Enhanced p53 gene transfer to human ovarian cancer cells using the cationic nonviral vector, DDC.
OBJECTIVE
Previously we have formulated a new cationic liposome, DDC, composed of dioleoyltrimethylamino propane (DOTAP), 1,2-dioeoyl-3-phosphophatidylethanolamine (DOPE), and cholesterol (Chol), and it efficiently delivered plasmid DNA into ovarian cancer cells. Mutations in the p53 tumor suppressor gene are the most common molecular genetic abnormalities to be described in ovarian cancer. However, there has been so far no report of nonviral vector-mediated p53 gene deliveries in ovarian cancer. In this study, wild-type p53 DNA was transfected into the ovarian cancer cells, using the DDC as a nonviral vector and the expression and activity of p53 gene were evaluated both in vitro and in vivo.


METHODS
DDC liposomes were prepared by mixing DOTAP:DOPE:Chol in a 1:0.7:0.3 molar ratio using the extrusion method. Plasmid DNA (pp53-EGFP) and DDC complexes were transfected into ovarian carcinoma cells (OVCAR-3 cells) and gene expression was determined by reverse transcription-polymerase chain reaction and Western blot analysis. The cellular growth inhibition and apoptosis of DDC-mediated p53 transfection were assessed by trypan blue exclusion assay and annexin-V staining, respectively. The OVCAR-3 cells treated with DDC/pp53-EGFP complexes were inoculated into female balb/c nude mice and tumor growth was observed.


RESULTS
The transfection of liposome-complexed p53 gene resulted in a high level of wild-type p53 mRNA and protein expressions in OVCAR-3 cells. In vitro cell growth assay showed growth inhibition of cancer cells transfected with DDC/pp53-EGFP complexes compared with the control cells. The reestablishment of wild-type p53 function in ovarian cancer cells restored the apoptotic pathway. Following the inoculation of DDC/pp53-EGFP complexes, the volumes of tumors in nude mice were significantly reduced more than 60% compared to the control group.


CONCLUSIONS
The DDC-mediated p53 DNA delivery may have the potential for clinical application as nonviral vector-mediated ovarian cancer therapy due to its effective induction of apoptosis and tumor growth inhibition.
Kim
Chong-Kook
CK
College of Pharmacy, Seoul National University, San 56-1, Shinlim-Dong, Kwanak-Gu, Seoul 151-742, South Korea. ckkim@plaza.snu.ac.kr
Choi
Eun-Jeong
EJ
Choi
Sung-Hee
SH
Park
Jeong-Sook
JS
Haider
Khawaja Hasnain
KH
Ahn
Woong Shick
WS
D000230:Adenocarcinoma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology / Q000628:therapy; D017209:Apoptosis / Q000235:genetics; D002455:Cell Division / Q000235:genetics; D002784:Cholesterol / Q000008:administration & dosage; D005229:Fatty Acids, Monounsaturated / Q000008:administration & dosage; D005260:Female; D016158:Genes, p53 / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D008081:Liposomes / Q000008:administration & dosage; D010051:Ovarian Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology / Q000628:therapy; D010714:Phosphatidylethanolamines / Q000008:administration & dosage; D010957:Plasmids / Q000008:administration & dosage / Q000235:genetics; D000644:Quaternary Ammonium Compounds / Q000008:administration & dosage; D014162:Transfection / Q000379:methods*; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis / Q000235:genetics
C410262:1,2-dioleoyl-glycero-3-phosphatidyl ethanolamine; D005229:Fatty Acids, Monounsaturated; D008081:Liposomes; D010714:Phosphatidylethanolamines; D000644:Quaternary Ammonium Compounds; D016159:Tumor Suppressor Protein p53; D002784:Cholesterol; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane