Development of Inhibitor-Directed Enzyme Prodrug Therapy (IDEPT) for Prostate Cancer

ABSTRACT: Prostate cancer (PCa) is the second most common cause of cancer death among American men after lung cancer. Unfortunately, current therapies do not provide effective treatments for patients with advanced, metastatic, or hormone refractory disease. Therefore, we seek to generate therapeutic agents for a novel PCa treatment strategy by delivering a suicide enzyme (yCDtriple) to a cell membrane bound biomarker found on PCa cells (prostate-specific membrane antigen (PSMA)). This approach has resulted in a new PCa treatment strategy reported here as inhibitor-directed enzyme prodrug therapy (IDEPT). The therapeutic agents described were generated using a click chemistry reaction between the unnatural amino acid (p-azidophenylalanine (pAzF)) incorporated into yCDtriple and the dibenzylcyclooctyne moiety of our PSMA targeting agent (DBCO-PEG4-AH2-TG97). After characterization of the therapeutic agents, we demonstrate significant PCa cell killing of PSMA-positive cells. Importantly, we demonstrate that this click chemistry approach can be used to efficiently couple a therapeutic protein to a targeting agent and may be applicable to the ablation of other types of cancers and/or malignancies

Functional prostate-specific membrane antigen is enriched in exosomes from prostate cancer cells

Developing simple and effective approaches to detect tumor markers will be critical for early diagnosis or prognostic evaluation of prostate cancer treatment. Prostate‑specific membrane antigen (PSMA) has been validated as an important tumor marker for prostate cancer progression including angiogenesis and metastasis. As a type II membrane protein, PSMA can be constitutively internalized from the cell surface into endosomes. Early endosomes can fuse with multivesicular bodies (MVB) to form and secrete exosomes (40-100 nm) into the extracellular environment. Herein, we tested whether some of the endosomal PSMA could be transferred to exosomes as an extracellular resource for PSMA. Using PSMA-positive LNCaP cells, the secreted exosomes were collected and isolated from the cultured media. The vesicular structures of exosomes were identified by electron microscopy, and exosomal marker protein CD9 and tumor susceptibility gene (TSG 101) were confirmed by western blot analysis. Our present data demonstrate that PSMA can be enriched in exosomes, exhibiting a higher content of glycosylation and partial proteolysis in comparison to cellular PSMA. An in vitro enzyme assay further confirmed that exosomal PSMA retains functional enzymatic activity. Therefore, our data may suggest a new role for PSMA in prostate cancer progression, and provide opportunities for developing non-invasive approaches for diagnosis or prognosis of prostate cancer

From AR to c-Met: androgen deprivation leads to a signaling pathway switch in prostate cancer cells

Elucidating the role of androgen deprivation in the transition from androgen-dependence to independence may enable the development of more specific therapeutic strategies against prostate cancer. Our previous in vitro model was employed to further assess the effects of continuous androgen‑deprivation on prostate cancer cells (LNCaP) with respect to both androgen receptor (AR) and c-Met expression. The results indicated that long-term androgen deprivation resulted in a signaling pathway switch from AR to c-Met in androgen-sensitive cells, which was confirmed by immunofluorescence imaging and western blot analysis. This signaling pathway switch may be predictive of a more aggressive disease state following androgen deprivation therapy

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

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 (α-/β-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 α-/β-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

A method to determine the mode of binding for GCPII inhibitors using bio-layer interferometry

<p>The rapid dilution of the enzyme-inhibitor complex assay to monitor the recovery of enzyme activity is a well-established assay to determine the reversibility of inhibition. Our laboratory has previously employed this method to ascertain the reversibility of known glutamate carboxypeptidase II (GCPII)-targeting agents. Due to the tedious and time-consuming nature of the assay, we sought to develop a facile method to determine the reversibility of well-characterized GCPII inhibitors using bio-layer interferometry (BLI). The results from the BLI assay are in agreement with the rapid dilution method. Herein, we report for the first time, a rapid, novel real-time BLI method to determine reversibility of inhibition.</p

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

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. Prostate 69:585–594, 2009. © 2009 Wiley‐Liss, Inc