Gowda A S Prakasha, Suo Zucai, Spratt Thomas E
Department of Biochemistry and Molecular Biology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine , Hershey, Pennsylvania 17033, United States.
Department of Chemistry and Biochemistry, The Ohio State University , Columbus, Ohio 43210, United States.
Chem Res Toxicol. 2017 Feb 20;30(2):715-725. doi: 10.1021/acs.chemrestox.6b00451. Epub 2017 Jan 19.
A major concept to sensitize cancer cells to DNA damaging agents is by inhibiting proteins in the DNA repair pathways. X-family DNA polymerases play critical roles in both base excision repair (BER) and nonhomologous end joining (NHEJ). In this study, we examined the effectiveness of honokiol to inhibit human DNA polymerase β (pol β), which is involved in BER, and DNA polymerase λ (pol λ), which is involved in NHEJ. Kinetic analysis with purified polymerases showed that honokiol inhibited DNA polymerase activity. The inhibition mode for the polymerases was a mixed-function noncompetitive inhibition with respect to the substrate, dCTP. The X-family polymerases, pol β and pol λ, were slightly more sensitive to inhibition by honokiol based on the K value of 4.0 μM for pol β, and 8.3 μM for pol λ, while the K values for pol η and Kf were 20 and 26 μM, respectively. Next we extended our studies to determine the effect of honokiol on the cytotoxicity of bleomycin and temozolomide in human cancer cell lines A549, MCF7, PANC-1, UACC903, and normal blood lymphocytes (GM12878). Bleomycin causes both single strand DNA damage that is repaired by BER and double strand breaks that are repaired by NHEJ, while temozolomide causes methylation damage repaired by BER and O-alkylguanine-DNA alkyltransferase. The greatest effects were found with the honokiol and bleomycin combination in MCF7, PANC-1, and UACC903 cells, in which the EC values were decreased 10-fold. The temozolomide and honokiol combination was less effective; the EC values decreased three-fold due to the combination. It is hypothesized that the greater effect of honokiol on bleomycin is due to inhibition of the repair of the single strand and double strand damage. The synergistic activity shown by the combination of bleomycin and honokiol suggests that they can be used as combination therapy for treatment of cancer, which will decrease the therapeutic dosage and side effects of bleomycin.
使癌细胞对DNA损伤剂敏感的一个主要概念是抑制DNA修复途径中的蛋白质。X家族DNA聚合酶在碱基切除修复（BER）和非同源末端连接（NHEJ）中都起着关键作用。在本研究中，我们检测了厚朴酚抑制参与BER的人类DNA聚合酶β（pol β）和参与NHEJ的DNA聚合酶λ（pol λ）的有效性。对纯化聚合酶的动力学分析表明厚朴酚抑制DNA聚合酶活性。聚合酶的抑制模式对于底物dCTP而言是一种混合功能非竞争性抑制。基于pol β的K值为4.0 μM，pol λ的K值为8.3 μM，X家族聚合酶pol β和pol λ对厚朴酚抑制的敏感性略高，而pol η和Kf的K值分别为20 μM和26 μM。接下来，我们扩展研究以确定厚朴酚对博来霉素和替莫唑胺在人癌细胞系A549、MCF7、PANC - 1、UACC903以及正常血液淋巴细胞（GM12878）中的细胞毒性的影响。博来霉素会导致通过BER修复的单链DNA损伤和通过NHEJ修复的双链断裂，而替莫唑胺会导致通过BER和O - 烷基鸟嘌呤 - DNA烷基转移酶修复的甲基化损伤。在MCF7、PANC - 1和UACC903细胞中，厚朴酚与博来霉素联合使用时效果最为显著，其中半数效应浓度（EC）值降低了10倍。替莫唑胺与厚朴酚联合使用效果较差；联合使用导致EC值降低了3倍。据推测，厚朴酚对博来霉素的更大作用是由于抑制了单链和双链损伤的修复。博来霉素与厚朴酚联合显示出的协同活性表明它们可作为联合疗法用于癌症治疗，这将降低博来霉素的治疗剂量和副作用。
