Enhancing Direct Tumor Cell Cytotoxicity By Manipulating Growth Factor Signaling

本申请的广泛的长期目标是通过以下方式优化Serbine光动力疗法（PDT）： 抑制信号转导途径的相关组分，从而改善 癌症胸膜和腹膜扩散的患者。表皮生长因子受体的激活 EGFR信号通路被认为限制了PDT作为治疗方法的有效性。初步 数据表明，EGFR的抑制将增加PDT的治疗指数，增加肿瘤细胞杀伤， 而不伴随正常组织毒性的增加。此外，虽然PDT被认为主要杀死细胞， 通过细胞凋亡机制，通过抑制EGFR信号传导增加癌细胞死亡可能 通过刺激坏死/非凋亡细胞死亡途径发生。因此，本项目将重点关注 定义和表征抑制生长因子信号传导对信号传导、细胞毒性和 BPD介导的卵巢癌和肺癌细胞PDT后的细胞死亡机制。最大化 外部效度、普遍性和临床适用性，将进行实验 使用一组卵巢癌（QvCa）和非小细胞肺癌（NSCLC）细胞系，包括 使用0 VCAR-5和H460以及还使用0 VCAR-5和H460肿瘤异种移植物。我们将使用化学抑制剂 和siRNA抑制EGFR及其受体后信号分子，探讨其分子机制 由此抑制EGFR信号传导增强BPD介导的PDT细胞毒性。我们亦会研究 PDT和EGFR通路抑制的测序和定时，以最大限度地增强 BPD介导的PDT细胞毒性在这些研究中将分析的具体内涵是：1）水平 EGFR及其相关信号蛋白的核质分布及其与EGFR的关系 与核STAT 3的关联：3）细胞和肿瘤的克隆形成存活。最后，我们将确定 调节生长因子信号对BPD介导的PDT后细胞死亡机制的影响 为了进一步表征这种新的治疗方法，并进一步确定增强 细胞毒在这些研究中将分析的具体终点是：1）具有以下特征的细胞的百分比： 凋亡与坏死细胞死亡表型; 2}细胞死亡相关蛋白的表达和活化：3） 对p53的影响Bax和巴克蛋白表达对克隆形成细胞存活率和具有 凋亡与坏死细胞死亡表型这些研究应有助于我们了解潜在的临床 联合PDT +生长因子抑制作为治疗血清型糖尿病患者的方法的应用 恶性肿瘤。考虑到卵巢癌扩散的患者预后极差， 腹腔内或胸腔内的非小细胞肺癌，成功应用于临床 可能会为这些人群带来新的临床护理标准。

The broad, long-term goal of this application is to optimize serosal photodynamic therapy (PDT) through the inhibition of relevant components of the signal transduction pathways thereby improving the outcome of patients with pleural and peritoneal spread of cancers. Activation of the epidermal growth factor receptor (EGFR) signaling pathway is thought to limit the effectiveness of PDT as a therapeutic approach. Preliminary data suggest that inhibition of EGFR will increase the therapeutic index of PDT, increasing tumor cell kill without a concomitant increase in normal tissue toxicity. Moreover, while PDT is thought to kill cells primarily through apoptotic mechanisms, augmentation of cancer cell death through inhibition of EGFR signaling may occur through stimulation of necrotic/non-apoptotic cell death pathways. Therefore, this project will focus on defining and characterizing the impact of inhibiting growth factor signaling on the signaling, cytotoxicity and mechanisms of cell death following BPD-mediated PDT of ovarian and lung cancer cells. To maximize the external validity, generalizabilitv and clinical applicability of these findings, experiments will be performed using a panel of ovarian cancer (QvCa) and non-small cell lung cancer (NSCLC) cell lines that includes OVCAR-5 and H460 and also using OVCAR-5 and H460 tumor xenografts. We will use chemical inhibitors and siRNA to inhibit EGFR and its post-receptor signaling partners to investigate the molecular mechanism by which the inhibition of EGFR signaling enhances BPD-mediated PDT cytotoxicity. We will also examine the sequencing and timing PDT and EGFR pathway inhibition in order to maximize the enhancement of BPD-mediated PDT cvtotoxicitv. The specific endooints that will be analyzed in these studies are: 1) Levels and activation of EGFR and related signaling proteins 2} The nucleo-cvtoplasmic distribution of EGFR and its association with nuclear STAT3: 3) The clonogenic survival of cells and tumors. Lastly, we will determine the effect of modulating growth factor signaling on the mechanism of cell death following BPD-mediated PDT in order to further characterize this novel treatment approach and to further identify areas for enhancement of cytotoxicity. The specific endpoints that will be analyzed in these studies are: 1) The percent of cells with apoptotic vs necrotic cell death pheotvpes; 2} The expression and activation of cell death-related proteins: 3) The effect on p53. Bax and Bak protein expression on clonogenic cell survival and the percent of cells with apoptotic vs necrotic ceil death pheotypeslhese studies should help us to understand the potential clinical application of combination PDT + growth factor inhibition as a therapeutic approach for patients with serosal malignancies. Given the extremely poor prognosis for patients with spread of either ovarian cancer within the abdominal cavity or NSCLC within the chest cavity, successful clinical application of this treatment approach could lead to a new standard of clinical care in these populations.