Targeted RNA interference of PI3K pathway components sensitizes colon cancer cells to TNF-related apoptosis-inducing ligand (TRAIL)
Targeted RNA interference of PI3K pathway components sensitizes colon cancer cells to TNF-related apoptosis-inducing ligand (TRAIL)
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DOI：
10.1016/j.surg.2005.05.012
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发表时间：
2005-08-01
期刊：
影响因子：
3.8
通讯作者：
Evers, BM
中科院分区：
文献类型：
--
作者：
Rychahou, PG;Murillo, CA;Evers, BM
背景资料。磷脂酰肌醇3-激酶(PI3K/Akt)通路是生长因子信号转导通路。此前，我们发现了PI3K/Akt在结肠癌进展中的重要作用。本研究的目的是确定(1)针对PI3K/Akt组分的短干扰RNA(SiRNA)是否能使结肠癌细胞对肿瘤坏死因子相关的凋亡诱导配体(TRAIL)敏感；(2)提高敏感性的细胞机制。将针对PI3K P85α调节亚基的siRNA或非靶向对照序列导入人结肠癌细胞KM20和KM12C(均为TRAIL抗性)，然后用TRAIL(100 ng/mL)或赋形剂处理。核糖核酸酶保护法检测TRAIL受体表达的变化。提取蛋白，免疫印迹分析TRAIL受体(死亡受体4和5)、半胱氨酸天冬氨酸蛋白酶3、半胱氨酸天冬氨酸蛋白酶8和Bid裂解产物的表达。DNA片段酶联免疫吸附试验检测细胞凋亡率。P85α(或Akt1 siRNA和TRAIL)联合作用增加KM20和KM12C细胞的凋亡率，与单独使用TRAIL相比，这些结果被caspase-3抑制剂Z-乙酰-Asp-Glu-Val-Asp-(DEVD)-fmk完全抑制。此外，sRNiA介导的PI3K通路抑制导致TRAIL死亡受体4和5的表达增加。RNA干扰抑制PI3K/Akt通过诱导TRAIL受体和激活选择性靶向的caspase-3和caspase-8药物，使耐药结肠癌细胞对TRAIL诱导的细胞死亡敏感。PI3K/Akt通路可能会增强化疗药物的疗效，并为部分结肠癌提供新的辅助治疗。
Background. The phosphoinasilide 3-kinase (PI3K/Akt) pathway transduces signals initiated from growth factors. Previously, we identified an important role for PI3K/Akt in colon cancer progression. The purpose of this study was to determine (1) whether short interfering RNA (siRNA) directed to PI3K/Akt components can render colon cancer cells sensitive to treatment with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and (2) the cellular mechanisms contributing to the enhanced sensitivity.Methods. Human colon cancer cells KM20 and KM12C (both TRAIL resistant) were transfected with siRNA directed against the PI3K p85 alpha regulatory subunit AN] or nontargeting control sequence and then treated with TRAIL (100 ng/mL) or vehicle. A ribonuclease protection assay was performed to assess changes in TRAIL receptor expression. Protein was extracted and analyzed by Western, blot for expression of cleavage of TRAIL receptors (death receptor (DR) 4 and 5), caspase 3, caspase-8, and BID. Apoptosis was measured by enzyme-linked immunosorbent assay of DNA fragmentation.Results. Combination treatment with P85 alpha( or Akt1 siRNA and TRAIL increased apoptosis in KM20 and KM12C cells, compared with TRAIL alone, these results were corrobarated further by complete inhibition of apoptosis by Z-acetyl-Asp-Glu-Val-Asp-(DEVD)-fmk a caspase-3 inhibitor. Furthermore, sRNiA-mediated PI3K pathway inhibition resulted in increased expression of the TRAIL death receptors 4 and 5.Conclusions. Inhibition of PI3K/Akt by RNA interference sensitizes resistant colon cancer cells to TRAIL-induced cell death through the induction of TRAIL receptors and activation of caspase-3 and caspase-8 Agents that selectively target. the PI3K/Akt pathway may enhance the effects of chemotherapeutic agents and provide novel adjuvant treatment for selected colon cancers.