稳恒强磁场通过影响细胞“铁代谢”促进骨肉瘤细胞“铁死亡”的作用及机制

Osteosarcoma is an orthopedic malignant tumor with highly lethal rate. Moreover, because osteosarcoma is not particularly sensitive to radiotherapy and chemotherapy, surgery is the only option for definitive tumor removal. Many studies showed that malignant tumor cells have a common characteristic of high intracellular iron content resulted from the “iron metabolism” disorders. Recently, “Ferroptosis” of cancer cells is identified as a novel form of programmed cell death with the mechanism of iron-involved imbalance of redox homeostasis, and the increase of ROS in the cells. Magnetic field and iron have a natural connection. We have found that High static magnetic fields (HiSMF) affect some genes expression of “iron metabolism” and cell biological characteristics of human osteosarcoma cells lines. Also, HiSMF can increase iron uptake of human osteosarcoma cells lines and promote cells “Ferroptosis”. Based on the rencent progress in this field at home and abroad, and also combined with the disicoveries of our present research work, we put forward a hypothesis that HiSMF increases the intracellular iron content by promoting the iron uptake of osteosarcoma cells, and the high level intracellular iron are involved in the production of ROS in osteosarcoma cells, then induce “Ferroptosis”. We intend to use HiSMF and different osteosarcoma cell lines to investigate: 1) the process of iron uptake, process, storage and transport of osteosarcoma cells under HiSMF; 2) the effects of HiSMF on the imbalance of redox homeostasis caused by high levels intracellular iron and the production progress of ROS; 3) the biological processes of “Ferroptosis” caused by ROS under HiSMF; 4) the positive/negative regulation of a variety of clinical drugs and tool reagents on the above biological processes.

骨肉瘤是致死性极高的恶性肿瘤,目前除手术治疗外,其它治疗方法疗效甚微。恶性肿瘤细胞具有“铁代谢”异常特性，细胞内铁含量升高。细胞“铁坏死”(Ferroptosis)是新近提出的一种细胞内铁参与的细胞氧化还原稳态失衡导致ROS升高,引起的细胞程序性死亡。磁场对铁有着直接的作用,申请人研究发现：稳恒强磁场影响骨肉瘤细胞“铁代谢”相关基因表达和细胞生物学特性；并对诱导性骨肉瘤细胞“铁死亡”具有促进作用。结合国内外相关研究进展，提出研究假设：稳恒强磁场通过促进骨肉瘤细胞的“铁代谢”，细胞内铁含量升高，高含量铁参与骨肉瘤细胞内的ROS产生，从而引起细胞“铁死亡”。采用稳恒强磁场和多种骨肉瘤细胞研究：1) 细胞对“铁”的摄取、加工、储存和转运过程；2) 细胞内高含量铁参与氧化还原稳态失衡和ROS产生过程；3) ROS引起细胞“铁死亡”的细胞生物学过程；4) 多种药物对上述过程的正/负向调控作用。

骨肉瘤是具有极高的致死性的恶性骨肿瘤，目前除手术治疗外，其它治疗方法疗效甚微。已有研究表明：恶性肿瘤细胞一般具有铁代谢异常的特性；稳恒磁场对多种肿瘤具有一定的抑制作用，并且影响细胞内的铁水平。因此，本项目提出了“稳恒强磁场可通过调节细胞铁代谢从而发挥抗骨肉瘤效应的研究假设”。我们建立了2T-12T稳恒强磁场下的动物饲养系统，并利用该装置研究了2T-12T 稳恒强磁场长期曝露对小鼠安全性的影响，结果表明稳恒强磁场下连续曝露对小鼠体重、血常规及主要组织器官无明显影响，但可以影响组织器官内铁、镁、锌、钙、铜元素的含量及分布。基于稳恒强磁场的生物安全性，我们研究了12T稳恒强磁场对骨肉瘤细胞生物学效应及其对骨肉瘤细胞铁代谢、氧化还原水平的影响。结果表明12T稳恒强磁场可以通过调控骨肉瘤细胞的铁代谢，从而导致细胞内游离铁含量升高引起过量活性氧 (ROS) 的累积，进而导致细胞周期阻滞并抑制骨肉瘤细胞的增殖和活力。而且外源性铁制剂可促进12T 稳恒强磁场的抗骨肉瘤作用；铁螯合剂可抑制其抗骨肉瘤作用。研究发现1T-2T稳恒强磁场本身可抑制骨肉瘤细胞的增殖，但是对成骨细胞的增殖及活力无显著影响。进一步研究发现，与12T 稳恒强磁场不同，1T-2T稳恒强磁场通过降低细胞内铁含量和ROS 水平发挥抗骨肉瘤作用，而且可以增强铁螯合剂去铁胺的抗肿瘤作用。1T-2T稳恒强磁场下肿瘤局部曝露，不但可以介导铁制剂Feraheme及枸橼酸铁铵（FAC）在骨肉瘤小鼠肿瘤组织中的浓聚，而且磁场与Feraheme联合或与FAC及二甲双胍联合均具有明显的抗骨肉瘤作用。1T-2T 稳恒强磁场局部曝露，虽然可以增强高剂量抗坏血酸的抗骨肉瘤作用，但并未增强高剂量抗坏血酸联合Feraheme的抗骨肉瘤作用。虽然16T稳恒强磁场在部分骨肉瘤细胞系中表现出抗骨肉瘤作用，但其对双氢青蒿素联合铁制剂的抗骨肉瘤作用无明显影响。本研究结果为深入探究稳恒强磁场对肿瘤的作用机制提供了新的实验条件和研究方法，同时也为稳恒强磁场作为骨肉瘤治疗的物理辅助手段奠定了应用基础。