不同的 BTK 抑制剂可诱导明显不同的细胞凋亡，但对套细胞淋巴瘤的趋化作用和脂质堆积的抑制作用相似。
Distinct BTK inhibitors differentially induce apoptosis but similarly suppress chemotaxis and lipid accumulation in mantle cell lymphoma.
机构信息
Interdisciplinary Institute of Cancer Diagnosis and Treatment, Beijing Advanced Innovation Center for Biomedical Engineering, Beihang University, Beihang University, Beijing, 100083, China.
School of Biological Science and Medical Engineering, Beihang University, Beijing, 100083, China.
出版信息
BMC Cancer. 2021 Jun 26;21(1):732. doi: 10.1186/s12885-021-08475-3.
BACKGROUND
The more selective second-generation BTK inhibitors (BTKi) Acalabrutinib and Zanubrutinib and the first-generation BTKi Ibrutinib are highlighted by their clinical effectiveness in mantle cell lymphoma (MCL), however, similarities and differences of their biological and molecular effects on anti-survival of MCL cells induced by these BTKi with distinct binding selectivity against BTK remain largely unknown.
METHODS
AlamarBlue assays were performed to define cytotoxicity of BTKi against MCL cells, Jeko-1 and Mino. Cleaved PARP and caspase-3 levels were examined by immunoblot analysis to study BTKi-induced apoptotic effects. Biological effects of BTKi on MCL-cell chemotaxis and lipid droplet (LD) accumulation were examined in Jeko-1, Mino and primary MCL cells via Transwell and Stimulated Raman scattering imaging analysis respectively. Enzyme-linked immunoassays were used to determine CCL3 and CCL4 levels in MCL-cell culture supernatants. RNA-seq analyses identified BTKi targets which were validated by quantitative RT-PCR (qRT-PCR) and immunoblot analysis.
RESULTS
Acalabrutinib and Zanubrutinib induced moderate apoptosis in Ibrutinib high-sensitive JeKo-1 cells and Ibrutinib low-sensitive Mino cells, which was accompanied by cleaved PARP and caspase-3. Such effects might be caused by the stronger ability of Ibrutinib to upregulate the expression of pro-apoptotic genes, such as HRK, GADD45A, and ATM, in JeKo-1 cells than in Mino cells, and the expression of such apoptotic genes was slightly changed by Acalabrutinib and Zanubrutinib in both JeKo-1 and Mino cells. Further, Acalabrutinib, Zanubrutinib and Ibrutinib reduced MCL-cell chemotaxis with similar efficiency, due to their similar abilities to downmodulate chemokines, such as CCL3 and CCL4. Also, these three BTKi similarly suppressed MCL-cell LD accumulation via downregulating lipogenic factors, DGAT2, SCD, ENPP2 and ACACA without significant differences.
CONCLUSION
BTKi demonstrated differential capacities to induce MCL-cell apoptosis due to their distinct capabilities to regulate the expression of apoptosis-related genes, and similar biological and molecular inhibitory effects on MCL-cell chemotaxis and LD accumulation.
背景
第二代选择性布鲁顿酪氨酸激酶抑制剂（BTKi）阿卡鲁替尼和泽布替尼以及第一代 BTKi 伊布替尼在套细胞淋巴瘤（MCL）中的临床疗效显著，然而，这些 BTKi 对 MCL 细胞抗生存的生物学和分子效应的异同，以及它们对 BTK 具有不同结合选择性，在很大程度上仍然未知。
方法
通过 AlamarBlue 测定法测定 BTKi 对 MCL 细胞（Jeko-1 和 Mino）的细胞毒性。通过免疫印迹分析检测裂解的 PARP 和 caspase-3 水平，以研究 BTKi 诱导的凋亡作用。通过 Transwell 和受激拉曼散射成像分析分别研究 BTKi 对 Jeko-1、Mino 和原发性 MCL 细胞的趋化作用和脂滴（LD）积累的生物学效应。酶联免疫吸附测定法测定 MCL 细胞培养上清液中 CCL3 和 CCL4 的水平。RNA-seq 分析鉴定 BTKi 靶标，并用定量 RT-PCR（qRT-PCR）和免疫印迹分析进行验证。
结果
阿卡鲁替尼和泽布替尼在伊布替尼高敏感的 Jeko-1 细胞和伊布替尼低敏感的 Mino 细胞中诱导适度凋亡，伴有裂解的 PARP 和 caspase-3。这种作用可能是由于伊布替尼在 Jeko-1 细胞中上调促凋亡基因（如 HRK、GADD45A 和 ATM）的能力强于在 Mino 细胞中，而阿卡鲁替尼和泽布替尼在 Jeko-1 和 Mino 细胞中对这些凋亡基因的表达影响较小。此外，阿卡鲁替尼、泽布替尼和伊布替尼以相似的效率降低 MCL 细胞的趋化性，这是由于它们下调趋化因子（如 CCL3 和 CCL4）的能力相似。同样，这三种 BTKi 通过下调脂生成因子（如 DGAT2、SCD、ENPP2 和 ACACA）相似地抑制 MCL 细胞 LD 积累，没有显著差异。
结论
BTKi 由于其调节凋亡相关基因表达的不同能力，对 MCL 细胞凋亡的诱导能力不同，以及对 MCL 细胞趋化性和 LD 积累的相似的生物学和分子抑制作用，显示出不同的能力。
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