| ObjectivesLung cancer,also known as primary bronchial lung cancer,is a most common malignant tumor worldwide.The treatments include surgery,chemotherapy,radiotherapy,interventional therapy,immunotherapy,and targeted chemotherapy.However,the morbidity and mortality of primary lung cancer remain highest among all cancers in China.The antitumor effect of magnetic field(MF)with specific frequency and intensity has been widely reported,and is being applied in clinical practice,but the underlying mechanism remains unraveled.The aim of this study is to use cultured lung cancer cells to investigate the antitumor effect of a magnetic field previously found to inhibit several other types of cancers,and to investigate the relevant cellular mechanisms.MF applied in this study is a 50 Hz extremely low frequency electromagnetic field modulated by static MF with time-averaged intensity of 5.1 mT.Previous studies have found that this MF exposure protocol could inhibit the growth of adult breast cancer,colon cancer,as well as childhood malignancies including neuroblastoma and nephroblastoma.The anti-proliferative mechanism of this MF was explored mainly focusing on the aspect of free radical generation and the subsequent DNA damage pathways.Changes on the cellular tubulin network and autophagy levels were also examined.MethodsThe cell lines used in this study include human lung adenocarcinoma cell A549,human non-small cell lung cancer cell H460,H1975,H1650,mouse lung adenocarcinoma cell LLC,immortalized human lung bronchial epithelial cell Beas-2B,wild-type mouse embryonic fibroblast(MEF),NBS1 deficient MEF(NBS1-/-MEF),and MDC1 deficient MEF(MDC1-/-MEF).The equipment generating electromagnetic field was developed in our laboratory.The MF exposure condition was as follows:the cells cultured in the incubator were exposed to 50 Hz extremely low frequency electromagnetic fields modulated static magnetic field,with time-average intensity of 5.1 mT,daily exposure time was 2 hours,and cells were exposed for 3 consecutive days;otherwise the cells were continuously exposed under the MF described above for 0.5h or longer.1.Inhibitory effect of MF on cultured cells:cultured cells were exposed to MF based on standard protocol,and co-cultured with desired drugs;to determine cell viability,both cell counter and CCK8 kit were used;cell proliferation and cell inhibition curves were drawn based on cell viability data;cell proliferation was analyzed by BrdU incorporation assay;cell apoptosis was determined by flow cytometry;2.Generation of free radicals upon MF exposure:DCFH-DA probes were used to detect the level of reactive oxygen species(ROS);a free radical scavenger,N-acetylcysteine(NAC),was used to further investigate the participatory role of ROS in the antitumor mechanism of MF;3.DNA damage induced by MF exposure and the dependence on free radicals:DNA alkaline and neutral comet assay were performed to detect DNA single and double strand breaks,and NAC was used to analyze whether DNA damage was dependent on free radicals;q-PCR was used to analyze the expression of the DNA damage repair genes,and NBS1-/-MEF、MDC1-/-MEF was used to verify whether the anti-proliferative effect of MF was related to DNA damage;4.Morphological changes of microtubule upon MF exposure:immunofluorescence of β-tubulin was used to observe the morphological changes of the microtubule network;5.Regulation of cellular autophagy by MF:expression level of autophagy related proteins was determined by Western blotting;distribution of of autophagy related proteins in the cytosol was observed by immunofluorescence;fluorescent lysotracker probe was used to reveal the morphology of lysosomes.ResultsAfter being exposed to MF described above,the viability of cultured lung cancer cell lines A549,LLC,H460 were significantly decreased from day 2,and the inhibition rate was 20-30%after 3 days’ exposure.There was no significant inhibition in human lung bronchial epithelial cell Beas-2B and human non-small cell lung cancer cell H1975,H1650 with the same MF exposure protocol.MF worked synergistically with chemotherapeutic drugs cisplatin and paclitaxel to inhibit lung cancer cell A549.It was further found that this MF inhibited cell proliferation,but did not induce apoptosis.Using A549 cell as the experimental model,the level of ROS was found to be significantly increased after MF exposure.Free radical scavenger NAC could protect A549 from MF exposure and reduced the inhibitory effect.DNA single and double strand breaks could be induced by MF exposure.MEF cells deficient of DNA damage repair system were more sensitive to MF exposure compared with wild-type MEF cells.NAC could significantly reduce DNA damage,indicating that MF-induced DNA damage was related to free radicals.Tubulin immunofluorescence showed that after MF exposure,the cellular microtubule structure disappeared,and the intercellular junctions decreased,suggestting that MF might hamper the morphology and the function of the spindle,leading to mitosis arrest.By detection of autophagy biomarkers LC3II protein by Western blotting and fluorescent imaging,cellular autophagy level was found to be elevated after MF exposure.In conclusion,the characteristics MF exposure protocol applied in this study inhibited the proliferation of lung cancer cell by generating ROS production,and causing subsequent DNA damage.At the same time,abnormalties in the microtubule system and autophagy malregulation could both contribute to the biological effect of this MF.Conclusion1.The MF protocol used in this study could specifically inhibit proliferation of a number of lung cancer cell lines,and could further increase the inhibitory rate when being combined with traditional chemotherapeutics;2.This MF could generate ROS production,leading to subsequent DNA damage,this mechanism could be the major contributing factor of the anti-proliferative effect of the MF applied;3.Both microtubules and autophagy could be the biological targets of the MF applied,leading to decrease of cell viability after MF exposure. |
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