摘要
目的研究低剂量顺铂和低浓度天花粉蛋白（trichosanthin，TCS）联合作用抑制喉癌细胞（Hep．2细胞株和AMC．HN．8细胞株）增殖的作用及其机制。方法设立不同的实验组，包括低浓度顺铂组（3μg/m1）、高浓度顺铂组（10μg／m1）、TCS组（5μg/m1）、TCS和顺铂联合作用组（5μg／mlTCS和3μg／ml顺铂）和阴性对照组；连续5d使用细胞增殖检测法（cellcountingkit-8，CCK-8）和乳酸脱氢酶法检测不同实验组对喉癌细胞的抑制作用和细胞毒性；流式细胞仪和Hochest33258染色观察细胞凋亡；Westernblot检测C—Jun氨基端激酶（JNK），磷酸化JNK、核因子KB（nuclearfactorKB，NF—KB）、p38、磷酸化p38、KB抑制蛋白（inhibitorofKB，I-KB）及磷酸化I—KB的变化。结果与TCS和低剂量顺铂单独作用相比，TCS和顺铂联合作用可明显抑制喉癌细胞的生长（P值均〈0．01），凋亡细胞数量增加；联合作用组的抑制效果与高浓度顺铂组差异无统计学意义，细胞毒性同TCS和顺铂单独作用相比差异无统计学意义（P〉0．05），而毒性低于高浓度顺铂组（P〈0．01）；Western blot显示顺铂增强NF．KB转录因子活性，抑制JNK信号通路，TCS组主要激活JNK信号通路而抑制NF—KB，TCS和顺铂联合作用后磷酸化JNK保持高水平抑制NF—KB活性。结论TCS通过激活JNK信号通路，同时抑制NF—KB转录因子活性，增强顺铂诱导喉癌细胞凋亡比例，从而降低其对细胞的毒性并增强其抗肿瘤疗效，为中药天花粉蛋白联合顺铂应用于喉癌临床治疗奠定了实验基础。
Objective To investigate the combined effect of thrichosanthin （TCS） and cisplatin on Hep-2 and AMC-HN-8 cell proliferation. Methods Hep-2 and AMC-HN-8 cells were treated with low （3 I^g/ml） or high （10 i^g/ml） concentration of cisplatin（10 Ixg/ml）, TCS（5 ;~g/ml） , or the combination TCS （5 ~g/ml） and cisplatin （3 I^g/ml）, the cells no exposure to the drugs as control. Cell proliferation was detected by CCK-8. Toxicity of drugs to cells was evaluated by LDH assay. Flow cytometry was used to detect apoptosis. Western blot was performed to detect the expressions of JNK, phosphor-JNK, p38, phosphor-p38, NF-KB, inhibitor of KB （I-KB）, and phospho-I-KB. Results Compared with TCS （5 tzg/ml） or cisplatin （3 [zg/ml） alone, the combination of them had more significant inhibitory effect on the proliferation of Hep-2 and AMC-HN-8 cells （P 〈 O. O1 ）, but with no significant increase in cytotoxieity （ P 〉 0.05 ）. Western blot showed the expression of p-JNK/SAPK significantly increased in the cells treated with 5 Ixg/ml TCS for 48 hours, while the expression of NF-KB and phospho-I-KB increased in the cells treated with 3 tzg/ml cisplatin. However in the cells treated with 5 txg/ml TCS combined with 3 txg/ml cisplatin, the expression of p-JNK stayed at a high level and the expressions of NF-KB and phospho-l-KB decreased dramatically compared to the cells treated with 3 μg/ml cisplatin alone. Conclusions TCS could enhance cisplatin-induced apoptosis in Hep-2 and AMC-HN-8, at least in part, by inhibiting the NF-KB signaling pathway and activating JNK/SAPK signaling pathway and thus strengthening the antitumor effects of cisplatin, which highlights the possibility of combined application of TCS and cisplatin in the treatment of laryngeal carcinoma.
出处
《中华耳鼻咽喉头颈外科杂志》
CAS
CSCD
北大核心
2013年第4期322-328,共7页
Chinese Journal of Otorhinolaryngology Head and Neck Surgery
基金
国家自然科学基金（30801283）
上海市科技人才计划项目（09QAl401000）
上海市卫生系统优秀人才培养计划（XYQ2011055）
