Garcia Diogo Gomes, de Castro-Faria-Neto Hugo Caire, da Silva Camila Ignácio, de Souza e Souza Kauê Francisco Correa, Gonçalves-de-Albuquerque Cassiano Felippe, Silva Adriana Ribeiro, de Amorim Lidia Maria da Fonte, Freire Aline Soares, Santelli Ricardo Erthal, Diniz Luan Pereira, Gomes Flávia Carvalho Alcantara, Faria Mauro Velho de Castro, Burth Patrícia
Laboratório de Imunofarmacologia, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil.
Departamento de Biologia Celular e Molecular, Instituto de Biologia, Universidade Federal Fluminense, Niterói, RJ, Brazil.
Mol Cancer. 2015 May 15;14:105. doi: 10.1186/s12943-015-0374-5.
Na/K-ATPase (NKA) is inhibited by perillyl alcohol (POH), a monoterpene used in the treatment of tumors, including brain tumors. The NKA α1 subunit is known to be superexpressed in glioblastoma cells (GBM). This isoform is embedded in caveolar structures and is probably responsible for the signaling properties of NKA during apoptosis. In this work, we showed that POH acts in signaling cascades associated with NKA that control cell proliferation and/or cellular death.
NKA activity was measured by the amount of non-radioactive Rb(+) incorporation into cultured GBM cell lines (U87 and U251) and non-tumor cells (mouse astrocytes and VERO cells). Cell viability was measured by lactate dehydrogenase levels in the supernatants of POH-treated cells. Activated c-Jun N-terminal Kinase (JNK) and p38 were assessed by western blotting. Apoptosis was detected by flow cytometry and immunocytochemistry, and the release of interleukins was measured by ELISA.
All four cell types tested showed a similar sensitivity for POH. Perillic acid (PA), the main metabolite of POH, did not show any effect on these cells. Though the cell viability decreased in a dose-dependent manner when cells were treated with POH, the maximum cytotoxic effect of PA obtained was 30% at 4 mM. 1.5 mM POH activated p38 in U87 cells and JNK in both U87 and U251 cells as well as mouse astrocytes. Dasatinib (an inhibitor of the Src kinase family) and methyl β-cyclodextrin (which promotes cholesterol depletion in cell membranes) reduced the POH-induced activation of JNK1/2 in U87 cells, indicating that the NKA-Src complex participates in this mechanism. Inhibition of JNK1/2 by the JNK inhibitor V reduced the apoptosis of GBM cells that resulted from POH administration, indicating the involvement of JNK1/2 in programmed cell death. 1.5 mM POH increased the production of interleukin IL-8 in the U251 cell supernatant, which may indicate a possible strategy by which cells avoid the cytotoxic effects of POH.
A signaling mechanism mediated by NKA may have an important role in the anti-tumor action of POH in GBM cells.
钠钾ATP酶（NKA）受到紫苏醇（POH）的抑制，紫苏醇是一种用于治疗肿瘤（包括脑肿瘤）的单萜类化合物。已知NKA α1亚基在胶质母细胞瘤细胞（GBM）中过表达。这种同工型嵌入小窝结构中，可能在细胞凋亡过程中负责NKA的信号传导特性。在本研究中，我们表明POH作用于与NKA相关的信号级联反应，这些信号级联反应控制细胞增殖和/或细胞死亡。
通过测量非放射性Rb(+)掺入培养的GBM细胞系（U87和U251）以及非肿瘤细胞（小鼠星形胶质细胞和VERO细胞）中的量来测定NKA活性。通过测量POH处理细胞上清液中的乳酸脱氢酶水平来测定细胞活力。通过蛋白质印迹法评估活化的c-Jun氨基末端激酶（JNK）和p38。通过流式细胞术和免疫细胞化学检测细胞凋亡，并通过酶联免疫吸附测定法测量白细胞介素的释放。
所测试的所有四种细胞类型对POH表现出相似的敏感性。POH的主要代谢产物紫苏酸（PA）对这些细胞没有任何影响。虽然用POH处理细胞时细胞活力呈剂量依赖性下降，但在4 mM时获得的PA的最大细胞毒性作用为30%。1.5 mM POH激活U87细胞中的p38以及U87和U251细胞以及小鼠星形胶质细胞中的JNK。达沙替尼（Src激酶家族抑制剂）和甲基β-环糊精（促进细胞膜中胆固醇消耗）降低了POH诱导的U87细胞中JNK1/2的活化，表明NKA-Src复合物参与了这一机制。JNK抑制剂V对JNK1/2的抑制减少了POH给药导致的GBM细胞凋亡，表明JNK1/2参与程序性细胞死亡。1.5 mM POH增加了U251细胞上清液中白细胞介素IL-8的产生，这可能表明细胞避免POH细胞毒性作用的一种可能策略。
由NKA介导的信号传导机制可能在POH对GBM细胞的抗肿瘤作用中起重要作用。
