Department of Women's Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA.
J Gynecol Oncol. 2012 Jan;23(1):35-42. doi: 10.3802/jgo.2012.23.1.35. Epub 2012 Jan 9.
The BCL2 family proteins are critical mediators of cellular apoptosis and, as such, have been implicated as determinants of cancer cell chemo-sensitivity. Recently, it has been demonstrated that the phosphorylation status of the BCL2 antagonist of cell death (BAD) protein may influence ovarian cancer (OVCA) cell sensitivity to cisplatin. Here, we sought to evaluate how kinase and phosphatase components of the BAD apoptosis pathway influence OVCA chemo-sensitivity.
Protein levels of cyclin-dependent kinase 1 (CDK1) and protein phosphatase 2C (PP2C) were measured by immunofluorescence in a series of 64 primary advanced-stage serous OVCA patient samples. In parallel, levels of cAMP-dependent protein kinase (PKA), AKT, and PP2C were quantified by Western blot analysis in paired mother/daughter platinum-sensitive/resistant OVCA cell lines (A2008/C13, A2780S/A2780CP, Chi/ChiR). BAD pathway kinase CDK1 was depleted using siRNA transfection, and the influence on BAD phosphorylation and cisplatin-induced apoptosis was evaluated.
OVCA patient samples that demonstrated complete responses to primary platinum-based therapy demonstrated 4-fold higher CDK1 (p<0.0001) and 2-fold lower PP2C (p=0.14) protein levels than samples that demonstrated incomplete responses. Protein levels of PP2C were lower in the platinum-resistant versus that shown in the platinum-sensitive OVCA cell line sub-clones. Levels of PKA were higher in all platinum-resistant than in platinum-sensitive OVCA cell line sub-clones. Selective siRNA depletion of CDK1 increased sensitivity to cisplatin-induced apoptosis (p<0.002).
BAD pathway kinases and phosphatases, including CDK1 and PP2C, are associated with OVCA sensitivity to platinum and may represent therapeutic opportunities to enhance cytotoxic efficacy.
BCL2 家族蛋白是细胞凋亡的关键介质，因此被认为是癌症细胞化疗敏感性的决定因素。最近，已经证明 BCL2 凋亡拮抗剂（BAD）蛋白的磷酸化状态可能影响卵巢癌（OVCA）细胞对顺铂的敏感性。在这里，我们试图评估 BAD 凋亡途径的激酶和磷酸酶成分如何影响 OVCA 的化疗敏感性。
通过免疫荧光法在 64 例晚期浆液性 OVCA 患者的一系列样本中测量细胞周期蛋白依赖性激酶 1（CDK1）和蛋白磷酸酶 2C（PP2C）的蛋白水平。同时，通过 Western blot 分析在配对的母/子铂敏感/耐药 OVCA 细胞系（A2008/C13、A2780S/A2780CP、Chi/ChiR）中定量测定 cAMP 依赖性蛋白激酶（PKA）、AKT 和 PP2C 的水平。使用 siRNA 转染耗尽 BAD 途径激酶 CDK1，并评估其对 BAD 磷酸化和顺铂诱导的细胞凋亡的影响。
对初次铂类基础治疗有完全反应的 OVCA 患者样本显示 CDK1 蛋白水平高出 4 倍（p<0.0001），而对不完全反应的患者样本则高出 2 倍（p=0.14），而 PP2C 蛋白水平则较低。在铂耐药的 OVCA 细胞系亚克隆中，PP2C 蛋白水平低于在铂敏感的 OVCA 细胞系亚克隆中。所有铂耐药的 OVCA 细胞系亚克隆中 PKA 水平均高于铂敏感的 OVCA 细胞系亚克隆。选择性 siRNA 耗尽 CDK1 可增加对顺铂诱导的细胞凋亡的敏感性（p<0.002）。
BAD 途径激酶和磷酸酶，包括 CDK1 和 PP2C，与 OVCA 对铂的敏感性相关，可能代表增强细胞毒性疗效的治疗机会。
