Abstract：
BACKGROUND: Transforming growth factor-beta (TGF-β), an immunosuppressive cytokine, is often elevated in various tumors and inhibits the immune system\'s ability to combat tumor cells. Despite promising results from TGF-β inhibitor therapies, their clinical efficacy remains limited.
OBJECTIVE: This study aimed to enhance the antitumor capabilities of natural killer (NK) cells in the presence of TGF-β by exploring the potential of asiaticoside, a natural compound with established clinical safety.
METHODS: The effects of asiaticoside on NK cells were investigated to determine its potential to counteract TGF-β-induced immunosuppression and elucidate the underlying mechanisms.
METHODS: Natural compounds were screened using a Luminex assay to identify those promoting Interferon-γ (IFN-γ) secretion from NK cells. Asiaticoside-pretreated NK cells\' cytotoxicity was assessed against K562, OVCAR8, and A2780 cells using organoids from ascites-derived ovarian cancer (OC) cells. In vivo efficacy was evaluated with B16 melanoma lung metastasis and subcutaneous tumor models in C57BL/6 mice, using asiaticoside as a 50 mg/kg injection. The compound\'s ability to enhance NK cell-driven anti-neoplastic responses was further assessed in an OC murine model. Effects on TGF-β/SMAD pathways and mitochondrial functions were examined through various microscopy and metabolomic techniques. The involvement of the mTOR/DRP1 axis in asiaticoside-mediated restoration of mitochondrial oxidation in NK cells after TGF-β suppression was determined using the mTOR inhibitor rapamycin and the DRP1 inhibitor Mdivi-1.
RESULTS: Asiaticoside-treated NK cells retained their ability to suppress tumor growth and metastasis despite TGF-β presence. Asiaticoside downregulated TGF-β receptors 1 (TGFBR1) expression, impaired the protein stability of TGFBR1 and TGF-β receptors 2 (TGFBR2), and reduced SMAD2 phosphorylation, preventing SMAD2 translocation from the mitochondria. This preserved mitochondrial respiration and maintained NK cell antitumor activity.
CONCLUSIONS: The study concludes that asiaticoside has significant potential as a strategy for \"priming\" NK cells in cellular immunotherapy. By demonstrating that asiaticoside degrades the TGF-β receptor, leading to reduced phosphorylation of SMAD2 and preventing its mitochondrial translocation, thereby maintaining mitochondrial integrity. Meantime, asiaticoside counteracts TGF-β-induced suppression of mitochondrial oxidative and aerobic respiration through the mTOR/DRP1 pathways. The research uncovers a previously unreported pathway for preserving mitochondrial respiration and NK cell functionality. A detailed mechanistic insight into how asiaticoside functions at the molecular level was explored. Its ability to counteract the immunosuppressive effects of TGF-β makes it a valuable candidate for enhancing the effectiveness of immunotherapies in treating a variety of tumors with elevated TGF-β levels.
OBJECTIVE: This study aimed to enhance the antitumor capabilities of natural killer (NK) cells in the presence of TGF-β by exploring the potential of asiaticoside, a natural compound with established clinical safety.
METHODS: The effects of asiaticoside on NK cells were investigated to determine its potential to counteract TGF-β-induced immunosuppression and elucidate the underlying mechanisms.
METHODS: Natural compounds were screened using a Luminex assay to identify those promoting Interferon-γ (IFN-γ) secretion from NK cells. Asiaticoside-pretreated NK cells\' cytotoxicity was assessed against K562, OVCAR8, and A2780 cells using organoids from ascites-derived ovarian cancer (OC) cells. In vivo efficacy was evaluated with B16 melanoma lung metastasis and subcutaneous tumor models in C57BL/6 mice, using asiaticoside as a 50 mg/kg injection. The compound\'s ability to enhance NK cell-driven anti-neoplastic responses was further assessed in an OC murine model. Effects on TGF-β/SMAD pathways and mitochondrial functions were examined through various microscopy and metabolomic techniques. The involvement of the mTOR/DRP1 axis in asiaticoside-mediated restoration of mitochondrial oxidation in NK cells after TGF-β suppression was determined using the mTOR inhibitor rapamycin and the DRP1 inhibitor Mdivi-1.
RESULTS: Asiaticoside-treated NK cells retained their ability to suppress tumor growth and metastasis despite TGF-β presence. Asiaticoside downregulated TGF-β receptors 1 (TGFBR1) expression, impaired the protein stability of TGFBR1 and TGF-β receptors 2 (TGFBR2), and reduced SMAD2 phosphorylation, preventing SMAD2 translocation from the mitochondria. This preserved mitochondrial respiration and maintained NK cell antitumor activity.
CONCLUSIONS: The study concludes that asiaticoside has significant potential as a strategy for \"priming\" NK cells in cellular immunotherapy. By demonstrating that asiaticoside degrades the TGF-β receptor, leading to reduced phosphorylation of SMAD2 and preventing its mitochondrial translocation, thereby maintaining mitochondrial integrity. Meantime, asiaticoside counteracts TGF-β-induced suppression of mitochondrial oxidative and aerobic respiration through the mTOR/DRP1 pathways. The research uncovers a previously unreported pathway for preserving mitochondrial respiration and NK cell functionality. A detailed mechanistic insight into how asiaticoside functions at the molecular level was explored. Its ability to counteract the immunosuppressive effects of TGF-β makes it a valuable candidate for enhancing the effectiveness of immunotherapies in treating a variety of tumors with elevated TGF-β levels.
摘要：
背景：转化生长因子-β（TGF-β），一种免疫抑制细胞因子，通常在各种肿瘤中升高，并抑制免疫系统对抗肿瘤细胞的能力。尽管TGF-β抑制剂治疗有希望的结果，其临床疗效仍然有限。
目的：本研究旨在通过探索积雪草苷的潜力，增强自然杀伤（NK）细胞在TGF-β存在下的抗肿瘤能力，一种具有既定临床安全性的天然化合物。
方法：研究积雪草苷对NK细胞的影响，以确定其对抗TGF-β诱导的免疫抑制的潜力，并阐明潜在的机制。
方法:使用Luminex测定法筛选天然化合物以鉴定促进从NK细胞分泌干扰素-γ(IFN-γ)的那些。使用来自腹水来源的卵巢癌（OC）细胞的类器官，评估了积雪草苷预处理的NK细胞对K562，OVCAR8和A2780细胞的细胞毒性。用B16黑色素瘤肺转移和皮下肿瘤模型在C57BL/6小鼠中评估体内疗效，使用积雪草苷作为50mg/kg注射剂。在OC鼠模型中进一步评估化合物增强NK细胞驱动的抗肿瘤反应的能力。通过各种显微镜和代谢组学技术检查对TGF-β/SMAD途径和线粒体功能的影响。使用mTOR抑制剂雷帕霉素和DRP1抑制剂Mdivi-1确定了在TGF-β抑制后积雪草苷介导的NK细胞线粒体氧化恢复中mTOR/DRP1轴的参与。
结果：积雪草苷处理的NK细胞保留了抑制肿瘤生长和转移的能力，尽管存在TGF-β。积雪草苷下调TGF-β受体1（TGFBR1）表达，TGFBR1和TGF-β受体2（TGFBR2）的蛋白质稳定性受损，减少SMAD2磷酸化，防止SMAD2从线粒体易位。这保留了线粒体呼吸并保持了NK细胞抗肿瘤活性。
结论：该研究得出结论，积雪草苷在细胞免疫疗法中作为“引发”NK细胞的策略具有重要潜力。通过证明积雪草苷降解TGF-β受体，导致SMAD2磷酸化减少并防止其线粒体易位，从而保持线粒体的完整性。同时,积雪草苷通过mTOR/DRP1途径抵消TGF-β诱导的线粒体氧化和有氧呼吸抑制。该研究揭示了以前未报道的保留线粒体呼吸和NK细胞功能的途径。探索了积雪草苷如何在分子水平上发挥作用的详细机理。其抵消TGF-β的免疫抑制作用的能力使其成为增强免疫疗法在治疗具有升高的TGF-β水平的多种肿瘤中的有效性的有价值的候选者。
目的：本研究旨在通过探索积雪草苷的潜力，增强自然杀伤（NK）细胞在TGF-β存在下的抗肿瘤能力，一种具有既定临床安全性的天然化合物。
方法：研究积雪草苷对NK细胞的影响，以确定其对抗TGF-β诱导的免疫抑制的潜力，并阐明潜在的机制。
方法:使用Luminex测定法筛选天然化合物以鉴定促进从NK细胞分泌干扰素-γ(IFN-γ)的那些。使用来自腹水来源的卵巢癌（OC）细胞的类器官，评估了积雪草苷预处理的NK细胞对K562，OVCAR8和A2780细胞的细胞毒性。用B16黑色素瘤肺转移和皮下肿瘤模型在C57BL/6小鼠中评估体内疗效，使用积雪草苷作为50mg/kg注射剂。在OC鼠模型中进一步评估化合物增强NK细胞驱动的抗肿瘤反应的能力。通过各种显微镜和代谢组学技术检查对TGF-β/SMAD途径和线粒体功能的影响。使用mTOR抑制剂雷帕霉素和DRP1抑制剂Mdivi-1确定了在TGF-β抑制后积雪草苷介导的NK细胞线粒体氧化恢复中mTOR/DRP1轴的参与。
结果：积雪草苷处理的NK细胞保留了抑制肿瘤生长和转移的能力，尽管存在TGF-β。积雪草苷下调TGF-β受体1（TGFBR1）表达，TGFBR1和TGF-β受体2（TGFBR2）的蛋白质稳定性受损，减少SMAD2磷酸化，防止SMAD2从线粒体易位。这保留了线粒体呼吸并保持了NK细胞抗肿瘤活性。
结论：该研究得出结论，积雪草苷在细胞免疫疗法中作为“引发”NK细胞的策略具有重要潜力。通过证明积雪草苷降解TGF-β受体，导致SMAD2磷酸化减少并防止其线粒体易位，从而保持线粒体的完整性。同时,积雪草苷通过mTOR/DRP1途径抵消TGF-β诱导的线粒体氧化和有氧呼吸抑制。该研究揭示了以前未报道的保留线粒体呼吸和NK细胞功能的途径。探索了积雪草苷如何在分子水平上发挥作用的详细机理。其抵消TGF-β的免疫抑制作用的能力使其成为增强免疫疗法在治疗具有升高的TGF-β水平的多种肿瘤中的有效性的有价值的候选者。
