Quan Xingguo, Kwak Beom Seok, Lee Ji-Young, Park Jin Hee, Lee Anbok, Kim Tae Hyun, Park SaeGwang
Department of Microbiology and Immunology, College of Medicine, Inje University, Busan 47392, Republic of Korea.
Department of Surgery, Ilsan Hospital, College of Medicine, Dongguk University, Ilsan 10326, Republic of Korea.
Evid Based Complement Alternat Med. 2020 Sep 3;2020:9053274. doi: 10.1155/2020/9053274. eCollection 2020.
has been widely used as a traditional medicine in East Asia. Its effects against breast cancer have been reported previously. However, whether -induced breast cancer cell death is immunogenic remains unelucidated. This study aimed to determine whether ethanolic extracts of (CM-EE) could induce immunogenic cell death (ICD) in breast cancer immunotherapy to improve the efficacy of immune checkpoint inhibitors. Human and mouse breast cancer cells were treated with various concentrations of CM-EE for 72 h, and cytotoxicity was measured using the sulforhodamine B assay. Flow cytometry was used to assess cell death with annexin V/7-AAD staining and measure the surface exposure of damage-associated molecular pattern (DAMP) molecules including calreticulin, HSP70, and HSP90. Western blot for cleaved poly (ADP-ribose) polymerase (PARP) was used to confirm apoptotic cell death. The immunogenicity of CM-EE-induced dead cells was evaluated using the CFSE dilution assay. CM-EE reduced the viability of human (MCF7, MDA-MB-231, HS578T, and SKBR3) and mouse (4T1-neu-HA, TUBO-HA, and TUBO-P2J-HA) breast cancer cells. The IC was 25-50 g/ml in human breast cancer cells and 10-50 g/ml in mouse breast cancer cells at 72 h. CM-EE-treated breast cancer cells were positively stained by annexin V, cleaved PARP, and cleaved caspase 3/7 which were increased upon CM-EE treatment. Surface exposure of DAMP molecules was increased in dose- and time-dependent manners. The CFSE dilution assay revealed that dendritic cells fed with CM-EE-treated breast cancer cells successfully stimulated tumor-specific T cell proliferation without inhibiting DC function and T cell proliferation. The expression of PD-L1 mRNA and protein level was increased in dose-dependent manners. In addition, CM-EE also potentiated the cytotoxic activity of tumor-specific T cells. CM-EE can induce immunogenic and apoptotic cell death in breast cancer cells, and it is a good candidate for cancer immunotherapy and may improve the efficacy of immune checkpoint inhibitors.
在东亚地区，它作为一种传统药物已被广泛使用。此前已有关于其抗乳腺癌作用的报道。然而，[药物名称]诱导的乳腺癌细胞死亡是否具有免疫原性仍未阐明。本研究旨在确定[药物名称]乙醇提取物（CM-EE）是否能在乳腺癌免疫治疗中诱导免疫原性细胞死亡（ICD），以提高免疫检查点抑制剂的疗效。用不同浓度的CM-EE处理人源和小鼠乳腺癌细胞72小时，采用磺酰罗丹明B法检测细胞毒性。通过膜联蛋白V/7-氨基放线菌素D染色利用流式细胞术评估细胞死亡情况，并检测包括钙网蛋白、热休克蛋白70（HSP70）和热休克蛋白90（HSP90）在内的损伤相关分子模式（DAMP）分子的表面暴露情况。使用裂解的聚（ADP-核糖）聚合酶（PARP）的蛋白质免疫印迹法来确认凋亡细胞死亡。使用羧基荧光素二乙酸琥珀酰亚胺酯（CFSE）稀释试验评估CM-EE诱导的死亡细胞的免疫原性。CM-EE降低了人源（MCF7、MDA-MB-231、HS578T和SKBR3）和小鼠（4T1-neu-HA、TUBO-HA和TUBO-P2J-HA）乳腺癌细胞的活力。在72小时时，人乳腺癌细胞中的半数抑制浓度（IC）为25 - 50μg/ml，小鼠乳腺癌细胞中的IC为10 - 50μg/ml。经CM-EE处理的乳腺癌细胞被膜联蛋白V、裂解的PARP和裂解的半胱天冬酶3/7阳性染色，且在CM-EE处理后增加。DAMP分子的表面暴露以剂量和时间依赖性方式增加。CFSE稀释试验表明，用经CM-EE处理的乳腺癌细胞喂养的树突状细胞成功刺激了肿瘤特异性T细胞增殖，而不抑制树突状细胞功能和T细胞增殖。程序性死亡受体配体1（PD-L1）mRNA和蛋白水平以剂量依赖性方式增加。此外，CM-EE还增强了肿瘤特异性T细胞的细胞毒性活性。CM-EE可诱导乳腺癌细胞发生免疫原性和凋亡性细胞死亡，它是癌症免疫治疗的良好候选药物，可能会提高免疫检查点抑制剂的疗效。
