Abstract：
OBJECTIVE: Combing PD-1/PD-L1 immune checkpoint inhibitors with natural products has exhibited better efficacy than monotherapy. Hence, the purpose of this research was to examine the anti-cancer effects of brusatol, a natural quassinoid-terpenoid derived from Brucea javanica, when used in conjunction with an anti-mouse-PD-1 antibody in a murine head and neck squamous cell carcinoma (HNSCC) model and elucidate underlying mechanisms.
METHODS: A murine HNSCC model and an SCC-15 cell xenograft nude mouse model were established to investigate the anti-cancer effects of brusatol and anti-PD-1 antibody. Mechanistic studies were performed using immunohistochemistry. Cell proliferation, migration, colony formation, and invasion were evaluated by MTT, migration, colony formation, and transwell invasion assays. PD-L1 levels in oral squamous cell carcinoma (OSCC) cells were assessed through qRT-PCR, flow cytometry, and western blotting assays. The impact of brusatol on Jurkat T cell function was assessed by an OSCC/Jurkat co-culture assay.
RESULTS: Brusatol improved tumor suppression by anti-PD-1 antibody in HNSCC mouse models. Mechanistic studies revealed brusatol inhibited tumor cell growth and angiogenesis, induced apoptosis, increased T lymphocyte infiltration, and reduced PD-L1 expression in tumors. Furthermore, in vitro assays confirmed brusatol inhibited PD-L1 expression in OSCC cells and suppressed cell migration, colony formation, and invasion. Co-culture assays indicated that brusatol\'s PD-L1 inhibition enhanced Jurkat T cell-mediated OSCC cell death and reversed the inhibitory effect induced by OSCC cells.
CONCLUSIONS: Brusatol improves anti-PD-1 antibody efficacy by targeting PD-L1, suggesting its potential as an adjuvant in anti-PD-1 immunotherapy.
METHODS: A murine HNSCC model and an SCC-15 cell xenograft nude mouse model were established to investigate the anti-cancer effects of brusatol and anti-PD-1 antibody. Mechanistic studies were performed using immunohistochemistry. Cell proliferation, migration, colony formation, and invasion were evaluated by MTT, migration, colony formation, and transwell invasion assays. PD-L1 levels in oral squamous cell carcinoma (OSCC) cells were assessed through qRT-PCR, flow cytometry, and western blotting assays. The impact of brusatol on Jurkat T cell function was assessed by an OSCC/Jurkat co-culture assay.
RESULTS: Brusatol improved tumor suppression by anti-PD-1 antibody in HNSCC mouse models. Mechanistic studies revealed brusatol inhibited tumor cell growth and angiogenesis, induced apoptosis, increased T lymphocyte infiltration, and reduced PD-L1 expression in tumors. Furthermore, in vitro assays confirmed brusatol inhibited PD-L1 expression in OSCC cells and suppressed cell migration, colony formation, and invasion. Co-culture assays indicated that brusatol\'s PD-L1 inhibition enhanced Jurkat T cell-mediated OSCC cell death and reversed the inhibitory effect induced by OSCC cells.
CONCLUSIONS: Brusatol improves anti-PD-1 antibody efficacy by targeting PD-L1, suggesting its potential as an adjuvant in anti-PD-1 immunotherapy.
摘要：
目的：将PD-1/PD-L1免疫检查点抑制剂与天然产物组合比单药治疗具有更好的疗效。因此，这项研究的目的是检查布拉沙醇的抗癌作用，一种来自鸦胆子的天然类木素-萜类化合物，当与小鼠头颈部鳞状细胞癌（HNSCC）模型中的抗小鼠PD-1抗体结合使用时，并阐明了潜在的机制。
方法：建立小鼠HNSCC模型和SCC-15细胞异种移植裸鼠模型，以研究Brusatol和抗PD-1抗体的抗癌作用。使用免疫组织化学进行机制研究。细胞增殖，迁移,菌落形成,通过MTT评估和入侵，迁移,菌落形成,和transwell入侵测定。通过qRT-PCR评估口腔鳞状细胞癌（OSCC）细胞中的PD-L1水平，流式细胞术,和蛋白质印迹分析。通过OSCC/Jurkat共培养测定评估了Brusatol对JurkatT细胞功能的影响。
结果：Brusatol在HNSCC小鼠模型中通过抗PD-1抗体改善肿瘤抑制。机制研究表明，布鲁沙洛尔抑制肿瘤细胞生长和血管生成，诱导细胞凋亡，T淋巴细胞浸润增加，并降低肿瘤中PD-L1的表达。此外，体外试验证实，Brusatol抑制OSCC细胞中PD-L1的表达并抑制细胞迁移，菌落形成,和入侵。共培养实验表明，Brusatol的PD-L1抑制作用增强了JurkatT细胞介导的OSCC细胞死亡，并逆转了OSCC细胞诱导的抑制作用。
结论：Brusatol通过靶向PD-L1提高抗PD-1抗体的疗效，提示其作为抗PD-1免疫治疗的佐剂的潜力。
方法：建立小鼠HNSCC模型和SCC-15细胞异种移植裸鼠模型，以研究Brusatol和抗PD-1抗体的抗癌作用。使用免疫组织化学进行机制研究。细胞增殖，迁移,菌落形成,通过MTT评估和入侵，迁移,菌落形成,和transwell入侵测定。通过qRT-PCR评估口腔鳞状细胞癌（OSCC）细胞中的PD-L1水平，流式细胞术,和蛋白质印迹分析。通过OSCC/Jurkat共培养测定评估了Brusatol对JurkatT细胞功能的影响。
结果：Brusatol在HNSCC小鼠模型中通过抗PD-1抗体改善肿瘤抑制。机制研究表明，布鲁沙洛尔抑制肿瘤细胞生长和血管生成，诱导细胞凋亡，T淋巴细胞浸润增加，并降低肿瘤中PD-L1的表达。此外，体外试验证实，Brusatol抑制OSCC细胞中PD-L1的表达并抑制细胞迁移，菌落形成,和入侵。共培养实验表明，Brusatol的PD-L1抑制作用增强了JurkatT细胞介导的OSCC细胞死亡，并逆转了OSCC细胞诱导的抑制作用。
结论：Brusatol通过靶向PD-L1提高抗PD-1抗体的疗效，提示其作为抗PD-1免疫治疗的佐剂的潜力。
