Objective: To investigate the anti-tumor effect of the Histone Deacetylase 6 (HDAC6) inhibitor ACY-738 and its underlying mechanisms in Diffuse Large B-cell Lymphoma (DLBCL) . Methods: The expression of HDAC6 in various tumors and DLBCL was analyzed using bioinformatics. DLBCL cells were treated with different concentrations of ACY-738. Cell viability, DNA synthesis, and clone formation were assessed by CCK-8 assay, EdU assay, and soft agar assay, respectively. Intracellular reactive oxygen species (ROS) levels were detected by fluorescence microscopy. Morphological changes in cells were observed using transmission electron microscopy (TEM). Mitochondrial ROS levels and apoptosis were measured by flow cytometry. The expression levels of apoptosis-related and autophagy-related proteins were detected by Western blotting. Results: HDAC6 was highly expressed in DLBCL (P<0.05). ACY-738 inhibited the proliferation, DNA synthesis, and colony formation of DLBCL cells in a dose-dependent manner (P<0.05). Treatment with ACY-738 increased intracellular and mitochondrial ROS levels in DLBCL cells in a dose-dependent manner (P<0.05). TEM revealed that after ACY-738 treatment, mitochondria in cells were swollen and ruptured, mitochondrial cristae were reduced or absent, autolysosomes appeared, and features characteristic of apoptosis were observed. Western blotting showed that after ACY-738 treatment, the expression of the anti-apoptotic protein BCL-2 was downregulated, while the expression of Cleaved-PARP, Cleaved caspase-3, and BAX was upregulated (P<0.05). The expression of autophagy-related proteins Atg7, Atg3, LC3B, and P62 was downregulated, and the expression of acetylated P53 protein was upregulated (P<0.05) . Conclusion: The HDAC6 inhibitor ACY-738 induces mitochondria-dependent apoptosis and autophagy in DLBCL cells by acetylating P53, thereby inhibiting DLBCL cell proliferation.
目的： 初步探讨组蛋白去乙酰化酶6（HDAC6）抑制剂ACY-738在弥漫大B细胞淋巴瘤（DLBCL）中的抗肿瘤作用及其机制。 方法： 生信分析HDAC6在不同肿瘤及DLBCL中的表达。不同浓度ACY-738处理DLBCL细胞后，CCK-8检测细胞活力；EdU检测细胞DNA合成能力；软琼脂实验检测细胞克隆形成；荧光显微镜检测细胞内活性氧（ROS）的水平；透射电镜观察细胞形态的变化；流式细胞术检测线粒体ROS和细胞凋亡水平；蛋白质印迹法（Western blotting）检测细胞凋亡相关蛋白及自噬相关蛋白的表达水平。 结果： HDAC6在DLBCL中高表达（P<0.05）。ACY-738抑制DLBCL细胞增殖、DNA合成及克隆形成，且呈剂量依赖性（P<0.05）。ACY-738处理后DLBCL细胞内ROS水平及线粒体ROS水平增加，且呈剂量依赖性（P<0.05）。电镜观察细胞形态，发现ACY-738处理后，细胞线粒体肿胀破裂，线粒体嵴减少或消失，出现自噬溶酶体，并观察到细胞凋亡形态；Western blotting结果显示ACY-738处理后，凋亡相关蛋白BCL-2表达下调，Cleaved-PARP、Cleaved caspase-3、BAX表达上调（P<0.05），自噬相关蛋白Atg7、Atg3、LC3B、P62表达下调，乙酰化P53蛋白表达上调（P<0.05）。 结论： HDAC6抑制剂ACY-738通过乙酰化P53诱导线粒体依赖性细胞凋亡和自噬，从而抑制DLBCL细胞生长。.
Keywords: Apoptosis; Autophagy; HDAC6; Lymphoma, large B-cell, diffuse.