The Hebrew University of Jerusalem, Biological Chemistry, Jerusalem 91904, Israel.
J Transl Med. 2010 Dec 16;8:134. doi: 10.1186/1479-5876-8-134.
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.
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.
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.
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.
人类 IGF2-P4 和 H19 启动子在多种人类癌症（包括膀胱癌）中高度活跃，而在周围正常组织中几乎检测不到。先前曾使用受 IGF2-P4 或 H19 调控序列控制的单个启动子载体表达白喉毒素 A 片段（DTA）（IGF2-P4-DTA 和 H19-DTA）在细胞系、动物模型中，最近在患有浅表膀胱癌的人类患者中（用 H19-DTA 治疗）取得了成功。然而，这种靶向药物治疗方法可能会受到限制，因为并非所有癌症患者都表达高水平的 H19。因此，创建了一个双启动子 DTA 表达载体，在单个构建体上携带两个来自不同调控序列的单独基因，这两个基因表达白喉毒素 A 片段（DTA），这两个调控序列是从癌症特异性启动子 H19 和 IGF2-P4 中选择的。
通过原位杂交（ISH）和实时定量 PCR（qRT-PCR）检测膀胱癌中转形细胞癌（TCC）样本中的 H19 和 IGF2-P4 基因表达。在 TCC 细胞系以及异位和原位膀胱癌动物模型中测试了双启动子毒素载体 H19-DTA-IGF2-P4-DTA 的治疗潜力。
ISH 和 qRT-PCR 确定，几乎 100%的 TCC 患者高度表达 IGF2-P4 和 H19。与单启动子表达载体相比，该双启动子载体在细胞系以及异位和原位膀胱癌中表现出更优异的肿瘤生长抑制活性。
我们的研究结果表明，膀胱癌可以通过膀胱内灌注双启动子载体 H19-DTA-IGF2-P4-DTA 成功治疗。总体而言，与单独携带任一基因的单启动子表达载体相比，双启动子载体表现出增强的抗癌活性。
