The Underlying Mechanisms Of Inhibition On A549 Cell By Shikonin And The Effect On Tumor Inhibition Of Adriamycin

Background Recent epidemiological data has shown that lung cancer has taken the first place of malignant tumors,seriously endangering human health.Among them,80-85% is non-small cell lung cancer(NSCLC),including adenocarcinoma,squamous cell carcinoma and large cell carcinoma,etc.The survival rate of lung cancer is still low despite the continuous improvement of lung cancer treatment.Chemotherapy and radiotherapy are common methods for the treatment of these patients.Drug resistance is one of the most important problems encountered in chemotherapy,which involves various factors,such as the potency of anticancer drugs,the drug response of tumor cells,the tumor microenvironment,the heterogeneity of tumor cells and so on.Therefore,the search for highly effective and low toxic drugs has become a hot topic in the field of cancer research.In the treatment of patients with NSCLC,adriamycin as a anticancer drug,has been selected in some refractory cases.However,with the development of modern medicine and new chemotherapeutic drugs,adriamycin has gradually been forgotten in the treatment of lung cancer.However,two factors limit the clinical application of adriamycin.On the one hand,it has some side effects such as bone marrow suppression,myocardial injury and myocardial toxicity;on the other hand,adriamycin resistance is an important factor.Like many traditional anticancer drugs,how to overcome the drug resistance of adriamycin is an urgent problem to be solved.Research on adriamycin has never been stopped.In recent years,more and more attention has been paid to the anticancer effect of Chinese herbal medicine.Chinese herbal medicine is a powerful weapon in theprevention and treatment of disease.It has been found that Chinese medicine has a good effect in the treatment of malignant tumors,and it can improve the effect of chemotherapy,improve the quality of life and reduce the clinical symptoms.It has been proven that some traditional Chinese medicine combined with traditional chemotherapy drugs have unique advantages,can effectively alleviate the symptoms of patients with advanced NSCLC,reduce adverse reactions and improve the quality of life of patients.Shikonin is a naphthoquinone compounds extracted from Arnebia Root.The study found that shikonin could inhibit the growth of tumor cells of liver cancer,breast cancer and other tumors.It is not very clear about the anti-cancer effect of shikonin on NSCLC and the related specific mechanism.Objective In this study,through observation on the proliferation of A549 cell inhibitory effect,we further observed the effect of A549 cell apoptosis,cell cycle,invasion and migration after shikonin combined with adriamycin.On this basis,we explore the possible mechanism of antitumor effect on lung cancer A549 cells by shikonin in combination with adriamycin.Methods 1.MTT assay was used to detect the inhibition rate of A549 cells in different drugs.2.Transwell method was used to detect the invasion of A549 cells.3.Cell scratch assay was used to detect the migration rate of A549 cells.4.Proliferation of A549 cells was detected by colony assay.5.PI staining was used to detect apoptosis.6.Cell cycle was detected by flow cytometry.7.Enzyme linked immunosorbent assay(ELISA)was used to detect the expression of MMP-2 and MMP-9.8.DHE fluorescent probe was used to detect the ROS effect of drugs on A549 cells.9.ATP assay kit was used to detect the changes of intracellular ATP levels.10.The potential change of mitochondrial membrane was observed under fluorescence microscope.11.Flow cytometry was used to determine the concentration of adriamycin in A549 cells.12.Living cells were used to observe the intracellular adriamycin fluorescence intensity.13.Expression of ABC transporter related protein was detected by Westem-blot.Results 1.More than 0.8μmol/L shikonin showed inhibitory effect on A549 cells,so we choose 0.8μmol/L as the initial concentration of drug in the follow-up experiment.2.With more than 0.8μmol/L of shikonin on A549 cells after 48 h,the cell number decreased,cells became round,and narrow,and cell spacing increased,which were obvious apoptosis phenomenons.3.Compared with the control group,A549 cell invasion and migration rate number decreased after different concentrations of shikonin treatment,there was significant difference.In shikonin combined with adriamycin group,invasion cell number and the migration rate decreased significantly.Compared with the control group,the expression levels of MMP-2 and MMP-9 were decreased.MMP-2 and MMP-9 were the lowest in shikonin combined with adriamycin group.4.A549 cells were treated by different concentrations of shikonin and adriamycin respectively,MTT results showed that shikonin and adriamycin inhibit the proliferation of human lung cancer A549 cells in a dose and time dependent manner.The differences were statistically significant compared with the control group.There was no obvious difference between shikonin and adriamycin in the proliferation of lung cancer A549 cells.Cell survival rate was 53.77% after treatment by shikonin combined with adriamycin,which showed less activity than the single application of shikonin oradriamycin treated cells.5.Colony formation rate decreased significantly in shikonin combined with adriamycin group on lung cancer A549 cell.6.Shikonin in combination with adriamycin can effectively induce apoptosis of lung cancer A549 cells,which induced apoptosis was significantly higher than the control group,shikonin group,adriamycin group.7.The percentage of cells in S phase increased,G0/G1 phase cell percentage decreased in shikonin combined with adriamycin group compared with adriamycin group.The effect of two drugs on cell cycle was the most obvious.8.Shikonin can induce A549 cells to produce ROS.9.After treatment with different drugs,the ATP levels in A549 cells of each group were lower than those in the control group.In shikonin group and adriamycin group,ATP decreased rate reached 80% and 81% respectively.In shikonin combined with adriamycin treatment group,ATP decreased most obviously,which was reaching 60%.10.Mitochondrial membrane potential decreased significantly in shikonin combined with adriamycin group.11.Flow cytometry showed that the concentration of adriamycin in A549 cells was significantly higher in shikonin combined with adriamycin group,which suggested that shikonin could significantly increase the level of adriamycin in A549 cells.12.Live cell station displayed shikonin can significantly enhance the fluorescence intensity of adriamycin in A549 cells.13.Westem-blot showed that the expression levels of MRP1,BCRP,and P-gp were different in the control group.After shikonin or adriamycin treatment,the expression level of these resistant protein decreased than before.The decrease was significantly largest in shikonin combined with adriamycin treatment group.Conclusions1.Different concentrations of shikonin can inhibit the proliferation of A549 cells;shikonin can enhance the inhibitory effect of adriamycin on proliferation of A549 cells.2.Different concentrations of shikonin can induce apoptosis of A549 cells;shikonin can enhance the apoptosis of A549 cells induced by adriamycin.3.Shikonin can inhibit the invasion and migration effect of lung cancer A549 cell;Shikonin can enhance the inhibitory effect of adriamycin on migration and invasion of lung cancer A549 cell.These effects may be related to the inhibition of the expression of MMP-2 and MMP-9.4.Shikonin can interfere with the cell cycle of A549 cell.5.Shikonin can enhance the potential damage of adriamycin to A549 cells mitochondrial membrane,inhibit the production of ATP,and increase ROS level in A549 cells.6.Shikonin can reduce adriamycin efflux and improve the concentration of adriamycin aggregated in A549 cells.7.Shikonin can inhibit the expression level of ABC transporter.