Dhivya Rajakumar, Jaividhya Paramasivam, Riyasdeen Anvarbatcha, Palaniandavar Mallayan, Mathan Ganeshan, Akbarsha Mohammad Abdulkader
Department of Biomedical Science, Bharathidasan University, Tiruchirappalli, Tamil Nadu, 620024, India.
Mahatma Gandhi-Doerenkamp Center for Alternatives to Use of Animals in Life Science Education, Bharathidasan University, Tiruchirappalli, Tamil Nadu, 620024, India.
Biometals. 2015 Oct;28(5):929-43. doi: 10.1007/s10534-015-9877-1. Epub 2015 Sep 3.
In the background that there is concerted effort to discover newer metal-based cancer chemotherapeutic agents that could overcome the limitations in cisplatin and that copper, a biocompatible and redox-active metal, offers potential as alternative to cisplatin, the present study was undertaken to investigate the in vitro anti-proliferative properties of the mononuclear copper(II)complex [Cu(L)(diimine)] + where LH = 2-[(2-dimethylaminoethylimino)methyl]phenol and diimine = dipyrido[3,2-a:2',3'-c]phenazine (dppz) using breast cancer cell lines MCF-7 (ER(+ve) and p53(WT)) and MDA-MB-231(ER(-ve) and p53(mutant)) when cisplatin was used as positive control. The complex affected the viability of both the cell lines in dose-as well as duration-dependent manner as revealed in the MTT assay. The 24 and 48 h IC50 of the complex were several times lesser than those of cisplatin, and within this huge difference the efficacy of the complex was much superior with MCF-7 cell compared to MDA-MB-231 cell. The cell death was preferentially apoptosis, though necrosis also occurred to a certain extent. These inferences were substantiated by AO/EB fluorescent staining, Hoechst staining, assessment of mitochondrial transmembrane potential, comet assay for DNA damage, DCFH assay for reactive oxygen species (ROS) generation and Western blot of apoptosis-related proteins. Thus, the copper(II) dppz complex under investigation is much more efficient than cisplatin in affecting viability of the breast cancer cells. The underlying mechanism appears to be DNA damage-primed (in view of the known intercalation mode of binding of the complex with DNA) and ROS-associated mitochondria-mediated intrinsic apoptosis to a great extent but necrosis also has a role to a certain extent, which may also be a PARP-mediated cell death independent of apoptosis. Within the purview of this conclusion, the results indicate that the ER and/or p53 genotypes have a bearing on the efficacy of the complex as a cytotoxic agent since the response in the ER(-ve) and p53(mutant) MDA-MB-231 cell was not so prominent as in ER(+ve) and p53(WT) MCF-7 cell. Taken together, the complex has been shown to be a potential DNA damaging agent and, in the light of the superiority of the complex over cisplatin, we are further investigating the possibility of targeted nano-delivery of the complex to the tumor cells. When tested on a normal cell, 3T3, Cu(II)dppz was found to affect its viability but at concentrations very high compared to those for the breast cancer cells. Yet, this is a cause of concern and, therefore, we are working out a strategy for targeted delivery of this complex to the cancer cells only.
在人们齐心协力寻找能够克服顺铂局限性的新型金属基癌症化疗药物，且生物相容性和具有氧化还原活性的金属铜有望替代顺铂的背景下，本研究旨在使用乳腺癌细胞系MCF-7（雌激素受体阳性和p53野生型）和MDA-MB-231（雌激素受体阴性和p53突变型），以顺铂作为阳性对照，研究单核铜（II）配合物[Cu(L)(二亚胺)]⁺的体外抗增殖特性，其中LH = 2-[(2-二甲基氨基乙基亚氨基)甲基]苯酚，二亚胺 = 二吡啶并[3,2-a:2',3'-c]吩嗪（dppz）。MTT试验结果表明，该配合物以剂量和时间依赖性方式影响两种细胞系的活力。该配合物24小时和48小时的半数抑制浓度（IC50）比顺铂低几倍，且在这一巨大差异范围内，与MDA-MB-231细胞相比，该配合物对MCF-7细胞的疗效要优越得多。细胞死亡优先为凋亡，尽管也有一定程度的坏死。吖啶橙/溴化乙锭荧光染色、Hoechst染色、线粒体跨膜电位评估、DNA损伤彗星试验、活性氧（ROS）生成的2',7'-二氯荧光素二乙酸酯（DCFH）试验以及凋亡相关蛋白的蛋白质印迹法证实了这些推断。因此，所研究的铜（II）dppz配合物在影响乳腺癌细胞活力方面比顺铂高效得多。潜在机制似乎在很大程度上是由DNA损伤引发的（鉴于该配合物与DNA已知的嵌入结合模式）以及与ROS相关的线粒体介导的内源性凋亡，但坏死在一定程度上也起作用，这也可能是一种聚（ADP-核糖）聚合酶（PARP）介导的与凋亡无关的细胞死亡。在这一结论范围内，结果表明雌激素受体和/或p53基因型对该配合物作为细胞毒性剂的疗效有影响，因为雌激素受体阴性和p53突变型的MDA-MB-231细胞中的反应不如雌激素受体阳性和p53野生型的MCF-7细胞中那么显著。综上所述，该配合物已被证明是一种潜在的DNA损伤剂，鉴于该配合物优于顺铂，我们正在进一步研究将该配合物靶向纳米递送至肿瘤细胞的可能性。当在正常细胞3T3上进行测试时，发现铜（II）dppz会影响其活力，但所需浓度比乳腺癌细胞高得多。然而，这是一个值得关注的问题，因此，我们正在制定一种仅将该配合物靶向递送至癌细胞的策略。
