ASCORBATE-INDUCED RADIOSENSITIZATION IN PANCREATIC CANCER

描述(申请人提供)：胰腺癌是美国癌症死亡的第四大原因，发病率正在增加；预后仍然很差。我们建议研究一种全新的方法，使用药理抗坏血酸作为放射治疗的辅助手段，来治疗胰腺癌。静脉注射抗坏血酸(即抗坏血酸、维生素C)，而不是口服抗坏血酸，会产生较高的血浆浓度，对肿瘤细胞具有细胞毒性。我们小组的初步研究表明，抗坏血酸在胰腺癌细胞中诱导氧化应激和细胞毒性；这种细胞毒性在肿瘤细胞中似乎比正常细胞更大。我们已经确定，在体外和体内，药理抗坏血酸是一种通过自氧化传递过氧化氢(H_2O_2)的前体药物。我们之前资助的R21赠款CA137230的研究表明，在人类可以达到的剂量下，抗坏血酸通过过氧化氢介导的机制降低所有被检查的胰腺癌细胞系的存活率和增殖。目前的应用扩展了这项临床前工作，以确定该策略是否可以有效地用作胰腺癌放射治疗的辅助手段。如果胰腺癌细胞(相对于正常细胞)更容易受到抗坏血酸诱导的细胞毒性的影响，这是因为金属离子催化的抗坏血酸自氧化导致过氧化氢的产生增加，那么使用抗坏血酸作为佐剂将有望使胰腺癌细胞对标准治疗方法敏感，例如增加活性氧水平的电离辐射。我们将使用生物化学/分子生物学技术在体外和体内确定对抗坏血酸诱导的放射增敏和氧化应激的相对敏感性；我们将使用荧光标记胸苷正电子发射断层扫描(Flt-PET)作为体内细胞增殖的无创性指标。这项建议侧重于在胰腺癌的治疗中使用辅助方法(高剂量抗坏血酸)来提高标准抗癌疗法(电离辐射)的治疗比率。目前的提议将在体外和体内验证金属离子催化抗坏血酸自氧化生成过氧化氢诱导人胰腺癌细胞辐射增敏的假设。我们将通过以下两个具体目标来验证我们的假设：1.确定抗坏血酸是否通过金属离子催化的抗坏血酸自氧化产生的H_2O_2通量选择性地在体外诱导人胰腺癌细胞(与正常细胞相比)的放射增敏。2.确定抗坏血酸是否在体内选择性地诱导人胰腺癌细胞的放射增敏。如果我们能够严格地证明由药理抗坏血酸介导的放射增敏在人胰腺癌细胞中诱导优先的氧化应激和随后的细胞毒性，那么这一应用的结果将为合理设计对NCCAM的RFA PA-09-167高度响应的联合方式癌症治疗提供基础，该RFA-09-167题为“癌症护理补充方法的发展项目”。 公共卫生相关性：静脉注射抗坏血酸可产生较高的血浆浓度，对胰腺肿瘤细胞具有细胞毒性。药理抗坏血酸被认为是过氧化氢(H_2O_2)形成的前体药物。我们的建议研究了通过药物抗坏血酸治疗产生的过氧化氢通量来增强人胰腺癌细胞辐射敏感性的机制。

DESCRIPTION (provided by applicant): Adenocarcinoma of the pancreas is the fourth leading cause of cancer death in the United States and is increasing in incidence; the prognosis remains dismal. We propose to investigate an entirely new approach, using pharmacological ascorbate as an adjuvant to radio-therapy, to treat pancreas cancer. Intravenous ascorbate (i.e., ascorbic acid, vitamin C), but not oral ascorbate, produces high plasma concentrations, which are in the range that are cytotoxic to tumor cells. Preliminary studies from our group have demonstrated that ascorbate induces oxidative stress and cytotoxicity in pancreatic cancer cells; this cytotoxicity appears to be greater in tumor vs. normal cells. We have firmly established that pharmacological ascorbate is a pro-drug for delivery of hydrogen peroxide (H2O2) in vitro and in vivo via its autoxidation. Studies from our previously funded R21 grant CA137230 "Mechanisms of ascorbate-induced cytotoxicity in pancreatic cancer" have demonstrated that ascorbate, in doses achievable in humans, decreases viability and proliferation in all pancreatic cancer cell lines examined, via a H2O2-mediated mechanism. The current application extends this preclinical work to determine if this strategy can be used effectively as an adjuvant in radiotherapy for pancreatic cancer. If pancreatic cancer cells (relative to normal cells) are more susceptible to ascorbate-induced cytotoxicity due to increased metal ion catalyzed ascorbate autoxidation leading to increased production of H2O2, then using ascorbate as an adjuvant would be expected to sensitize pancreatic tumor cells to standard therapies such as ionizing radiation that increase levels of reactive oxygen species. We will use biochemistry/molecular biology techniques to determine relative susceptibility to ascorbate-induced radiosensitization and oxidative stress in vitro and in vivo; we will employ FLT-PET (fluoro-labeled thymidine positron emission tomography) as a non- invasive in vivo index of cell proliferation. This proposal focuses on improvement of the therapeutic ratio of a standard anti-cancer therapy (ionizing radiation) using a complementary approach (high dose ascorbate), in the treatment of pancreatic cancer. The current proposal will test the hypothesis that metal ion catalyzed H2O2 generation from the auto-oxidation of ascorbate induces radiosensitization of human pancreatic cancer cells in vitro and in vivo. We will test our hypotheses with the following two Specific Aims: 1. Determine if ascorbate selectively induces radio-sensitization of human pancreatic cancer cells (vs. normal cells) in vitro via the flux of H2O2 generated from metal ion catalyzed auto-oxidation of ascorbate. 2. Determine if ascorbate selectively induces radio-sensitization of human pancreatic cancer cells in vivo. If we can rigorously demonstrate that the radiosensitization mediated by pharmacological ascorbate induces preferential oxidative stress and subsequent cytotoxicity in human pancreatic cancer cells, then the results of this application will provide a foundation for the rational design of a combined modality cancer therapy that is highly responsive to the RFA PA-09-167 from NCCAM entitled "Developmental Projects in Complementary Approaches to Cancer Care". PUBLIC HEALTH RELEVANCE: Intravenous ascorbate produces high plasma concentrations in the range that is cytotoxic to pancreatic tumor cells. Pharmacological ascorbate has been hypothesized to be a pro-drug for formation of hydrogen peroxide (H2O2). Our proposal investigates mechanisms to enhance radiosensitivity of human pancreatic cancer cells by the flux of H2O2 generated by treatment with pharmacological ascorbate.