Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla (Departamento de Fisiología Médica y Biofísica), Seville, Spain.
J Cell Physiol. 2014 Nov;229(11):1787-801. doi: 10.1002/jcp.24632.
AQP3 has been correlated with higher transport of glycerol, increment of ATP content, and larger proliferation capacity. Recently, we described the gold(III) complex Auphen as a very selective and potent inhibitor of AQP3's glycerol permeability (Pgly ). Here we evaluated Auphen effect on the proliferation of various mammalian cell lines differing in AQP3 expression level: no expression (PC12), moderate (NIH/3T3) or high (A431) endogenous expression, cells stably expressing AQP3 (PC12-AQP3), and human HEK293T cells transiently transfected (HEK-AQP3) for AQP3 expression. Proliferation was evaluated in the absence or presence of Auphen (5 μM) by counting number of viable cells and analyzing 5-bromo-2'-deoxyuridine (BrdU) incorporation. Auphen reduced ≈50% the proliferation in A431 and PC12-AQP3, ≈15% in HEK-AQP3 and had no effect in PC12-wt and NIH/3T3. Strong arrest in the S-G2/M phases of the cell cycle, supported by analysis of cyclins (A, B1, D1, E) levels, was observed in AQP3-expressing cells treated with Auphen. Flow-cytometry of propidium iodide incorporation and measurements of mitochondrial dehydrogenases activity confirmed absence of cytotoxic effect of the drug. Functional studies evidenced ≈50% inhibition of A431 Pgly by Auphen, showing that the compound's antiproliferative effect correlates with its ability to inhibit AQP3 Pgly . Role of Cys-40 on AQP3 permeability blockage by Auphen was confirmed by analyzing the mutated protein (AQP3-Ser-40). Accordingly, cells transfected with mutated AQP3 gained resistance to the antiproliferative effect of Auphen. These results highlight an Auphen inhibitory effect on proliferation of cells expressing AQP3 and suggest a targeted therapeutic effect on carcinomas with large AQP3 expression.
AQP3 与甘油转运增加、ATP 含量增加和增殖能力增强有关。最近，我们描述了金（III）配合物 Auphen 是 AQP3 甘油通透性（Pgly）的非常选择性和有效抑制剂。在这里，我们评估了 Auphen 对不同 AQP3 表达水平的各种哺乳动物细胞系增殖的影响：无表达（PC12）、中度（NIH/3T3）或高（A431）内源性表达、稳定表达 AQP3 的细胞（PC12-AQP3）和瞬时转染 AQP3 的人 HEK293T 细胞（HEK-AQP3）。通过计数活细胞数量和分析 5-溴-2'-脱氧尿苷（BrdU）掺入来评估有无 Auphen（5μM）存在时的增殖。Auphen 使 A431 和 PC12-AQP3 的增殖减少了约 50%，使 HEK-AQP3 的增殖减少了约 15%，对 PC12-wt 和 NIH/3T3 没有影响。用 Auphen 处理表达 AQP3 的细胞时，观察到细胞周期 S-G2/M 期明显停滞，细胞周期蛋白（A、B1、D1、E）水平分析支持这一结果。碘化丙啶掺入的流式细胞术和线粒体脱氢酶活性测量证实了药物无细胞毒性作用。功能研究表明，Auphen 对 A431 Pgly 的抑制率约为 50%，表明该化合物的抗增殖作用与其抑制 AQP3 Pgly 的能力相关。通过分析突变蛋白（AQP3-Ser-40），证实了 Cys-40 在 Auphen 阻断 AQP3 通透性中的作用。因此，转染突变 AQP3 的细胞对 Auphen 的抗增殖作用具有抗性。这些结果突出了 Auphen 对表达 AQP3 的细胞增殖的抑制作用，并表明在 AQP3 表达量较大的癌中具有靶向治疗作用。
