Piechocki Marie P, Yoo George H, Dibbley Susan K, Amjad Esmael H, Lonardo Fulvio
Department of Otolaryngology-Head and Neck Surgery, Wayne State University and Karmanos Cancer Center, Detroit, MI 48201, USA.
Int J Cancer. 2006 Jul 15;119(2):441-54. doi: 10.1002/ijc.21837.
Understanding the role of signal transduction in regulating pathways responsible for cell growth, survival and apoptosis is critical for cancer therapy. We developed and characterized a HER2/neu and Fas overexpressing cell line (BNT.888 ACA2) from a salivary gland adenocarcinoma that arose in a HER2/neu transgenic mouse. We evaluated the effects of Iressa on signal transduction networks downstream of the activated HER2 and the impact on proliferation, cell cycle and apoptosis. Iressa treatment diminished phosphorylation of the HER2/neu and EGFR. Phosphorylation of STAT-3 also decreased and mitogenic signaling through the MAPK pathways was greatly reduced. Cyclin D1 levels decreased, and cells were arrested in G0 and failed to enter S-phase because of hypophosphorylation of Rb and to traverse the G2M checkpoint because of degradation of cyclin B1. Cytostasis occurred within 48 hr at 250-500 nM Iressa. Levels of proapoptotic factors (bim and bax) increased and levels of antiapoptotic factors (bcl-2 and bcl-xL) decreased in a dose-dependent manner. Higher doses of Iressa diminished phosphorylation of Akt slightly, but failed to induce apoptosis. Fas antibody was a potent agonist of apoptosis. Pretreatment with Iressa (1 microM, 24 hr) greatly enhanced Fas-mediated apoptosis as determined by Annexin V binding, cleavage of caspase-3 and PARP. Augmentation of apoptosis was associated with increased Fas expression and membrane localization. Iressa pretreatment increased bid activation, cleavage of caspases -3, -9 and -12 and stress signaling via c Jun. These data showing that Iressa induces cytostasis and primes the extrinsic (Fas) and intrinsic (mitochondrial and endoplasmic reticulum) apoptotic pathways should lead to the development of novel therapeutic targets and strategies.
了解信号转导在调节细胞生长、存活和凋亡相关通路中的作用对于癌症治疗至关重要。我们从一只HER2/neu转基因小鼠发生的涎腺腺癌中开发并鉴定了一种HER2/neu和Fas过表达的细胞系（BNT.888 ACA2）。我们评估了易瑞沙对活化的HER2下游信号转导网络的影响以及对增殖、细胞周期和凋亡的影响。易瑞沙处理可减少HER2/neu和EGFR的磷酸化。STAT-3的磷酸化也降低，并且通过MAPK通路的促有丝分裂信号大大减少。细胞周期蛋白D1水平降低，细胞因Rb的低磷酸化而停滞在G0期，无法进入S期，并且由于细胞周期蛋白B1的降解而无法通过G2M检查点。在250 - 500 nM易瑞沙作用下，48小时内出现细胞生长停滞。促凋亡因子（bim和bax）水平呈剂量依赖性增加，抗凋亡因子（bcl-2和bcl-xL）水平降低。更高剂量的易瑞沙可轻微减少Akt的磷酸化，但未能诱导凋亡。Fas抗体是凋亡的有效激动剂。如通过膜联蛋白V结合、caspase-3和PARP的裂解所测定，用易瑞沙（1 microM，24小时）预处理可大大增强Fas介导的凋亡。凋亡的增强与Fas表达增加和膜定位有关。易瑞沙预处理增加了bid激活、caspases -3、-9和-12的裂解以及通过c-Jun的应激信号。这些数据表明易瑞沙诱导细胞生长停滞并启动外源性（Fas）和内源性（线粒体和内质网）凋亡途径，这应该会导致新的治疗靶点和策略的开发。
