Abstract
Our objective was to evaluate cell growth and death effects by inhibiting Murine Double Minute 2 (MDM2) expression in human prostate cancer cells overexpressing the wild-type (WT) p53 gene. Prostate PC-3 tumor cells were transfected with a plasmid containing either mdm2 small interfering (Si-mdm2) or the WT p53 gene (Pp53) alone, or both (Pmp53), using Lipofectamine in vitro and attenuated Salmonella enterica serovar Typhi vaccine strain Ty21a (Salmonella Typhi Ty21a) in vivo. Cell growth, apoptosis, and the expression of related genes and proteins were examined in vitro and in vivo by flow cytometry and Western blot assays. We demonstrated that human prostate tumors had increased expression of MDM2 and mutant p53 proteins. Transfection of the PC-3 cells with the Pmp53 plasmid in vitro offered significant inhibition of cell growth and an increase in apoptotic cell death compared with that of the Si-mdm2 or Pp53 group. These effects were associated with up-regulation of p21 and down-regulation of hypoxia-inducible factor 1α expression in Pmp53-transfected cells. To validate the in vitro findings, the nude mice implanted with PC-3 cells were treated with attenuated Salmonella Typhi Ty21a carrying the plasmids, which showed that the Pmp53 plasmid significantly inhibited the tumor growth rate in vivo compared with that of the Si-mdm2 or Pp53 plasmid alone. Tumor tissues from mice treated with the Pmp53 plasmid showed increased expression of p21 and decreased expression of hypoxia-inducible factor 1α proteins, with an increased apoptotic effect. These results suggest that knockdown of mdm2 expression by its specific small interfering RNA with overexpression of the WT p53 gene offers synergistic inhibition of prostate cancer cell growth in vitro and in vivo.
Footnotes
This work was supported by the Research Fund for the Doctoral Program of Higher Education of China [Grant 20070183012]; and the Scientific and Technological Development Plan Project in Jilin Province [Grant 200705360].
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.111.180364.
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ABBREVIATIONS:
- MDM2
- Murine Double Minute 2
- WT
- wild-type
- MT
- mutated
- PI
- propidium iodide
- PBS
- phosphate-buffered saline
- MTT
- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
- siRNA
- small interfering RNA
- PCNA
- proliferating cell nuclear antigen
- pRb
- hypophosphorylated retinoblastoma
- PCR
- polymerase chain reaction
- RT-PCR
- reverse transcription polymerase chain reaction
- CFU
- colony-forming unit
- TUNEL
- terminal deoxynucleotidyl transferase dUTP nick-end labeling
- CDK
- cyclin-dependent kinase
- HIF-1α
- hypoxia-inducible factor 1α
- FCM
- flow cytometry
- Si-mdm2
- plasmid containing mdm2 siRNA alone
- Pp53
- plasmid containing wild-type p53 gene alone
- Pmp53
- plasmid containing mdm2 siRNA and the wild-type p53 gene
- Si-scramble
- plasmid containing the scrambled small interfering RNA.
- Received February 7, 2011.
- Accepted March 21, 2011.
- U.S. Government work not protected by U.S. copyright
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