Cationic polymeric gene delivery of beta-glucuronidase for doxorubicin prodrug therapy.
BACKGROUND
An approach to improve current chemotherapy is the selective transduction of tumor cells with suicide genes to sensitize these cells to prodrugs of cytostatic agents.


METHODS
In this study, gene transfer was accomplished with the cationic polymer poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA), able to condense plasmid-DNA by electrostatic interaction. OVCAR-3 cells were transfected with plasmids encoding E. coli-derived or human beta-glucuronidase and the transfection efficiency and inhibition by serum was determined. Next, we measured the sensitivity of OVCAR-3 cells transiently expressing beta-glucuronidase to the glucuronide prodrug of doxorubicin (DOX-GA3) or to doxorubicin.


RESULTS
OVCAR-3 cells were efficiently transfected with a plasmid encoding E. coli-derived beta-glucuronidase. The degree of transfection (30% of cells) was higher than that achieved with commercially available cationic lipids (DOTAP, Lipofectamine) without inhibition by serum. OVCAR-3 cells transiently expressing beta-glucuronidase were equally sensitive to the glucuronide prodrug of doxorubicin (DOX-GA3) or to doxorubicin itself, indicating complete conversion of prodrug to drug. Similar studies were performed with the plasmid encoding for human beta-glucuronidase, which is likely to be less immunogenic. Also in this case, OVCAR-3 cells showed an increased sensitivity to the prodrug DOX-GA3, although less pronounced than when the bacterial enzyme was used. A strong bystander effect was observed when OVCAR-3 cells transfected with beta-glucuronidase were mixed with non-transfected cells at different ratios. Complete tumor cell growth inhibition was already observed when only 15% of the cells expressed the activating enzyme.


CONCLUSIONS
These studies suggest that beta-glucuronidase gene therapy using PDMAEMA as a carrier system and DOX-GA3 as the prodrug has a potential application in cancer gene therapy.
Fonseca
M J
MJ
Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, The Netherlands.
Storm
G
G
Hennink
W E
WE
Gerritsen
W R
WR
Haisma
H J
HJ
D000903:Antibiotics, Antineoplastic / Q000008:administration & dosage* / Q000378:metabolism / Q000493:pharmacokinetics; D000964:Antimetabolites, Antineoplastic / Q000008:administration & dosage / Q000378:metabolism / Q000493:pharmacokinetics; D001711:Biotransformation; D002277:Carcinoma / Q000473:pathology*; D002411:Cation Exchange Resins; D016923:Cell Death; D002455:Cell Division / Q000187:drug effects; D016895:Culture Media, Serum-Free; D043525:Cytosine Deaminase; D004274:DNA, Recombinant / Q000737:chemistry / Q000187:drug effects; D004317:Doxorubicin / Q000008:administration & dosage* / Q000031:analogs & derivatives* / Q000378:metabolism / Q000493:pharmacokinetics; D004337:Drug Carriers / Q000008:administration & dosage / Q000494:pharmacology; D004354:Drug Screening Assays, Antitumor; D005260:Female; D005437:Flucytosine / Q000008:administration & dosage / Q000378:metabolism / Q000493:pharmacokinetics; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics; D005965:Glucuronates / Q000008:administration & dosage* / Q000378:metabolism / Q000493:pharmacokinetics; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008055:Lipids; D008689:Methacrylates / Q000494:pharmacology*; D009700:Nucleoside Deaminases / Q000235:genetics; D009757:Nylons / Q000494:pharmacology*; D010051:Ovarian Neoplasms / Q000473:pathology*; D010316:Particle Size; D010957:Plasmids / Q000008:administration & dosage*; D011355:Prodrugs / Q000008:administration & dosage* / Q000378:metabolism / Q000493:pharmacokinetics; D055672:Static Electricity; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000201:enzymology
D000903:Antibiotics, Antineoplastic; D000964:Antimetabolites, Antineoplastic; D002411:Cation Exchange Resins; D016895:Culture Media, Serum-Free; D004274:DNA, Recombinant; D004337:Drug Carriers; D005965:Glucuronates; D008055:Lipids; C086724:Lipofectamine; D008689:Methacrylates; C407038:N-(4-doxorubicin-N-carbonyl(oxymethyl)phenyl)-O-glucuronyl carbamate; D009757:Nylons; D011355:Prodrugs; C407037:poly(2-(dimethylamino)ethyl methacrylate); D004317:Doxorubicin; D005437:Flucytosine; D005966:Glucuronidase; D009700:Nucleoside Deaminases; D043525:Cytosine Deaminase