The long-term goal of this proposal is to develop ceramides as novel therapeutic approaches to cancer treatment. The sphingolipid ceramide has recently emerged as a key regulator of apoptosis in response to multiple inducers such as chemotherapeutic agents, UV radiation, and tumor necrosis factor alpha. Exogenous ceramides in turn, are potent inducers of cell death, the mechanism of which primarily involves disrupting mitochondria, leading to cytochrome c release, caspase activation, and execution of apoptosis. Key questions remain as to mechanisms by which endogenous ceramide regulates apoptosis, the role of mitochondria, and ultimately whether and how ceramides (or analogues) can be applied as novel and potent pharmacologic approaches to cancer chemotherapy. Work in our laboratory funded by the two cycles of this grant led to elucidating mechanisms of ceramide-induced apoptosis, demonstrating the existence of a mitochondrial pool of ceramide and its function in apoptosis, and very recently the development of novel ceramide analogues (mito-ceramides) that preferentially target and accumulate in mitochondria and that are extremely potent inducers of cell death. This work has led us to propose the following hypothesis: generation of mitochondrial ceramide is key to induction of apoptotic cell death, and that mito-ceramides can be developed as novel and potent cancer chemotherapeutic agents. To test this hypothesis, we propose the following specific aims. Specific Aim 1. To determine mechanisms of ceramide generation and its regulation in mitochondria. We will measure activities of sphingomyelinase and ceramide synthase in mitochondrial fractions, study the regulation of mitochondrial ceramide by calcium in vitro and in cells, and identify at the molecular level the enzyme/s that cause ceramide generation in response to calcium in mitochondria. Specific Aim 2. To determine the mechanisms by which mito-ceramides induce apoptosis. This will be approached by (a) defining the apoptotic mechanism of mito-ceramide in cells and (b) determining the mechanisms of action of mito-ceramides in vitro. Specific Aim 3. To develop mito-ceramides as novel chemotherapeutic agents. We will determine the spectrum of action of mito-ceramides in tissue culture, define their specificity of action, optimize their structure-activity relationship, determine their cellular metabolism, and begin studies in animal models of cancer. These studies will set the stage for pre-clinical development of these molecules and will establish mito-ceramides as potential novel chemotherapeutic agents.