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Study of suppressions of invasion / metastasis by molecular targeted therapy using GFP exppessing transfectant of a high-invasion human tongue squamous cell carcinoma cells, SAS-H1.

使用表达高侵袭性人舌鳞状细胞癌细胞 SAS-H1 的 GFP 转染子进行分子靶向治疗抑制侵袭/转移的研究。

基本信息

批准号：
14571906
负责人：
OKUMURA Kazuhiko
金额：
$ 2.3万
依托单位：
Health Sciences University of Hokkaido
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

项目摘要

Objective We report the in viitmestablishment of a highly stable green fluorescent protein (GFP) -expressing transfectant of highly-invasive human tongue squamous cell carcinoma cells, SAS-Hi. The fluorescent cells permitted the visualization of tumor growth, local invasion, micrmetastasis, and eeevical lymph node metastasis aver submucosal injection into the tongue of nude mice. This model should be useful for studying the metastatic process and for evaluating anti-metastatic agents in pre-clinesl trials. Using GFP-tagged human tongue squamous cell carcinoma cells, we examined the LY294002 reduce local invasion and cervical lymph node metastasis in vivo. Additionally, using this model, eflect ofanti invasion and -metastasis by antimic mbial peptide hCAP18, and MEK inhibitorwas studied. Methods High invasion human tongue squamous cell carcinoma cell line, as SAS-H1 cells were transfected with the pEGFP-N1 plasmid (CLONTECH) using Nucleofector4device. Exponentially growing GFP-tagged tu … More mor cells suspension (2X10^6) was injected into the tongue of 12 KSN strained male athymic nude mice (4-6-week-old). Day 1 post-injection, mice were treated with LY294002 (0, 25, 50; or 100 mg/kg) i.p. for 4 weeks. At week 4, tumor-bearing mice were sacrificed and autopsied under light and fluorescent multi-angle digital microscope (KEYENCE). Furthermore, mice were treated with hCAP18 peptide (0, 50, 100 μg/ml) in local injection twice a week for 4weeks.Results At day 1 post-injection, all mice expressed fluorescent tongue tumors. At week 4, disseminated GFP-tagged cells around the primary tumor could be observed, and all tumors metastasized to the cervical lymph nodes in sacrificed mice. GFP-tagged tumor cells detected in fresh cervical lymph nodes. LY294002-treated group was reduced dose-dependently local invasion and lymph node metastasis than control group at 4 weeks. 100 mg/kg of LY294002 treated mice were being completely free from cervical metastasis. On one hand. hCAP 18-treated group was reduced dose dependently tumor volume in primary site, but not inhibited cervical lymph node metastasis.Conclusion These results that LY294002 inhibits local invasion, micrometastasis and cervical lymph node metastasis in viva, suggesting an important role of P13-K inhibitors as a potentially useful treatment for oral squamous cell carcinoma. Our data suggest that a combination of a P13K inhibitor and antimicrobial peptide, hCAP 18 may provide an effective approach to inhibiting tumor metastasis and tumor growth in oral squamous cell carcinoma. Less

目的建立稳定表达绿色荧光蛋白(GFP)的高侵袭性人舌鳞癌细胞系SAS-Hi。通过将荧光细胞注射到裸鼠舌粘膜下，可以观察到肿瘤的生长、局部侵袭、微转移和颈部淋巴结转移。该模型可用于研究转移过程和临床前试验中抗转移药物的评价。利用GFP标记的人舌鳞状细胞癌细胞，我们在体内检测了LY294002降低局部侵袭和颈淋巴转移的作用。此外，利用该模型研究了抗癌多肽hCAP18和MEK抑制剂抗肿瘤侵袭转移的作用。方法将pEGFP-N1真核表达载体(Clontech)导入高侵袭性人舌鳞状细胞癌细胞系SAS-H1。指数级增长的绿色荧光蛋白标记的TU…将更多的MOR细胞悬液(2X10^6)注射到12只4~6周龄KSN系雄性裸鼠的舌内。注射LY294002(0、25、50、100 mg/kg)后第1天，腹腔注射LY294002。为期4周。第4周处死荷瘤小鼠，在光镜和荧光多角度数字显微镜(Keyence)下解剖。给小鼠注射hCAP18多肽(0、50、100μg/ml)，每周2次，连续4周。术后4周，原发灶周围可见散在分布的GFP标记细胞，处死小鼠肿瘤全部转移至颈淋巴结。新鲜颈淋巴中检测到GFP标记的肿瘤细胞。LY294002治疗组在4周时局部侵袭和淋巴结转移明显低于对照组，且呈剂量依赖关系。100 mg/kg的LY294002治疗组小鼠完全没有颈部转移。一方面。结论LY294002可抑制口腔鳞癌的局部侵袭、微转移和颈淋巴转移，提示P13-K抑制剂在口腔鳞状细胞癌的治疗中具有重要作用。我们的数据表明，P13K抑制剂和抗菌肽的结合可能为抑制口腔鳞状细胞癌的肿瘤转移和肿瘤生长提供一种有效的途径。较少