| ObjectiveLung cancer is the malignant tumor with the second highest incidence rate and the first mortality rate,which seriously threatens the life and health of patients.At present,the treatment of lung cancer is mainly based on radical surgery in the early stage,andas the TNM stage develops to the advanced stage,the treatment method has gradually changed to a comprehensive treatment such as surgery cooperation with chemotherapy,targeted therapy,and immunotherapy.Itis significance to develop new drugs for lung cancer.Demethylzeylasteralis a nortriterpene compound extracted from Tripterygium wilfordii,which has been confirmed to have anti-inflammatory and immunosuppressant functions.In recent years,researches had found that demethylzeylasteral has a strong anti-tumor effect,but the research is mainly concentrated on a few cancer types such as gastric cancer,breast cancer,and colon cancer,and there is no further research for lung cancer.This study intended to fill the research gap in the field of lung cancer by verifying the antitumor effect of demethylzeylasteral in inhibiting proliferation and promoting apoptosis in lung adenocarcinoma cells,and had a further discusses in the mechanism of the inhibitory effect ofdemethylzeylasteral in lung adenocarcinoma.Methods1.A549 cells and H1975 cells were plated in 96-well plates,and complete medium containingdemethylzeylasteral with concentration of 0.3μM,0.6μM,0.9μM,1.2μM,1.5μM,and 1.8μM was added,and CCK-8 experiments were performed every 24 hours.OD450 was measured and the cell inhibition rate was calculated.GraphPad Prism was used to draw a curve to explore the time dependence of demethylzeylasteral.2.The CCK-8 experiment was performed on bronchial epithelial cells HBE,lung adenocarcinoma A549 cells and H1975 cells after adding complete medium containingdemethylzeylasteral with concentration of 0.4μM,0.8μM,1.2μM,1.6μM,2.0μM,2.4μM for 72 hours.The OD450 was sequenced and the cell inhibition rate was calculated.The dosing concentration dependence of demethylzelasteral was investigated by GraphPad Prism curve.3.After being treated with demethylzelasteral for 72 h,A549 cells and H1975 cells of lung adenocarcinoma were stained with Annexin V-FITC/PI.Flow cytometer was used to collect fluorescence data,FlowJo software was used for analysis,GraphPad Prism was used for statistics and histogram drawing.This experiment was designed to study the changes in apoptosis after drug applied.4.Expression levels of BAX,Bcl-2,caspase3 and cleaved caspase3 afteraddingdemethylzelasteral were detected by western blot,ImageJ converted the bands into gray values,GraphPad Prism was used for statistics and histogram drawing.This experiment was designed to study the expression changes of apoptosis-related proteins after drug applied.5.The colony formation experiment was performed.A549 cells and H1975 cells were spread in a 6-well plate according to 150 cells per well,and the cells were cultured to 5 days,then the demethylzelasteral was applied and cultured the cells to 10 th day for fixingand staining.The inhibition ability of the tumor proliferation was reflected by plate cloning experiment.6.The fluorescence intensity of A549 cells and H1975 cells which demethylzelasteral was applied in was measured by flow cytometry.The cell cycle was analyzed by FlowJo,and GraphPad Prism was used for statistics and histogram plotting to study the cell cycle changes of the two kinds of cell lines.7.CyclinD1 and PCNA were detected by Western Blots after demethylzelasteral was applied for 72 h.ImageJ converted the bands into gray values,GraphPad Prism was used for statistics and histogram drawing.This experiment was designed to study the changes of cell cycle-related proteins after drug applied.8.Transcriptome sequencing was performed to clarify the mechanism ofdemethylzelasteral.Total RNA was extracted and thelibrary was built after demethylzelasteralwasapplied to A549 cells for 72 hours,and RNA-Seq was performed.The count data was analyzed bythe DESeq2 package of R language,and the volcano map and MA-plot were drawn,and theheat-map was drawn by the pheatmap package.GSEA enrichment analysis was performed by clusterProfiler package.9.Western blotting was performed to detect the changes of MAPK pathway,AKT pathway and p53 pathway in A549 cells and H1975 cells after demethylzelasteral was applied.ERK1/2 and its phosphorylation level,AKT and its phosphorylation level,p85 PI3 K expression level,P53 and its phosphorylation level were detected.ImageJ converted the bands into gray values,GraphPad Prism was used for statistics.10.According to the results of the transcription group,further study of RRM1 and RRM2 were performed both before and after demethylzelasteral was applied.RT-qPCR method was used to verify the changes of mRNA transcription,and Western blotting was used to verify the changes of protein expression,the mechanism of demethylzelasteral anti-tumor was speculated after the whole experiment was finished.Results1.The inhibitory effect on A549 cells by demethylzelasteral reached its peak at the concentration of 0.3μM on the second day,and reached its peak at the concentration of 0.6μM to 1.5μM on the third day.The increase of inhibitory rate was significantly slowed down at the concentration of 1.8μM on the third day.The cell inhibition rate of H1975 cells in the 0.3μM and 0.6μM groups reached the peak at third day,and the cell inhibition rate of the rest concentrations increased rapidly in the first 2 days.After the third day,the cell inhibition rate entered a relatively smooth plateau.Compared with lung adenocarcinoma cells A549 and H1975,bronchial epithelial HBE cells are less sensitive to the effect of demethylzelasteral,suggesting that demethylzelasteral has anti-tumor effect.IC50 of HBE cells,A549 cells and H1975 cells They were 3.436μM,0.626μM,and 0.967μM,respectively.It suggesting thatthe anti-tumor effect of demethylzelasteral is time-dependent and concentration-dependent..2.Annexin V-FITC/PI double-staining to detect cell apoptosis after adding demethylzelasteral.The apoptosis of A549 cells before and after adding demethylzelasteral was 3.65±0.44% and 15.99±1.05% respectively,and the apoptosis of A549 cells in the early stage was 0.62±0.24% and 1.05±0.43%,respectively.The apoptotic cells in the late period were 3.03±0.27% and14.93±0.65%,respectively.The apoptotic cells in H1975 before and after adding demethylzelasteral was 8.76±1.27% and 18.02±2.21%,respectively.The apoptotic cells in the early period was 2.05±1.14% and 1.98±1.17%,respectively.The late apoptosis was 6.71±0.45% and 16.03±1.37%,respectively.The difference of the proportion of apoptotic cells and the proportion of late apoptotic cells between the two cell lines was statistically significant before and after adding the drug,suggesting that the pro-apoptotic effect of demethylzelasteral was mainly to induce late apoptosis.Further detection of changes in apoptosis-related proteins after demethylzelasteral was applied,it had found that BAX,caspase3 and cleaved-caspase3 were significantly increased,and the difference was statistically significant;Bcl-2 did not change significantly in A549 cells after treatment,but in H1975 cells,it decreased slightly.It was suggesting that demethylzelasteral has the ability to induce apoptosis of lung adenocarcinoma cells.3.The colony formation experiment showed that there were significant differences in the number and size of clones formed by A549 cells and H1975 cells before and after adding demethylzeylasteral.Further flow cytometry cell cycle analysis showed that both cells showed arrest in G1 phase and a decrease in the proportion of cells in S phase,the expressions of cell cyclerelated proteins CyclinD1 and PCNA were all decreased after adding demethylzeylasteral,suggesting that demethylzeylasteral has the ability to inhibit the proliferation of lung adenocarcinoma.4.Transcriptome analysis was performed after the addition of demethylzeylasteral,and 85 differential genes were obtained by threshold screening,of which 35 were up-regulated and 45 were down-regulated.Further GSEA analysis was performed,ordering by NES score,the changed genes are mainly enriched in cell cycle,DNA replication,homologous recombination and other pathways.5.Western blotting was performed to detect the changes in signaling pathways after adding demethylzeylasteral to lung adenocarcinoma cells,and it was found that the expression of ERK1/2 and its phosphorylation level,AKT and its phosphorylation level,and p85 PI3 K did not change significantly.There was no significant difference in p53 phosphorylation level either.The expression level of p53 protein was slightly up-regulated,but the up-regulation range was not high enough to explain the strong anti-tumor effects of demethylzeylasteral;6.After adding demethylzeylasteral to lung adenocarcinoma cells,the mRNA level and protein level of RRM1 have no significant changes.But the mRNA level and protein level of RRM2 was strongly suppressed.It is suggested that demethylzeylasteral may exert its anti-tumor effect by inhibiting RRM2.Conclusions1.Demethylzeylasteral has an anti-tumor effect on lung adenocarcinoma,and its killing of tumors has obvious dose and time dependence;2.Demethylzeylasteral induces apoptosis of lung adenocarcinoma cells,especially the proportion of late apoptotic cells increases significantly;3.Demethylzeylasteral inhibits the proliferation of lung adenocarcinoma cells,blocks the cell cycle and G1 phase,and the proportion of cells in the S phase decreases significantly;4.Transcriptome analysis found that the changed genes thatadding demethylzeylasteral 72 hto lung adenocarcinoma cells were mainly enriched in the cell cycle,DNA replication,homologous recombination and other pathways by GESA,suggesting the mechanism related to these signaling pathway;5.Demethylzeylasteral caused the upregulation of P53 protein expression in lung adenocarcinoma,but there was no significant difference in the phosphorylation level of P53,and the effect on MAPK pathway and AKT pathway was not significant either;6.Demethylzeylasteral has obvious inhibitory effect on RNR small subunitRRM2,suggesting that it has an anti-tumor effect by inhibiting of RRM2. |
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