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.

目的体外建立稳定表达绿色荧光蛋白（GFP）的人舌鳞癌细胞株SAS-Hi。通过舌粘膜下注射，荧光细胞可显示肿瘤生长、局部浸润、微转移和淋巴结转移。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，我们检测了LY 294002在体内降低局部侵袭和颈淋巴结转移的作用。并利用该模型研究了抗肿瘤肽hCAP 18和MEK类似物抗肿瘤侵袭转移的作用。方法采用Nucleofector 4转染装置，将pEGFP-N1质粒转染人舌鳞癌细胞株SAS-H1。指数增长的GFP标记的肿瘤 ...更多信息 将莫尔细胞悬液（2 × 106）注射到12只KSN紧张的雄性无胸腺裸鼠（4-6周龄）的舌中。注射后第1天，用LY 294002（0、25、50或100 mg/kg）腹膜内处理小鼠4周。在第4周，将荷瘤小鼠处死并在光和荧光多角度数字显微镜（KEYENCE）下尸检。结果注射hCAP 18肽后第1天，所有小鼠舌部肿瘤均呈荧光状，并在第2天开始出现荧光性肿瘤。在第4周，可以观察到原发性肿瘤周围散布的GFP标记的细胞，并且所有肿瘤都转移到处死小鼠的颈部淋巴结。在新鲜颈部淋巴结中检测到GFP标记的肿瘤细胞。LY 294002治疗组4周时局部浸润和淋巴结转移较对照组呈剂量依赖性减少。100 mg/kg LY 294002处理的小鼠完全没有颈部转移。一方面。结论LY 294002能抑制口腔鳞癌的局部浸润、微转移和颈淋巴结转移，提示P13-K抑制剂在口腔鳞癌的治疗中可能发挥重要作用。我们的数据表明，P13 K抑制剂和抗菌肽hCAP 18的组合可以提供一种有效的方法来抑制口腔鳞状细胞癌的肿瘤转移和肿瘤生长。少

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