LF-MF inhibits iron metabolism and suppresses lung cancer through activation of P53-miR-34a-E2F1/E2F3 pathway

www.nature.com/scientificreports OPEN Received: 16 December 2016 Accepted: 16 March 2017 Published: xx xx xxxx LF-MF inhibits iron metabolism and suppresses lung cancer through activation of P53-miR-34a-E2F1/ E2F3 pathway Jing Ren1, Liang Ding1, Qianyun Xu1, Guoping Shi1, Xiaojing Li1, Xiujun Li Dongya Zhang1, Yaping Wang3, Tingting Wang1,2 & Yayi Hou1,2 1 , Jianjian Ji1, Our previous studies showed that low frequency magnetic fields (LF-MF) suppressed tumor growth and influenced the function of immune system. Nevertheless the mechanisms behind the effect of LF-MF still remain to be elucidated. In this study, Tumor- bearing mice subcutaneously inoculated with Lewis lung cancer cells were exposed to a LF-MF (0.4T, 7.5 Hz) for 35 days and Survival rate, tumor growth and the tumor markers were measured. Results showed that tumor growth was obviously inhibited with a prolonged survival of tumor- bearing mice by LF-MF exposure. In vitro experiments, LF-MF was found to induce cell growth arrest, cell senescence and inhibit iron metabolism of lung cancer cells. Moreover, LF-MF stabilized p53 protein via inhibiting cell iron metabolism and the stabilized p53 protein enhanced miR-34a transcription. Furthermore, increased expression of miR-34a induced cell proliferation inhibition, cell cycle arrest and cell senescence of lung cancer cells by targeting E2F1/E2F3. We also detected the relevant indicator in tumor tissue such as the iron content, the level of miR-34a and related protein, corresponding results were obtained. Taken together, these observations imply that LF-MF suppressed lung cancer via inhibiting cell iron metabolism, stabilizing p53 protein and activation P53miR-34a-E2F1/E2F3 pathway. Lung cancer is one of the most common causes of cancer-related morbidity and mortality, representing 13% of newly diagnosed cancers worldwide1, 2. Although radiotherapy and chemotherapy provide better therapeutic effects over the last decades, the toxicity and side effects are hard to tolerate for patients. The development of novel strategies for lung cancer is still critical3, 4. Biological effect of magnetic fields (MF) on tumor development has been widely investigated 5, 6. Epidemiological studies suggest that increased childhood leukemia risk is associated with residential magnetic fields7. While, most animal studies results that combined MFs with known carcinogenic agents have produce equivocal results and have not provide evidence of the enhancement of carcinogenesis by MF exposure8, 9. In a toxicity pilot human study, patients with heavily pre-treated advanced cancer treated with different schedules of time exposure to LF-MF and no toxicity and adverse side effects were observed10. Of note, LF-MF, with property of the non-invasive, non-ionizing and non-thermal effects on cells and tissues, has been used to study the influence of various diseases, including cancer, pain, and spasticity reduction5, 11, 12. LF-MF inhibited cell growth and induced cell apoptosis and cell cycle arrest of prostate cancer mediated by ROS in vitro13. Several in vivo studies proved the anti-tumor effects of LF-MF with decreased tumor volume and longer survival time14, 15. Meanwhile, a 15-mT and 50-Hz LF-MF was introduced as a tumor necrosis agent16. A 5.5 mT and 50-Hz LF-MF was showed to have synergistic activity with chemotherapy (cisplatin) against lung cancer in vivo17. Interestingly, LF-MF induced germ cell apoptosis while had no effect on prenatal development18. In our previous studies, we examined the inhibitory effect of LF-MF with different parameters on gastric carcinoma cells and chose 7.5 Hz as the suitable frequency of LF-MF in our magnetic field exposure system19. We also found that the LF-MF (0.4T, 1 The State Key Laboratory of Pharmaceutical Biotechnology, Division of Immunology, Medical School, Nanjing University, Nanjing, 210093, China. 2Jiangsu Key Laboratory of Molecular Medicine, Nanjing, 210093, China. 3 Department of Medical Genetics, Nanjing University School of Medicine, Nanjing, 210093, China. Correspondence and requests for materials should be addressed to T.W. (email: ) or Y.H. (email: yayihou@nju. edu.cn) Scientific Reports | 7: 749 | DOI:10.1038/s41598-017-00913-2 1 www.nature.com/scientificreports/ Figure 1. Low frequency magnetic fields inhibit tumor growth in a Lewis lung cancer murine model. C57BL/6 mice (n = 12 each group) subcutaneously inoculated with lewis lung cancer cells (5 × 105) were exposed to LF-MF (0.4T, 7.5 Hz) or Sham MF for 35 days (2 h per day). (A) Representative images of LLC tumors after treatment of MF or Sham MF. (B) Growth curve of LLC tumors was calculated during treatment of MF or Sham MF. (C) Representative images of hematoxylin and eosin staining (10×) of tumors. (D) Survival curve of mice was calculated during treatment of MF or Sham MF. (E,F) Representative images of immunohistochemical (IHC) staining for Ki-67. Scale bars, 100 µm. (F) Positive cells of Ki-67 were counted using Image Pro Plus software 6.0. Data represent one of two independent experiments. Data represent Mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001. 7.5 Hz) inhibited the growth of gastric cancer, hepatocellular carcinoma and melanoma cancer cells and improved immune function in tumor-bearing mice19–22. However, the detailed anti-tumor mechanisms of LF-MF still need to be clarified. MicroRNAs are a group of short single-stranded non-coding RNAs that exert biological functions by regulating transcription and/or translation of protein-coding genes23. miR-34a was reported to be down-regulated in several cancer cells including lung cancer24–27. The ectopic expression of miR-34a inhibited cell growth and induced apoptosis26, 27 and therapeutic delivery of miR-34a could inhibit lung tumor growth25. Iron (Fe) is an essential element for all living organisms. It is involved in several fundamental biological processed28. Accumulation of iron in tissues increases the risk of cancer and TfR is frequently expressed multiple carcinoma cell lines29. The deficiency iron results in cell proliferation reduction and G1/S arrest of tumor cell. Depriving essential nutrient iron of cells by chelators has been used as an approach for cancer treatment30. Interestingly, previous study showed that LF-MF significantly changed iron concentration in liver and kidney31. However, to date it is not reported whether the interaction between iron and LF-MF may have an effect on cancer. In this study, we found that LF-MF inhibited tumor growth in lewis lung cancer cells (LLC) mouse model. LF-MF also induced cell growth arrest and cell senescence in lung cancer cells. Specially, LF-MF enhanced the transcription of miR-34a and decreased the expression of E2F1/E2F3, which affect cell proliferation and cell senescence. We also confirmed that LF-MF suppresses the iron metabolism of lung cancer cells to stabilize p53 protein, which in turn enhance the transcription of miR-34a. Results LF-MF inhibit (...truncated)