Qiu Dan, Yan Xianxin, Xiao Xinqin, Zhang Guijuan, Wang Yanqiu, Cao Jingyu, Ma Ruirui, Hong Shouyi, Ma Min
School of Traditional Chinese Medicine, Jinan University, No. 601, West Huangpu Avenue, Guangzhou, 510632, Guangdong, China.
School of Nursing, Jinan University, No. 601, West Huangpu Avenue, Guangzhou, 510632, Guangdong, China.
Cancer Cell Int. 2021 Nov 27;21(1):632. doi: 10.1186/s12935-021-02345-5.
The precancerous disease of breast cancer is an inevitable stage in the tumorigenesis and development of breast neoplasms. Quercetin (Que) has shown great potential in breast cancer treatment by inhibiting cell proliferation and regulating T cell function. γδ T cells are a class of nontraditional T cells that have long attracted attention due to their potential in immunotherapy. In this study, we revealed the immunomodulatory function of Que through regulation of the JAK/STAT1 signaling pathway, which was followed by the synergistic killing of breast cancer cells.
In the experimental design, we first screened target genes with or without Que treatment, and we intersected the Que target with the disease target by functional enrichment analysis. Second, MCF-10A, MCF-10AT, MCF-7 and MDA-MB-231 breast cancer cell lines were treated with Que for 0 h, 24 h and 48 h. Then, we observed the expression of its subsets by coculturing Que and γδ T cells and coculturing Que and γδ T cells with breast tumor cells to investigate their synergistic killing effect on tumor cells. Finally, Western blotting was used to reveal the changes in proteins related to the JAK/STAT1 signaling pathway after Que treatment in MCF-10AT and MCF-7 cells for 48 h.
The pathway affected by Que treatment was the JAK/STAT1 signaling pathway and was associated with precancerous breast cancer, as shown by network pharmacology analysis. Que induced apoptosis of MCF-10AT, MCF-7 and MDA-MB-231 cells in a time- and concentration-dependent manner (P < 0.05). Most importantly, Que promoted the differentiation of γδ T cells into the Vδ2 T cell subpopulation. The best ratio of effector cells to target cells (E/T) was 10:1, the killing percentages of γδ T cells against MCF-10A, MCF-10AT, MCF-7, and MDA-MB-231 were 61.44 ± 4.70, 55.52 ± 3.10, 53.94 ± 2.74, and 53.28 ± 1.73 (P = 0.114, P = 0.486, and P = 0.343, respectively), and the strongest killing effect on precancerous breast cancer cells and breast cancer cells was found when the Que concentration was 5 μM and the E/T ratio was 10:1 (64.94 ± 3.61, 64.96 ± 5.45, 55.59 ± 5.98, and 59.04 ± 5.67, respectively). In addition, our results showed that Que increased the protein levels of IFNγ-R, p-JAK2 and p-STAT1 while decreasing the protein levels of PD-L1 (P < 0.0001).
In conclusion, Que plays a synergistic role in killing breast cancer cells and promoting apoptosis by regulating the expression of IFNγ-R, p-JAK2, p-STAT1 and PD-L1 in the JAK/STAT1 signaling pathway and promoting the regulation of γδ T cells. Que may be a potential drug for the prevention of precancerous breast cancer and adjuvant treatment of breast cancer.
乳腺癌癌前疾病是乳腺肿瘤发生发展过程中不可避免的阶段。槲皮素（Que）通过抑制细胞增殖和调节T细胞功能，在乳腺癌治疗中显示出巨大潜力。γδT细胞是一类非传统T细胞，因其在免疫治疗中的潜力长期以来备受关注。在本研究中，我们通过调节JAK/STAT1信号通路揭示了Que的免疫调节功能，随后实现了对乳腺癌细胞的协同杀伤。
在实验设计中，我们首先筛选了有无Que处理时的靶基因，并通过功能富集分析将Que靶标与疾病靶标进行交集分析。其次，用Que处理MCF-10A、MCF-10AT、MCF-7和MDA-MB-231乳腺癌细胞系0小时、24小时和48小时。然后，通过将Que与γδT细胞共培养以及将Que与γδT细胞和乳腺肿瘤细胞共培养，观察其亚群的表达，以研究它们对肿瘤细胞的协同杀伤作用。最后，采用蛋白质印迹法揭示Que处理48小时后MCF-10AT和MCF-7细胞中与JAK/STAT1信号通路相关的蛋白质变化。
网络药理学分析表明，Que处理影响的信号通路是JAK/STAT1信号通路，且与乳腺癌癌前病变相关。Que以时间和浓度依赖性方式诱导MCF-10AT、MCF-7和MDA-MB-231细胞凋亡（P<0.05）。最重要的是，Que促进γδT细胞分化为Vδ2 T细胞亚群。效应细胞与靶细胞的最佳比例（E/T）为10:1，γδT细胞对MCF-10A、MCF-10AT、MCF-7和MDA-MB-231的杀伤率分别为61.44±4.70、55.52±3.10、53.94±2.74和53.28±1.73（P分别为0.114、0.486和0.343），当Que浓度为5μM且E/T比例为10:1时，对乳腺癌癌前细胞和乳腺癌细胞的杀伤作用最强（分别为64.94±3.61、64.96±5.45、55.59±5.98和59.04±5.67）。此外，我们的结果表明，Que增加了IFNγ-R、p-JAK2和p-STAT1的蛋白水平，同时降低了PD-L1的蛋白水平（P<0.0001）。
总之，Que通过调节JAK/STAT1信号通路中IFNγ-R、p-JAK2、p-STAT1和PD-L1的表达以及促进γδT细胞的调节，在杀伤乳腺癌细胞和促进细胞凋亡方面发挥协同作用。Que可能是预防乳腺癌癌前病变和辅助治疗乳腺癌的潜在药物。
