CAJ | 학술논문

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(3 μg/ml)、高浓度顺铂组(10 μg/ml)、TCS组(5μg/ml)、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(cell counting kit-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；Western blot检测C-Jun氨基端激酶(JNK),磷酸化JNK、核因子κB (nuclear factor κB,NF-κB)、p38、磷酸化p38、κB抑制蛋白(inhibitor of κB,I-κB)及磷酸化I-κB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)；Westem blot显示顺铂增强NF-κB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-κB,TCS和顺铂联合作用后磷酸化JNK保持高水平抑制NF-κB活性.结论 TCS通过激活JNK信号通路,同时抑制NF-κB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.
목적 연구저제량순박화저농도천화분단백(trichosanthin,TCS)연합작용억제후암세포(Hep-2세포주화AMC-HN-8세포주)증식적작용급기궤제.방법 설립불동적실험조,포괄저농도순박조(3 μg/ml)、고농도순박조(10 μg/ml)、TCS조(5μg/ml)、TCS화순박연합작용조(5μg/mlTCS화3μg/ml순박)화음성대조조；련속5d사용세포증식검측법(cell counting kit-8,CCK-8)화유산탈경매법검측불동실험조대후암세포적억제작용화세포독성；류식세포의화Hochest33258염색관찰세포조망；Western blot검측C-Jun안기단격매(JNK),린산화JNK、핵인자κB (nuclear factor κB,NF-κB)、p38、린산화p38、κB억제단백(inhibitor of κB,I-κB)급린산화I-κB적변화.결과 여TCS화저제량순박단독작용상비,TCS화순박연합작용가명현억제후암세포적생장(P치균＜0.01),조망세포수량증가；연합작용조적억제효과여고농도순박조차이무통계학의의,세포독성동TCS화순박단독작용상비차이무통계학의의(P＞0.05),이독성저우고농도순박조(P＜0.01)；Westem blot현시순박증강NF-κB전록인자활성,억제JNK신호통로,TCS조주요격활JNK신호통로이억제NF-κB,TCS화순박연합작용후린산화JNK보지고수평억제NF-κB활성.결론 TCS통과격활JNK신호통로,동시억제NF-κB전록인자활성,증강순박유도후암세포조망비례,종이강저기대세포적독성병증강기항종류료효,위중약천화분단백연합순박응용우후암림상치료전정료실험기출.
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 μg/ml) or high (10 μg/ml) concentration of cisplatin(10 μg/ml),TCS(5 μg/ml),or the combination TCS (5 μg/ml) and cisplatin (3 μg/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-κB,inhibitor of κB (I-κB),and phospho-I-κB.Results Compared with TCS (5 μg/ml) or cisplatin (3 μg/ml) alone,the combination of them had more significant inhibitory effect on the proliferation of Hep-2 and AMC-HN-8 cells (P ＜0.01),but with no significant increase in cytotoxicity (P ＞ 0.05).Western blot showed the expression of p-JNK/SAPK significantly increased in the cells treated with 5 μg/ml TCS for 48 hours,while the expression of NF-κB and phospho-I-κB increased in the cells treated with 3 μg/ml cisplatin.However in the cells treated with 5 μg/ml TCS combined with 3 μg/ml cisplatin,the expression of p-JNK stayed at a high level and the expressions of NF-κB and phospho-I-κB 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-κB 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.