Bargiela-Iparraguirre J, Prado-Marchal L, Pajuelo-Lozano N, Jiménez B, Perona R, Sánchez-Pérez I
a Departamento Bioquimica ; Facultad Medicina ; UAM ; Madrid , Spain.
Cell Cycle. 2014;13(22):3590-601. doi: 10.4161/15384101.2014.962952.
Aneuploidy and chromosomal instability (CIN) are common features of gastric cancer (GC), but their contribution to carcinogenesis and antitumour therapy response is still poorly understood. Failures in the mitotic checkpoint induced by changes in expression levels of the spindle assembly checkpoint (SAC) proteins cause the missegregation of chromosomes in mitosis as well as aneuploidy. To evaluate the possible contribution of SAC to GC, we analyzed the expression levels of proteins of the mitotic checkpoint complex in a cohort of GC cell lines. We found that the central SAC proteins, Mad2 and BubR1, were the more prominently expressed members in disseminated GC cell lines. Silencing of Mad2 and BubR1 in MKN45 and ST2957 cells decreased their cell proliferation, migration and invasion abilities, indicating that Mad2 and BubR1 could contribute to cellular transformation and tumor progression in GC. We next evaluated whether silencing of SAC proteins could affect the response to microtubule poisons. We discovered that paclitaxel treatment increased cell survival in MKN45 cells interfered for Mad2 or BubR1 expression. However, apoptosis (assessed by caspase-3 activation, PARP proteolysis and levels of antiapoptotic Bcl 2-family members), the DNA damage response (assessed by H2Ax phosphorylation) and exit from mitosis (assessed by Cyclin B degradation and Cdk1 regulation) were activated equally between cells, independently of Mad2 or BubR1-protein levels. In contrast, we observed that the silencing of Mad2 or BubR1 in MKN45 cells showed the induction of a senescence-like phenotype accompanied by cell enlargement, increased senescence-associated β-galactosidase activity and increased IL-6 and IL-8 expression. In addition, the senescent phenotype is highly increased after treatment with PTX, indicating that senescence could prevent tumorigenesis in GC. In conclusion, the results presented here suggest that Mad2 and BubR1 could be used as prognostic markers of tumor progression and new pharmacological targets in the treatment for GC.
非整倍体和染色体不稳定（CIN）是胃癌（GC）的常见特征，但其在致癌作用和抗肿瘤治疗反应中的作用仍知之甚少。纺锤体组装检查点（SAC）蛋白表达水平的变化诱导的有丝分裂检查点功能障碍会导致有丝分裂过程中染色体的错误分离以及非整倍体的产生。为了评估SAC对GC的可能作用，我们分析了一组GC细胞系中有丝分裂检查点复合物蛋白的表达水平。我们发现，核心SAC蛋白Mad2和BubR1在播散性GC细胞系中是表达更为突出的成员。在MKN45和ST2957细胞中沉默Mad2和BubR1会降低它们的细胞增殖、迁移和侵袭能力，表明Mad2和BubR1可能在GC的细胞转化和肿瘤进展中发挥作用。接下来，我们评估了沉默SAC蛋白是否会影响对微管毒物的反应。我们发现，在干扰Mad2或BubR1表达的MKN45细胞中，紫杉醇处理可提高细胞存活率。然而，细胞凋亡（通过半胱天冬酶-3激活、PARP蛋白水解和抗凋亡Bcl-2家族成员水平评估）、DNA损伤反应（通过H2Ax磷酸化评估）以及有丝分裂退出（通过细胞周期蛋白B降解和Cdk1调节评估）在细胞之间的激活程度相同，与Mad2或BubR1蛋白水平无关。相反，我们观察到在MKN45细胞中沉默Mad2或BubR1会诱导一种衰老样表型，伴有细胞增大、衰老相关β-半乳糖苷酶活性增加以及IL-6和IL-8表达增加。此外，用PTX处理后衰老表型显著增加，表明衰老可能会阻止GC的肿瘤发生。总之，本文给出的结果表明，Mad2和BubR1可作为肿瘤进展的预后标志物以及GC治疗中的新药理学靶点。
