Polypurine reverse Hoogsteen hairpins as a gene therapy tool against survivin in human prostate cancer PC3 cells in vitro and in vivo.
As a new approach for gene therapy, we recently developed a new type of molecule called polypurine reverse Hoogsteen hairpins (PPRHs). We decided to explore the in vitro and in vivo effect of PPRHs in cancer choosing survivin as a target since it is involved in apoptosis, mitosis and angiogenesis, and overexpressed in different tumors. We designed four PPRHs against the survivin gene, one of them directed against the template strand and three against different regions of the coding strand. These PPRHs were tested in PC3 prostate cancer cells in an in vitro screening of cell viability and apoptosis. PPRHs against the promoter sequence were the most effective and caused a decrease in survivin mRNA and protein levels. We confirmed the binding between the selected PPRHs and their target sequences in the survivin gene. In addition we determined that both the template- and the coding-PPRH targeting the survivin promoter were interfering with the binding of transcription factors Sp1 and GATA-3, respectively. Finally, we conducted two in vivo efficacy assays using the Coding-PPRH against the survivin promoter and performing two routes of administration, namely intratumoral and intravenous, in a subcutaneous xenograft tumor model of PC3 prostate cancer cells. The results showed that the chosen Coding-PPRH proved to be effective in decreasing tumor volume, and reduced the levels of survivin protein and the formation of blood vessels. These findings represent the preclinical proof of principle of PPRHs as a new silencing tool for cancer gene therapy.
Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, 08028 Barcelona, Spain. Electronic address: laura.rodriguez@ub.edu.
Laura
L
Rodríguez
Xenia
X
Villalobos
Sheila
S
Dakhel
Laura
L
Padilla
Rosa
R
Hervas
Jose Luis
JL
Hernández
Carlos J
CJ
Ciudad
Véronique
V
Noé
D000818:Animals; D017209:Apoptosis / Q000235:genetics; D020029:Base Pairing; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000235:genetics; D004247:DNA / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic; D018390:Gene Targeting; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000235:genetics*; D008297:Male; D051379:Mice; D008819:Mice, Nude; D011471:Prostatic Neoplasms / Q000235:genetics / Q000473:pathology / Q000628:therapy*; D011685:Purine Nucleotides / Q000235:genetics*; D000077022:Survivin; D023041:Xenograft Model Antitumor Assays
C107489:BIRC5 protein, human; D051034:Inhibitor of Apoptosis Proteins; D011685:Purine Nucleotides; D000077022:Survivin; D004247:DNA
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; CRPC; DOTAP; EMSA; Gene silencing; MTT; N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate; NE; PPRH; PPRHs; Polypurine Reverse Hoogsteen hairpins; Prostate cancer; Survivin; Xenograft; aODN; antisense oligonucleotide; castration-resistant prostatic cancer; electrophoretic mobility shift assay; nuclear extract