,−2,−3,−4,−5,−8, and−9 contribute to one or more processes, generally potentiating cancer invasion and metastasis by boosting tumor angiogenesis, enhancing cell volume regulation, regulating cell-cell and cellmatrix adhesions, interacting with the actin cytoskeleton, regulating proteases and ECM degrading molecules, contributing to the regulation of epithelial-mesenchymal transition in cancer cells, and interacting with specific signaling pathways important in cancer cell motility and invasions. Pharmacological agents for aquaporin channels have therapeutic promise for improving cancer treatment, and include derivatives of bumetanide, organic metal compounds, plant medicinal agents, and other small molecule compounds. Although conflicting evidence has been raised for some compounds, there is nevertheless a compelling need to continue identifying novel candidates for AQP-specific modulators relevant not only for the treatment of cancer, but other pathological conditions. In conclusion, although much remains to be defined for molecular mechanisms in cancer invasion and metastasis, the roles of AQP channel function in cancer progression will inspire new therapeutic targets for improving treatment of malignant and invasive carcinomas.

## AUTHOR CONTRIBUTIONS

MD: wrote the manuscript; AY: reviewed and edited the manuscript.

### FUNDING

This work was supported by a Discovery Project grant from Australian Research Council (ARC DP160104641).

### ACKNOWLEDGMENTS

Thanks to Dr. Jinxin Pei for assisting with molecular structure diagrams (**Table 2**).

### REFERENCES

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in human gastric carcinoma SGC7901 cells. Nat. Rev. Cancer 30:86. doi: 10.1186/1756-9966-30-86

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 De Ieso and Yool. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Identification of Loop D Domain Amino Acids in the Human Aquaporin-1 Channel Involved in Activation of the Ionic Conductance and Inhibition by AqB011

Mohamad Kourghi, Michael L. De Ieso, Saeed Nourmohammadi, Jinxin V. Pei and Andrea J. Yool\*

Aquaporin Physiology and Drug Discovery Program, Adelaide Medical School, University of Adelaide, Adelaide, SA, Australia

### Edited by:

Graça Soveral, Universidade de Lisboa, Portugal

#### Reviewed by: Eric Beitz,

Christian-Albrechts-Universität zu Kiel, Germany Oscar Moran, Istituto di Biofisica (CNR), Italy

\*Correspondence:

Andrea J. Yool<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Chemical Biology, a section of the journal Frontiers in Chemistry

Received: 15 February 2018 Accepted: 12 April 2018 Published: 27 April 2018

#### Citation:

Kourghi M, De Ieso ML, Nourmohammadi S, Pei JV and Yool AJ (2018) Identification of Loop D Domain Amino Acids in the Human Aquaporin-1 Channel Involved in Activation of the Ionic Conductance and Inhibition by AqB011. Front. Chem. 6:142. doi: 10.3389/fchem.2018.00142 Aquaporins are integral proteins that facilitate the transmembrane transport of water and small solutes. In addition to enabling water flux, mammalian Aquaporin-1 (AQP1) channels activated by cyclic GMP can carry non-selective monovalent cation currents, selectively blocked by arylsulfonamide compounds AqB007 (IC50 170µM) and AqB011 (IC50 14µM). In silico models suggested that ligand docking might involve the cytoplasmic loop D (between AQP1 transmembrane domains 4 and 5), but the predicted site of interaction remained to be tested. Work here shows that mutagenesis of two conserved arginine residues in loop D slowed the activation of the AQP1 ion conductance and impaired the sensitivity of the channel to block by AqB011. Substitution of residues in loop D with proline showed effects on ion conductance amplitude that varied with position, suggesting that the structural conformation of loop D is important for AQP1 channel gating. Human AQP1 wild type, AQP1 mutant channels with alanines substituted for two arginines (R159A+R160A), and mutants with proline substituted for single residues threonine (T157P), aspartate (D158P), arginine (R159P, R160P), or glycine (G165P) were expressed in Xenopus laevis oocytes. Conductance responses were analyzed by two-electrode voltage clamp. Optical osmotic swelling assays and confocal microscopy were used to confirm mutant and wild type AQP1-expressing oocytes were expressed in the plasma membrane. After application of membrane-permeable cGMP, R159A+R160A channels had a significantly slower rate of activation as compared with wild type, consistent with impaired gating. AQP1 R159A+R160A channels showed no significant block by AqB011 at 50µM, in contrast to the wild type channel which was blocked effectively. T157P, D158P, and R160P mutations had impaired activation compared to wild type; R159P showed no significant effect; and G165P appeared to augment the conductance amplitude. These findings provide evidence for the role of the loop D as a gating domain for AQP1 ion channels, and identify the likely site of interaction of AqB011 in the proximal loop D sequence.

Keywords: major intrinsic protein, MIP, AQP1, water channel, non-selective cation channel, cyclic GMP, arylsulfonamide

## INTRODUCTION

Aquaporins (AQPs) are a diverse family of channels for water and solutes, classified as major intrinsic proteins (MIPs) (Benga et al., 1986; Agre et al., 1993; Reizer et al., 1993). In mammals, classes of AQPs are differentially expressed in endothelial, epithelial and other cell types, and comprise key components of mechanisms for fluid homeostasis in single cells, barrier tissues, and organs (Nielsen et al., 1993; Boassa and Yool, 2005; Hachez and Chaumont, 2010). Some classes of aquaporin channels have been found shown to transport molecules other than water across the cell membrane, including glycerol, ammonia, urea, protons, as well as CO2 and O2 gases (Madeira et al., 2014; Kitchen et al., 2015), and ions (Yool, 2007; Yool and Campbell, 2012).

Aquaporin ion channel functions have been described for multiple members of the MIP family. Recent work has shown that a plant aquaporin channel (AtPIP2;1) serves as a nonselective cation channel that is sensitive to Ca2+ and pH (Byrt et al., 2016), addressing a mystery regarding the molecular basis of a Ca2+-inhibited leak current known to be involved in environmental stress responses of roots (Demidchik and Tester, 2002). The insect aquaporin Big Brain (BIB) channel in Drosophila (Yanochko and Yool, 2002) and mammalian lens MIP (AQP0) have been characterized as ion channels (Zampighi et al., 1985; Ehring et al., 1990); their importance of these channels is evident from the consequences of genetic knockouts resulting in impaired nervous system development (Rao et al., 1992) and cataract formation (Berry et al., 2000), respectively. However the precise roles of their ion channel activities in cell signaling and development remain to be determined.

Controversy on the role of AQP1 as an ion channel, first proposed in 1996 (Yool et al., 1996), stemmed from a paradigm which stated AQP1 was nothing but a water channel (Tsunoda et al., 2004). An extensive body of work published since has shown: (i) AQP1 is a dual water and cation channel with a unitary conductance of 150 pS under physiological conditions, permeable to Na+, K+, and Cs+, and gated by the binding of cGMP at the intracellular loop D domain (Anthony et al., 2000; Yu et al., 2006). (ii) AQP1 carries water through the individual intra-subunit pores, whereas cations pass through the central pore of the tetramer (Yu et al., 2006; Campbell et al., 2012). (iii) Single channel activity of natively expressed AQP1 is selectively lost after small interfering knockdown of AQP1 expression (Boassa et al., 2006). (iv) The availability of AQP1 to be activated as an ion channel is regulated by tyrosine kinase phosphorylation of the carboxyl terminal domain (Campbell et al., 2012). (v) AQP1 ion channel properties are altered by site-directed mutagenesis of the central pore domain, which changes the cationic selectivity of the current, and creates a gain-of-function blocking site by Hg2+ via introduction of a cysteine residue at the extracellular side (Campbell et al., 2012). (vi) Mutations of the carboxyl terminal domain of hAQP1 alter the efficacy of cGMP in activating the ionic conductance (Boassa and Yool, 2003). (vii) Molecular dynamic simulations confirmed it was theoretically feasible to move Na+ ions through the AQP1 central pore and identified the cytoplasmic loop D domain as involved in gating of the ion channel; mutation of key loop D residues impaired ion channel activation without preventing water channel activity (Yu et al., 2006).

The ability to change specific ion channel properties of activation, ion selectivity, and block using site-directed mutations of the AQP1 amino acid sequence have provided convincing evidence that AQP1 directly mediates the observed ionic current (Anthony et al., 2000; Boassa and Yool, 2003; Yu et al., 2006; Campbell et al., 2012). The alternative suggestion that responses were due to unidentified native ion channels translocated into the membrane along with AQP1 was ruled out by these studies, which showed that the altered ion channel functions associated with mutations of AQP1 did not prevent normal assembly and plasma membrane expression of AQP1 channels as evidenced by immunolabeling, western blot, and measures of osmotic water permeability.

While the ion channel function of AQP1 was confirmed independently by other groups (Saparov et al., 2001; Zhang et al., 2007), the physiological relevance of AQP1 ion channel function remained uncertain, given the low proportion of ion conducting channels observed in reconstituted membrane assays. Mathematical modeling tested the premise, assuming only a tiny fraction of AQP1 acted as ion channels, and showed the predicted effects were sufficient for a meaningful impact on net transport in epithelial cells (Yool and Weinstein, 2002). Interestingly the relative amplitudes of ion currents and water fluxes for mammalian AQP6, also thought to be a dual water and ion channel (Yasui et al., 1999), were similar to those of AQP1, suggesting AQP6 similarly has a low proportion of functioning ion channels within the total population. Although high densities of water channels might be needed to move substantial fluid volumes, the apparently low ratios for aquaporins reinforce a basic concept in the ion channel field; relatively few chargeselective ion channels are needed to alter transmembrane voltage gradients (Hille, 2001).

With development of the first selective AQP1 ion channel inhibitor AqB011 (Kourghi et al., 2016), the question of the physiological function of the AQP1 ion channel could be directly addressed. Kourghi and colleagues showed AqB011 selectively inhibited migration in AQP1-expressing cancer cell lines, but not in those without AQP1, demonstrating that the AQP1 ion conductance can serve an essential role in cellular functions such as migration. Of the pharmacological inhibitors of AQP1 ion channel identified thus far, AqB011 is the most potent (IC50 14µM). Osmotic water fluxes in hAQP1-expressing oocytes were not altered by 200µM AqB011, indicating the block is selective for AQP1 ion channel activity. Molecular docking models suggested loop D domain as a candidate binding site for the AqB011 (Kourghi et al., 2016), but the prediction remained to be tested.

The role of AQP1 loop D residues in ion conductance activation and in mediating block by AqB011 was tested here using site-directed mutations of amino acids. Conserved arginine residues at positions 159 and 160 in human AQP1 were mutated to alanines. As compared with wild type, the cGMP-mediated activation of the AQP1 ionic conductance

FIGURE 1 | Human AQP1 ionic conductances activated by cGMP differ in sensitivity to the inhibitor AqB011 in wild type and R159A+R160A expressing oocytes. (A) Electrophysiology traces showing currents recorded in control non-AQP oocytes, and in hAQP1 wild type and R159A+R160A expressing oocytes. The current traces are shown prior to stimulation (initial), after the first maximal response to CPT-cGMP (1st cGMP), and after the second maximal response (2nd cGMP) following a 2 h incubation with 50µM AqB011 or vehicle (DMSO). (B) Trend plots show the ionic conductance amplitudes for individual oocytes through each series of treatments for AQP1 wild type, mutant, and non AQP-expressing control oocytes, measured before stimulation (initial), after the first CPT-cGMP (1st cGMP), after 2-h recovery in cGMP-free saline containing vehicle or 50µM AqB011 ("incubat"), and after the second CPT-cGMP (2nd cGMP). (C) Compiled box plot data illustrate statistically significant block of AQP1 wild type but not R159A+R160 ion conductances following incubation in 50µM AqB011. n values are above the x-axis. Boxes show 50% of data points; error bars show the full range; horizontal bars show median values. \*\*\*\*p < 0.0001.

response was significantly slower in R159A+R160A channels, the maximal amplitude of the activated current in the mutant construct was reduced as compared to wild type, and the mutant was insensitive to the inhibitor AqB011. Human AQP1 mutant constructs in which proline was substituted for conserved single residues threonine (T157P), aspartate (D158P), arginine (R159P, R160P), and glycine (G165P) showed differential effects on conductance activation depending on position, which suggested the conformation of loop D is important for AQP1 ion channel gating. Proline enables tight bends in peptide structures (Vanhoof et al., 1995). These results support the role of conserved loop D residues in AQP1 ion channel activation and inhibition

FIGURE 2 | Rates of activation of ion conductance responses to CPT-cGMP in oocytes expressing AQP1 wild type or R159A+R160A channels. (A) Ion current responses were monitored after application of CPT-cGMP using repeated series of brief steps to +40, 0, and −80 mV from a holding potential of −40 mV (10 per minute; 150 ms each). Traces are shown at 4 min intervals for clarity. Numbers indicate time in minutes post-application of CPT-cGMP. (B) The plot of steady state current amplitudes at +40 mV as a function of time after application of CPT-cGMP at time zero illustrates the difference in latency to activation in representative examples of AQP1 wild type and R159A+R160A expressing oocytes.

by AqB011, and provide further support for the concept that loop D is a gating domain for the AQP1 central ion pore.

FIGURE 3 | Confirmation of expression of AQP1 wild type and AQP1 R159A+R160 mutant channels in oocyte plasma membranes by significantly increased osmotic water permeabilities as compared to non-AQP1 expressing controls. (A) Osmotic water permeabilities (mean ± SEM) assessed by quantitative swelling assays for AQP1 wild type (open circles) and AQP1 R159A+R160 mutant (squares) compared with non-AQP1 expressing control oocytes (filled circles). Relative volumes as a function of time after introduction into 50% hypotonic saline at time zero were measured from video-imaged cross-sectional areas. (n = 6 per group). (B) Box plot data showing osmotic swelling rates were higher in oocytes expressing AQP1 wild type and AQP1 R159A+R160 mutants than non-AQP1 expressing controls (one way ANOVA; post-hoc Bonferroni tests). \*p < 0.05; \*\*\*\*p < 0.0001; n = 6 per group. Boxes show 50% of data points; error bars show the full range; horizontal bars show median values.

## MATERIALS AND METHODS

### Oocyte Preparation and Injection