紫草素对鼻咽癌CNE1细胞免疫逃逸的影响

Effect of Shikonin on the Immune Escape of CNE1 Cells from Nasopharyngeal Carcinoma

【目的】探讨紫草素对鼻咽癌细胞的治疗作用及机制。【方法】(1)体外研究:将人鼻咽癌CNE1细胞分为阴性对照组,紫草素1.2、2.4、3.6μmol/L组及紫杉醇2.5μmol/L组。采用四甲基偶氮唑盐(MMT)法检测CNE1细胞活性,Hoechst法检测CNE1细胞凋亡情况,Western Blot法检测CNE1细胞叉头样转录因子3(Foxp3)、维甲酸相关孤核受体γt(RORγt)蛋白表达。(2)体内研究:将48只裸鼠分为正常组,模型组,紫草素低、中、高剂量组及紫杉醇组,每组8只。除正常组外,其余各组裸鼠建立鼻咽癌模型。紫草素低、中、高剂量组分别给予1.0、2.0、3.0 mg/kg紫草素灌胃,紫杉醇组腹腔注射2.0 mg/kg的紫杉醇。测量瘤体质量,计算抑瘤率,提取巨噬细胞采用流式细胞仪检测程序性死亡受体1配体(PD-L1)表达,Western Blot法检测Foxp3、RORγt蛋白表达。【结果】与阴性对照组比较,紫草素1.2、2.4、3.6μmol/L组及紫杉醇2.5μmol/L组的细胞活性均降低,细胞凋亡率增加(均P<0.05),并存在紫草素浓度依赖性,且Foxp3蛋白表达水平升高、RORγt蛋白表达水平降低(均P<0.05)。与正常组比较,模型组裸鼠巨噬细胞中PD-L1、肿瘤组织RORγt蛋白表达水平升高,肿瘤组织Foxp3蛋白表达水平降低(均P<0.05);与模型组比较,紫草素低、中、高剂量组及紫杉醇组肿瘤质量减小,PD-L1、RORγt蛋白表达水平降低,Foxp3蛋白表达水平升高(均P<0.05);紫草素高剂量组各指标与紫杉醇组比较,差异均无统计学意义(P>0.05)。【结论】紫草素可以抑制鼻咽癌细胞增殖,抑制瘤体生长,其机制可能与降低肿瘤微环境巨噬细胞的PD-L1活性,激活Foxp3并抑制RORγt表达,进而减少肿瘤细胞免疫逃逸有关。

Objective To investigate the therapeutic effects and mechanisms of shikonin on nasopharyngeal carcinoma cells.Methods(1)In vitro study:Human nasopharyngeal carcinoma CNE1 cells were divided into the negative control group,shikonin 1.2,2.4,3.6μmol/L groups and taxol(TAX)2.5μmol/L group.CNE1 cell activity was detected by methyl thiazolyl tetrazolium(MTT)assay,CNE1 cell apoptosis was detected by Hoechst assay,and the protein expressions of CNE1 cell forkhead-like transcription factor 3(Foxp3)and retinoid orphan nuclear receptorγt(RORγt)were determined by Western Blot assay.(2)In vivo study:Forty-eight nude mice were divided into the normal group,the model group,the shikonin low-,medium-,and high-dose groups,and TAX group,with 8 mice in each group.Except for the normal group,the nasopharyngeal carcinoma nude mice models in all groups were established.The shikonin low-,medium-,and high-dose groups were gavaged with 1.0,2.0,3.0 mg/kg of shikonin,respectively,and the TAX group was injected intraperitoneally with 2.0mg/kg of TAX.The tumor mass and tumor suppression rate were calculated.The expression of programmed death receptor 1 ligand(PD-L1)was detected by flow cytometry in extracted macrophages,and the protein expressions of Foxp3 and RORγt were detected by Western Blot assay.Results Compared with the negative control group,cell activity was decreased and apoptosis rate was increased in the shikonin 1.2,2.4,3.6μmol/L group and TAX 2.5μmol/L group(all P<0.05)in a shikonin concentration dependence,and Foxp3 protein expression level was increased as well as RORγt protein expression level was decreased(all P<0.05).Compared with the normal group,the protein expression levels of PD-L1 in macrophages of nude mice and tumor tissue RORγt were increased and tumor tissue Foxp3 protein expression level was decreased in the model group(all P<0.05);compared with the model group,tumor mass was reduced,protein expression levels of PD-L1 and RORγt were decreased and Foxp3 protein expression level was increased in the shikonin low-,medium-and high-dose groups and the TAX group(all P<0.05),and the levels of indexes in the shikonin high-dose group and TAX group were not significantly different(P>0.05).Conclusion Shikonin inhibits the proliferation of nasopharyngeal carcinoma cells and suppress tumor growth by a mechanism that may be related to the reduction of PD-L1 activity in tumor microenvironment macrophages,activation of Foxp3 and inhibition of RORγt expression,thereby reducing tumor cell immune escape.