Koedrith Preeyaporn, Seo Young Rok
Department of Life Science, Dongguk University-Seoul, Seoul 100-715, Republic of Korea.
Exp Ther Med. 2011 Sep;2(5):873-878. doi: 10.3892/etm.2011.304. Epub 2011 Jun 30.
Thioredoxin reductase 1 (Trr1) is an antioxidant and redox regulator that functions in governing the cellular redox state and survival against oxidative insults in mammals. However, this selenoprotein is also overexpressed in various forms of malignant cancers, leading to the hypothesis that Trr1 may be a potential target for cancer therapy. A quinone anti-cancer drug, mitomycin C (MMC), has been clinically used in the treatment of several types of tumors, including those of the colon. MMC exerts its activity via ROS induction and further results in DNA cross-linkage. To evaluate the significant role of Trr1 in MMC resistance in human colon cancer (RKO) cells, specific reduction in the expression of Trr1 was achieved using short-hairpin RNA (shRNA)-based interference. Our results showed that stable Trr1 shRNA knockdown manifested higher cellular susceptibility to MMC in comparison to that in wild-type cells. In addition, increased intracellular ROS accumulation appeared in the Trr1 shRNA knockdown cells compared to the RKO wild-type cells, in proportion to a relatively higher fraction of the DNA damage reporter protein phosphorylated histone 'γ-H2AX'. Notably, a neutral comet assay demonstrated that DNA double-strand breaks were highly induced in the Trr1-deficient cancer cells in the presence of MMC, presumably stimulating cancer cell death. Our results also revealed that MMC-induced apoptosis was associated with enhancement of oxidative damage to DNA. These results suggest that the specific knockdown of Trr1 expression via shRNA vector interference technology may be a potent molecular strategy by which to enhance the effectiveness of MMC-mediated killing in human colon cancer cells, through acceleration of double-strand DNA damage-oxidative stress as a trigger for apoptosis. This implies that Trr1 may be a prime target for enhancing the effectiveness of MMC chemotherapy in combination with specific RNA interference.
硫氧还蛋白还原酶1（Trr1）是一种抗氧化和氧化还原调节因子，在维持哺乳动物细胞氧化还原状态以及抵抗氧化损伤中发挥作用。然而，这种硒蛋白在多种恶性肿瘤中也呈过表达，这引发了一种假说，即Trr1可能是癌症治疗的潜在靶点。一种醌类抗癌药物丝裂霉素C（MMC）已在临床上用于治疗多种类型的肿瘤，包括结肠癌。MMC通过诱导活性氧（ROS）发挥作用，并进一步导致DNA交联。为了评估Trr1在人结肠癌（RKO）细胞对MMC耐药中的重要作用，利用基于短发夹RNA（shRNA）的干扰技术特异性降低了Trr1的表达。我们的结果表明，与野生型细胞相比，稳定敲低Trr1的shRNA使细胞对MMC的敏感性更高。此外，与RKO野生型细胞相比，Trr1 shRNA敲低细胞中细胞内ROS积累增加，这与DNA损伤报告蛋白磷酸化组蛋白γ-H2AX的相对较高比例成正比。值得注意的是，中性彗星试验表明，在MMC存在的情况下，Trr1缺陷型癌细胞中DNA双链断裂被高度诱导，这可能会刺激癌细胞死亡。我们的结果还表明，MMC诱导的细胞凋亡与DNA氧化损伤的增强有关。这些结果表明，通过shRNA载体干扰技术特异性敲低Trr1表达可能是一种有效的分子策略，通过加速双链DNA损伤-氧化应激作为细胞凋亡的触发因素，增强MMC介导的对人结肠癌细胞的杀伤效果。这意味着Trr1可能是增强MMC化疗效果并结合特异性RNA干扰的主要靶点。
