Combined Effect of Cold Atmospheric Plasma and Curcumin in Melanoma Cancer.

Curcumin (CUR) has interesting properties to cure cancer. Cold atmospheric plasma (CAP) is also an emerging biomedical technique that has great potential for cancer treatment. Therefore, the combined effect of CAP and CUR on inducing cytotoxicity and apoptosis of melanoma cancer cells might be promising. Here, we investigated the combined effects of CAP and CUR on cytotoxicity and apoptosis in B16-F10 melanoma cancer cells compared to L929 normal cells using MTT method, acridine orange/ethidium bromide fluorescence microscopic assay, and Annexin V/PI flow cytometry. In addition, the activation of apoptosis pathways was evaluated using BCL2, BAX, and Caspase-3 (CASP3) gene expression and ratio of BAX to BCL2 (BAX/BCL2). Finally, in silico study was performed to suggest the molecular mechanism of this combination therapy on melanoma cancer. Results showed that although combination therapy with CUR and CAP has cytotoxic and apoptotic effects on cancer cells, it did not improve apoptosis rate in melanoma B16-F10 cancer cells compared to monotherapy with CAP or CUR. In addition, evaluation of gene expression in cancer cell line confirmed that CUR and CAP concomitant treatment did not enhance the expression of apoptotic genes. In silico analysis of docked model suggested that CUR blocks aquaporin- (AQP-) 1 channel and prevents penetration of CAP-induced ROS into the cells. In conclusion, combination therapy with CAP and CUR does not improve the anticancer effect of each alone.

Xinshuitong Capsule extract attenuates doxorubicin-induced myocardial edema via regulation of cardiac aquaporins in the chronic heart failure rats.

Doxorubicin (Dox), an effective antineoplastic drug, was limited use for cardiotoxicity. Xinshuitong Capsule (XST), a patented herbal formula, showed desirable beneficial effects in the treatment of chronic heart failure (CHF) patients. However, the drug on Dox-induced cardiotoxicity remains unclear. Ninety male Sprague-Dawley rats were randomized into two groups: 15 rats were selected as the normal group and 75 rats were injected intraperitoneally with Dox to establish CHF rat models, the success ones were randomly divided into five groups: low XST (LXST), medium XST (MXST) or high XST (HXST) (4.9, 9.8, or 19.6 g/kg d) administrated intragastrically twice a day for 4 weeks, with the captopril-treated group and the model group as comparison. The model group showed the cardiac functions generally impaired, and CHF mortality rate higher (47%) than those in the XST-treated groups (averaged 24%, P < 0.05). Compared with XST-treated groups, myocardial remodeling, inflammation and desarcomerization, and higher water content more severe in the cardiac tissue in the model group (P < 0.05), which was associated with higher expressions of mRNA or protein levels of AQP1, 4 and 7. Dox-impaired cardiac functions, cardiac remodeling and myocardial edema could be dose-dependently reverted by XST treatment. XST could inhibit AQP1, 4 and 7 at mRNA levels or at protein levels, which was associated with the attenuation of myocardial edema and cardiac remodeling, decreasing the ventricular stiffness and improving the cardiac functions and rats' survival. AQPs is involved in cardiac edema composed one of the mechanisms of Dox-induced cardiotoxicity, XSTvia inhibition of AQPs relieved the Dox-induced side effects.

Acetazolamide ameliorates the severity of collagen-induced arthritis in rats: Involvement of inducing synovial apoptosis and inhibiting Wnt/ß-catenin pathway.

We previously revealed that the overexpression of synovial aquaporin 1 (AQP1) aggravated collagen-induced arthritis (CIA) in rats via regulating ß-catenin signaling. This study was to demonstrate the therapeutic effect of acetazolamide (AZ, an AQP1 inhibitor) on rat CIA and explored its underlying mechanisms. Paw swelling, arthritis index, pathological assessments, and serum levels of collagen type II (Col II) antibody, IL-1ß and TNF-α were measured to evaluate the anti-arthritic effect of AZ on rat CIA. Ki67 immunohistochemistry and TUNEL assay were performed to reveal the anti-proliferative and pro-apoptotic effects of AZ on synovial cells in vivo. The protein levels of apoptosis-related genes and Wnt/ß-catenin pathway key members were detected by western blot. We found that AZ treatment on CIA rats could inhibit paw swelling, reduce arthritis index, alleviate the pathologic changes of ankle joint and decrease the serum levels of Col II antibody, TNF-α and IL-1ß. AZ could reduce Ki67 expression and increase apoptosis index in CIA synovial tissues by reducing Bcl-2 protein level, increasing Bax and caspase 3 protein levels and normalizing Bcl-2/Bax ratio. Moreover, AZ could reduce the protein levels of Wnt1, ß-catenin, p-GSK-3ß (Ser9), c-myc, cyclin D1 and MMP9, while increase GSK-3ß protein level in CIA synovial tissues. Importantly, these mentioned effects of AZ (60 mg/kg) on CIA rats could be reversed by the combined use of lithium chloride (LiCl), an activator of Wnt/ß-catenin pathway. In short, AZ exerted potent anti-arthritic effects on CIA rats by inducing synovial apoptosis and inhibiting Wnt/ß-catenin pathway.

Aquaporin-1 plays a key role in erythropoietin-induced endothelial cell migration.

Water influx through aquaporin-1 (AQP-1) has been linked to the ability of different cell types to migrate, and therefore plays an important part in processes like metastasis and angiogenesis. Since the erythroid growth factor erythropoietin (Epo) is now recognized as an angiogenesis promoter, we investigated the participation of AQP-1 as a downstream effector of this cytokine in the migration of endothelial cells. Inhibition of AQP-1 with either mercury ions (Hg2+) or a specific siRNA led to an impaired migration of EA.hy926 endothelial cells exposed to Epo (wound-healing assays). Epo also induced the expression of AQP-1 at mRNA and protein levels, an effect which was dependent on the influx of extracellular calcium through L-type calcium channels as well as TRPC3 channels. The relationship between Epo and AQP-1 was further confirmed at shorter exposure times, as the cytokine was unable to trigger calcium influxes in cells where AQP-1 had previously been knocked down. Moreover, Epo promoted changes in the subcellular localization of AQP-1 as well as rearrangements in the actin cytoskeleton, which are consistent with a migratory phenotype. Worthy of note, carbamylated erythropoietin (cEpo), the non-erythropoietic and non-promigratory derivative of Epo, was incapable of AQP-1 modulation. The therapeutical implications of aquaporin targeting in angiogenesis-related diseases highlight the importance of the present results in the context of the relationship between AQP-1 and Epo.

Personalized Treatment Response Assessment for Rare Childhood Tumors Using Microcalorimetry-Exemplified by Use of Carbonic Anhydrase IX and Aquaporin 1 Inhibitors.

We present a novel approach to a personalized therapeutic concept for solid tumors. We illustrate this on a rare childhood tumor for which only a generalized treatment concept exists using carbonic anhydrase IX and aquaporin 1 inhibitors. The use of microcalorimetry as a refined in vitro method for evaluation of drug susceptibility in organotypic slice culture has not previously been established. Rapid microcalorimetric drug response assessment can refine a general treatment concept when it is applied in cases in which tumors do not respond to conventional chemo-radiation treatment. For solid tumors, which do not respond to classical treatment, and especially for rare tumors without an established protocol rapid microcalorimetric drug response testing presents an elegant novel approach to test alternative therapeutic approaches. While improved treatment concepts have led to improved outcome over the past decades, the prognosis of high risk disease is still poor and rethinking of clinical trial design is necessary. A small patient population combined with the necessity to assess experimental therapies for rare solid tumors rather at the time of diagnosis than in relapsed or refractory patients provides great challenges. The possibility to rapidly compare established protocols with innovative therapeutics presents an elegant novel approach to refine and personalize treatment.

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

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

Inhibitory effect of AQP1 silencing on adhesion and angiogenesis in ectopic endometrial cells of mice with endometriosis through activating the Wnt signaling pathway.

The development mechanism of endometriosis remains unknown. Water channel aquaporin-1 (AQP1) enhances water flux across cell membranes, which is highly expressed and associated with cell migration, metastasis and angiogenesis in some human cancers. In this study, the role of the Wnt signaling pathway mediated by AQP1 in endometriosis was investigated, in a bid to provide new therapeutic targets for endometriosis. Microarray expression profiles were screened to acquired differentially expressed genes related to endometriosis. Mouse models with endometriosis were established and grouped. The level of endometriosis was evaluated by measurement of the volume of ectopic region. The expression of AQP1, pathway-related factors (Wnt1 and Wnt4), adhesion molecules (VCAM-1 and ICAM-1), invasive factors (MMP-2, MMP-9, TIMP-1 and TIMP-2), angiogenic factors (VEGF-A, VEGFR1 and VEGFR2) and apoptotic factors (Caspase-3, Caspase-9, Bax and BcL-2) was measured by RT-qPCR and western blot analysis. Furthermore, the role of AQP1 in adhesion, invasion, angiogenesis, and apoptosis of ectopic endometrial cells was determined by transfection of si-AQP1 plasmid. AQP1 was robustly expressed in endometriosis. AQP1 gene silencing alleviated the progression of endometriosis by activating the Wnt signaling pathway in mice with endometriosis. Specifically, silencing of AQP1 gene inhibited ectopic endometrial cell adhesion and invasion abilities, suppressed angiogenesis while promoted apoptosis. Collectively, the present study highlights the role of AQP1 in the regulation of the Wnt signaling pathway in endometriosis mouse models, suggesting that AQP1 could represent a new target aimed at improving the survival of patients with endometriosis.

Bumetanide-Derived Aquaporin 1 Inhibitors, AqB013 and AqB050 Inhibit Tube Formation of Endothelial Cells through Induction of Apoptosis and Impaired Migration In Vitro.

AqB013 and AqB050 compounds inhibit aquaporin 1 (AQP1), a dual water and ion channel implicated in tumour angiogenesis. We tested AqB013 and AqB050 either as monotherapy or in combination on tube formation of murine endothelial cells (2H-11 and 3B-11) and human umbilical vascular endothelial cells (HUVECs). The mechanism underlying their anti-tubulogenic effect was explored by examining cell viability, induction of apoptosis and migration using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, Annexin V/propidium iodide apoptosis assay and scratch wound assay. Tube formation of all the cell lines was inhibited by AqB013, AqB050 and the combination of the two compounds. The inhibition of 2H-11 and 3B-11 was frequently accompanied by impaired migration, whereas that of HUVEC treated with AqB050 and the combination was associated with reduced cell viability due to apoptosis. AqB013 and AqB050 exhibited an anti-tubulogenic effect through inhibition of AQP1-mediated cell migration and induction of apoptosis. Together with previously reported anti-tumour cell effect of AqB013 and AqB050, our findings support further evaluation of these compounds as potential cancer therapeutics.

Therapeutic efficacy of zingerone against vancomycin-induced oxidative stress, inflammation, apoptosis and aquaporin 1 permeability in rat kidney.

Vancomycin (VCM) is a glycopeptidic broad-spectrum antibiotic against methicillin-resistant Staphylococcus aureus, though it has some adverse effects, including nephrotoxicity, that limit its usefulness. Zingerone (ZO), a component of dry ginger root, has several pharmacological activities due to its antioxidant, anti-inflammatory and antiapoptotic properties. The aim of this study was to determine the therapeutic efficacy of ZO against VCM-induced oxidative stress, inflammation, apoptosis and kidney aquaporin 1 (AQP1) levels in rats. Intraperitoneal administration of VCM (200 mg/kg body weight) for seven days increased kidney lipid peroxidation and decreased antioxidant enzyme activities, including kidney superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). VCM increased serum creatinine and urea levels and induced histopathological changes while causing a decrease in AQP1 protein level. VCM also increased the levels of the inflammatory markers nuclear factor kappa B (NF-κB), B-cell lymphoma-3(Bcl-3), interleukin-1ß (IL-1ß), interleukin-33 (IL-33), tumor necrosis factor-α (TNF-α), prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS), myeloperoxidase (MPO) and cyclooxygenase-2 (COX-2). Moreover, it activated the apoptotic pathway by increasing the expression levels of p53, Bcl-2 associated X protein (Bax), cysteine aspartate specific protease-3 (caspase-3) and 8-hydroxy-2'-deoxyguanosine (8-OHdG), which is a marker of oxidative DNA damage. Treatment with ZO (25 and 50 mg/kg body weight) at both doses prevented nephrotoxicity by ameliorating the histopathological alterations, oxidative stress, inflammation, apoptosis, oxidative DNA damage and renal AQP1 levels. The findings of the present study suggested that ZO attenuates VCM-induced nephrotoxicity.

Polypharmacology Approach Against Migraine with Aura and Brain Edema for the Development of an Efficient Inhibitor and its Analogues.

BACKGROUND: Polypharmacology is a design or use of pharmaceutical agents in which single drug is used to treat multiple diseases. Aquaporin proteins are identified to treat migraine with aura and brain edema. This study focuses on Aquaporin-1 and Aquaporin-4. AQP-1 is expressed in small afferent sensory nerve fibers. Over-expression of peripheral nervous system causes migraine. METHODS: AQP-4 is an abundant channel water protein in brain that regulates water transport to prevent homeostasis. Over-expression of AQP-4 contributes to water imbalance in ischemic pathology resulting in cerebral edema. Purpose of this study is to identify a potent inhibitor for the treatment of migraine with aura and brain edema. RESULTS: As in the recent studies, no conventional methodologies have been focused through the approach of polypharmacology. Structures of AQP-1 and AQP- 4 proteins were retrieved from PDB (Protein Data Bank). PyRx software was used to perform molecular docking. CONCLUSION: Analogues of ligands were analyzed which contained significant similarities of associated proteins to get efficient inhibitor. Toxicity of lead compound was measured through admetSAR. A lead compound was predicted to treat these diseases.

The Aquaporin 1 Inhibitor Bacopaside II Reduces Endothelial Cell Migration and Tubulogenesis and Induces Apoptosis.

Expression of aquaporin-1 (AQP1) in endothelial cells is critical for their migration and angiogenesis in cancer. We tested the AQP1 inhibitor, bacopaside II, derived from medicinal plant Bacopa monnieri, on endothelial cell migration and tube-formation in vitro using mouse endothelial cell lines (2H11 and 3B11) and human umbilical vein endothelial cells (HUVEC). The effect of bacopaside II on viability, apoptosis, migration and tubulogenesis was assessed by a proliferation assay, annexin-V/propidium iodide flow cytometry, the scratch wound assay and endothelial tube-formation, respectively. Cell viability was reduced significantly for 2H11 at 15 µM (p = 0.037), 3B11 at 12.5 µM (p = 0.017) and HUVEC at 10 µM (p < 0.0001). At 15 µM, the reduced viability was accompanied by an increase in apoptosis of 38%, 50% and 32% for 2H11, 3B11 and HUVEC, respectively. Bacopaside II at ≥10 µM significantly reduced migration of 2H11 (p = 0.0002) and 3B11 (p = 0.034). HUVECs were most sensitive with a significant reduction at ≥7.5 µM (p = 0.037). Tube-formation was reduced with a 15 µM dose for all cell lines and 10 µM for 3B11 (p < 0.0001). These results suggest that bacopaside II is a potential anti-angiogenic agent.

Therapeutic effect of acetazolamide, an aquaporin 1 inhibitor, on adjuvant-induced arthritis in rats by inhibiting NF-κB signal pathway.

OBJECTIVES: Previous studies have shown that aquaporin 1 (AQP1) is up-regulated in synovium and cartilage of rheumatoid arthritis (RA) patients and that AQP1 may be involved in joint swelling and synovial inflammation. This study was aimed to investigate the potential therapeutic effect of acetazolamide (AZ, an AQP1 inhibitor) on rat adjuvant-induced arthritis (AIA) and explore its related mechanisms. MATERIALS AND METHODS: Rat AIA was induced by complete Freund's adjuvant. The effect of AZ on rat AIA was evaluated by secondary hind paw swelling, arthritis index, TNF-α and IL-1ß serum levels and histological examination of ankle joint. Proteoglycans expression and mRNA levels of type-II collagen (COII) and aggrecan in cartilage were measured by alcian blue staining and real-time PCR, respectively. The protein levels of AQP1, IκBα, phospho-IκBα (p-IκBα), NF-κB p65 and phospho-NF-κB p65 (p-NF-κB p65) in synovial tissues were detected by western blot. RESULTS: AZ treatment could inhibit secondary hind paw swelling and arthritis index, reduce serum levels of TNF-α and IL-1ß, and ameliorate pathological changes of ankle joint in AIA rats. AZ increased proteoglycans production and mRNA levels of COII and aggrecan in cartilage tissues. Moreover, AZ decreased AQP1 protein level and suppressed the activation of NF-κB pathway in synovium, indicated by inhibiting the degradation and phosphorylation of IκBα and reducing p-NF-κB p65 protein level. CONCLUSIONS: AZ as an AQP1 inhibitor has a powerful therapeutic effect on rat AIA via inhibiting NF-κB activation, suggesting AQP1 inhibition might be of potential clinical interest in RA treatment.

Association of aquaporin­1 with tumor migration, invasion and vasculogenic mimicry in glioblastoma multiforme.

The present study aimed to assess the expression and functional role of aquaporin-1 (AQP1) in glioblastoma multiforme (GBM) migration, invasion and vasculogenic mimicry (VM). In the primary human gliomas and human glioma­derived cell lines tested, it was observed that the expression of AQP1 was upregulated. In addition, it was demonstrated that silencing of AQP1 expression resulted in decreased migration and invasion, in addition to vasculogenic mimicry in vitro. It was additionally observed that silencing of AQP1 expression resulted in in vivo inhibition of tumor growth, a decrease in the expression of invasion­associated protein, and suppression of VM formation. Based on these data, it was concluded that AQP1 may serve a role in GBM migration, invasion and VM formation, and that it may serve as a novel diagnostic/prognostic biomarker and a potential therapeutic target.

The Effect of Aquaporin 1-Inhibition on Vasculogenic Mimicry in Malignant Mesothelioma.

Malignant mesothelioma (MM) is an aggressive malignancy of the serosal membranes, with poor overall survival and quality of life. Limited targeted treatment strategies exist due to restricted knowledge of pathogenic pathways. Vasculogenic mimicry (VM) is a newly described phenomenon associated with increased aggressiveness in other malignancies, and has been characterized in MM. Normal mesothelium expresses aquaporin 1 (AQP1) and retained expression has been associated with improved survival in MM. AQP1 is expressed by normal vascular endothelium and is involved in mediating MM cell motility and proliferation. We investigated the role of AQP1 in VM, and its interaction with the pro-angiogenic factor vascular endothelial growth factor A (VEGFA), which is variably expressed in MM. Matrigel VM assays were performed using NCI-H226 and NCI-H28 MM cell lines and primary cells in hypoxia and normoxia. The synthetic blocker AqB050 and siRNA were used to inhibit AQP1, and bevacizumab was used to inhibit VEGF. Inhibition of AQP1 resulted in increased VEGFA secretion by MM cells and reduced VM in MM cell lines in hypoxia but not normoxia. No change in VM was seen in MM primary cells. Combined inhibition of AQP1 and VEGF had no effect on VM in normoxia. In a heterotopic xenograft mouse model, AqB050 treatment did not alter vessel formation. AQP1 may interact with VEGFA and play a role in VM, especially under hypoxic conditions, but the heterogeneity of MM cells may result in different dominant pathways between patients.

Inhibition of AQP1 Hampers Osteosarcoma and Hepatocellular Carcinoma Progression Mediated by Bone Marrow-Derived Mesenchymal Stem Cells.

The complex cross-talk between tumor cells and their surrounding stromal environment plays a key role in the pathogenesis of cancer. Among several cell types that constitute the tumor stroma, bone marrow-derived mesenchymal stem cells (BM-MSCs) selectively migrate toward the tumor microenvironment and contribute to the active formation of tumor-associated stroma. Therefore, here we elucidate the involvement of BM-MSCs to promote osteosarcoma (OS) and hepatocellular carcinoma (HCC) cells migration and invasion and deepening the role of specific pathways. We analyzed the function of aquaporin 1 (AQP1), a water channel known to promote metastasis and neoangiogenes. AQP1 protein levels were analyzed in OS (U2OS) and HCC (SNU-398) cells exposed to conditioned medium from BM-MSCs. Tumor cell migration and invasion in response to BM-MSC conditioned medium were evaluated through a wound healing assay and Boyden chamber, respectively. The results showed that the AQP1 level was increased in both tumor cell lines after treatment with BM-MSC conditioned medium. Moreover, BM-MSCs-mediated tumor cell migration and invasion were hampered after treatment with AQP1 inhibitor. These data suggest that the recruitment of human BM-MSCs into the tumor microenvironment might cause OS and HCC cell migration and invasion through involvement of AQP1.

Differential Inhibition of Water and Ion Channel Activities of Mammalian Aquaporin-1 by Two Structurally Related Bacopaside Compounds Derived from the Medicinal Plant Bacopa monnieri.

Aquaporin-1 (AQP1) is a major intrinsic protein that facilitates flux of water and other small solutes across cell membranes. In addition to its function as a water channel in maintaining fluid homeostasis, AQP1 also acts as a nonselective cation channel gated by cGMP, a property shown previously to facilitate rapid cell migration in a AQP1-expressing colon cancer cell line. Here we report two new modulators of AQP1 channels, bacopaside I and bacopaside II, isolated from the medicinal plant Bacopa monnieri Screening was conducted in the Xenopus oocyte expression system, using quantitative swelling and two-electrode voltage clamp techniques. Results showed bacopaside I blocked both the water (IC50 117 µM) and ion channel activities of AQP1 but did not alter AQP4 activity, whereas bacopaside II selectively blocked the AQP1 water channel (IC50 18 µM) without impairing the ionic conductance. These results fit with predictions from in silico molecular modeling. Both bacopasides were tested in migration assays using HT29 and SW480 colon cancer cell lines, with high and low levels of AQP1 expression, respectively. Bacopaside I (IC50 48 µM) and bacopaside II (IC50 14 µM) impaired migration of HT29 cells but had minimal effect on SW480 cell migration. Our results are the first to identify differential AQP1 modulators isolated from a medicinal plant. Bacopasides could serve as novel lead compounds for pharmaceutic development of selective aquaporin modulators.

Fragment Screening of Human Aquaporin 1.

Aquaporins (AQPs) are membrane proteins that enable water transport across cellular plasma membranes in response to osmotic gradients. Phenotypic analyses have revealed important physiological roles for AQPs, and the potential for AQP water channel modulators in various disease states has been proposed. For example, AQP1 is overexpressed in tumor microvessels, and this correlates with higher metastatic potential and aggressiveness of the malignancy. Chemical modulators would help in identifying the precise contribution of water channel activity in these disease states. These inhibitors would also be important therapeutically, e.g., in anti-cancer treatment. This perceived importance contrasts with the lack of success of high-throughput screens (HTS) to identify effective and specific inhibitors of aquaporins. In this paper, we have screened a library of 1500 "fragments", i.e., smaller than molecules used in HTS, against human aquaporin (hAQP1) using a thermal shift assay and surface plasmon resonance. Although these fragments may not inhibit their protein target, they bound to and stabilized hAQP1 (sub mM binding affinities (KD), with an temperature of aggregation shift ΔTagg of +4 to +50 °C) in a concentration-dependent fashion. Chemically expanded versions of these fragments should follow the determination of their binding site on the aquaporin surface.

Experimental Evaluation of Proposed Small-Molecule Inhibitors of Water Channel Aquaporin-1.

The aquaporin-1 (AQP1) water channel is a potentially important drug target, as AQP1 inhibition is predicted to have therapeutic action in edema, tumor growth, glaucoma, and other conditions. Here, we measured the AQP1 inhibition efficacy of 12 putative small-molecule AQP1 inhibitors reported in six recent studies, and one AQP1 activator. Osmotic water permeability was measured by stopped-flow light scattering in human and rat erythrocytes that natively express AQP1, in hemoglobin-free membrane vesicles from rat and human erythrocytes, and in plasma membrane vesicles isolated from AQP1-transfected Chinese hamster ovary cell cultures. As a positive control, 0.3 mM HgCl2 inhibited AQP1 water permeability by >95%. We found that none of the tested compounds at 50 µM significantly inhibited or increased AQP1 water permeability in these assays. Identification of AQP1 inhibitors remains an important priority.

Pharmacological blockade of aquaporin-1 water channel by AqB013 restricts migration and invasiveness of colon cancer cells and prevents endothelial tube formation in vitro.

BACKGROUND: Aquaporins (AQP) are water channel proteins that enable fluid fluxes across cell membranes, important for homeostasis of the tissue environment and for cell migration. AQP1 knockout mouse models of human cancers showed marked inhibition of tumor-induced angiogenesis, and in pre-clinical studies of colon adenocarcinomas, forced over-expression of AQP1 was shown to increase angiogenesis, invasion and metastasis. We have synthesized small molecule antagonists of AQP1. Our hypothesis is that inhibition of AQP1 will reduce migration and invasiveness of colon cancer cells, and the migration and tube-forming capacity of endothelial cells in vitro. METHODS: Expression of AQP1 in cell lines was assessed by quantitative (q) PCR, western blot and immunofluorescence, while expression of AQP1 in human colon tumour tissue was assessed by immunohistochemistry. The effect of varying concentrations of the AQP1 inhibitor AqB013 was tested on human colon cancer cell lines expressing high versus low levels of AQP1, using wound closure (migration) assays, matrigel invasion assays, and proliferation assays. The effect of AqB013 on angiogenesis was tested using an endothelial cell tube-formation assay. RESULTS: HT29 colon cancer cells with high AQP1 levels showed significant inhibition of migration compared to vehicle control of 27.9% ± 2.6% (p < 0.0001) and 41.2% ± 2.7 (p <0.0001) treated with 160 or 320 µM AqB013 respectively, whereas there was no effect on migration of HCT-116 cells with low AQP1 expression. In an invasion assay, HT29 cells treated with 160 µM of AqB013, showed a 60.3% ± 8.5% decrease in invasion at 144 hours (p < 0.0001) and significantly decreased rate of invasion compared with the vehicle control (F-test, p = 0.001). Almost complete inhibition of endothelial tube formation (angiogenesis assay) was achieved at 80 µM AqB013 compared to vehicle control (p < 0.0001). CONCLUSION: These data provide good evidence for further testing of the inhibitor as a therapeutic agent in colon cancer.

Influence of AQP1 on cell adhesion, migration, and tumor sphere formation in malignant pleural mesothelioma is substratum- and histological-type dependent.

Malignant pleural mesothelioma (MPM) is an aggressive cancer. MPM cells express aquaporin-1 (AQP1) that in other cancers has been shown to participate in the tumor metastasis processes. However, in MPM patients AQP1 overexpression is an independent prognostic factor favoring survival. In this study we aimed at evaluating the role of AQP1 in cell adhesion, migration, and tumor sphere formation in nonmalignant mesothelial cells (MeT-5A) and in epithelioid (M14K) and sarcomatoid (ZL34) MPM cell lines. We used fibronectin (FN) or homologous cell-derived extracellular martrix (ECM) substratum to investigate the role of AQP1 in these experimental phenotypes, inhibiting AQP1 by 10(-5) M mercury chloride (MC). Deposited ECM during cell culture exhibited significant concentration differences among cell types. ZL34 cell adhesion was significantly higher than MeT-5A or M14K cells on FN and ECM. MeT-5A and M14K cell adhesion on FN was sensitive to AQP1 inhibition, whereas AQP1 inhibition on ECM was limited to M14K cells. Wound healing in ZL34 cells was significantly higher than MeT-5A and M14K cells on FN and ECM. AQP1 inhibition significantly lowered cell migration in ZL34 cells on FN and ECM. Sphere formation was not dependent on FN or ECM in the media. AQP1 inhibition in FN media reduced sphere formation in M14K cells, whereas, in ECM, all three cell types were sensitive to AQP1 inhibition.