Sphingolipids in Cell Death after Photodamage Abstract The long-term objective of our research is to augment the efficacy of the cancer treatment photodynamic therapy (PDT). In PDT, tumor-localizing photosensitizers are activated by highly-focused laser light to induce oxidative stress in a cellular target that can lead to apoptotic cell death. Recently, protection by autophagy after low PDT doses and lack of protection by autophagy after high PDT doses has been shown. The balance between the proapoptotic (pro-death) sphingolipid (SL) ceramide and the antiapoptotic SL sphingosine-1- phosphate (S1P) constitutes the SL rheostat. The regulation of the SL rheostat has been proposed to be critical in triggering cell death by apoptosis and autophagy. The details of regulation of pro-death SLs and their role in cancer cell death after PDT are not understood, and represent a significant barrier to effective combination of PDT with ceramide-enhancing agents to augment cancer cell killing and PDT therapeutic value. Our novel discoveries strongly support the notion that resetting of the SL rheostat in favor of pro-death SLs, i.e., ceramide and dihydroceramide (DHceramide), is a contributor to the killing of tumor cells by PDT. Moreover, our pilot study shows that the combination of PDT with a SL analog improves PDT tumor response. The objective of this application is to exploit the regulation of pro-death SLs, their role in total cell death via apoptosis and autophagy, and in promoting tumor ablation after PDT. The central hypothesis is: resetting the SL rheostat in favor of pro-death SLs promotes cancer cell killing and tumor response after PDT. Three specific aims will test the hypothesis: (1) to demonstrate that cell killing after PDT requires a definitive, enzyme-controlled, SL profile. Selective increases in ceramide and DHceramide relative to S1P will be identified as a key factor in promoting overall cell death in apoptosis- and/or autophagy-competent cells. Dihydroceramide synthase and dihydroceramide desaturase will be characterized and validated as PDT targets in increasing the levels of pro-death SLs and in enhancing photosensitization. (2) To demonstrate that SL analogs govern sensitivity of cells to PDT. SL analogs that target mitochondria and which are all effective in killing cancer cells will be tested for their role in promoting photosensitivity. (3) To demonstrate that PDT tumor response requires a definitive SL profile and that co-treatment with SL analogs improves the response. Selective in vivo increases in ceramide and DHceramide relative to S1P will be verified as critical for PDT tumor response and the combination of PDT with SL analogs for improvement of PDT tumor response. The proposed work is expected to identify SL analogs and/or promote developing strategies that will selectively target enzymes that increase cellular levels of pro-death SLs to advance PDT therapeutic success. PUBLIC HEALTH RELEVANCE: Photodynamic therapy (PDT), a type of cancer treatment that bypasses most of the problems associated with resistance to chemotherapy, can produce in cells certain lipids called "sphingolipids" that are critical for destroying cancer cells. In the proposed novel studies we will discover key molecules and use drugs that are responsible for increasing the amounts of those sphingolipids that help killing cancer cells after PDT and promote PDT tumor response. Thus, the proposal holds the promise of improving clinical PDT.