Aquaporins permeability to hydrogen peroxide may control oxidative stress

Transmembrane water channel proteins, known as aquaporins (AQPs), play a pivotal role in many biological processes including volume regulation, cell migration and proliferation, and adipocyte metabolism. Recently, it has been demonstrated the involvement of some AQPs, known as “peroxiporins”, in the transport of hydrogen peroxide (H2O2). H2O2 is the most abundant and stable reactive oxygen species (ROS) in living cells and H2O2 can have different effects depending on its concentration. Physiological levels of H2O2 induce positive adaptive responses acting as second messenger, while excessive levels provoke negative effects as apoptosis and cell death. Since AQPs allow the diffusion of H2O2 across plasma membranes to the extracellular fluid, they have been considered as a possible ROS scavenging mechanism. Until now, AQP1, 3, 5, 8, 9 and 11 are the mammalian AQPs involved in H2O2 diffusion. This project aims to understand the mechanism of AQPs as peroxiporins in mediating H2O2 diffusion through cellular plasma membrane. The guiding thread of this thesis was, on the one hand, the analysis of the role of peroxiporins in some pathophysiological conditions and, on the other, the identification and characterization of new gating modulators. In particular this thesis aimed: 1. to investigate the negative role of human Papillomavirus (HPV) infection on the aquaporin-mediated hydrogen peroxide elimination which affects human sperm functioning; 2. to clarify AQPs/peroxiporins involvement in malignant pleural mesothelioma progression (MPM); 3. to identify and characterize new aquaporin modulators to counteract the oxidative stress. As a whole, AQPs permeability alteration (and sensitivity to oxidative stress) in HPV infection and in MPM seems to reduce the fertility of sperm cells and make MPM cells resistant to conventional chemotherapy, respectively. The possibility to modify the gating of the AQPs and ROS scavenging opens to new therapeutic strategies for the treatment of debilitating diseases involving oxidative stress namely neurodegenerative diseases and cancer

Propolis Induces AQP3 Expression: A Possible Way of Action in Wound Healing

Propolis is the generic name of a complex of resinous compound collected by honeybees and it has been utilized for many years in folk medicine. As other products generated by honeybees (such as royal jelly, pollen, honey), propolis has great therapeutic properties, but very little scientific information is available. Therefore, this study was aimed at exploring the potential wound healing properties of propolis. To that end, we utilized an in vitro scratch wound healing model consisting of human immortalized keratinocytes. Our scratch wound data clearly demonstrated that propolis induced a pronounced increase in the wound repair abilities of keratinocytes. A cell migration assay showed that propolis stimulated keratinocytes to close the wound. We revealed the role of H2O2 as the main mediator of propolis regenerative properties. We showed that this extracellularly released H2O2 could pass across the plasma membrane through a specific aquaporin (i.e., AQP3) modulating intracellular responses. The data offer a biological characterization of propolis positive effects suggesting that propolis could also be utilized in wound treatment within clinical settings

Aquaporin-6 May Increase the Resistance to Oxidative Stress of Malignant Pleural Mesothelioma Cells

Malignant pleural mesothelioma (MPM) is an aggressive cancer of the pleural surface and is associated with previous asbestos exposure. The chemotherapy drug is one of the main treatments, but the median survival ranges from 8 to 14 months from diagnosis. The redox homeostasis of tumor cells should be carefully considered since elevated levels of ROS favor cancer cell progression (proliferation and migration), while a further elevation leads to ferroptosis. This study aims to analyze the functioning/role of aquaporins (AQPs) as a hydrogen peroxide (H2O2) channel in epithelial and biphasic MPM cell lines, as well as their possible involvement in chemotherapy drug resistance. Results show that AQP-3, -5, -6, -9, and -11 were expressed at mRNA and protein levels. AQP-6 was localized in the plasma membrane and intracellular structures. Compared to normal mesothelial cells, the water permeability of mesothelioma cells is not reduced by exogenous oxidative stress, but it is considerably increased by heat stress, making these cells resistant to ferroptosis. Functional experiments performed in mesothelioma cells silenced for aquaporin-6 revealed that it is responsible, at least in part, for the increase in H2O2 efflux caused by heat stress. Moreover, mesothelioma cells knocked down for AQP-6 showed a reduced proliferation compared to mock cells. Current findings suggest the major role of AQP-6 in providing mesothelioma cells with the ability to resist oxidative stress that underlies their resistance to chemotherapy drugs

Regulation of aquaporin functional properties mediated by the antioxidant effects of natural compounds

Some aquaporins (AQPs) have been recently demonstrated to facilitate the diffusion of hydrogen peroxide (H₂O₂) from the producing cells to the extracellular fluid, and their reactive oxygen species scavenging properties have been defined. Nevertheless, the identification of different AQPs acting as peroxiporins, their functional role in eustress and distress, and the identification of antioxidant compounds able to regulate AQP gating, remain unsolved. This study aims to investigate, in HeLa cells: (1) the expression of different AQPs; (2) the evaluation of naringenin, quercetin, (R)-aloesaponol III 8-methyl ether, marrubiin, and curcumin antioxidant profiles, via α,α-diphenyl-β-picrylhydrazyl assay; (3) the effect of the compounds on the water permeability in the presence and in the absence of oxidative stress; and (4) the effect of pre- and post-treatment with the compounds on the H₂O₂ content in heat-stressed cells. Results showed that HeLa cells expressed AQP1, 3, 8, and 11 proteins. The oxidative stress reduced the water transport, and both pre- and post-treatment with the natural compounds recovering the water permeability, with the exception of curcumin. Moreover, the pre- and post-treatment with all the compounds reduced the H₂O₂ content of heat-stressed cells. This study confirms that oxidative stress reduced water AQP-mediated permeability, reversed by some chemical antioxidant compounds. Moreover, curcumin was shown to regulate AQP gating. This suggests a novel mechanism to regulate cell signaling and survival during stress, and to manipulate key signaling pathways in cancer and degenerative diseases

Cerium Oxide Nanoparticles Regulate Oxidative Stress in HeLa Cells by Increasing the Aquaporin-Mediated Hydrogen Peroxide Permeability

Some aquaporins (AQPs) allow the diffusion of hydrogen peroxide (H2O2), the most abundant ROS, through the cell membranes. Therefore, the possibility of regulating the AQP-mediated permeability to H2O2, and thus ROS scavenging, appears particularly important for controlling the redox state of cells in physiological and pathophysiological conditions. Several compounds have been screened and characterized for this purpose. This study aimed to analyze the effect of cerium oxide nanoparticles (CNPs) presenting antioxidant activity on AQP functioning. HeLa cells express AQP3, 6, 8, and 11, able to facilitate H2O2. AQP3, 6, and 8 are expressed in the plasma membrane and intracellularly, while AQP11 resides only in intracellular structures. CNPs but not cerium ions treatment significantly increased the water and H2O2 permeability by interacting with AQP3, 6, and especially with AQP8. CNPs increased considerably the AQP-mediated water diffusion in cells with oxidative stress. Functional experiments with silenced HeLa cells revealed that CNPs increased the H2O2 diffusion mainly by modulating the AQP8 permeability but also the AQP3 and AQP6, even if to a lesser extent. Current findings suggest that CNPs represent a promising pharmaceutical agent that might potentially be used in numerous pathologies involving oxidative stress as tumors and neurodegenerative disease

Sigma-1 Receptor Agonists Acting on Aquaporin-Mediated H2O2 Permeability: New Tools for Counteracting Oxidative Stress

Sigma1 Receptor (S1R) is involved in oxidative stress, since its activation is triggered by oxidative or endoplasmic reticulum stress. Since specific aquaporins (AQP), called peroxiporins, play a relevant role in controlling H2O2 permeability and ensure reactive oxygen species wasted during oxidative stress, we studied the effect of S1R modulators on AQP-dependent water and hydrogen peroxide permeability in the presence and in the absence of oxidative stress. Applying stopped-flow light scattering and fluorescent probe methods, water and hydrogen peroxide permeability in HeLa cells have been studied. Results evidenced that S1R agonists can restore water permeability in heat-stressed cells and the co-administration with a S1R antagonist totally counteracted the ability to restore the water permeability. Moreover, compounds were able to counteract the oxidative stress of HeLa cells specifically knocked down for S1R. Taken together these results support the hypothesis that the antioxidant mechanism is mediated by both S1R and AQP-mediated H2O2 permeability. The finding that small molecules can act on both S1R and AQP-mediated H2O2 permeability opens a new direction toward the identification of innovative drugs able to regulate cell survival during oxidative stress in pathologic conditions, such as cancer and degenerative diseases

Regulation of Aquaporin Functional Properties Mediated by the Antioxidant Effects of Natural Compounds

Some aquaporins (AQPs) have been recently demonstrated to facilitate the diffusion of hydrogen peroxide (H2O2) from the producing cells to the extracellular fluid, and their reactive oxygen species scavenging properties have been defined. Nevertheless, the identification of different AQPs acting as peroxiporins, their functional role in eustress and distress, and the identification of antioxidant compounds able to regulate AQP gating, remain unsolved. This study aims to investigate, in HeLa cells: (1) the expression of different AQPs; (2) the evaluation of naringenin, quercetin, (R)-aloesaponol III 8-methyl ether, marrubiin, and curcumin antioxidant profiles, via α,α-diphenyl-β-picrylhydrazyl assay; (3) the effect of the compounds on the water permeability in the presence and in the absence of oxidative stress; and (4) the effect of pre- and post-treatment with the compounds on the H2O2 content in heat-stressed cells. Results showed that HeLa cells expressed AQP1, 3, 8, and 11 proteins. The oxidative stress reduced the water transport, and both pre- and post-treatment with the natural compounds recovering the water permeability, with the exception of curcumin. Moreover, the pre- and post-treatment with all the compounds reduced the H2O2 content of heat-stressed cells. This study confirms that oxidative stress reduced water AQP-mediated permeability, reversed by some chemical antioxidant compounds. Moreover, curcumin was shown to regulate AQP gating. This suggests a novel mechanism to regulate cell signaling and survival during stress, and to manipulate key signaling pathways in cancer and degenerative diseases

Some natural compounds exert their antioxidant effect by altering aquaporins functional properties

The ability of some aquaporins (AQPs) to facilitate the diffusion of H2O2 from the producing cells to the extracellular fluid has been recently demonstrated. Accordingly, this significant AQPs property may have a beneficial effect in promoting Reactive Oxygen Species (ROS) scavenging. Nevertheless, some AQPs aspects remain unsolved and, in this scenario, the identification and localization of different AQPs acting as peroxiporins, their functional role in eustress and distress, as well as the identification of chemical antioxidant compounds capable to regulate AQP gating are still the main features to disclose. In light of these considerations, this study aimed at investigating the potential effect of well-known natural compounds in modulating the activity of AQPs. From an experimental standpoint, in this work the expression of mRNA and proteins of different AQPs in HeLa cells, the functional experiments of water (and H2O2) permeability using stopped-flow light scattering method and the gating of AQPs in the presence/absence of oxidative stress have been evaluated. The obtained results suggest that oxidative stress reduced water AQP-mediated permeability and the effectiveness of the analyzed compounds to promote AQPs gating. Therefore, this study could lead to the identification of a novel therapeutic target for the treatment of cancer and degenerative diseases, since the positive regulation of AQPs avoids the accumulation of ROS within the cells