MDA-7/IL-24 and free radicals in renal cancer therapy

描述(申请人提供)：肾细胞癌(RCC)是最致命和最难治疗的肿瘤之一，特别是当疾病已经扩散到肾脏以外并扩散到周围筋膜的时候；即使是密集的放化疗组合也不能治愈，对患者的生存只产生轻微的影响。存在着对替代治疗方式的重大需求。在许多不同类型的肿瘤细胞中，MDA-7/IL-24的表达导致生长停滞和凋亡，而在未转化的细胞中，它既不改变细胞生长，也不改变细胞存活。以前的研究表明，无论是作为病毒(Ad.mda-7)、作为纯化的融合蛋白(GST-MDA-7)，还是在条件培养液中，MDA-7都能抑制肿瘤细胞的生长。在肾癌中，我们发现低浓度的丙二醛-7(0.5-1.5 nM)抑制生长而不杀死细胞，而高浓度的丙二醛-7(&gt；20 nM)抑制生长并促进细胞死亡。低水平的MDA-7增强了RCC对几种产生自由基的试剂的敏感性。原代培养的肾细胞未观察到丙二醛-7和自由基的抗增殖和细胞毒作用。MDA-7抑制RCC增殖并与自由基相互作用以杀死RCC的机制尚不完全清楚。具体目标1将确定GST-MDA-7是否以剂量依赖的方式导致RCC中p38MAPK和JNK1/2的激活增加，其信号转导被认为是高浓度(&gt；30 nM)GST-MDA-7引起细胞因子诱导的细胞凋亡的原因。此外，我们将证明或驳斥[三氧化二砷(As_2O_3)和N-(4-羟基苯基)维甲酰胺(4-HPR)]这两种产生活性氧物种的试剂是否能增强低浓度GST-MDA-7(0.5-1.5 nM)导致p38和JNK1/2通路长时间激活的能力。具体目标2将确定PI3激酶和MEK1/2抑制剂联合处理肾细胞癌增强纯化的MDA-7蛋白对细胞的杀伤作用的机制。此外，我们将证明或驳斥[三氧化二砷(As_2O_3)和N-(4-羟基苯基)维甲酰胺(4-HPR)]这两种产生活性氧物种的试剂是否通过导致ERK1/2失活来提高低浓度GST-MDA-7的致命性。具体目标3将确定向先前存在的肿瘤中注入Ad.mda-7或MDA-7蛋白是否可以减少肾癌生长，并增强肿瘤对As_2O_3和4-HPR的敏感性。

DESCRIPTION (provided by applicant): Renal Cell Carcinoma (RCC) is among the most lethal and difficult tumors to treat, particularly when disease has spread beyond the kidney and into the surrounding fascia; even intensive combinations of radio- and chemotherapy are not curative and yield only a modest impact on patient survival. There is a major need for alternative therapeutic modalities. Expression of MDA-7/IL-24 in many different tumor cell types causes growth arrest and apoptosis whereas in non-transformed cells it alters neither cell growth nor cell survival. Previous studies have shown that MDA-7 administered either as a virus (Ad.mda-7), as a purified fusion protein (GST-MDA-7), or in conditioned media, suppressed the growth of tumor cells. In RCC we found that low concentrations of MDA-7 (0.5-1.5 nM) suppressed growth without killing cells whereas higher levels of MDA-7 (> 20 nM) suppressed growth and enhanced cell death. Low levels of MDA-7 enhanced the sensitivity of RCCs to several agents that generate free radicals. The anti-proliferative and cytotoxic effects of MDA-7 and free radicals were not observed in primary renal cells. The mechanisms by which MDA-7 inhibits RCC proliferation and interacts with free radicals to kill RCCs are not fully understood. Specific aim 1 will determine whether GST-MDA-7, in a dose-dependent fashion, causes increasing amounts of p38 MAPK activation and JNK1/2 activation, in RCCs, whose signaling is believed to be responsible for cytokine-induced apoptosis at high (> 30 nM) GST-MDA-7 concentrations. Additionally, we will prove or refute whether [arsenic trioxide (As2O3) and N-(4-hydroxyphenyl) retinamide (4-HPR)], agents that generate reactive oxygen species, enhance the ability of low GST-MDA-7 concentrations (0.5-1.5 nM) to cause prolonged activation of the p38 and JNK1/2 pathways. Specific aim 2 will determine the mechanisms by which combined treatment of RCCs with PI3 kinase and MEK1/2 inhibitors enhance cell killing by purified MDA-7 protein. Additionally, we will prove or refute whether [arsenic trioxide (As2O3) and N-(4-hydroxyphenyl) retinamide (4-HPR)], agents that generate reactive oxygen species, enhance the lethality of low GST-MDA-7 concentrations by causing inactivation of ERK1/2. Specific aim 3 will determine whether infusion of Ad.mda-7 or MDA-7 protein, into a pre-existing tumor, reduces RCC growth and enhances tumor sensitivity to As2O3 and 4-HPR.