In vitro targeted photodynamic therapy with a pyropheophorbide--a conjugated inhibitor of prostate-specific membrane antigen

Abstract

Background: The lack of specific delivery of photosensitizers (PSs), represents a significant limitation of photodynamic therapy (PDT) of cancer. The biomarker prostate-specific membrane antigen (PSMA) has attracted considerable attention as a target for imaging and therapeutic applications for prostate cancer. Although recent efforts have been made to conjugate inhibitors of PSMA with imaging agents, there have been no reports on PS-conjugated PSMA inhibitors for targeted PDT of prostate cancer. The present study focuses on the use of a PSMA inhibitor-conjugate of pyropheophorbide-a (Ppa-conjugate 2) for targeted PDT to achieve apoptosis in PSMA+ LNCaP cells.

Methods: Confocal laser scanning microscopy with a combination of nuclear staining and immunofluorescence methods were employed to monitor the specific imaging and PDT-mediated apoptotic effects on PSMA-positive LNCaP and PSMA-negative (PC-3) cells.

Results: Our results demonstrated that PDT-mediated effects by Ppa-conjugate 2 were specific to LNCaP cells, but not PC-3 cells. Cell permeability was detected as early as 2 hr by HOE33342/PI double staining, becoming more intense by 4 hr. Evidence for the apoptotic caspase cascade being activated was based on the appearance of poly-ADP-ribose polymerase (PARP) p85 fragment. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay detected DNA fragmentation 16 hr post-PDT, confirming apoptotic events.

Conclusions: Cell permeability by HOE33342/PI double staining as well as PARP p85 fragment and TUNEL assays confirm cellular apoptosis in PSMA+ cells when treated with PS-inhibitor conjugate 2 and subsequently irradiated. It is expected that the PSMA targeting small-molecule of this conjugate can serve as a delivery vehicle for PDT and other therapeutic applications for prostate cancer.

Figure 1

Structures of phosphoramidate peptidomimetic inhibitor core 1, Ppa, and its Ppa-conjugate 2.

Figure 2

TUNEL staining of PDT-treated cells. PDT-treated LNCaP cells at 1, 2.5, or 5 μM Ppa-conjugate 2 (A, B, C). PDT-treated PC-3 cells at 1, 2.5, or 5 μM Ppa-conjugate 2 (E, F, G). PDT-treated LNCaP and PC-3 cells with 2.5 μM free Ppa (D, H), as control. Cellular nuclei were counterstained by DAPI. Bar scale is 20 μm.

Figure 3

HOE33342/PI Double-Staining. Post PDT-treated LNCaP were stained by Hoechst 33342 (Top panels) and Propium Iodide (Bottom panels) before fixation. Controls A and B: 4 h after light exposure; cells were subjected to 10 min light exposure but were not pre-treated with Ppa-conjugate 2. C-F: cells were pre-treated with 2.5 μM Ppa-conjugate 2 prior to 10 min light exposure. C and D: 2 h post-PDT treatment. E and F: 4 h post-PDT treatment. G and H: 4 h after light exposure but first pre-treated 30 min with 100 μM Inhibitor core 1 prior to pre-treatment with Ppa-conjugate 2. Double-stained cells were fixed for microscopy visualization. Bar scale is 20 μm.

Figure 4

Cytoimmunofluorescence detection of PARP cleavage p85 fragment following PDT treatment with Ppa-conjugate 2 (2.5 μM). Control cells (light treatment only, no Ppa-conjugate 2 applied (A, B, and C). 2 h post-PDT treatement (D, E, and F). 4 h post-PDT (G, H, and I). Cellular nuclei were counterstained by DAPI. A, D, G: DAPI staining. B, E, H: p85 staining. C, F, I: merged DAPI and p85 staining. Bar scale is 20 μm.