PSMA-positive LNCaP Cells Research Articles

PSMA specific single chain antibody-mediated targeted knockdown of Notch1 inhibits human prostate cancer cell proliferation and tumor growth

The down-regulation of Notch1 by small interfering RNA (siRNA) can significantly inhibit human prostate cancer cell growth. The delivery of siRNA into specific cells is a key requirement for its clinical application. Recent reports have indicated that antibody-mediated siRNA delivery is an effective approach for targeted knockdown of specific genes in appropriate cells. Prostate-specific membrane antigen (PSMA) is regarded as an ideal target for the delivery of therapeutic agents to prostate cancer cells. The purpose of the present study was to evaluate whether siRNA can be efficiently delivered into PSMA-positive prostate cancer cells using two fusion proteins, s-tP and sFH-tP. These fusion proteins are composed of an anti-PSMA single chain antibody (scFv, abbreviated as an “s”) and a truncated protamine (tP); and in sFH-tP a furin cleavage site and an HA2 fragment sequence (FH) were inserted between the scFv and tP domains. Our results showed that siRNA can be specifically delivered into PSMA-positive LNCaP cells by these two fusion proteins, with the sFH-tP fusion protein being more effective. Efficient knockdown of Notch1 by siNotch1 delivered by either fusion protein was observed in PSMA-positive LNCaP cells and in LNCaP xenografted nude mice. Further experiments confirmed that the fusion protein-delivered siNotch1 could efficiently inhibit PSMA-positive LNCaP cell proliferation and promote apoptosis both in vitro and in vivo. Our data describe a promising strategy for the targeted delivery of siRNA to PSMA-positive prostate cancer cells using anti-PSMA scFv fusion proteins.

Targeting prostate cancer cells with PSMA inhibitor-guided gold nanoparticles

Prostate-specific membrane antigen (PSMA) is a notable biomarker for diagnostic and therapeutic applications in prostate cancer. Gold nanoparticles (AuNPs) provide an attractive nanomaterial platform for combining a variety of targeting, imaging, and cytotoxic agents into a unified device for biomedical research. In this study, we present the generation and evaluation of the first AuNP system functionalized with a small molecule phosphoramidate peptidomimetic inhibitor for the targeted delivery to PSMA-expressing prostate cancer cells. The general approach involved the conjugation of streptavidin-coated AuNPs with a biotin-linked PSMA inhibitor (CTT54) to generate PSMA-targeted AuNPs. In vitro evaluations of these targeted AuNPs were conducted to determine PSMA-mediated and time-dependent binding to PSMA-positive LNCaP cells. The PSMA-targeted AuNPs exhibited significantly higher and selective binding to LNCaP cells compared to control non-targeted AuNPs, thus demonstrating the feasibility of this approach.

PSMA‐targeted SPECT agents: Mode of binding effect on in vitro performance

The enzyme-biomarker prostate-specific membrane antigen (PSMA) is an active target for imaging and therapeutic applications for prostate cancer. The internalization of PSMA has been shown to vary with inhibitors' mode of binding: irreversible, slowly reversible, and reversible. In the present study, PSMA-targeted clickable derivatives of an irreversible phosphoramidate inhibitor DBCO-PEG(4) -CTT-54 (IC(50) = 1.0 nM) and a slowly reversible phosphate inhibitor, DBCO-PEG(4) -CTT-54.2 (IC(50) = 6.6 nM) were clicked to (99m) Tc(CO)(3) -DPA-azide to assemble a PSMA-targeted SPECT agent. The selectivity, percent uptake, and internalization of these PSMA-targeted SPECT agents were evaluated in PSMA-positive and PSMA-negative cells. In vitro studies demonstrated that PSMA-targeted SPECT agents exhibited selective cellular uptake in the PSMA-positive LNCaP cells compared to PSMA-negative PC3 cells. More importantly, it was found that (99m) Tc(CO)(3) -DPA-DBCO-PEG(4) -CTT-54 based on an irreversible PSMA inhibitor core, exhibited greater uptake and internalization than (99m) Tc(CO)(3) -DPA-DBCO-PEG(4) -CTT-54.2 constructed from a slowly reversible PSMA inhibitor core. We have demonstrated that a PSMA-targeted SPECT agent can be assembled efficiently using copper-less click chemistry. In addition, we demonstrated that mode of binding has an effect on internalization and percent uptake of PSMA-targeted SPECT agents; with the irreversible targeting agent demonstrating superior uptake and internalization in PSMA+ cells. The approach demonstrated in this work now supports a modular approach for the assembly of PSMA-targeted imaging and therapeutic agents.

Small molecule inhibitors of PSMA incorporating technetium-99m for imaging prostate cancer: Effects of chelate design on pharmacokinetics

Single amino acid chelate (SAAC) systems for the complexation of the M(CO)3 moiety (M=Tc/Re) have been successfully incorporated into novel synthetic strategies for radiopharmaceuticals and evaluated in a variety of biological applications. However, the lipophilicity of the first generation of 99mTc(CO)3 complexes has resulted in substantial hepatobiliary uptake when examined either as lysine derivatives or integrated into biologically active small molecules and peptides. Here, we designed, synthesized, and evaluated novel polar functionalized imidazole derived SAAC systems (SAAC II) which have been chemically modified to promote overall 99mTc(CO)3L3 complex hydrophilicity with the intent of reducing non-target effects and enhancing renal clearance of prostate specific membrane antigen (PSMA) targeting small molecules. The 99mTc-labeled compounds were prepared, purified, and evaluated for stability, lipophilicity, and tissue distribution in LNCaP xenograft mice. The Glu-urea-Lys-C11 analogs were prepared with a variety of chelators to form (19R,23S)-1-(X)-2-((Y)methyl)-13,21-dioxo-2,14,20,22-tetraazapentacosane-19,23,25-tricarboxylic acid where X=Y=(methyl)pyridin-2-yl (6), X=Y=(methyl)-1H-imidazol-2-yl (7), X=(methyl)pyridin-2-yl, Y=carboxymethyl (8), X=Y=1-(carboxymethyl)-1H-imidazol-2-yl (9), X=1-(carboxymethyl)-1H-imidazol-2-yl, Y=carboxymethyl (10), and X=Y=1-(1-(2-(bis(carboxymethyl)amino)-2-oxoethyl)-1H-imidazol-2-yl)-2-((1-(2-(bis(carboxymethyl)amino)-2-oxoethyl)-1H-imidazol-2-yl) (11). 99mTc labeling was achieved at ligand concentrations as low as 10−6M and the complexes were stable (>90%) for 24h. These new SAAC II chelators were evaluated for their influence on binding of the Glu-urea-Lys-C11 analogs to PSMA-positive LNCaP cells and compared to pyridine- and N-methylimidazole-containing SAAC ligands. Tissue distribution of the 99mTc-complexes containing the more polar chelators, 9 and 11, demonstrated decreased liver (<2% ID/g) and increased LNCaP tumor (>11% ID/g) accumulation at 1h post-injection.

Tc-99m labeled small-molecule inhibitors of prostate-specific membrane antigen (PSMA): New molecular imaging probes to detect metastatic prostate adenocarcinoma (PC).

173 Background: Sensitive and specific imaging remains a clinically-relevant problem for men with PC. PSMA is a well established target for imaging of PC with therapeutic implications. We have recently developed novel 99mTc-labeled small molecule inhibitors of the enzymatic domain of PSMA based on glutamate-urea-glutamate and glutamate-urea-lysine pharmacophores, and contain a bis-imidazole chelator to complex Tc-99m. Preclinical studies with PSMA positive LNCaP cells and xenografts demonstrate that 99mTc-MIP-1404 and 99mTc-MIP-1405 bind to PSMA with high affinity and localize in tumors rapidly. This study reports the first human data in men with metastatic PC and in healthy male subjects. Methods: Under an exploratory IND, using a cross-over design, the pharmacokinetics, biodistribution, and tumor uptake of 99mTc-MIP-1404 and 99mTc-MIP-1405 were compared in 6 healthy men and 6 men with radiographic evidence of metastatic PC. Whole body images were obtained at 10 min, 1, 2, 4 and 24 hr. Single photon emission computed tomography (SPECT) was performed between 3–4 hours post injection. Results: Both agents cleared the blood rapidly with MIP-1404 demonstrating significantly lower urinary activity (7%) compared to MIP-1405 (26%). Both agents showed persistent uptake in the salivary, lacrimal and parotid glands. Uptake in liver and kidney was acceptable for imaging at 1-2 hr post injection (PI). In men with PCa, both agents rapidly localized in bone and lymph node lesions as early as 1 hr PI. SPECT demonstrated excellent lesion contrast. Good correlation was seen with bone and CT scans, In majority of patients, more lesions including sub-cm lymph nodes were seen with 99mTc-MIP-1404 and 99mTc-MIP-1405. The high contrast images exhibited signal:noise ratios from 3:1 to 28:1 at 4 and 24 hr. Conclusions: 99mTc-MIP-1404 and 99mTc-MIP-1405 identified a greater number of lesions than bone scans and rapidly detected soft tissue PC lesions including sub-cm lymph nodes. Since 99mTc-MIP-1404 has minimal activity in the bladder, further work is planned to correlate imaging findings with histopathology in patients with high risk clinically-localized PC.

Abstract A117: Rapid uptake of a streptavidin conjugate into prostate cancer cells: A model of macromolecule targeting delivery.

Abstract Introduction: Prostate-specific membrane antigen (PSMA), a type II membrane glycoprotein, has been confirmed to be an important tumor-marker indicating prostate cancer progression, and is becoming an active target for imaging or therapy for prostate cancer. Furthermore, the streptavidin-biotin coupling pair has been successfully employed in pretargeting therapy for multiple cancers. In the present study, we describe the synthesis and in vitro performance evaluation of a biotinylated PSMA inhibitor (biotin-PEG12-CTT54) possessing two functional motifs separated by a PEG linker: 1) a PSMA targeting core (CTT-54) that specifically binds enzyme-biomarker PSMA and 2) biotin for binding streptavidin. Results and Discussion: Biotin-PEG12-CTT54 was confirmed by MALDI-TOF analysis: calculated 1267.52, found 1267.5221. This bifunctional compound was further evaluated for its inhibitory potency against purified PSMA and in vitro cell imaging efficiency. PSMA inhibition study confirmed that conjugation of CTT-54 to biotin through PEG linker (biotinylated inhibitor, IC50 = 10 nM) had no adverse effect on the inhibitory potency of the parent inhibitor core CTT-54 (IC50 = 14 nM). In a two-step pre-targeting imaging experiment, 3-day grown PSMA-positive LNCaP cells was treated with this biotinylated compound, then followed by the incubation with Cy5-conjugated streptavidin, our data demonstrated specifically labeling PSMA-positive prostate cancer cells, but not for cells treated with Cy5-conjugated streptavidin only. Additionally, the complex between the biotinylated compound and Cy5-conjugated streptavidin was prepared and purified prior to cell labeling. The purified pre-formed complex was found to contain three biotinylated inhibitors for each streptavidin using a newly-developed digitally-operated linear quadrupole ion trap orthogonal acceleration time-of-flight mass spectrometer, which is designed to analyze non-covalently bound complex by confidentially maintaining its integrity through the analytical process. The preformed complex was tested for its inhibitory potency against purified PSMA and the cell-labeling of PSMA-positive cells, the experimental results revealed an improved inhibitory potency (IC50 = 1.9 nM) against PSMA and rapid uptake of streptavidin into PSMA-positive prostate cancer cells within 10 min. To better understand the intracellular fate of Cy5-streptavidin once internalized in LNCaP cells, co-localization studies were conducted with transferrin-fluorescein, which can be tracked as an endosomal marker for the ligand-receptor internalization via clathrin-coated pits. Co-localization of internalized streptavidin-Cy5 with transferrin-fluorescein appeared strongly in perinuclear region after 2h-incubation. The pattern of PSMA internalization induced by preformed complex is consistent with antibody or small-molecule inhibitors alone. Conclusions: We have successfully demonstrated that macromolecules such as streptavidin can be specifically delivered into prostate cancer cells. These results support a proof-of-concept that a combination of streptavidin (serving as a drug or imaging agent carrier) and a biotinylated PSMA inhibitor may lead to the development of a novel strategy of tumor-targeting imaging or drug delivery for prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A117.

123I-MIP-1072, a Small-Molecule Inhibitor of Prostate-Specific Membrane Antigen, Is Effective at Monitoring Tumor Response to Taxane Therapy

Because traditional endpoints in oncology trials are not always applicable for metastatic prostate cancer, better ways of following response to treatment are needed. Prostate-specific membrane antigen (PSMA) is a transmembrane protein expressed in normal human prostate epithelium and is upregulated in prostate cancer. (S)-2-(3-((S)-1-carboxy-5-((4-(123)I-iodobenzyl)amino)pentyl)ureido)pentanedioic acid, (123)I-MIP-1072, targets PSMA and was evaluated for monitoring the growth of PSMA-positive LNCaP cells in vitro and as xenografts after paclitaxel therapy. LNCaP and 22Rv1 cells were treated with paclitaxel (0-100 nM) for 48 h, after which binding of (123)I-MIP-1072 was examined. Cell number was determined by MTS assay, and PSMA expression was analyzed by Western blotting. LNCaP xenograft-bearing mice were treated with paclitaxel (6.25 mg/kg) for 3.5 cycles of 5 d on and 2 d off. Tissue distribution of (123)I-MIP-1072 was determined on days 2 and 23 from the start of paclitaxel treatment. Paclitaxel (10-100 nM) inhibited LNCaP and 22Rv1 cell growth after 48 h, and binding of (123)I-MIP-1072 was proportional to cell number. Western blot analysis verified there was no paclitaxel-dependent change in PSMA expression. Treatment of LNCaP xenografts with paclitaxel resulted in a decrease in tumor volume (-21%), compared with an increase in the untreated xenografts (+205%) by day 23. Tumor uptake of (123)I-MIP-1072 was proportional to changes in tumor mass: decreased by paclitaxel treatment and increased in untreated mice. Treatment of LNCaP cells or xenograft tumors with paclitaxel resulted in growth inhibition, which was detected with (123)I-MIP-1072. The high specificity of (123)I-MIP-1072 for prostate cancer may allow monitoring of tumor progression in patients before, during, and after chemotherapy.

Multi-component Polymeric System for Tumour Cell-Specific Gene Delivery Using a Universal Bungarotoxin Linker

A new universal tool for specific, non-covalent and non-destructive attachment of a recombinant antibody fragment to a polymer-modified adenovirus has been utilised to regulate the tropism of adenoviral gene delivery vector. We have prepared a multivalent reactive N-(2-hydroxypropyl)methacrylamide-based copolymer (PHPMA) bearing an α-bungarotoxin-binding peptide (BTXbp). The copolymer was used for covalent surface modification of adenoviral vectors (Ad). The α-bungarotoxin protein (BTX) has a nanomolar binding affinity for BTXbp, allowing non-covalent linkage of BTX fusion proteins. A single chain variable fragment of anti-PSMA antibody bearing BTX (scFv-BTX) binding to the prostate-specific membrane antigen (PSMA) was conjugated with the copolymer-coated adenovirus to enable specific infection of prostate cancer cells via PSMA receptors. As shown by ELISA, the copolymer-coated virus exhibited much reduced binding to anti-Ad antibodies. Infection of PC-3 and LNCaP prostate cancer cells was ∼100-fold less efficient with copolymer-coated Ad than with un-modified Ad. Conjugation of scFv-BTX with Ad-PHPMA-BTXbp led to 5-10-fold restoration of infection in PSMA-positive LNCaP cells. In PSMA-negative PC-3 cells, the conjugation of scFv-BTX with Ad-PHPMA-BTXbp gave no enhancement of infection. We have shown that the presented Ad-PHPMA-BTXbp/scFv-BTX system can be used as a universal tool for a receptor-specific virotherapy.

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

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.

Prostate-Specific Membrane Antigen Targeted Imaging and Therapy of Prostate Cancer Using a PSMA Inhibitor as a Homing Ligand

Prostate cancer (PCa) is a major cause of mortality and morbidity in Western society today. Current methods for detecting PCa are limited, leaving most early malignancies undiagnosed and sites of metastasis in advanced disease undetected. Major deficiencies also exist in the treatment of PCa, especially metastatic disease. In an effort to improve both detection and therapy of PCa, we have developed a PSMA-targeted ligand that delivers attached imaging and therapeutic agents selectively to PCa cells without targeting normal cells. The PSMA-targeted radioimaging agent (DUPA-(99m)Tc) was found to bind PSMA-positive human PCa cells (LNCaP cell line) with nanomolar affinity (K(D) = 14 nM). Imaging and biodistribution studies revealed that DUPA-(99m)Tc localizes primarily to LNCaP cell tumor xenografts in nu/nu mice (% injected dose/gram = 11.3 at 4 h postinjection; tumor-to-muscle ratio = 75:1). Two PSMA-targeted optical imaging agents (DUPA-FITC and DUPA-rhodamine B) were also shown to efficiently label PCa cells and to internalize and traffic to intracellular endosomes. A PSMA-targeted chemotherapeutic agent (DUPA-TubH) was demonstrated to kill PSMA-positive LNCaP cells in culture (IC(50) = 3 nM) and to eliminate established tumor xenografts in nu/nu mice with no detectable weight loss. Blockade of tumor targeting upon administration of excess PSMA inhibitor (PMPA) and the absence of targeting to PSMA-negative tumors confirmed the specificity of each of the above targeted reagents for PSMA. Tandem use of the imaging and therapeutic agents targeted to the same receptor could allow detection, staging, monitoring, and treatment of PCa with improved accuracy and efficacy.

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

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. 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. 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. 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.

Targeting Gene Therapy for Prostate Cancer Cells by Liposomes Complexed with Anti-Prostate-Specific Membrane Antigen Monoclonal Antibody

Prostate-specific membrane antigen (PSMA) is a membrane-bound antigen expressed on the surface of prostate cancer cells, and this paper describes the use of an antibody against PSMA for targeting gene therapy. We coupled anti-PSMA monoclonal antibody with poly-L-lysine and then incubated it with plasmids. These complexes were then transfected with cationic liposomes into cells. The transfection efficiency of anti-PSMA- liposome complex was higher than that of normal IgG-liposome complex in PSMA-positive LNCaP cells. Furthermore, anti-PSMA-liposome complex containing a suicide gene, thymidine kinase, demonstrated a selective growth-inhibitory effect on LNCaP cells in vitro, but did not exert a significant effect on PSMA-negative cells. In an in vivo xenograft model of LNCaP cells in nu/nu mice, we administered the complexes via the tail vein. Judging on the basis of both 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) staining and luciferase assay findings, a significant enrichment of plasmid DNA was observed in LNCaP xenografts with anti-PSMA-liposome complex in comparison with normal IgG-liposome complex. However, the distribution of plasmid DNA did not change substantially in any other organs including the liver, kidney, lung, and spleen. Moreover, in suicide gene therapy, anti-PSMA-liposome complex exerted a significant inhibitory effect on the growth of LNCaP xenograft, in contrast to normal IgG-liposome complex.

Development of gene therapy using prostate-specific membrane antigen promoter/enhancer with Cre Recombinase/LoxP system for prostate cancer cells under androgen ablation condition.

To enhance the efficacy of suicide gene therapy for prostate cancer under androgen deprivation, we designed a promoter system that consists of the prostate‐specific membrane antigen (PSMA) promoter/enhancer (PEPM) and Cre‐loxP DNA recombination system. We constructed two kinds of plasmids. One plasmid contains a Cre recombinase (Cre) under the control of PEPM and the other expresses CMV‐lox‐luciferase/herpes simplex virus thymidine kinase (TK). In PSMA‐positive LNCaP cells, the promoter activity of the PEPM‐Cre plus CMV‐lox‐luciferase demonstrated 800‐fold greater activity compared with that of the PSMA promoter alone. However, no enhancement of the promoter activity was observed in the PSMA‐negative cells. Furthermore, in contrast to prostate specific antigen promoter/enhancer (PP), the promoter activity of PEPM did not decrease when the LNCaP cells were cultured in charcoal‐stripped fetal bovine serum (CFBS). In an in vitro gene therapy model with LNCaP cells, the cell growth inhibition in the presence of ganciclovir (GCV) was more evident in the cells transfected with the PEPM‐Cre plus CMV‐lox‐TK than in the cells with the PP‐TK, and the difference in efficacy between the two plasmids was more remarkable when the cells were maintained in CFBS medium. The therapeutic effect of PEPM‐Cre plus CMV‐lox‐TK was also observed in xenografted LNCaP cells on nude mice when the plasmids were directly injected into tumors and GCV was administered intraperitoneally. These findings indicate that the combination of the PSMA promoter/enhancer and the Cre‐loxP system can enhance the PSMA promoter activity even under androgen ablation conditions and can exert its anti‐tumor effect both in vitro and in vivo.