Department of Stem Cell Transplantation and Cellular Therapy, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
Exp Hematol. 2012 Oct;40(10):800-10. doi: 10.1016/j.exphem.2012.06.001. Epub 2012 Jun 9.
Hematopoietic stem cell transplantation is used for treatment of lymphoma. In an attempt to design an efficacious and safe prehematopoietic stem cell transplantation conditioning regimen, we investigated the cytotoxicity of the combination of busulfan (B), melphalan (M), and gemcitabine (G) in lymphoma cell lines in the absence or presence of drugs that induce epigenetic changes. Cells were exposed to drugs individually or in combination and analyzed by the MTT proliferation assay, flow cytometry, and Western blotting. We used IC(10) drug concentrations (57 μM B, 1 μM M and 0.02 μM G), which individually did not have major effects on cell proliferation. Their combination resulted in 50% inhibition of proliferation. Reduction to almost half concentration (20 μM B, 0.7 μM M and 0.01 μM G) did not have significant effects, but addition of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (0.6 μM) to this combination resulted in a marked (65%) growth inhibition. The cytotoxicity of these combinations correlates with the activation of the ataxia telangiectasia mutated-CHK2 pathway, phosphorylation of KRAB-associated protein-1, epigenetic changes such as methylation and acetylation of histone 3, and activation of apoptosis. The relevance of epigenetic changes is further shown by the induction of DNA methyltransferases in tumor cells with low constitutive levels of DNMT3A and DNMT3B. The addition of 5-aza-2'-deoxycytidine to (BMG+suberoylanilide hydroxamic acid) further enhances cell killing. Overall, BMG combinations are synergistically cytotoxic to lymphoma cells. Epigenetic changes induced by suberoylanilide hydroxamic acid and 5-aza-2'-deoxycytidine further enhance the cytotoxicity. This study provides a rationale for an ongoing clinical trial in our institution using (BMG+suberoylanilide hydroxamic acid) as pre-hematopoietic stem cell transplantation conditioning for lymphoma.
造血干细胞移植用于治疗淋巴瘤。为了设计一种有效且安全的造血干细胞移植预处理方案，我们研究了在没有或存在诱导表观遗传改变的药物的情况下，联合应用白消安（B）、美法仑（M）和吉西他滨（G）对淋巴瘤细胞系的细胞毒性。细胞单独或联合暴露于药物，并通过 MTT 增殖测定、流式细胞术和 Western blot 进行分析。我们使用了约 IC(10)药物浓度（57 μM B、1 μM M 和 0.02 μM G），这些浓度单独使用对细胞增殖没有明显影响。它们的联合使用导致增殖抑制率达到 50%。将浓度降低到近一半（20 μM B、0.7 μM M 和 0.01 μM G）并没有显著影响，但将组蛋白去乙酰化酶抑制剂 suberoylanilide hydroxamic acid（0.6 μM）加入到该联合用药中，会导致明显的（约 65%）生长抑制。这些组合的细胞毒性与共济失调毛细血管扩张症突变-CHK2 途径的激活、KRAB 相关蛋白-1 的磷酸化、表观遗传变化（如组蛋白 3 的甲基化和乙酰化）以及凋亡的激活相关。表观遗传变化的相关性还通过诱导肿瘤细胞中低组成型 DNMT3A 和 DNMT3B 水平的 DNA 甲基转移酶进一步证明。在（BMG+suberoylanilide hydroxamic acid）中加入 5-aza-2'-脱氧胞苷进一步增强细胞杀伤作用。总体而言，BMG 联合用药对淋巴瘤细胞具有协同细胞毒性。suberoylanilide hydroxamic acid 和 5-aza-2'-脱氧胞苷诱导的表观遗传变化进一步增强了细胞毒性。这项研究为我们机构正在进行的一项临床试验提供了依据，该试验使用（BMG+suberoylanilide hydroxamic acid）作为淋巴瘤造血干细胞移植预处理。
