Aquaporins

Abstract

Aquaporins are a family of integral membrane proteins that are expressed in all living organisms and play vital roles in transcellular and transepithelial water movement. Cell viability and motility are critical for progression of cancer. Cell survival requires the suitable concentration of water and solutes. The balance is largely maintained by aquaporins whose major function is the transport of water and small solutes across the plasma membrane. The important role of aquaporins has received more and more attention in the recent years. A number of recent studies have revealed that aquaporins may be involved in cell migration and angiogenesis. This review will highlight the expression of aquaporins in different malignant neoplasms. Remarkably, we will summarize the influence of drugs on aquaporins, not only the traditional Chinese medicine but also the Western medicine. Therapeutic targeting of aquaporins may thus be advantageous for blocking the mechanism common for a number of key cancer phenotypes.

Introduction

The aquaporins (AQPs) are a family of small (∼30 kDa/monomer), hydrophobic, integral membrane proteins that are expressed widely in the plant and animal kingdoms. Aquaporins are membrane proteins that transport water and small solutes such as glycerol. Aquaporins increase cell plasma membrane water permeability 5 to 50 times compared to that in membranes where water moves primarily through the lipid bilayer. There are 13 AQPs (AQP 0-12) in mammals expressed in large amount of epithelia, endothelia, and other types of cells. Functionally, AQPs are divided into 2 subclasses: the subfamily that includes AQPs 1, 2, 4, 5, and 8, which are water-selective channels and are permeable to water but not to small organic and inorganic molecules, and the subfamily that includes AQPs 3, 7, 9, and 10, which are nonselective water channels and transport glycerol and possibly other small solutes and water. ¹ All kinds of metabolism of tumor need to depend on the participation of the water molecules that are quickly and specifically transferred by AQPs. Meanwhile, a number of recent studies have revealed the great roles of mammalian AQPs in tumor angiogenesis and metastasis. With a rapid extension of the knowledge in AQP structure and functional regulation, new avenues for manipulation of AQP channels are likely to be discovered. In this review, we will focus on that AQPs not only are expressed in various types of tumors but also will be as targets for novel therapy of cancer.

Expression of AQPs in Cancer

Cancer, also known as a malignant tumor, is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. Tumor cells can move into neighboring space occupied by another tissue (invasion) or migrate to distant site(s) (metastasis). All kinds of metabolism of tumor are closely related to the flow across the biological membrane of the water molecules, and the fluid transport mediated by the AQPs is the main way of water in and out of the cells. For this reason, AQPs may play a great role in the angiogenesis, growth, and metastasis of tumors.

Aquaporin 1 was first discovered in human erythrocytes as a water channel for high osmotic water permeability. ^2,3 Aquaporin 1 was highly expressed in nasopharyngeal carcinoma, colon cancer, human glial tumors, breast cancer, epithelial ovarian tumors, cerebellar cystic hemangioblastomas, and cervical carcinoma ^{4 –10} and was absent in the case of choroid plexus carcinoma. ¹¹ Moreover, a recent study found AQP1 is an independent prognostic factor in pleural malignant mesothelioma. ¹² They found the expression of AQP1 by ≥50% of tumor cells was associated with significantly enhanced survival, and the suggested immunohistochemical labeling for AQP1 should be included in the routine histopathologic workup. It is pretty remarkable that a significant decrease in angiogenesis and neoplasticity following AQP1 gene disruption in animal models was reported. ¹³ Xenograft models of subcutaneous melanoma tumors showed decreased tumor growth and reduced microvascularization in AQP1^−/− mice compared to tumors in wild-type mice. The AQP1 knockout mice were observed to have significantly longer survival times than the wild-type animals. Significant tumor necrosis was also observed in xenograft models using AQP1 null mice. These findings support a fundamental role of water channels in cell migration, which is central to diverse biological phenomena including angiogenesis, wound healing, tumor spread, and organ regeneration. Besides, Tan et al showed that AQP1 was significantly hypomethylated in adenoid cystic carcinoma compared to the controls. On univariate analysis, increased AQP1 methylation is associated with improved prognosis. ¹⁴ In view of the above-mentioned reports, AQP1 may be a practical target for oncotherapy.

Aquaporin 2 is the most important water channel in the apical plasma membrane. ¹⁵ Recent studies have linked that the expression of AQP2 was increased in endometrial tissues from patients with endometrial carcinoma (EC) and endometriosis. ¹⁶ Aquaporin 2 mediated estradiol (E2)-enhanced migration, invasion, and adhesion by altering the expression levels of F-actin and annexin 2. Moreover, it was found AQP2 might be related to chemotherapy response in patients with lung cancer. In their study, the SNP rs7314734 of AQP2 showed more association with chemotherapy response in nonsmoking subgroup. ¹⁷

Aquaporin 3, the most studied and the well-validated AQP in the skin, was first reported in keratinocytes of rat epidermis. ¹⁸ It is expressed strongly in skin squamous cell carcinomas (SCCs), suggesting involvement of AQP3-facilitated glycerol transport in cell proliferation during tumorigenesis. ¹⁹ Of course, AQP3 is not only expressed in skin but also expressed in other tissues. A research showed that the significantly higher levels of expression of AQP3 were observed in tumor areas of human primary SCC such as esophageal and lingual cancers than that in nontumor areas. ²⁰ Aquaporin 3 was also expressed in the human gastric cancer cell lines, AGS and SGC-7901. ²¹ Their findings provided for the first time that AQP3 played a critical role in human epidermal growth factor (hEGF)-induced cancer cell migration and proliferation and that hEGF induces AQP3 expression via extracellular signal-regulated kinase (ERK) signal transduction pathways. In recent years, a few studies found that prostate cancer was also closely related to the expression of AQP3. The expression of AQP3 messenger RNA (mRNA) was seen in both normal and cancerous epithelia of human prostate tissues but not in the mesenchyme, ²² and the inhibition of the AQP3 increased the sensitivity of prostate cancer cells to cryotherapy. ²³ Moreover, someone demonstrated that several AQPs were expressed in human urothelial carcinoma for the first time and indicated that there was a correlation between AQP3 protein expression and tumor stage and grade. ²⁴ In addition, AQP3 is also closely related to other malignant solid tumors such as non-small-cell lung cancer (NSCLC), hepatocellular carcinoma (HCC), breast cancer, cervical carcinoma, thyroid cancer, and bladder cancer. ^{10,25 –29} With the increasing reports of AQP3, it received significant attention in the recent years. Therefore, we concluded that AQP3 might constitute a prognostic marker for progression to malignant disease.

Aquaporin 4 is expressed as 2 principal isoforms: a long isoform with translation initiation at Met-1 (M1–AQP4) and a short isoform with translation initiation at Met-23 (M23–AQP4). ³⁰ Aquaporin 4 is a key molecule involved in maintaining ion and water homeostasis in the central nervous system and has been recently reported to play a role in cell migration in addition to its well-known function in brain edema. ³¹ In high-grade astrocytoma, there is massive upregulation of AQP4 expression throughout the cytoplasm of neoplastic astrocytes and that of reactive astrocytes at the periphery of the tumor. Furthermore, there was a significant (P < .0001) correlation between the degree of contrast enhancement on computed tomography and AQP4 immunolabeling of tissue from the same patient. With developing of the research, researchers demonstrated the possible mechanisms by which AQP4 functions in the process of glioblastoma cell invasion. They showed the downregulation of matrix metalloprotease 2 expression in glioblastoma LN-229 cells with AQP4 reduction coinciding with decreased cell invasive ability, and the expression of β-catenin and connexin 43 was increased in AQP4-downregulated LN229 cells consistent with their enhanced cell–cell adhesion ability. ³² Certainly, research regarding AQP4 is not limited to brain tissue. More interestingly, the expression of AQP4 seems to be higher in well-differentiated tissues. It was reported that higher transcript and protein levels of AQP4 in well-differentiated lung adenocarcinomas suggested an association with a more favorable prognosis. ³³ Moreover, AQP4 protein and mRNA expression levels in gastric cancer tissue were significantly lower than those in normal gastric tissue. ³⁴ The expression of AQP4 was also different in normal, hyperplastic, and neoplastic thyroid tissues in conjunction with human thyroid cancer cell lines. Aquaporin 4 was demonstrated in 100% of follicular adenomas, 90% of follicular carcinomas, and 85% of papillary carcinomas, whereas it was negative in all medullary carcinomas and undifferentiated carcinomas. ²⁸

Aquaporin 5 may be an important member in the family of AQPs, and it appears to be a potential target for oncotherapy. Woo et al provided the first remarkable data that AQP5 could play a vital role in human carcinogenesis. ³⁵ They demonstrated that treatment with AQP5 small-interfering RNA could decrease cell proliferation in NSCLC and colon cancer cell lines overexpressing AQP5, and ectopic expression of AQP5 led to colony formation in vitro and tumor growth in mice. A study developed the idea further, they had previously demonstrated that AQP5 was overexpressed in colon cancer cell lines and colon cancer tissues and AQP5 mediated cell proliferation through activating ERK phosphorylation. ³⁶ The expression of AQP5 was associated with cell proliferation and migration. A recent study found the AQP5 protein was upregulated in prostate cancer and was closely related to advanced ABCD stage, lymph node metastasis, circulating tumor cells, and poor prognosis. ³⁷ Likewise, the expression of AQP5 was increased in breast cancer, cervical cancer, lung adenocarcinoma, gastric cancer cells, chronic myelogenous leukemia (CML), pancreatic adenocarcinoma, and gall bladder carcinoma as well as in HCC. ^{38 –44,26} In addition to the above-mentioned expression studies, there have been some other studies on the role of AQP5 in human carcinogenesis that have been alluded. It is reported that AQP5 expression has also been linked to the human ovarian cancer. ⁴⁵ The expression of AQP5 in ovarian malignant and borderline tumors was significantly higher than that of benign tumors and normal tissue. Moreover, increased AQP5 protein level was associated with lymph node metastasis.

Extracellular signal-regulated kinases 1/2 (ERK1/2) are associated with tumorigenesis and cancer progression. The expression of AQP8 was associated with the depth of invasion of cervical cancer cells and positively correlated with ERK1/2 expression in cervical cancer. ⁴⁶ The expression of AQP8 and ERK1/2 was highest in cervical intraepithelial neoplasia (CIN) samples, and the expression in cervical carcinoma samples was higher than in normal tissues. Additionally, Chang et al demonstrated for the first time that AQP8 was expressed in human esophageal cancer cells and that epidermal growth factor induced AQP8 expression and cell migration via the epidermal growth factor receptor (EGFR)/ERK1/2 signal transduction pathway in these cells. ⁴⁷ Aquaporin 8 was weakly distributed in mammalian brains in the past. A recent study showed that the expression of AQP8 was related to the pathological grade of astrocytomas. The expression levels of AQP8 significantly increased in low-grade astrocytomas and further increased in high-grade astrocytomas, especially in glioblastoma, compared with normal brain tissue. ⁴⁸ However, unlike the above-mentioned studies, AQP8 was expressed only in normal colonic tissue and not, or to a much lesser extent, in the adenomas, carcinomas, and cancer cell lines. ⁴⁹ The result suggests that the expression of AQP8 is a marker of normal proliferating colonic epithelial cells. And interestingly enough, AQP8 expression was also significantly decreased in HCC versus normal liver. ⁵⁰ These studies indicated that AQP8 induced tumor cell apoptosis and downregulated expression in carcinoma.

Aquaporin 9, a member of the AQP protein family, transports glycerol and possibly other small solutes and water. The mammalian AQPs AQP7 and AQP9 were shown to transport arsenite and antimonite. ⁵¹ It was found that AQP9 is critical to arsenic transportation in treating human acute promyelocytic leukemia (APL), ⁵² and tanshinone IIA (T) and indirubin (I) can facilitate arsenic uptake through upregulation of AQP9. Miao et al found increased AQP9 expression significantly enhanced arsenic uptake and may play a critical role in the development of arsenic resistance in human lung cancer cells. ⁵³ Aquaporin 9 was not only important to arsenic resistance in human lung cancer cells by enhancing arsenic uptake but also plays a critical role in the development of platinum-based chemotherapy response. Aquaporin 9 might be significantly associated with chemotherapy response for patients with NSCLC but not for patients with small-cell lung cancer. ¹⁷ As with AQP4, AQP9 was markedly more highly expressed in glioblastoma cells. ⁵⁴ In support of AQP9 expression in malignant glioma, another group found positive correlation between enhanced AQP9 expression and astrocytoma grade in immunoblots of astrocytoma tissue. ⁵⁵

The Influence of Drugs on AQPs

Water movement across the cell membrane mediated by the AQPs was closely related to the angiogenesis, growth, and metastasis of tumor cells. The structure and function of AQPs, the importance of AQPs in the physiology of water and solute movement as well as the evidence in support of AQPs as drug targets are now clear. ^56,57 Over the past decade, significant advances have been made in understanding the fundamental roles of AQPs in the various types of cancers. There have been numerous reports of drugs that modulate the expression of AQPs to regulate water flux. Consequently, AQPs are attractive targets for the development of novel drug therapies for disorders that involve aberrant water movement, such as edema and kidney disease, especially the tumors.

The Traditional Chinese Medicines

The culture of traditional Chinese medicines (TCM) is broad and profound, and the action mechanism of many traditional Chinese herbal medicines has not yet been discovered. Those compounds are valuable for gaining insight into the effect of AQP modulation on a cellular level, and unfortunately, they are unattractive substrates for drug discovery efforts. It was found that curcumin, the active ingredient from the spice turmeric (Curcuma longa Linn), downregulated AQP3 expression and reduced cell migration in CaOV3, a cell line of human ovarian cancer. And, curcumin reduced cell migration by its inhibitory effects on EGFR and downstream protein kinase B/ERK activation. ⁵⁸ Realgar-Indigo naturalis formula (RIF) has been proven to be very effective in treating human APL. ⁵² The main components of RIF are realgar, Indigo naturalis, and Salvia miltiorrhiza, with tetraarsenic tetrasulfide (A), I, and T as major active ingredients, respectively. The ATI which is the components of tetrasulfide (A), tanshinone IIA (T) and indirubin (I) enhances the expression of AQP9 and facilitates the transportation of A into APL cells, which in turn intensifies A-mediated promyelocytic leukemia protein–retinoic acid receptor α degradation and therapeutic efficacy. As mentioned previously, abnormal expression of AQP5 is associated with ovarian cancer. Epigallocatechin gallate inhibited the proliferation and induced the apoptosis of ovarian cancer SKOV3 cells in a dose- and time-dependent manner by downregulated expression of AQP5, which may be associated with nuclear transcription factor, nuclear factor kappa B (NF-κB). ⁵⁹ Ginsenoside Rg3 (Rg3), one of the bioactive extracts found in ginseng root, was reported to have anticancer activity in various cancer models such as malignant melanoma ⁶⁰ and esophageal SCC. ⁶¹ It is also reported that Rg3 effectively suppressed migration of PC-3M cell, which was a highly metastatic prostate cancer cell line, by downregulating AQP1 expression through p38 mitogen-activated protein kinases pathway and some transcription factors acting on the AQP1 promoter. ⁶² In addition, some other herbal medicines or their extracts such as rhein, ⁶³ Polyporus umbellatus, ⁶⁴ also play an increasingly important role in regulating the expression of AQPs.

The Western Medicine

In the years since the article written by Benga et al describing water channel inhibition, ⁶⁵ there have been a lot of reports of chemical compounds that modulate AQP-mediated water flux. Acetazolamide, N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)-acetamide, is a nonbacteriostatic sulfonamide with a chemical structure and pharmacological activity different from the sulfonamide antimicrobials. Acetazolamide is potentially useful as an angiogenic inhibitor by regulating AQP-1 water channel function and protein expression in the Lewis lung carcinoma model. ⁶⁶ It can also suppress xenograft tumor growth of colon cancer in nude mice, in part by inhibiting the AQP-1 gene expression. ⁶⁷ Tetraethylammonium (TEA⁺) was also reported to be a reversible inhibitor of AQP1 ⁶⁸ and later found to be a blocker of AQP1, AQP2, and AQP4. ⁶⁹ A tentative TEA⁺ binding site was proposed based on the resistance of a mutant AQP1 (with a mutation at the Tyr186 site) to TEA⁺. Ma et al found that topiramate significantly reduced the growth of primary tumor of Lewis lung carcinoma model, at least in part by direct suppression of AQP-1 expression. ⁷⁰ At the same time, these authors reported a decreased vasculature in topiramate-treated tumors. Many years ago, it clearly demonstrated that dexamethasone increases mRNA levels of AQP1 in erythroleukemia cells. ⁷¹ The article also showed a gradual increase in AQP1 protein, reaching a maximum level after 2 to 4 days in dexamethasone. However, the news that glucocorticoids did not affect AQP1 was reported the following year. ⁷² Moreover, the study by Hayashi et al focused on the effects of glycolytic conditions on AQP1 expression, the possibility that glucocorticoids increased the expression of AQP1 had also been raised in glioma cells. ⁷³ Another alternative view is that the suppression of brain tumor permeability by dexamethasone was mediated by the glucocorticoid receptor but not by AQP1. ⁷⁴ These suggest that more research are needed into the relationship between glucocorticoids and the AQP1.

Estradiol (E2) dose dependently increased AQP2 expression and significantly increased the migration, invasion, adhesion, and proliferation of Ishikawa cells in human EC, which was blocked by the estrogen receptor inhibitor ICI 182780. ¹⁶ Besides, both dexamethasone and ambroxol stimulated the expression of AQP3 and AQP5 at the mRNA and protein levels in a human airway epithelial cell line (A549 cells). ⁷⁵

Bumetanide showed a modest blocking effect on AQP4-mediated osmotic swelling in Xenopus laevis oocytes. Using bumetanide as a starting scaffold, they created a novel synthetic derivative, AqB013, and demonstrated that it was an effective inhibitor of AQP1 and AQP4. ⁷⁶ Subsequent studies have suggested that bumetanide exerted its neuroprotective and antiedema effects partly via blockade of the perivascular pool of AQP4 and may have therapeutic potential for ischemic stroke in the clinical setting. ⁷⁷

The expression level of AQP5 was relatively higher among patients with CML who gained imatinib mesylate resistance at chronic phase in paired samples. ⁴² Yang et al showed that AQP5 protein had a positive relationships with cell growth rate, and the expression of AQP5 could be induced to decrease by cisplatin in a concentration-dependent manner in human ovarian cancer CAOV3 cells. ⁷⁸ In addition, AQP5 expression negatively correlated with the proliferation inhibition rate of ovarian cancer SKOV3 cells induced by topotecan and positively correlated with NF-κB p65 and its receptor IκBα expression. ⁷⁹

Cyclophosphamide is clinically useful in treating malignancy and rheumatologic disease. A research found that the proximal nephron might be involved in cyclophosphamide-induced water retention via AQP1 and AQP7 water channels. ⁸⁰ For this reason, further studies are required to demonstrate intracellular mechanisms to increase the protein abundance of AQP1 in response to cyclophosphamide administration.

All in all, there remain broad opportunities for the development of AQP-based diagnostics and therapeutics. Disease-relevant AQP polymorphisms are beginning to be explored. There will be a great promise in the development of small-molecule AQP modulators for therapy of cancer.

The Chemical Composition

Multiple AQPs are inhibited by sulfhydryl-reactive mercurials such as mercury and gold, ⁸¹ and these metal ions are nonselective in their action and very toxic. Inhibition of the AQPs by HgCl₂ increased the sensitivity of both PC-3 and DU145 prostate cancer cells to cryoinjury, and there was a complete loss of cell viability at −10°C (P < .01). ²³ Treatment of the cell line of poorly differentiated human gastric adenocarcinoma (MKN45) stably expressing AQP5 with HgCl₂, an inhibitor of AQPs, significantly decreased the proportion of differentiated cells and the activity of alkaline phosphatase. ⁴¹

Arsenic and antimony are metalloid elements. At physiological pH, the trivalent metalloids behave as molecular mimics of glycerol and are conducted through AQP channels. ⁸² Despite their toxicity, both metalloids are used as chemotherapeutic agents for the treatment of cancer and protozoan parasitic diseases. The metalloid homeostasis property of AQPs can be a mixed blessing. Although inorganic arsenite (iAs [III]) was known to be toxic to most cells, it is reported that iAs (III) was much less cytotoxic than dimethylmonothioarsinic acid (V) in human bladder cancer EJ-1 cells. ⁸³ It is because AQP3, AQP7, and AQP9 were not detected in EJ-1 cells, and its expression is concerned with arsenic uptake.

Besides, other candidate blockers of AQPs that are heavy metals have also been reported, including copper chelator ATN-224 ⁸⁴ and zinc. ⁸⁵ Inhibitors of AQPs with translational potential are needed. Blockers found thus far are handicapped by toxicity, low efficacy, and lack of specificity. More and more inhibitors of AQPs are found, but their toxicity limits therapeutic value.

Conclusion and Perspective

Facilitated water transport across the cell membrane is a significant mechanism associated with a variety of processes in human physiology and pathophysiology. The importance of AQPs in the physiology of water and solute movement has been established. ⁸⁶ Following the formal identification of AQP1, there has been considerable interest in the development of AQP modulators for use in pharmacotherapy. A large body of reports have demonstrated the conceivable areas where AQP modulators could be useful in treating human disease. Targeted pharmacological modulation of water and solute transport using AQPs would appear to provide novel opportunities for therapeutic interventions in a variety of human disorders. There are few pharmacological relevant modulators of AQPs that have been identified to date, and those that are known lack specificity or are toxic. Therefore, it is a formidable and fundamental task to discovering new AQP modulators for thousands of researchers.

With recent data supporting further research into this area, the possibility of functionally significant AQP polymorphisms has received more and more attention. In this article, for the broad tissue expression of AQP subtypes in human tumor, we have made a more comprehensive summary. And more importantly, we summarized and presented the most comprehensive influence of drugs on AQPs. Through summing up these data, we can make the bold hypothesis: since both the TCM and Western medicine showed favorable effects on tumors by regulating the expression of AQPs, we may combine the Chinese herbal drugs with Western medicine in the treatment program for the disease, which could show better clinical efficacy than use of any one alone. It may also reduce the adverse reactions and enhance the therapeutic effects. A study indicated that puerarin and 5-fluorouracil (5-FU) produced a significant synergic effect on gastric cancer cells. ⁸⁷ Puerarin may potentiate the antiproliferative effect of 5-FU and reduce the therapeutically required dose of 5-FU, without increasing the toxicity. Of course, while combining the Chinese herbal drugs with Western medicine, the result that it will create antagonism, addition, or synergism exactly needs further research. We hope all these recent discoveries might have an important implication in tumor therapy.

Although the development of drugs that target modulation of AQP function is at an early stage, success in such an endeavor could open the door to treatments of life-threatening disorders. The modulation of AQPs in oncology treatment represents a potentially significant application area, with both treatment and diagnosis being possible scenarios for AQP ligands. Consequently, search for drugs that selectively antagonize the particular AQPs is a step forward in developing new therapeutic strategies to combat cancer cell invasion and metastasis and drug irresponsiveness, the phenotypes that drive the disease to its fatal stage.