Anti-hepatocarcinoma activity of TT-1, an analog of melittin, combined with interferon-α via promoting the interaction of NKG2D and MICA

Abstract

Hepatocarcinoma is one of the malignant cancers with significant morbidity and mortality. Immunotherapy has emerged in clinical treatment, owing to the limitation and severe side effects of chemotherapy. In the immune system, natural killer (NK) cells are important effectors required to eliminate malignant tumor cells without the limitation of major histocompatibility complex (MHC) molecule issues. Hence, treatment which could stimulate NK cells is of great interest. Here, we investigated the efficacy of the combined therapy of TT-1 (a mutant of melittin) and interferon-α (IFN-α) on NK cells and human liver cancer HepG-2/Huh7 cells in vitro and in vivo, as well as the mechanism involved. The combination therapy significantly inhibited the growth of HepG-2/Huh7 cells in vivo, but this effect was impaired after depleting NK cells. TT-1 not only up-regulated MHC class I-related chain molecules A (MICA) expression, but also prevented the secretion of soluble MICA (sMICA). Both the mRNA and protein of a disintegrin and metallopeptidase 10 (ADAM 10) in HepG-2/Huh7 cells were decreased after TT-1 treatment. The combined therapy of TT-1 and IFN-α could suppress the growth of HepG-2/Huh7 xenografted tumor effectively via promoting the interaction of NK group 2, member D (NKG2D) and MICA, indicating that TT-1+IFN-α would be a potential approach in treating liver cancer.

摘要

目的

评估干扰素α(IFN-α)和TT-1(一种蜂毒肽的 类似物)联合用药的抗肿瘤效果,并初步研究联 合用药的抗肿瘤及免疫调节机制。

创新点

为了增强蜂毒肽的抗肿瘤效果,本课题组在其基 础上进行改造,合成了一种新的化合物TT-1。该 研究第一次将蜂毒肽类似物和免疫细胞因子 IFN-α 联合使用,并通过实验证实联合用药可以 通过激活免疫调节来增强TT-1 的抗肿瘤效果。

方法

首先通过MTT 实验验证TT-1 对HepG-2/Huh7 细 胞的增殖抑制作用。接着建立HepG-2/Huh7 小鼠 移植瘤模型,考察TT-1+IFN-α 的体内抗肿瘤效 果;使用anti-asialo GM-1 抗体消除自然杀伤 (NK)细胞,验证NK 细胞在联合用药中的关键 作用。使用流式细胞术和酶联免疫吸附法 (ELISA)验证TT-1 对HepG-2/Huh7 细胞MHC I 链相关分子A(MICA)表达的影响,并用实时 聚合酶联反应(RT-PCR)和蛋白质印迹(Western blot)对其机制进行探究;通过细胞毒性实验考察TT-1+IFN-α 是否可以增强NK细胞对HepG-2/ Huh7 细胞的特异性杀伤作用。最后使用免疫组 化的方法考察TT-1+IFN-α 联合用药对肿瘤组织 中MICA 和NKG2D 的表达量的影响。

结论

MTT 实验表明TT-1 可以在体外有效地抑制 HepG-2/Huh7 细胞的增殖。小鼠移植瘤模型实验 结果显示TT-1+IFN-α 联合用药比TT-1 单独给药 更能有效地抑制HepG-2/Huh7 移植瘤的生长,但 是在消除NK 细胞之后该效应明显减弱,说明 TT-1+IFN-α 的抗肿瘤效应是通过NK细胞特异性 介导的。TT-1 不仅可以上调肿瘤细胞表面MICA 的表达量,而且可以减少可溶性MICA 的分泌; 进一步研究表明,TT-1 通过抑制去整合素金属蛋 白酶10(ADAM 10)的表达来阻止MICA 从肿 瘤细胞表面脱落。细胞毒性实验表明,TT-1+IFN-α 可以显著增强NK细胞对HepG-2/Huh7 细胞的杀 伤作用。免疫组化实验结果显示,TT-1+IFN-α 联 合用药可以明显增加肿瘤组织中肿瘤细胞表面 MICA 和NK 细胞NKG2D 的表达量。综上所述, TT-1+IFN-α 联合用药可以通过增强MICA 和 NKG2D 的相互作用达到显著的抗肿瘤效果。