Development of targeted therapy for bladder cancer mediated by a double promoter plasmid expressing diphtheria toxin under the control of H19 and IGF2-P4 regulatory sequences

J Transl Med. 2010 Dec 16:8:134. doi: 10.1186/1479-5876-8-134.

Abstract

Background: The human IGF2-P4 and H19 promoters are highly active in a variety of human cancers (including bladder cancer), while existing at a nearly undetectable level in the surrounding normal tissue.Single promoter vectors expressing diphtheria toxin A-fragment (DTA) under the control regulation of IGF2-P4 or H19 regulatory sequences (IGF2-P4-DTA and H19-DTA) were previously successfully used in cell lines, animal models and recently in human patients with superficial cell carcinoma of the bladder (treated with H19-DTA). However this targeted medicine approach could be limited, as not all cancer patients express high levels of H19. Hence, a double promoter DTA-expressing vector was created, carrying on a single construct two separate genes expressing the diphtheria toxin A-fragment (DTA), from two different regulatory sequences, selected from the cancer-specific promoters H19 and IGF2-P4.

Methods: H19 and IGF2-P4 gene expression was tested in samples of Transitional Cell Carcinoma (TCC) of the bladder by in-situ hybridization (ISH) and by quantitative Real-Time PCR (qRT-PCR). The therapeutic potential of the double promoter toxin vector H19-DTA-IGF2-P4-DTA was tested in TCC cell lines and in heterotopic and orthotopic animal models of bladder cancer.

Results: Nearly 100% of TCC patients highly expressed IGF2-P4 and H19, as determined by ISH and by qRT-PCR. The double promoter vector exhibited superior tumor growth inhibition activity compared to the single promoter expression vectors, in cell lines and in heterotopic and orthotopic bladder tumors.

Conclusions: Our findings show that bladder tumors may be successfully treated by intravesical instillation of the double promoter vector H19-DTA-P4-DTA.Overall, the double promoter vector exhibited enhanced anti-cancer activity relative to single promoter expression vectors carrying either gene alone.

MeSH terms

  • Administration, Intravesical
  • Animals
  • Cell Line, Tumor
  • Cell Proliferation
  • Diphtheria Toxin / metabolism*
  • Disease Models, Animal
  • Gene Expression Regulation, Neoplastic
  • Genetic Vectors / administration & dosage
  • Humans
  • In Situ Hybridization
  • Insulin-Like Growth Factor II / genetics*
  • Insulin-Like Growth Factor II / metabolism
  • Luciferases / genetics
  • Mice
  • Mice, Nude
  • Peptide Fragments / metabolism*
  • Plasmids / genetics*
  • Promoter Regions, Genetic*
  • RNA, Long Noncoding
  • RNA, Untranslated / genetics*
  • RNA, Untranslated / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Subcutaneous Tissue / pathology
  • Urinary Bladder Neoplasms / genetics
  • Urinary Bladder Neoplasms / pathology
  • Urinary Bladder Neoplasms / therapy*
  • Xenograft Model Antitumor Assays

Substances

  • Diphtheria Toxin
  • H19 long non-coding RNA
  • Peptide Fragments
  • RNA, Long Noncoding
  • RNA, Untranslated
  • diphtheria toxin fragment A
  • Insulin-Like Growth Factor II
  • Luciferases