Background

Natural killer (NK) cells play important roles in immune responses and have been wildly used in immunotherapy. Nevertheless, some limitations remain. It is urgent to explore novel and safe strategies to enhance NK cell activity.

Purpose

The aim of this study was to investigate the immuno-stimulatory effects and to reveal the molecular mechanism of LJ101019C, a derivative of a natural small-molecule compounds cajanine, on NK cells.

Methods

Cell proliferation was examined by CCK8 assay, then we used the cytotoxicity detection kit to detect the cytotoxicity of NK cells. The change of cell cycle, intracellular reactive oxygen species (ROS) level and mitochondrial mass were evaluated by FACS and Operetta high-content image analysis, respectively. Furthermore, the IFN-γ secretion of NK cells were measured by ELISA. The Kv1.3 protein expression and function were detected by western blot and patch-clamp technique, respectively. The role of Kv1.3 in AKT/mTOR pathway activation was determined by western blot.

Results

The results showed that LJ101019C at relatively low concentrations (0.05–0.1 µM) significantly increased the proliferation of NK cells. And 1 µM LJ101019C could elevate the proportion of NK cells in the S-phase of the cell cycle (*p < 0.1). Furthermore, the cytotoxic effects of NK cells targeting MIA PaCa-2 cells were significantly enhanced by 0.1 and 1 µM LJ101019C, and were associated with the enhanced secretion of IFN-γ by NK cells (*p < 0.1; **p < 0.05). 0.1 and 1 µM LJ101019C increased intracellular levels of ROS (**p < 0.05), and 0.1 µM LJ101019C elevated mitochondrial mass (*p < 0.1). Electrophysiological recordings indicated that LJ101019C led to a remarkably increase the Kv1.3 current density. Moreover, western blot results indicated that LJ101019C elevated Kv1.3 protein expression and activated AKT/mTOR signaling via increasing the expression of Kv1.3 in NK cells.

Conclusion

LJ101019C increases the proliferation and the cytotoxicity of NK cells at relatively low concentrations. The mechanism is the activation of AKT/mTOR signaling pathway driven by up-regulation of Kv1.3 in NK cells. These suggest LJ101019C is a promising candidate for improving the efficacy of NK cell-based immunotherapies.

中文翻译：

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木瓜碱衍生物LJ101019C通过Kv1.3通道驱动的AKT / mTOR途径激活来调节增殖并增强NK细胞的活性

背景

天然杀伤（NK）细胞在免疫反应中起重要作用，并已广泛用于免疫治疗中。但是，仍然存在一些局限性。迫切需要探索新颖，安全的策略来增强NK细胞的活性。

目的

这项研究的目的是研究免疫刺激作用，并揭示天然小分子化合物cajanine的衍生物LJ101019C在NK细胞上的分子机制。

方法

通过CCK8检测细胞增殖，然后使用细胞毒性检测试剂盒检测NK细胞的细胞毒性。通过FACS和Operetta高内涵图像分析分别评估细胞周期，细胞内活性氧（ROS）水平和线粒体质量的变化。此外，通过ELISA测量NK细胞的IFN-γ分泌。分别通过蛋白质印迹和膜片钳技术检测Kv1.3蛋白的表达和功能。Western blot检测Kv1.3在AKT / mTOR途径激活中的作用。

结果

结果表明，相对较低的浓度（0.05–0.1 µM）的LJ101019C显着提高了NK细胞的增殖。1 µM LJ101019C可以提高NK细胞在细胞周期S期的比例（* p  <0.1）。此外，靶向MIA PaCa-2细胞的NK细胞的细胞毒性作用被0.1和1 µM LJ101019C显着增强，并且与NK细胞的IFN-γ分泌增强相关（* p <  0.1; ** p <  0.05） 。0.1和1 µM LJ101019C增加了细胞内ROS的水平（** p <  0.05），而0.1 µM LJ101019C增加了线粒体的质量（* p < 0.1）。电生理记录表明，LJ101019C导致Kv1.3电流密度显着增加。此外，蛋白质印迹结果表明，LJ101019C通过增加NK细胞中Kv1.3的表达来提高Kv1.3的蛋白表达并激活AKT / mTOR信号传导。

结论

LJ101019C在相对较低的浓度下会增加NK细胞的增殖和细胞毒性。其机制是由NK细胞中Kv1.3的上调驱动AKT / mTOR信号通路的激活。这些表明LJ101019C是提高基于NK细胞的免疫疗法功效的有前途的候选者。