Assessing pancreatic cancer susceptibility to ceramide-mediated cell death.

描述（由申请人提供）：胰腺癌是最致命的癌症之一，死亡率接近100%。绝大多数胰腺癌被发现得太晚而无法切除，并且对当前的化疗方案没有反应。由于手术通常提供很少的治愈性，胰腺癌的医疗管理一直是一个不断增长的探索领域。这项R21试点项目申请的长期目标是确定使用增强细胞神经酰胺水平的药物是否是治疗胰腺癌的可行方法。一些药物，特别是与某些其他药物联合使用时，通过刺激正常细胞蜡（称为神经酰胺）的过度产生来选择性地杀死癌症。胰腺癌的这一研究领域尚未探索。神经酰胺激活内在细胞死亡级联反应，促进下游产生活性氧（ROS），半胱天冬酶活性，表达促凋亡Bim（Bcl-2相互作用的死亡介质），并下调生存素。这些事件导致细胞凋亡、自噬和细胞衰老。为了确定靶向神经酰胺代谢是否是治疗胰腺癌的有效途径，将研究两种促进胰腺癌细胞中神经酰胺形成的药物。这些药物是芬维A胺（4-HPR），一种维生素A类似物，和Valspodar（PCS 833），一种环孢菌素A类似物。本研究将采用培养的人胰腺癌细胞系和SV-40转化的永生化胰腺细胞系。总的假设是胰腺癌细胞对神经酰胺急性敏感，该假设的推论是增强神经酰胺或神经酰胺的特定分子种类的水平会沿着沿着特定途径推进细胞毒性。这项研究有三个具体的目的，旨在评估胰腺癌细胞对神经酰胺和神经酰胺控制的细胞死亡级联的脆弱性。具体目标1将通过使用C6-神经酰胺（天然神经酰胺的短链、细胞可渗透类似物）表征胰腺癌细胞对神经酰胺的反应，并确定是否通过引入阻断神经酰胺代谢的酶抑制剂增强细胞毒性反应。具体目标2将表征4-HPR和PSC 833对细胞活力的影响，并确定细胞毒性是否与神经酰胺相关。本文包括响应于4-HPR和PSC 833产生的二氢神经酰胺和神经酰胺的质谱分析，以确定特定分子种类是否引起特定反应，例如自噬和衰老。具体目标3将评估响应4-HPR和PSC 833产生的神经酰胺对与内源性细胞死亡激活相关的生化事件的影响。这种方法的创新性在于可以操纵神经酰胺途径，为提高疗效提供了现成的平台。胰腺癌现有疗法的疗效非常有限，治疗失败的发生率非常高，这是寻求新方法的有力理由。 公共卫生相关性：该项目将通过研究无毒维生素A类似物（4-HPR）的影响来测试治疗胰腺癌的新方法。4-HPR可以通过刺激正常细胞蜡（称为神经酰胺）的过度产生来选择性地杀死癌细胞，过量的神经酰胺对恶性细胞而不是正常细胞是致命的。由于胰腺癌细胞可以将神经酰胺代谢为无毒的副产物，我们将研究包括阻断神经酰胺代谢的“伙伴药物”，以微调这种新的治疗方法。

DESCRIPTION (provided by applicant): Pancreatic cancer is one of the deadliest cancers, with a mortality rate of nearly 100%. The vast majority of pancreatic cancers are discovered too late to resect and do not respond to current chemotherapeutic regimens. Because surgery generally has little to offer curatively, medical management of pancreatic cancer has been a growing area of exploration. The long-term objective of this R21 pilot project application is to determine whether the use of agents that enhance cellular ceramide levels will be a viable approach for treating pancreatic cancer. Some drugs, especially in combination with certain other drugs, selectively kill cancers by stimulating over-production of normal cellular waxes known as ceramides. This area of investigation in pancreatic cancer is unexplored. Ceramides activate intrinsic cell death cascades that promote downstream generation of reactive oxygen species (ROS), caspase activity, expression of proapoptotic Bim (Bcl-2-interacting mediator of death), and downregulate survivin. These events contribute to apoptosis, autophagy, and cellular senescence. To determine whether targeting ceramide metabolism is an effective avenue for treating pancreatic cancer, two agents that promote ceramide formation in pancreatic cancer cells will be studied. These agents are fenretinide (4-HPR), a vitamin A analog, and Valspodar (PCS 833), a cyclosporin A analog. This study will employ cultured human pancreatic cancer cell lines and an SV-40- transformed, immortalized pancreatic cell line. The overall hypothesis is that pancreatic cancer cells are acutely sensitive to ceramide with the corollary to this hypothesis being that enhancing either the level of ceramide or specific molecular species of ceramide propels cytotoxicity along specific routes. This study has three specific aims designed to assess pancreatic cancer cell vulnerability to ceramide and ceramide-governed cell death cascades. Specific Aim 1 will characterize pancreatic cancer cell response to ceramide by using C6- ceramide, a short-chain, cell permeable analog of natural ceramide, and determine if cytotoxic responses are enhanced by the introduction of enzyme inhibitors that block ceramide metabolism. Specific Aim 2 will characterize the effect of 4-HPR and PSC 833 on cell viability and determine if cytotoxicity is linked to ceramide. Included here are mass spectroscopic analysis of dihydroceramides and ceramides generated in response to 4-HPR and PSC 833 to determine whether specific molecular species provoke specific responses, autophagy and senescence for example. Specific Aim 3 will assess the influence of ceramide generated in response to 4-HPR and PSC 833 on biochemical events associated with activation of intrinsic cell death. The innovative nature of this approach is that ceramide pathways can be manipulated, presenting a ready platform to improve efficacy. The very limited efficacy of available therapies for pancreatic cancer and the very high incidence of treatment failure are strong reasons to pursue new approaches. PUBLIC HEALTH RELEVANCE: This project will test a new approach to treat pancreatic cancer by studying the influence of a non-toxic vitamin A analog (4-HPR). 4-HPR can selectively kill cancer cells by stimulating over-production of normal cell waxes, called ceramides, which in excess are lethal to malignant cells and not normal cells. Because pancreatic cancer cells can metabolize ceramide to non-toxic byproducts, we will investigate inclusion of "partnering drugs" that block ceramide metabolism in order to fine-tune this novel approach to therapy.