Study On The Anti-tumor Effect Of Deoxyshikonin On Large Cell Lung Cancer NCI-H460 Cells

In order to explore the inhibitory effect of Chinese herbal monomer deoxyshikonin on human cell lung cancer NCI-H460,the following experiments were carried out in this study:(1)in the cytological experiments,will be in logarithmic phase of NPC cell lung cancer NCI-H460 cell lines were randomly divided into blank control group,the DMSO group,positive control group(5-Fu,5 fluorouracil,80μM)and different concentrations of deoxyshikonin groups [tetramethylthiazole Blue(MTT)assay,cell scar assay,Transwell cell invasion assay and cell apoptosis assay: 4,6,8,10 μM;plate clonal colony formation assay: 0.5,1,1.5 μM],the effects of different concentrations of deoxyshikonin on proliferation,migration,invasion and apoptosis of NCI-H460 cells were detected by MTT assay,cell plate cloning colony formation assay,cell scratches assay,Transwell assay and one-step TUNEL assay.(2)In animal experiments,male Balb/c nude mice were selected to construct a subcutaneous tumor carrying model of nude mice by subcutaneous inoculation of NCI-H460 cells.According to the mean tumor volume of each group about 140 mm3,nude mice were randomly divided into blank control group,positive control group and deoxyshikonin group,with 3 mice in each group.The patients were given0.5%CCMC-Na(100 μL),5-FU(25 mg/kg,100 μL)and 0.5%CCMC-Na+deoxyshikonin(5 mg/kg,100 μL)by gavage,respectively.The gavage cycle was 15 d,and serum and tumor tissues were collected after gavage.H&E staining and immunohistochemistry were used to investigate the inhibitory effect of deoxyshikonin on the tumor of NCI-H460 in animals.(3)Subsequently,this study analyzed the differentially expressed genes and potential signaling pathways of the effect of deoxyshikonin on NCI-H460 cells through network pharmacology and cell transcripomics sequencing,so as to initially explore the mechanism of action of deoxyshikonin in inhibiting NCI-H460 in human cell lung cancerThe experimental results are as follows:(1)The results of MTT experiment showed that compared with blank control group,the survival rate of NCI-H460 cells decreased gradually with the increase of deoxyshikonin concentration in a dose-dependent manner,and reached the lowest at 8μM.The results of cell plate cloning colony formation experiment showed that with the increase of deoxyshikonin concentration,the number of cell colonies decreased gradually,and the number of colony formation was the lowest at 1.5 μM.(2)The cell scratch test showed that,compared with the blank control group,the migration ability of NCI-H460 cells was significantly inhibited by different concentrations of deoxyshikonin,and the cell mobility was the lowest at 10 μM.(3)The results of Transwell cell invasion experiment showed that with the increase of deoxyshikonin concentration,the number of cells passing through the upper compartment to the lower compartment became less and less,showing an obvious dose-dependent,and the number of invasive cells was the least when the concentration of deoxyshikonin was 10 μM.(4)The results of TUNEL cell apoptosis test showed that the higher the concentration of deoxyshikonin,the stronger the green fluorescence intensity,indicating the more obvious apoptosis phenomenon,in an obvious dose-dependent manner,the green fluorescence appeared at 4 μM,the highest green fluorescence intensity at 10 μM,the most obvious apoptosis phenomenon.(5)The results of subcutaneous inoculation of NCI-H460 cells in Balb/c nude mice showed that the tumor volume and RTV value in the 5 mg/kg deoxyshikonin group were decreased compared with that in the blank control group,and the trend of body weight change in the deoxyshikonin group was basically the same as that in the blank control group.These results indicated that 5 mg/kg deoxyshikonin could effectively inhibit the proliferation of NCI-H460 tumor in Balb/c nude mice and had little effect on the normal physiological life of nude mice.H&E staining showed that,compared with the CMC-Na group,the tumor tissues in the deoxyshikonin group showed obvious tumor cell shrinkage,sparse arrangement,fragmentation,vacuolation and necrosis.Immunohistochemical results showed that the expression level of Ki-67 in the deoxyshikonin group was lower than that in the CMC-Na group,indicating that the proliferation ability of tumors was decreased after the deoxyshikonin treatment,while the expression level of E-Cad was higher than that in the CMC-Na group,indicating that the adhesion between tumor cells was increased after the deoxyshikonin treatment,thus inhibiting the migration ability of tumors.(6)The results of network pharmacological analysis showed that deoxyshikonin may act on 35 targets such as BCL-2,CCNE 1,CCNE 2 and CDC 25 A in non-small cell lung cancer and may have a tumor suppressive effect on NSCLC by inducing cell apoptosis.Cell transcriptome sequencing results showed that deoxyshikonin may inhibit NCI-H460 in human cell lung cancer mainly by affecting lipid and atherosclerosis,steroid hormone biosynthesis,apoptosis,and PPAR signaling pathway.In conclusion,deoxyshikonin can inhibit proliferation,migration and invasion of human cell lung cancer cell line NCI-H460 in cytological experiments,and apoptosis occurs at 4 μM.In vivo experiment of Balb/c nude mice,5 mg/kg deoxyshikonin can effectively inhibit the proliferation of NCI-H460 tumor and has little effect on normal physiological life of nude mice.H&E staining showed that the tumor cells were broken and vacuolated after deoxyshikonin treatment,and immunohistochemical results showed that the expression level of Ki-67 was decreased after deoxyshikonin treatment.These results indicated that the proliferation ability of tumor cells was inhibited,and the expression level of E-Cad was increased,and the adhesion between cells was increased,so the migration ability of tumor cells was inhibited.Transcriptome sequencing results showed that the mechanism of deoxyshikonin on NCI-H460 in human cell lung cancer may be related to lipid and atherosclerosis,steroid hormone biosynthesis,cell apoptosis and PPAR signaling pathway.In this study,cytological experiments,animal experiments,network pharmacological analysis and cell transcriptome sequencing were conducted to study the inhibitory effect of Chinese herbal monomer deoxyshikonin on human cell lung cancer NCI-H460 cells,in order to provide new experimental reference data for the treatment of Chinese herbal monomer deoxyshikonin on human cell lung cancer NCI-H460 cells.