Shanxi Key Laboratory for Modernization of TCVM, College of Veterinary Medicine, Shanxi Agricultural University, Jinzhong 030600, China.
Laboratory Animal Center, Shanxi Agricultural University, Jinzhong 030600, China.
Int J Mol Sci. 2023 Dec 30;25(1):540. doi: 10.3390/ijms25010540.
The canine mammary tumor model is more suitable for studying human breast cancer, and the safety concentrations of matrine and the biotin-labeled matrine probe were determined in canine primary mammary epithelial cells, and then selected canine mammary tumor cell lines CHMm and CHMp were incubated with matrine, and cell viability was detected by CCK-8. The biotin-labeled matrine probe was used to pull-down the targets of matrine in canine mammary tumor cells, and the targets were screened in combination with activity-based protein profiling (ABPP) and Genecards database, and verified by qPCR and western blot. The results showed that the maximum non-cytotoxic concentrations of matrine and biotin-labeled matrine probe in canine primary mammary epithelial cells were 250 μg/mL and 500 μg/mL, respectively. Matrine and biotin-labeled matrine probe had a proliferation inhibitory effect time-dependently on CHMm and CHMp cells within a safe concentration range, and induced autophagy in cells. Then BTF3 targets were obtained by applying ABPP and Genecards screening. Cellular thermal shift assay (CETSA) findings indicated that matrine could increase the heat stability of BTF3 protein. Pull-down employing biotin-labeled matrine probe with CHMm and CHMp cell lysates revealed that BTF3 protein was detected in the biotin-labeled matrine probe group and that BTF3 protein was significantly decreased by the addition of matrine. The qPCR and western blot findings of CHMm and CHMp cells treated with matrine revealed that matrine decreased the expression of the BTF3 gene and protein with the extension of the action time, and the impact was more substantial at the protein level, respectively.
犬乳腺肿瘤模型更适合研究人类乳腺癌，确定了苦参碱和生物素标记的苦参碱探针在犬原代乳腺上皮细胞中的安全浓度，然后用苦参碱孵育犬乳腺肿瘤细胞系 CHMm 和 CHMp，通过 CCK-8 检测细胞活力。用生物素标记的苦参碱探针拉取犬乳腺肿瘤细胞中的苦参碱靶标，结合活性蛋白质谱（ABPP）和 Genecards 数据库进行筛选，并用 qPCR 和 Western blot 进行验证。结果表明，苦参碱和生物素标记的苦参碱探针在犬原代乳腺上皮细胞中的最大非细胞毒性浓度分别为 250 μg/mL 和 500 μg/mL。苦参碱和生物素标记的苦参碱探针在安全浓度范围内对 CHMm 和 CHMp 细胞具有时间依赖性的增殖抑制作用，并诱导细胞自噬。然后通过 ABPP 和 Genecards 筛选获得 BTF3 靶标。细胞热转移分析（CETSA）结果表明，苦参碱可以增加 BTF3 蛋白的热稳定性。用生物素标记的苦参碱探针与 CHMm 和 CHMp 细胞裂解物进行下拉实验表明，BTF3 蛋白在生物素标记的苦参碱探针组中被检测到，并且苦参碱的加入使 BTF3 蛋白显著减少。用苦参碱处理的 CHMm 和 CHMp 细胞的 qPCR 和 Western blot 结果表明，苦参碱随着作用时间的延长降低了 BTF3 基因和蛋白的表达，在蛋白水平上的影响更为显著。
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