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

Abstract

Abstract 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.