Prostate-specific membrane antigen-targeted photodynamic therapy induces rapid cytoskeletal disruption

Abstract

Prostate-specific membrane antigen (PSMA), an established enzyme-biomarker for prostate cancer, has attracted considerable attention as a target for imaging and therapeutic applications. We aimed to determine the effects of PSMA-targeted photodynamic therapy (PDT) on cytoskeletal networks in prostate cancer cells. PSMA-targeted PDT resulted in rapid disruption of microtubules (alpha-/beta-tubulin), microfilaments (actin), and intermediate filaments (cytokeratin 8/18) in the cytoplasm of LNCaP cells. The collapse of cytoplasmic microtubules and the later nuclear translocation of alpha-/beta-tubulin were the most dramatic alternation. It is likely that these early changes of cytoskeletal networks are partly involved in the initiation of cell death.

Fig. 1

Structures of Ppa and Ppa-CTT-54.

Fig. 2

Changes of α-tubulin and β-tubulin in LNCaP cells after targeted PDT with Ppa-CTT-54.0. (A) Immunofluorescence analysis: targeted PDT induced the rearrangement of microtubules (red) to ring-like perinuclear structures following PDT (0, 15, 30 min). (B) The nucleolar tanslocation of α- and β-tubulin (red) at 1 h or 2 h post-PDT. The nuclei were counterstained with H342 (blue). The cellular imaging was visualized by confocal microscopy; distance scale is 20 µm.

Fig. 3

Changes of actin in LNCaP cells after targeted PDT with Ppa-CTT-54.0. (A) F/G actin staining: cytoplasmic G-actin (green) was immediately destroyed following PDT. In contrast, the F-actin (red) appeared mainly beneath the cellular membrane and persisted longer than G-actin. (B) Immunofluorescence detection of actin: normal cytoplamic and membrane distribution of actin (green) in the control cells with a rapid loss of cytoplasmic actin and slower loss of membrane-localized actin in PDT-treated cells with increasing time (0, 15 and 30 min) following PDT. The nuclei were stained with H342 (blue). The cellular imaging was visualized with a confocal microscopy; distance scale is 20 µm. (C) Western blot analysis: the amount of actin decreased in PDT-treated cells following PDT (0, 15, 30 min), compared to control cells (C). An increase of cleaved actin was observed in PDT-treated cells.

Fig. 4

Changes of cytokeratin 8 and 18 in LNCaP cells after targeted PDT with Ppa-CTT-54.0. (A) Immunofluorescence analysis: targeted PDT induced the immediate loss of cytoplasmic intermediate filament networks (cytokeratin 8 and 18, red). A small increase of membrane-localized Cytokeratin 8 and 18 was observed in PDT-treated cells (0, 15 and 30 min). Cell nuclei were stained with H342 (blue). The cellular imaging was visualized with a confocal microscopy; distance scale is 20 µm. (B) Western blot analysis: compared to the control sample, the amount of aggregated (irreversible) and cleaved cytokeratin 8 increased in PDT-treated cell samples following PDT (0, 15, 30 min). In contrast, cytokeratin 18 remained stable except for a small increase of aggregated forms (irreversible) in PDT-treated cell samples.

Figure 5