Translational Research Of A Tumor Suppressing Magnetic Field On Nehproblastoma

Background Exposure to electromagnetic field(EMF)generated by man-made devices is common in modern life,and understanding of its impact on human health is significant.To date,the association between EMF and cancer is not yet fully understood.On one hand,the international agency for research on cancer(IARC)has defined EMF in certain frequency range as potential carcinogen based on the results from some epidemiological studies.On the other hand,some studies have shown that EMF has antitumor effects.For example,Optune,a device generating 200 k Hz EMF,has been used in clinical treatment of glioblastoma.EMF with the frequency above 100 k Hz usually generates thermal effects,while low frequency EMF results in various biological effects.Understanding of the biological targets of EMF may elucidate the nature of the relationship between EMF and cancer.Nephroblastoma is the second most common abdominal solid tumor in children,with the incidence of 7-10 cases per 1 million in children under 15.It is an embryonic malignant tumor.Nephroblastoma can be caused by a single or multiple gene mutations.The most well-known is Wilms’ tumor gene-1(WT-1).The majority of cases(98%)of nephroblastoma occur in children under 10 years of age,with an average age of 3.5 years.The overall 5-year survival rate can be as high as 90% with combinational therapies of surgery,radiotherapy and chemotherapy.However,the clinical outcome of nephroblastoma depends on factors such as tumor stratification,molecular typing,etc.The 5-year survival rate was significantly low in high risk cases with intermediate variant,clear cell type,rhabdomyosarcoma type,stage IV,stage V,and chromosome 1 p and 16 q chromosome heterozygosity deletion.In this dissertation,EMF has been investigated as the adjuvant therapy of traditional chemotherapy for highly malignant nephroblastoma,and the potential cellular mechanisms are also studied.Method In this dissertation,a 50 Hz power frequency EMF modulated by the static magnetic field with an average intensity of 5.1 m T,termed as tumor suppressing magnetic field(MF),was used.We investigated the potency and the selectivity of this field on a variety of tumor cell lines,and further studied the tumor suppressing mechanisms using nephroblastoma cell G401 as the model.In the aspect of cell fate determination,Ed U incorporation method was performed to detect cell proliferation,flow cytometry and Western blotting was performed to detect apoptosis.To determine the level of autophagy,autophagy flux,autophagy protein LC3 expression and autolysosome numbers were analyzed.Ferroptosis was analyzed by using ferroptosis inhibitor Ferrostatin-1.In addition,cell cycle was analyzed by flow cytometry.In terms of the mechanism of tumor inhibition,we focused on the role of intracellular reactive oxygen species(ROS)generated by this field,and the subsequent DNA damage and repair pathways.Results 1.The inhibitory effect of the tumor suppressing magnetic field on nephroblastoma The 3-day cell growth curve obtained from cell viability assays showed that the field inhibited most cell lines.However,compared with the corresponding immortalized cells derived from normal tissues,tumor cell lines were more susceptible to field exposure.On the first day after 2 hours of exposure,the inhibition rates of G401,A549,SGC-7901 and PANC-1 were significantly higher than those corresponding normal cell lines.When the accumulated magnetic field exposure reached 4 and 6 hours,i.e.on the second and third day of the experiment,the inhibition effect persisted and reached the peak: 22% inhibition rate in SGC-7901 cells on the second day,29% in G401 cells on the third day,30% in A549 on the third day,and 24% in PANC-1 cells on the third day.In another experiment,G401 cells were exposed to magnetic field or sham exposure for 0.5,1,or 2 hours for 3 consecutive days.The results showed that the inhibition rate increased with the increase of daily exposure time,suggesting dose-dependent inhibitory effect.To study the effect of tumor suppressing magnetic field on chemosensitization,two categories of chemotherapy drugs were used in G401 and A549 cells.The results showed that combinational use of MF exposure significantly sensitized tumor cells to chemotherapy with antimetabolic(5-fluorouracil,5-FU and cisplatin,DDP)and antirproliferation(vincristine,VCR and paclitaxel,PTX)drug.MF exposure increased the sensitivity of G401 cells to 5-FU and VCR,and increased the sensitivity of A549 cells to DDP and PTX.From the first day of treatment,the field had exercised significant sensitization effect on chemotherapeutic drugs,and the increased sensitization effect persisted to the second and third day of exposure.In in vivo G401 nephroblastoma model,compared with the sham exposed group,the combination of MF exposure and DDP reduced the tumor volume significantly.The toxicity and the side effects on key organs of the tumor suppressing magnetic field were studied in vivo.In G401 nephroblastoma tumor model in young nude mice,the growth of the animals exposed to magnetic field or DDP was normal,and there was no significant difference in the body weight between the different experimental groups.Blood analysis indicated no significant liver and kidney damage after field exposure.However,the levels of AST and ALT in the combined treatment group were about 10% higher than those in the DDP only group.Our preliminary study indicated MF exposure as a relatively safe adjuvant antitumor treatment option with low toxicity and side effects. 2.The biological mechanism of the tumor suppressing magnetic field on nephroblastoma Edu incorporation assay showed that the proliferation rate of G401 cells on the second day was 34% in sham exposed group and 21% in MF group.On the third day,the proliferation rate of the sham exposed group was 31%,and that of MF group was 20% with a decrease of 11%.Flow cytometry showed that the apoptosis rate of G401 cells after MF exposure was significantly increased than that of the sham exposed group.In nude mice,Western blotting showed that the expression of cleaved caspase 3 from sectioned tumor tissues in the MF group was higher than that in the sham exposed group.The data indicated that MF exposure induced apoptosis both in vitro and in vivo.As for autophagy flux,when lysosome inhibitor Baf A-1 was applied to G401 cells together with MF exposure,the ratio of LC3-Ⅱ to LC3-I by Western blotting was significantly higher than that of Baf-A1 group,indicating activation of autophagy,which was more pronounced on the second and third day.The lysosomes were detected by a fluorescent Lysotracker probe,and it was found that the number of lysosomes was increased in the MF group compared with the sham exposed group.In addition,the expression and colocalization of LC3 and LAMP1 in G401 cells also increased after MF exposure.We also found that the m TOR signaling pathway was inhibited by MF exposure.m TOR phosphorylation level was found to be decreased at 0.5,1 and 1.5 hours following MF exposure,The phosphorylation of m TOR downstream target,Thr389 and Ser71 of P70 S6 kinase,was also inhibited at 0.5,1,1.5 and 2 hours following exposure.In summary,in G401 cells,tumor suppressing magnetic field promoted autophagy,increased the number of autolysosomes,while inhibiting the m TOR pathway.Cell cycle analysis showed that cells in the G2/M phase accounted for 7-10% in the sham exposed control group,but increased to 29% after 2 hours of MF exposure,and increased to 33% after 4 hours of exposure.In G401 cells with cell cycle synchronized by thymidine,the G2 / M phase of the sham exposed group accounted for 31-36%,but increased to 53% after 2 hours of MF exposure,and increased to 66% after 4 hours of MF exposure.Results above showed that the cells were blocked in G2 / M phase by MF exposure,and the percentage of cells blocked increased with accumulation of exposure time.Furthermore,the phosphorylation of Chk1 and Chk2 and the expression of Cyclin B1 both increased significantly in G401 cells after exposed to MF for 2 and 4 hours,which were in accordance with G2/M phase arrest.As for ferroptosis,significant increase of MDA and decrease of NADPH were found in G401 after MF exposure.When ferroptosis inhibitor Ferrostatin-1 was used in combination with MF exposure,the growth inhibition rate dropped from 28% to 13% on the third day,indicating ferroptosis-1 could partially rescue the growth inhibition induced by MF.Thus the tumor suppressing magnetic field could promote ferroptosis in G401 cells.ROS levels in G401 cells increased in 0.5,1,1.5 hours after MF exposure,and reached the peak in 0.5 hours.In addition,ROS level 24 hours after termination of MF exposure in the previous day was still significantly higher than that of the sham exposure group,indicating that the ROS level of G401 induced by tumor suppressive magnetic field was increased and sustained.Furthermore,the growth inhibition rate in G401 treated with ROC scavenger NAC together with MF was significantly lower than that of the MF group.On the first day,the inhibition rate induced by MF exposure was 17%,and decreased to 9% after adding NAC;on the second day,the inhibition rate induced by MF was 20%,and 3.74% when NAC was added;on the third day,the inhibition rate induced by MF was 31%,and was 9.11% when combined with NAC.The results showed that the free radical scavenger could partially reverse the growth inhibition of G401 cells induced by MF exposure.Comet assay showed that the MF exposure induced DNA fragmentation in G401 cells.The alkaline comet assay showed 40.7% of DNA fragmentation in the MF group on the second day,while the neutral comet assay showed 16.7% of DNA fragmentation;on the third day,the alkaline comet assay showed 56.7% fragmented DNA and the neutral comet assay showed 35.9% fragmented DNA induced by MF,indicating that both single strand and double strand DNA damage were induced in G401 by MF exposure.Furthermore,use of ROS scavenger NAC significantly decreased MF-induced DNA fragmentation on the second day and the third day of exposure.That is to say,MF-induced DNA damage was mediated by ROS.Fluorescent imaging of γH2AX,a DNA double strand damage marker,showed that the average number of γH2AX foci in G401 cells was significantly increased in the MF group on the second day of MF exposure.In G401 cells,the m RNA expression levels of BMI1,LIG4 and RAD9 B genes were significantly increased 2 hours after MF exposure.In dissected G401 tumor from nude mice,the expression of LIG4 and RAD9 B was significantly increased in the MF group compared with the sham exposed group.The expression level of DNA-PKcs protein,another marker of DNA repair activation following double strand breaks,was analyzed by flow cytometry.It was shown that MF exposure increased the expression level of DNA-PKCs in G401 cells.In conclusion,in nephroblastoma G401,MF exposure induced DNA damage both in vitro and in vivo,and subsequently activated DNA damage repair pathways.Conclusions Based on the findings above,the following conclusions are drawn in this dissertation: 1)the tumor suppressing magnetic field can selectively inhibit a variety of tumor cells,with relatively low effect on normal cells;2)the molecular mechanism of this suppressing magnetic field involves multiple targets and pathways,which include inhibition of proliferation,induction of autophagy,apoptosis,ferroptosis and cell cycle arrest,as shown in G401 nephroblastoma;3)in G401 nephroblastoma,this tumor suppressing magnetic field can increase intracellular ROS accumulation,which subsequently causes DNA double and single strand breaks and activates DNA damage response;4)this tumor suppressing magnetic field has sensitization effect on chemotherapy both in vitro and vivo with low systemic toxicity and side effects,which adds to its potential and advantage in translational research.The novelties of this dissertation The novel discoveries in this dissertation are as follows: 1)the tumor suppressing magnetic field investigated can exercise its inhibitory effect through multiple molecular targets and pathways;using G401 nephroblastoma cell,this is the first study showing that magnetic field exposure can induce ferroptosis;2)ROS plays a role in the tumor suppressing effect of this MF in nephroblastoma by mediating DNA damage;3)tumor suppressing magnetic field has sensitization effect on chemotherapy with low toxic side effects,which makes it a relatively safe adjuvant therapeutic strategy in translational research.