Department of Oncology, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China.
State Key Laboratory of Bioactive Substances and Function of Natural Medicine, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China.
Phytomedicine. 2020 Jan;66:153113. doi: 10.1016/j.phymed.2019.153113. Epub 2019 Nov 4.
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.
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.
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.
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.
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.
自然杀伤 (NK) 细胞在免疫反应中发挥着重要作用，并已广泛应用于免疫疗法。然而，仍存在一些局限性。因此，迫切需要探索新的、安全的策略来增强 NK 细胞的活性。
本研究旨在探讨 LJ101019C 的免疫刺激作用及其分子机制，LJ101019C 是一种天然小分子化合物 cajanine 的衍生物。
通过 CCK8 检测试剂盒检测细胞增殖，通过细胞毒性检测试剂盒检测 NK 细胞的细胞毒性。通过流式细胞术和 Operaetta 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 法检测 NK 细胞 IFN-γ 的分泌。通过 Western blot 和膜片钳技术分别检测 Kv1.3 蛋白的表达和功能。通过 Western blot 确定 Kv1.3 在 AKT/mTOR 通路激活中的作用。
结果表明，LJ101019C 在较低浓度（0.05-0.1µM）下可显著增加 NK 细胞的增殖。并且 1µM LJ101019C 可增加 NK 细胞在细胞周期 S 期的比例（*p<0.1）。此外，0.1 和 1µM LJ101019C 可显著增强 NK 细胞针对 MIA PaCa-2 细胞的细胞毒性作用，并且与 NK 细胞 IFN-γ 的分泌增强相关（*p<0.1；**p<0.05）。0.1 和 1µM LJ101019C 增加了细胞内 ROS 水平（**p<0.05），并且 0.1µM LJ101019C 增加了线粒体质量（*p<0.1）。电生理记录表明，LJ101019C 可显著增加 Kv1.3 电流密度。此外，Western blot 结果表明，LJ101019C 通过增加 NK 细胞中 Kv1.3 的表达，可提高 Kv1.3 蛋白表达并激活 AKT/mTOR 信号通路。
LJ101019C 以较低浓度增加 NK 细胞的增殖和细胞毒性。其机制是通过上调 NK 细胞中的 Kv1.3 激活 AKT/mTOR 信号通路。这些结果表明，LJ101019C 是一种有前途的候选药物，可用于改善基于 NK 细胞的免疫疗法的疗效。
