两个双硫仑衍生物的合成及其体外抗肿瘤活性研究

目的：双硫仑（DSF）衍生物的合成、表征及其体外抗肿瘤活性及机制的初步研究。方法：采用两步法路线合成了2个双硫仑衍生物（DSF-1和DSF-2），应用核磁共振氢谱（¹H NMR）、核磁共振碳谱（¹³C NMR）及质谱（MS）确证其结构。采用CCK-8实验比较DSF及其衍生物在含与不含FBS的两种情况下对MCF-7和A549细胞的增殖抑制作用，检测NAC对DSF衍生物抗肿瘤作用的影响；流式细胞术分析DSF及2个衍生物对细胞凋亡的影响；采用Western blot方法检测它们对MCF-7和A549细胞中相关凋亡蛋白表达的影响。结果：波谱解析结果表明2个衍生物的结构与预期完全相符。CCK-8实验结果表明有FBS存在时，2个衍生物对两种肿瘤细胞的抑制作用强于DSF，IC₅₀的差异均有统计学意义（P < 0.05）；DSF衍生物对两种肿瘤细胞的增殖抑制作用在加入NAC后明显减弱（与不含NAC时比较，P < 0.05）。流式细胞术结果显示，与DSF相比，DSF-1和DSF-2作用后的MCF-7和A549细胞凋亡比例均增加（P < 0.05或P < 0.01）。Western blot结果显示2个DSF衍生物可使MCF-7和A549细胞中BAX表达水平增加，PARP表达水平降低。结论：本研究合成的2个DSF衍生物具有体外抗肿瘤作用，在FBS存在的情况下对两种肿瘤细胞的抑制作用优于DSF。DSF衍生物体外抗肿瘤作用可能与影响凋亡相关蛋白的表达及增加细胞内ROS的产生相关。

Synthesis and antitumor activity in vitro of the two derivatives of disulfiram

OBJECTIVE: Synthesis and characterization of disulfiram (DSF) derivatives,and preliminary study on their antitumor activities in vitro. METHODS: Two derivatives were synthesized in two steps,and their structures were confirmed using ¹H NMR,¹³C NMR and LC-MS. CCK-8 cytotoxicity assay was used to compare the inhibitory effects of DSF and its derivatives on proliferation of MCF-7 and A549 cells with or without FBS. Effects of NAC on anti-tumor activities of these compounds were detected in the same way. Whether DSF and its derivatives could induce apoptosis were analyzed using flow cytometry. Expressions of related apoptotic proteins in MCF-7 and A549 cells were detected using Western blot. RESULTS: Results from the spectral analyses show that the structures of the derivatives were consistent with the expectation. The CCK-8 results show that the inhibitory effects of the two derivatives on these two kinds of tumor cells were stronger than that of DSF,and there were significant differences on the value of IC₅₀. There was an obvious decline in the proliferation inhibitory effect of the derivatives in the absence of FBS. In addition,the inhibitory effects on the proliferation of MCF-7 and A549 cells was significantly weakened after the addition of NAC (P < 0.05). Flow cytometry analyses show that the apoptosis rates of MCF-7 and A549 cells which were treated with the two derivatives increased compared with DSF (P < 0.05 or P < 0.01). The results from Western blot analyses show that the two derivatives increased the expression of BAX and decreased the expression of PARP in the MCF-7 and A549 cells. CONCLUSION: The two derivatives showed anti-tumor activities. In addition,the inhibitory effects on the two kinds of tumor cells in the presence of FBS were better than that of DSF. The mechanisms may be related to the expression of apoptosis-related proteins and the increase of intracellular ROS production.