Targeted enzyme prodrug therapy for metastatic prostate cancer – a comparative study of L-methioninase, purine nucleoside phosphorylase, and cytosine deaminase

Abstract

BackgroundEnzyme prodrug therapy shows promise for the treatment of solid tumors, but current approaches lack effective/safe delivery strategies. To address this, we previously developed three enzyme-containing fusion proteins targeted via annexin V to phosphatidylserine exposed on the tumor vasculature and tumor cells, using the enzymes L-methioninase, purine nucleoside phosphorylase, or cytosine deaminase. In enzyme prodrug therapy, the fusion protein is allowed to bind to the tumor before a nontoxic drug precursor, a prodrug, is introduced. Upon interaction of the prodrug with the bound enzyme, an anticancer compound is formed, but only in the direct vicinity of the tumor, thereby mitigating the risk of side effects while creating high intratumoral drug concentrations. The applicability of these enzyme prodrug systems to treating prostate cancer has remained unexplored. Additionally, target availability may increase with the addition of low dose docetaxel treatment to the enzyme prodrug treatment, but this effect has not been previously investigated. To this end, we examined the binding strength and the cytotoxic efficacy (with and without docetaxel treatment) of these enzyme prodrug systems on the human prostate cancer cell line PC-3.ResultsAll three fusion proteins exhibited strong binding; dissociation constants were 0.572 nM for L-methioninase-annexin V (MT-AV), 0.406 nM for purine nucleoside phosphorylase-annexin V (PNP-AV), and 0.061 nM for cytosine deaminase-annexin V (CD-AV). MT-AV produced up to 99% cell death (p < 0.001) with limited cytotoxicity of the prodrug alone. PNP-AV with docetaxel created up to 78% cell death (p < 0.001) with no cytotoxicity of the prodrug alone. CD-AV with docetaxel displayed up to 60% cell death (p < 0.001) with no cytotoxicity of the prodrug alone. Docetaxel treatment created significant increases in cytotoxicity for PNP-AV and CD-AV.ConclusionsStrong binding of fusion proteins to the prostate cancer cells and effective cell killing suggest that the enzyme prodrug systems with MT-AV and PNP-AV may be effective treatment options. Additionally, low-dose docetaxel treatment was found to increase the cytotoxic effect of the annexin V-targeted therapeutics for the PNP-AV and CD-AV systems.

Construction of Functional GFP-Neo Fusion Protein by Selection from a Peptide Library

“…The original gfp sequence in the pGFP was replaced with the gfp sequence from pEGFP-C1 (Clontech), in which the stop codon of the gfp gene was deleted. The neo gene was derived from pJZ419 [16] so that the start codon (ATG) of the neo gene was mutated into the NdeI site (CATATG). The gfp gene without a stop codon was bound at the 3' end by an XhoI site while the neo gene began with an NdeI site so that the gfp gene and the neo gene would be separated by the two restriction sites.…”

“…Plasmid DNAs of retroviral vectors were transfected into the retroviral helper cell line PA317 [18]. Viruses released from transfected PA317 cells were used to infect D17 cells [16]. The MOI (multiplicity of infection) was about 0.01.…”

“…The ds DNA was then isolated from an agarose gel and digested with XhoI and NdeI ( Figure IE). Figure 2C) is a retroviral vector similar to JZ419 [16], except that pJZ419 encoded a separated gfp gene, neo gene and an IRES sequence between the two genes, while the pML42 encodes the gfpneo fusion protein without an IRES. The BamHI-RsrII (BamHI is located upstream of the start codon of the gfp gene, while the RsrII is located within the neo gene) fragment from each clone of pML41 was inserted between the BamHI and RsrII sites of the pJZ419.…”

Targeted antitumor prodrug therapy using CNGRC-yCD fusion protein in combination with 5-fluorocytosine

“…The enzyme-prodrug system that produces active drugs from safer prodrugs at the tumor site is an attractive strategy for antitumor therapy [ 17 – 24 ]. The combined therapy employing cytosine deaminase (CD) and the nucleoside analog 5-fluorocytosine (5-FC) is an effective approach offered by the enzyme-prodrug system.…”

Annexin V-Directed Enzyme Prodrug Therapy Plus Docetaxel for the Targeted Treatment of Pancreatic Cancer

“…16 Docetaxel in combination with AV-directed EPT has been shown to increase EPT cytotoxic efficacy in vitro for prostate cancer cells without any cytotoxic effects of docetaxel alone. 17 We have developed 3 distinct AV-directed EPT systems, 18-20 each containing a different enzyme, as follows:…”