Therapeutic targeting of neuropilin-2 on colorectal carcinoma cells implanted in the murine liver
Therapeutic targeting of neuropilin-2 on colorectal carcinoma cells implanted in the murine liver
复制标题
DOI：
10.1093/jnci/djm279
复制
发表时间：
2008-01-16
期刊：
影响因子：
--
通讯作者：
Ellis, Lee M.
中科院分区：
文献类型：
--
作者：
Gray, Michael J.;Van Buren, George;Ellis, Lee M.
背景 Neuropilin - 2 (NRP2) 是血管内皮生长因子 (VEGF) 和信号蛋白 3F 的高亲和力激酶缺陷型受体。我们研究了其在人类结直肠癌中的功能。方法采用免疫组织化学和免疫印迹法分别评估结直肠肿瘤和结直肠癌细胞系中NRP2的表达水平。使用针对 NRP2 的短发夹 RNA (shRNA) 或对照 shRNA 稳定转染的 HCT-116 结直肠癌细胞，通过四唑盐 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基溴化四唑 (MTT) 测定法测定增殖情况，并通过免疫印迹法测定 VEGFR1 通路的激活情况。软琼脂测定、膜联蛋白 V 染色和博伊登室测定分别用于检查 HCT-116 转染子中的贴壁依赖性生长、缺氧反应的细胞凋亡以及细胞迁移/侵袭。在注射表达 shRNA 的 HCT-116 细胞的小鼠（每组 10 只）中分析肿瘤生长和转移。通过测量生物发光和最终肿瘤体积的变化，评估体内小干扰 RNA (siRNA) 靶向 NRP2 对表达荧光素酶的 HCT-116 细胞来源的肝结直肠肿瘤生长的影响。所有统计检验都是双边的。结果NRP2在肿瘤中的表达显着高于邻近粘膜中的表达。 NRP2 水平降低的 HCT-116 转染子也降低了 VEGFR1 信号传导，但增殖能力没有变化。不依赖锚定的生长、缺氧条件下的存活以及运动性/侵袭性也降低。在体内，与对照细胞相比，NRP2 减少的 HCT-116 转染子表现出肿瘤生长减少、转移减少和细胞凋亡增加。用 NRP2 siRNA 治疗的小鼠的肝结直肠肿瘤在统计学上显着小于用对照 siRNA 治疗的小鼠（植入后 28 天，平均对照 siRNA = 420 mm(3)，平均 NRP2 siRNA = 36 mm(3)，NRP2 与对照：差异 = 385 mm(3)，95% 置信区间 = 174 mm(3) 至 595 mm(3)，P =.005)。结论结直肠癌细胞上的NRP2对于肿瘤生长很重要，并且是其表达的人类癌症的潜在治疗靶点。
Background Neuropilin - 2 ( NRP2) is a high-affinity kinase-deficient receptor for vascular endothelial growth factor ( VEGF) and semaphorin 3F. We investigated its function in human colorectal cancers.Methods Immunohistochemistry and immunoblotting were used to assess NRP2 expression levels in colorectal tumors and colorectal cancer cell lines, respectively. HCT-116 colorectal cancer cells stably transfected with short hairpin RNA ( shRNAs) against NRP2 or control shRNAs were assayed for proliferation by the tetrazolium salt 3-( 4,5-dimethylthiazol-2-yl)- 2,5- diphenyltetrazolium bromide ( MTT) assay and for activation of the VEGFR1 pathway by immuno blotting. Soft agar assays, Annexin V staining, and Boyden chamber assays were used to examine anchorage-independent growth, apoptosis in response to hypoxia, and cell migration/ invasion, respectively, in HCT-116 transfectants. Tumor growth and metastasis were analyzed in mice ( groups of 10) injected with shRNA-expressing HCT-116 cells. The effect of in vivo targeting of NRP2 by small interfering RNA ( siRNA) on the growth of hepatic colorectal tumors derived from luciferase-expressing HCT-116 cells was assessed by measuring changes in bioluminescence and final tumor volumes. All statistical tests were two-sided.Results NRP2 expression was substantially higher in tumors than in adjacent mucosa. HCT-116 transfectants with reduced NRP2 levels had reduced VEGFR1 signaling, but proliferation was unchanged. Anchorage-independent growth, survival under hypoxic conditions, and motility/ invasiveness were also reduced. In vivo, HCT-116 transfectants with reduced NRP2 demonstrated decreased tumor growth, fewer metastases, and increased apoptosis compared with control cells. Hepatic colorectal tumors in mice treated with NRP2 siRNAs were statistically significantly smaller than those in mice treated with control siRNAs ( at 28 days after implantation, mean control siRNAs = 420 mm(3), mean NRP2 siRNAs = 36 mm(3), NRP2 vs control: difference = 385 mm(3), 95% confidence interval = 174 mm(3) to 595 mm(3), P =.005).Conclusion NRP2 on colorectal carcinoma cells is important for tumor growth and is a potential therapeutic target in human cancers where it is expressed.