Aconitine Regulates The EMT Microenvironment To Reduce The Lung Cancer Malignant

Lung cancer is one of the most common malignant tumors with high morbidity and mortality,which is a serious threat to human health.The methods of clinical treatment for lung cancer include radiotherapy,chemotherapy,interventional therapy,gene therapy,and so on.However,the prognosis of patients with lung cancer is still very poor.The 5 year survival rate of patients with advanced lung cancer is only about 15%.Compared with the treatment after tumor occurrence,tumor prevention is undoubtedly the best way to reduce tumor mortality.Previously,the treatment for tumor mainly focused on the tumor cells.In recent years,more and more studies have found that tumor microenvironment is equally important for tumor development.Tumor microenvironment provides material and energy for the tumor occurrence and development.Epithelial mesenchymal transition is an important feature in the tumorigenic process,which is also regulated by tumor microenvironment.The pharmacological effects of aconitine are making heart strong,analgesic,anti-inflammatory,anti-tumor and regulating immune.In clinical,aconitine is widely used in joint pain,cold and dampness and sciatica,and its analgesic effect is strong and not easily tolerated.In recent years,the reports on aconitine in the cancer treatment has also been increasing.It is generally believed that aconitine can promote the tumor cell apoptosis and enhance the effect of radiotherapy and chemotherapy,but there are few reports on aconitine in the tumor prevention and microenvironment regulation.The purpose of this study is to observe the contribution of the epithelial mesenchymal transition to aconitine reducing lung cancer malignant,with a view to provide new ideas for lung cancer prevention and treatment.In this study,we first optimized the lung cancer model induced by urethane,the effect of 3 times and 10 times of urethane injection on the lung cancer incidence was compared and the sensitivity of the strains to urethane was evaluated in Kunming mice,BALB/C mice and C57BL/6 mice by comparing the lung cancer incidence,the stability and the average tumor number.To determine the lung cancer occurrence in mice,immunohistochemistry was used to detect the expression of TTF-1 in the mouse lung tissue and the CEA content in plasma was detected by enzyme-linked immunosorbent assay(ELISA).Next,the effect of aconitine on the lung cancer bearing mice was observed.The body weight,autonomous activities,the lung index and the tumor nodules were compared in mice.The expression of epithelial mesenchymal transition-related proteins and the stem cell marker proteins were in lung tissue detected by immunofluorescence.In order to observe the effect of aconitine on the lung cancer cells,MTT method was used to select the aconitine concentration which inhibits LLC cells no more than 10%,which had been used to treat LLC cells for two weeks.PCNA immunofluorescence assay was used to detect cell proliferation,transwell was used to detect cell invasion,cell scratches was used to detect migration,the tumor sphere formation and soft agar colonies were used to test self-renewal ability and flow cytometry was used to detect tumor stem cell markers Oct-4,NANOG and PCNA.The expression of epithelial mesenchymal transition related protein and EGF,FGF,HGF,OSM were detected by Western blot.The antibodies against EGF,FGF,HGF and OSM were added to the culture medium to observe the effect of aconitine on LLC cells.Finally,we changed the level of cytokines in culture medium,and added EGF,FGF,HGF,OSM to simulate the aconitine-related microenvironment and to observe whether the artificial microenvironment had similar effect to aconitine.The results showed that intraperitoneal injection of 600mg/Kg urethane once a week for 10 times could result in the100% lung cancer incidence and low mortality in mice.Kunming mice were the most sensitive strain.In the lung cancer process,the expression of TTF-1 in lung tissue and the CEA content in plasma were increased.The results from aconitine-treated mice with lung cancer showed that aconitine could increase the body weight and autonomy activity in lung cancer bearing mice,reduce lung index and lung nodules,increase the expression of E-Cadherin and Cytokeratin18,decrease the expression of N-Cadherin and Vimentin,and reduce the expression of Oct-4,NANOG,PCNA.In vitro,the aconitine at dose which has no effect on LLC cell proliferation could decrease the invasion and migration,reduce the number of soft agar colonies and tumor sphere formation,inhibit the expression of Oct-4,NANOG,PCNA,increase the expression of E-cadherin and Cytokeratin 18,suppress the expression of N-cadherin and Vimentin and increased the expression of EGF,FGF,HGF,OSM in LLC cells.The addition of any cytokine antibody against EGF,FGF,HGF,OSM to medium could increase the cell proliferation,cell invasion,cell expression of epithelial mesenchymal transition-related proteins and stem cell-related proteins,and reduce the effect of aconitine-decreased LLC cell malignant.Finally,we found that the addition of EGF,FGF,HGF,OSM to the culture medium could alter the LLC cell morphology,decrease cell proliferation,invasion,migration and the number of soft agar clones and tumor sphere formation,the expression of E-Cadherin and Cytokeratin 18 were increased,the expression of N-cadherin and Vimentin,and the expression of Oct-4,NANOG,PCNA were reduced,indicating a similar role of the artificial microenvironment to aconitine.In summary,intraperitoneal injection of 600mg/Kg urethane once a week for 10 times is the best method for mice lung cancer model.Aconitine can reduce the lung cancer malignant and prevent the lung cancer development by regulating cytokine levels to change EMT microenvironment.