Zheng Zhong, Zhao Yan, Dong Li-Hua, Wang Li, Cheng Shu, Zhao Wei-Li
Department of Hematology, Shanghai Institute of Hematology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, State Key Laboratory of Medical Genomics, Shanghai 200025, China.
Department of Hematology, Shanghai Institute of Hematology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, State Key Laboratory of Medical Genomics, Shanghai 200025, China. E-mail:
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2013 Dec;21(6):1441-7. doi: 10.7534/j.issn.1009-2137.2013.06.014.
The aim of this study was to illustrate the mechanism of inhibiting the cell growth in diffuse large B-cell lymphoma by histone deacetylase inhibitor valproic acid (VPA) combined with mTOR inhibitor temsirolimus (TEM). MTT assay and Wright's stain were used to assess cell growth inhibition and to detect the cell morphological changes respectively. The cell apoptosis, cell cycle and cell autophagy were determined by flow cytometry. Ultrastructure changes were confirmed by electron microscopy. Protein changes were detected by Western blot. The results showed that both VPA and TEM alone inhibited cell proliferation and the effect was more obvious in the combination group. VPA combined with TEM induced cell arrest in G0/G1 phase and upregulated the expression of autophagy-related protein LC3, without cell apoptosis. Moreover, typical autophagosomes were observed, further confirming the presence of autophagy. Western blot showed the changes of proteins involved in autophagy signaling pathway. VPA decreased HDAC1 and HDAC3 expression and increased histone acetylation, suggesting that VPA also affected lymphoma cell proliferation through epigenetic modification. It is concluded that the combined treatment of VPA and TEM induces cell cycle arrest and cell autophagy, which provides a new clue for their clinical application in diffuse large B-cell lymphoma.
本研究旨在阐明组蛋白去乙酰化酶抑制剂丙戊酸（VPA）联合mTOR抑制剂替西罗莫司（TEM）抑制弥漫性大B细胞淋巴瘤细胞生长的机制。采用MTT法和瑞氏染色分别评估细胞生长抑制情况和检测细胞形态变化。通过流式细胞术测定细胞凋亡、细胞周期和细胞自噬。通过电子显微镜确认超微结构变化。通过蛋白质印迹法检测蛋白质变化。结果显示，单独使用VPA和TEM均能抑制细胞增殖，联合组效果更明显。VPA联合TEM诱导细胞停滞于G0/G1期，并上调自噬相关蛋白LC3的表达，且无细胞凋亡。此外，观察到典型的自噬体，进一步证实了自噬的存在。蛋白质印迹法显示了自噬信号通路中相关蛋白质的变化。VPA降低了HDAC1和HDAC3的表达并增加了组蛋白乙酰化，表明VPA还通过表观遗传修饰影响淋巴瘤细胞增殖。结论是，VPA和TEM联合治疗诱导细胞周期停滞和细胞自噬，为其在弥漫性大B细胞淋巴瘤中的临床应用提供了新线索。
