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Peroxiporins Involvement in Oxidative Stress and their Potential for Drug Targeting
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As aquaporinas (AQP) peroxiporinas são canais transmembranares que facilitam a permeação de H2O2 através da membrana plasmática. Em concentrações elevadas, o H2O2 desencadeia stress oxidativo. Uma vez que a peroxiporina AQP3 se encontra sobreexpressa em vários cancros, como o melanoma, é um importante interveniente na biologia do cancro.
Neste estudo usaram-se células MNT-1 e A375 como modelo de melanoma. Primeiro, analisou-se a expressão do mRNA (qPCR) e proteína (Western Blot) da AQP3. Posteriormente, avaliou-se o efeito do Auphen, inibidor da AQP3, e de uma nova serie de compostos de ouro (CCON, CNHN e CCH2N) na permeabilidade membranar à água, glicerol e H2O2 (microscopia de epifluorescência). O efeito dos compostos foi ainda avaliado por estudos de toxicidade, adesão, proliferação celular (ensaio colorimétrico de MTT), migração (fecho de ferida) e indução da apoptose (libertação de LDH).
Os resultados revelaram que a AQP3 é a AQP da membrana plasmática mais expressa nas linhas de melanoma. As células MNT-1 expressam mais mRNA, contudo, as A375 produzem mais proteína AQP3. O ensaio de toxicidade demonstrou que a concentração de composto que não compromete a viabilidade celular é 5 μM, sendo esta a concentração usada em ensaios subsequentes. Todos os compostos inibiram o transporte de glicerol via AQP3, sendo que CCON (MNT-1) e CCH2N (A375) se mostraram mais potentes. O CCON mostrou ser o melhor inibidor do transporte de H2O2. Por outro lado, o CCH2N reduziu mais a adesão celular. No caso das A375, o CNHN pareceu influenciar a redução da proliferação e migração celular. A proliferação das MNT-1 foi mais comprometida pelo CCH2N e a migração pelo CCON. O ensaio de LDH demonstrou que estes compostos não afetam a integridade membranar destas células.
Concluindo, a nova série de inibidores da AQP3 mostrou-se promissora e esta inibição parece afetar a progressão do melanoma, confirmando a AQP3 como um alvo promissor para terapias contra o cancro.
The aquaporins (AQP) named peroxiporins are transmembrane channels that facilitate the permeation of H2O2 across biological membranes. At high concentrations, H2O2 triggers oxidative stress. Since AQP3 is found overexpressed in several cancers, such as melanoma, it may have an important role in the biology of cancer. In this study, MNT-1 and A375 cells were used as melanoma cell models. First, the expression of AQP3 mRNA (qPCR) and protein (Western Blot) was analysed. Subsequently, the effect of Auphen, an AQP3 inhibitor, and a new series of gold compounds (CCON, CNHN and CCH2N) was evaluated on membrane permeability to water, glycerol and H2O2. These compounds were further evaluated for their toxicity and effect on cell adhesion, proliferation (MTT colorimetric assay), migration (wound closure) and apoptosis induction (LDH release). The results revealed that AQP3 is the most expressed plasma membrane AQP in melanoma lines. MNT-1 cells express more AQP3 mRNA, however, A375 cells produce more AQP3 protein. The toxicity test demonstrated that the concentration of compound that does not compromise cell viability is 5 μM, which was used for further experiments. All compounds inhibited glycerol transport via AQP3 with CCON (MNT-1) and CCH2N (A375) being more potent. CCON proved to be the best inhibitor of H2O2 transport. On the other hand, CCH2N reduce adherence the most. In the case of A375, CNHN appeared to influence the reduction of cell proliferation and migration. The proliferation of MNT-1 was more compromised by CCH2N and migration by CCON. The LDH assay demonstrated that these compounds do not affect the membrane integrity of these cells. Overall, our data reveal that the impairment of H2O2 and glycerol permeability via AQP3 can affect melanoma skin cancer progression, unveiling AQP3 a promising drug target for cancer therapies. In addition, this new series of gold compounds revealed good candidates for cancer treatment since they decrease melanoma cell proliferation and migration.
The aquaporins (AQP) named peroxiporins are transmembrane channels that facilitate the permeation of H2O2 across biological membranes. At high concentrations, H2O2 triggers oxidative stress. Since AQP3 is found overexpressed in several cancers, such as melanoma, it may have an important role in the biology of cancer. In this study, MNT-1 and A375 cells were used as melanoma cell models. First, the expression of AQP3 mRNA (qPCR) and protein (Western Blot) was analysed. Subsequently, the effect of Auphen, an AQP3 inhibitor, and a new series of gold compounds (CCON, CNHN and CCH2N) was evaluated on membrane permeability to water, glycerol and H2O2. These compounds were further evaluated for their toxicity and effect on cell adhesion, proliferation (MTT colorimetric assay), migration (wound closure) and apoptosis induction (LDH release). The results revealed that AQP3 is the most expressed plasma membrane AQP in melanoma lines. MNT-1 cells express more AQP3 mRNA, however, A375 cells produce more AQP3 protein. The toxicity test demonstrated that the concentration of compound that does not compromise cell viability is 5 μM, which was used for further experiments. All compounds inhibited glycerol transport via AQP3 with CCON (MNT-1) and CCH2N (A375) being more potent. CCON proved to be the best inhibitor of H2O2 transport. On the other hand, CCH2N reduce adherence the most. In the case of A375, CNHN appeared to influence the reduction of cell proliferation and migration. The proliferation of MNT-1 was more compromised by CCH2N and migration by CCON. The LDH assay demonstrated that these compounds do not affect the membrane integrity of these cells. Overall, our data reveal that the impairment of H2O2 and glycerol permeability via AQP3 can affect melanoma skin cancer progression, unveiling AQP3 a promising drug target for cancer therapies. In addition, this new series of gold compounds revealed good candidates for cancer treatment since they decrease melanoma cell proliferation and migration.
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aquaporins peroxiporins permeability inhibitors melanoma