Abstract
BackgroundThe 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.MethodsH19 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.ResultsNearly 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.ConclusionsOur 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.
Highlights
Bladder cancer is the fourth most commonly diagnosed malignancy in men and the ninth most commonly diagnosed malignancy in women, (NCI annual report 2009).Urinary bladder neoplasm can be grouped into three different categories: Superficial, invasive and metastatic
Expression of IGF2-P4 and H19 transcripts in human bladder carcinoma tissues determined by in-situ hybridization (ISH) or by RTPCR The human IGF2-P4 and H19 regulatory sequences are highly active in a variety of human cancers
In this study we present an approach for targeted therapy of bladder carcinoma by driving the diphtheria toxin A-fragment (DTA) expression under the control of IGF2-P4 and H19 regulatory sequences
Summary
Bladder cancer is the fourth most commonly diagnosed malignancy in men and the ninth most commonly diagnosed malignancy in women, (NCI annual report 2009).Urinary bladder neoplasm can be grouped into three different categories: Superficial, invasive and metastatic. Tumors are usually limited to the bladder mucosa (Ta) or submucosa (T1) These tumors can be removed by transurethral resection (TUR), but tend to recur in 50-70% of the patients. 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). This targeted medicine approach could be limited, as not all cancer patients express high levels of H19. 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
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