Amplification of cancer cell apoptosis in photodynamic therapy-treated tumors by adjuvant ceramide analog LCL29

Abstract

C6-ceramide analog LCL29 was recently shown to improve the cure rates of mouse tumors treated by photodynamic therapy (PDT), but the mechanism underlying the therapeutic gain remains unclear. Since LCL29 is a pro-apoptotic agent, the main objective of the present study was to determine how LCL29 affects cancer cell apoptosis in PDT-treated tumors. Mice bearing SCCVII tumors (syngeneic squamous cell carcinomas) were treated by PDT with photosensitizer Foscan. Adjuvant LCL29 treatment (40 mg/kg) was 24 hours before PDT. The tumors were excised 3 hours after PDT, disaggregated into single cell suspensions that were stained for flow cytometry to detect apoptosis-related events in cancer cells (caspase activation, loss of mitochondrial transmembrane potential, and intracellular calcium). In addition, phagocytic activity in tumor-associated macrophages was assessed by phalloidin staining. While 5-10% apoptotic cancer cells were found in tumors treated by PDT or LCL29 alone, close to 40% of such cells were found in tumors treated by LCL29 combined with PDT. Mitochondrial depolarization was detected in PDT, LCL29, and LCL29 + PDT groups, reaching similar values in all groups. Intracellular calcium release triggered by PDT was more pronounced when PDT was combined with LCL29. Macrophage phagocytic activity, negatively affected by PDT, was stimulated by adjuvant LCL29. Combining LCL29 treatment with PDT can enhance intracellular calcium release in cancer cells and strongly amplify their apoptosis.