NF KAPPA B MEDIATED CHEMORESISTANCE IN HUMAN LUNG CANCER

肺癌是导致男性和女性癌症死亡的最常见原因。在所有新诊断的肺癌中，80%是非小细胞肺癌(NSCLC)，超过75%的人患有晚期疾病。出于这个原因，以及在完全切除后容易出现远处复发，大多数患者都是单独进行化疗或与其他方法联合治疗。不幸的是，尽管采用了第二代或第三代化疗药物的治疗方案，但大多数非小细胞肺癌仍具有化疗耐药性。肿瘤化疗耐药被归因于细胞克服程序性细胞死亡(细胞凋亡)的能力。最近，已经证实化疗上调了核因子-kappaB转录因子，这与细胞对化疗诱导的细胞凋亡的抵抗有关。基于这一认识，我们进行了实验，以确定在加入吉西他滨和顺铂这两种通常用于治疗NSCLC的遗传毒性药物后，NF-kappaB是否在NSCLC细胞系中提供了类似的细胞生存信号。初步数据表明，这些化疗药物可诱导核因子-kappaB转录活性。更重要的是，核因子-kappaB的缺失使非小细胞肺癌细胞系对化疗诱导的细胞凋亡敏感。虽然化疗诱导的细胞死亡涉及某些细胞类型的死亡受体通路，但化疗诱导的非小细胞肺癌细胞凋亡不涉及依赖于Fas或caspase-8的死亡受体通路。相反，在化疗后，需要使用核因子-kappaB来克服线粒体介导的细胞凋亡。此外，神经酰胺还能抑制P13K/Akt抗凋亡通路，激活NFkappaB。由于核因子-kappaB活性的丧失使NSCLC细胞对化疗诱导的凋亡敏感，本研究的主要目的是研究化疗后细胞凋亡和化疗耐药的信号机制。为了实现这一目标，我们将使用(由P.I.建立的)缺乏核因子-kappaB活性的细胞系，并与对照组进行比较。这些细胞将暴露在化疗药物中，并将在体外检测凋亡和抗凋亡信号通路。该提案的具体目的是：1)确定化疗是否通过线粒体依赖的机制杀死非小细胞肺癌细胞，2)确定细胞的氧化还原状态是否对化疗反应中依赖核因子-kappaB的细胞生存起调节作用，以及3)确定神经酰胺在化疗后作为促凋亡和抗凋亡介质的作用。这些研究将对化疗如何激活细胞凋亡途径，以及肿瘤通过上调核因子-kappaB而变得耐药的机制提供重要的见解。这项研究的最终目的是为设计治疗晚期肺癌患者的新治疗策略的启动提供必要的背景。

Lung cancer is the most common cause of cancer death in both men and women. Eighty percent of all newly diagnosed lung cancers are non-small cell lung cancers (NSCLC), and over 75 percent present with advanced stage disease. For this reason as well as the predilection for distant recurrence after complete resection, most patients are treated with chemotherapy alone or in combination with other modalities. Unfortunately, the majority of NSCLC are chemoresistant despite treatment regimens with second or third generation of chemotherapeutic agents. Tumor chemoresistance has been attributed to the ability of cells to overcome programmed cell death (apoptosis). Recently, it has been established that chemotherapy upregulates the NF-kappaB transcription factor and this is associated with cellular resistance to chemotherapy-induced apoptosis. With this understanding, experiments were performed to determine whether NF-kappaB provided a similar cell survival signal in NSCLC cell lines following the addition of gemcitabine and cisplatin, two genotoxic agents commonly used to treat NSCLC. Preliminary data demonstrates that these chemotherapeutic agent induce NF-kappaB transcriptional activity. More importantly, the loss of NF- kappaB sensitizes NSCLC cell lines to chemotherapy-induced apoptosis. Although cell death induced by chemotherapy involves death receptor pathways in certain cell types, chemotherapy- induced apoptosis in NSCLC cells did not involve either Fas- or caspase-8-dependent death receptor pathways. In contrast, NF- kappaB was required to overcome mitochondrial-mediated apoptosis following chemotherapy addition. In addition, ceramide has recently been shown to inhibit the anti-apoptotic P13K/Akt pathway as well as activate NFkappaB. Since the loss of NF- kappaB activity sensitizes NSCLC cells to chemotherapy-induced apoptosis, the major goal of this proposal is to examine the cell signaling mechanisms governing apoptosis and chemoresistance following the addition of chemotherapy. To achieve this objective, we will use established (by the P.I.) cell lines which lack NF-kappaB activity and compare them to controls. These cells will be exposed to chemotherapeutic agents and apoptotic and anti-apoptotic signaling pathways will be examined in vitro. The specific aims of the proposal are to: 1) establish whether chemotherapy kills NSCLC cells through mitochondrial-dependent mechanisms, 2) determine whether the redox status of the cell is responsible for modulating NF-kappaB-dependent cell survival in response to chemotherapy, and 3) determine the role of ceramide as both a pro- and anti-apoptotic mediator following chemotherapy. These studies will provide important insight into how chemotherapy activates apoptotic pathways, as well as mechanisms by which tumors become chemoresistant through upregulation of NF-kappaB. The ultimate goal of this study is to provide the necessary background for the initiation of novel treatment strategies designed to treat patients with advanced lung cancer.