Enhancing Direct Tumor Cell Cytotoxicity By Manipulating Growth Factor Signaling

这一应用的广泛、长期目标是通过以下方式优化浆膜光动力疗法(Pdt) 抑制信号转导通路的相关成分，从而改善预后 胸膜和腹膜扩散的癌症患者。表皮生长因子受体的激活 (EGFR)信号通路被认为限制了PDT作为一种治疗方法的有效性。初步 数据表明，抑制EGFR将增加PDT的治疗指数，增加对肿瘤细胞的杀伤力 而不会伴随正常组织毒性的增加。此外，尽管光动力疗法被认为主要是杀死细胞 通过凋亡机制，通过抑制EGFR信号来增加癌细胞的死亡可能 通过刺激坏死性/非凋亡性细胞死亡途径发生。因此，这个项目将重点放在 定义和表征抑制生长因子信号对信号的影响，细胞毒性和 Bpd介导的卵巢癌细胞和肺癌细胞光动力治疗后细胞死亡的机制。最大限度地提高 这些发现的外部有效性、可推广性和临床适用性，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 OVCAR-5和H460，以及OVCAR-5和H460肿瘤移植瘤。我们将使用化学缓蚀剂 并探讨siRNA抑制EGFR及其受体后信号转导伙伴的分子机制 通过抑制EGFR信号增强BPD介导的PDT细胞毒作用。我们还将检查 对PDT和EGFR通路的抑制进行测序和计时，以最大限度地增强 BPD介导的PDT毒副作用。将在这些研究中分析的具体内含物是：1)水平 和EGFR及相关信号蛋白的激活2)EGFR及其相关信号蛋白的核质分布 与核状态的关系3)细胞和肿瘤的克隆性存活。最后，我们将确定 调控生长因子信号在BPD介导的光动力疗法后细胞死亡机制中的作用 以进一步确定这一新的治疗方法的特点，并进一步确定需要加强的领域 细胞毒性。将在这些研究中分析的特定终点是：1)具有 2)细胞死亡相关蛋白的表达和激活：3) 对P53的影响。BAX和BAK蛋白表达对克隆形成细胞存活和细胞百分率的影响 细胞凋亡型与坏死型的比较研究应该有助于我们理解潜在的临床 光动力疗法联合生长因子抑制治疗浆膜腔积液 恶性肿瘤。考虑到卵巢癌在任何一种情况下扩散到 腹膜腔内或胸腔内非小细胞肺癌，此治疗方法临床应用成功 可能导致这些人群的临床护理达到新的标准。

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.