Abstract A136: Lysosome‐targeted photodynamic therapy for prostate cancer cells with a pyropheophorbide‐α conjugated inhibitor of prostate‐specific membrane antigen

Purpose: The limitation of specific delivery of photosensitizers to tumor sites, represents a significant shortcoming of photodynamic therapy (PDT) application at present. Prostate ‐specific membrane antigen (PSMA), a well known tumor marker of prostate cancer, has attracted considerable attention as a target for imaging and therapeutic applications for prostate cancer. The present study focuses on the investigation of a PSMA inhibitor‐conjugate (Ppa conjugate 2.0) as a delivery vesicle for photosensitizer molecules to target lysosomes for PDT application and the initiation of lysosomal pathway of apoptosis in targeted PDT‐treated prostate cancer cells. Experimental Procedures: For the first purpose, considering the rapid photobleaching of Ppa during microscopy observation, a PSMA inhibitor‐conjugate with fluorescein (FAM‐X‐54) instead of Ppa conjugate 2.0 was to performed for colocalization study. PSMA‐positive LNCaP cells grown in 2‐well slide chambers for 3 days were incubated with FAM‐X‐54 (4 M) in phosphate‐free RPMI 1640 containing 1% fetal bovine serum (FBS) for 2.5 h under cell growth conditions, and fixed in 4% paraformaldehyde in PBS for 15 min at room temperature (RT), permeabilized in cold‐methanol for 5min at −20°C, sequentially incubated with primary antibody (anti‐LAMP‐1 antibody, 1:100) and second antibody (goat anti‐mouse antibody‐TRITC, 1:50) in 1% BSA, PBS for 1h at RT, respectively. The cellular nuclei were stained with Hoescht33342, and anti‐fade solution was mounted on cells. For the second purpose, 3 day‐grown LNCaP cells were incubated with Ppa‐conjugate 2.0 or free Ppa (5 M/L) in phosphate‐free RPMI 1640 containing 1% fetal bovine serum (FBS) for 2.5 h under cell growth conditions, and irradiated with white light (7.5 J/cm2, with 25 mW/cm2 fluence rate) for 10 min. The PDT‐ treated cells were replaced in pre‐warmed complete growth medium RPMI 1640 to recover for different times (15, 30 and 1h), and stain with 200 nM Lysotracker red at last 15‐min in the dark. The treated cells were fixed and stained with Hoescht33342 as above. Cellular fluorescent image was captured by a Confocal laser scanning microscope. Results: Our results demonstrate that FAM‐X‐54 was clearly colocalized with lysosomal marker LAMP‐1. Furthermore, following targeted PDT with Ppa‐conjugate 2.0, the loss of Lysotracker labeling happened quickly, 90% loss at 15min, and complete loss at 1h post‐PDT. In contrast, free Ppa‐mediated PDT induced the stronger labeling of Lysotracker in LNCaP cells even at 15min post‐PDT. This difference can contribute to involvement of different apoptotic pathways due to their different sub‐localization of lysosomes (Ppa‐conjugate 2.0) and intracellular membrane systems such as mitochondria (free Ppa) within cells. Conclusions: We have revealed that targeted PDT with Ppa‐conjugate 2.0 can specifically trigger rapid lysosomal membrane permeabilization of PSMA‐positive prostate cancer cells, which is well consistent with lysosomal localization study. This proof‐of concept work now serves as the basis for further development of targeted PDT for prostate cancer and an alternative to antibody‐based approaches. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A136.