Kaszubiak Alexander, Holm Per Sonne, Lage Hermann
Institute of Pathology, Charité Campus Mitte, Humboldt University Berlin, D-10117 Berlin, Germany.
Int J Oncol. 2007 Aug;31(2):419-30.
Simultaneous resistance of cancer cells to multiple cytotoxic drugs, multidrug resistance (MDR), is the major limitation to the successful chemotherapeutic treatment of disseminated neoplasms. The 'classical' MDR phenotype is conferred by MDR1/P-glycoprotein (MDR1/P-gp) that is expressed in almost 50% of human cancers. Recent developments in the use of small interfering RNAs for specific inhibition of gene expression have highlighted their potential use as therapeutic agents. DNA cassettes encoding RNA polymerase III promoter-driven siRNA-like short hairpin RNAs (shRNAs) allow long-term expression of therapeutic RNAs in targeted cells. A variety of viral vectors have been used to deliver such cassettes to mammalian cells. In this study, the construction of different adenoviruses for anti-MDR1/P-gp shRNA delivery in different human multidrug-resistant cancer cells was investigated. The efficiency of the shRNAs was compared to adenoviral delivery of an anti-MDR1/P-gp ribozyme construct. It could be demonstrated that MDR1/P-gp mRNA and protein expression could be completely inhibited by adenoviral delivery of anti-MDR1/P-gp shRNAs. This downregulation in mRNA and protein expression was accompanied by a complete inhibition of the pump activity of MDR1/P-gp and a reversal of the multidrug-resistant phenotype. By application of adenoviral encoded anti-MDR1/P-gp ribozyme construct merely weak effects on gene expression were observed. In conclusion, the data demonstrate that adenoviral delivery of shRNAs can chemosensitize human cancer cells, that adenoviral delivery of shRNAs is much more effective than adenoviral delivery of ribozymes, and that adenovirus-based vectors can be very effective agents for efficient delivery of therapeutic RNA molecules.
癌细胞对多种细胞毒性药物同时产生耐药性，即多药耐药性（MDR），是成功化疗治疗播散性肿瘤的主要限制因素。“经典”的MDR表型由MDR1/ P-糖蛋白（MDR1/ P-gp）赋予，该蛋白在近50%的人类癌症中表达。小干扰RNA用于特异性抑制基因表达的最新进展突出了其作为治疗剂的潜在用途。编码RNA聚合酶III启动子驱动的类似siRNA的短发夹RNA（shRNA）的DNA盒允许治疗性RNA在靶细胞中长期表达。多种病毒载体已被用于将此类盒递送至哺乳动物细胞。在本研究中，研究了构建不同的腺病毒以在不同的人多药耐药癌细胞中递送抗MDR1/ P-gp shRNA。将shRNA的效率与抗MDR1/ P-gp核酶构建体的腺病毒递送进行了比较。可以证明，抗MDR1/ P-gp shRNA的腺病毒递送可完全抑制MDR1/ P-gp mRNA和蛋白表达。mRNA和蛋白表达的这种下调伴随着MDR1/ P-gp泵活性的完全抑制和多药耐药表型的逆转。通过应用腺病毒编码的抗MDR1/ P-gp核酶构建体，仅观察到对基因表达的微弱影响。总之，数据表明，shRNA的腺病毒递送可使人类癌细胞对化疗敏感，shRNA的腺病毒递送比核酶的腺病毒递送更有效，并且基于腺病毒的载体可以是有效递送治疗性RNA分子的非常有效的试剂。
