Inhibition Of MAPK/STAT3/NF-κB Pathway-Mediated Proliferation And Migration Of Lung Adenocarcinoma A549 Cells By Daphnetin

Objective:Lung cancer is one of the leading cause of cancer death,accounting for about 18%of all cancer mortality.Despite the treatment methods such as surgical resection and radiotherapy,patients are mostly in advanced stage when diagnosed with lung cancer and the treatment effect is poor.Traditional Chinese medicine has outstanding anti-cancer effects with its unique advantages of high efficiency and low side effects.Daphnetin is a coumarin derivative isolated from Daphne odorifera,which has anti-tumor effects.MAPK,STAT3 and NF-κB signaling pathways are closely related to the pathogenesis of lung cancer.This study was to investigate the role of MAPK,STAT3 and NF-κB signaling pathways in the anti-tumor effects of daphnetin and its mechanisms by means of lung adenocarcinoma A549 cells,and to elucidate the effects of daphnetin on the invasion,proliferation,migration and clone formation of A549 cells.The results of the study provide a new research direction and theoretical basis for the use of daphnetin in inhibiting lung adenocarcinoma.Methods:1.Effect of daphnetin on inflammatory factors in lung adenocarcinoma A549 cellsProtein and RNA were extracted from A549 cells after adding different concentrations of daphnetin(0,5,10μg/mL)and DMSO(solvent control of daphnetin)for 24 hours,and the protein and mRNA expression of inflammatory factors IL-18、IL-1β and iNOS were detected by Western Blot and RT-qPCR techniques.2.Effects of ryanodine on MAPK,STAT3 and NF-κB signaling pathways in lung adenocarcinoma A549 cellsThe protein expression levels of MAPK signaling pathway related molecules(JNK,ERK1/2 and p38 and their phosphorylation)and STAT3 signaling pathway related molecules(JAK2 and its phosphorylation and STAT3 and its phosphorylation)and NF-κB signaling pathway-related molecules(NF-κBp105,NF-κBp65,IκB-α and its phosphorylation),in order to determine the optimal concentration of daphnetin.3.Effect of daphnetin on tumor biological behavior of lung adenocarcinoma A549 cells1)The proliferation of A549 cells was detected by CCK8 after adding DMSO and 0 and 10 μg/mL of daphnetin to A549 cells for 24 h,48h and 72 h,respectively.2)DMSO and 0,10μg/mL daphnetin were added to A549 cells and the effect of daphnetin on cell clone formation was examined by plate clone formation.3)DMSO and 0,10μg/mL daphnetin were added to A549 cells for 0h,24 h,48h and 72 h respectively to detect the effect of daphnetin on A549 cell migration by cell scratching assay.4)DMSO and 0,10 μg/mL daphnetin were added to A549 cells,and the effect of daphnetin on A549 cell invasion was detected by Transwell cell invasion assay.4.Effects of daphnetin and JNK inhibitor on STAT3 and NF-κB pathway in lung adenocarcinoma A549 cells.A549 cells were pretreated with JNK inhibitor for 1 h,then different concentrations of daphnetin(0,10μg/mL)and DMSO(solvent control of daphnetin)were added according to experimental groups for 24 h.The protein expression levels of JNK and its phosphorylated proteins were detected by Western Blot,and the protein expression levels of STAT3 pathway and NF-κB pathway were detected.Results:1)Compared with the control group,the protein and mRNA expressions of IL-18、iNOS and IL-1β in DMSO group were not significant different.2)Compared with DMSO group,the protein and mRNA expressions of IL-18(P<0.01)decreased significantly in Daph group,and the mRNA expressions of IL-1β(P<0.05)and iNOS(P<0.05)decreased significantly,but no significant differences were seen in protein expression.3)Compared with the control group,the protein expressions of MAPK signaling pathway molecules JNK,ERK1/2,p38 and p-JNK,p-ERK1/2,p-p38 in the DMSO group were not significant different.4)Compared with DMSO group,the protein expressions of MAPK signaling pathway molecules p-JNK(P<0.01),p-ERK1/2(P<0.01),and p-p38(P<0.05)were significantly decreased in Daph group,while the protein expressions of JNK,ERK1/2and p38 remained unchanged.5)Compared with the control group,the protein expressions of STAT3 signaling pathway molecules JAK2,p-STAT3,p-JAK2 and p-STAT3 in DMSO group were not significant different.6)Compared with DMSO group,the protein expression of STAT3 signaling pathway molecules JAK2 and STAT3 were unchanged in Daph group,but the expressions of phosphorylated proteins p-JAK2(P<0.01)and p-STAT3(P<0.01)were significantly decreased.7)Compared with the control group,the protein expressions of NF-κB pathway molecule NF-κB p105,NF-κB p65,IκB-α and p-NF-κBp105,p-NF-κBp65,p-IκB in the DMSO group were not significant different.8)Compared with DMSO group,the protein expressions of NF-κB pathway molecule NF-κB p105、NF-κB p65、IκB-α in Daph group were not different,but p-NF-κB p105(P<0.01),p-NF-κB p65(P<0.01),and p-IκB(P<0.01)were significantly decreased.9)The results of CCK8 assay showed that the concentration of daphnetin within the range of 20μg/mL had no significant effect on the viablity of A549 cells.10)The results of CCK8 assay for proliferation showed that compared with the control group,there was no significant difference in the effect of DMSO group on the proliferation of A549 cells at 24 h,48h and 72h;compared with the DMSO group,the Daph group was able to inhibit the proliferation of A549 cells,but there was no significant difference in the effect on the proliferation of A549 cells when the action was 24 h.There was a significant difference in the proliferation of A549 cells when the effect was 48h(P<0.05)and 72 h(P<0.01).11)The results of clone formation experiment showed that the DMSO group did not inhibit the colony formation of A549 cells compared with the control group;the number of clone-forming clusters was significantly reduced in the Daph group compared with the DMSO group(P<0.01),and there was a significant difference.12)The results of migration assay showed that there was no significant difference in the scratch healing rate in the DMSO group compared with the control group at 24 h,48h and 72h;compared with the DMSO group,the inhibition of scratch area in the Daph group was lessened at 24h(P<0.05),48h(P<0.01)and 72h(P<0.01)with significant difference.13)The results of Transwell invasion assay showed that there was no significant difference in the number of cells invading through the stromal gel in the DMSO group compared with the control group;the number of cells invading through the stromal gel in the Daph group was significantly reduced compared with the DMSO group,and there was a significant difference(P<0.01),and the invasion of A549 cells was inhibited.14)Compared with DMSO group,both Daph group and JNK inhibitor group significantly reduced p-JNK expression(P<0.01),and JNK inhibitor+Daph group significantly reduced p-JNK expression(P<0.01)15)Compared with DMSO group,both Daph group(P<0.05)and JNK inhibitor group(P<0.01)significantly reduced p-STAT3 expression,and JNK inhibitor+Daph group significantly reduced p-STAT3 expression(P<0.01).16)Compared with DMSO group,both Daph group and JNK inhibitor group significantly reduced the expression of p-NF-κB p105,p-NF-κB p65 and p-IκB(P<0.05),and JNK inhibitor+Daph group significantly reduced the expression of p-NF-κB p105,p-NF-κB p65 and p-IκB(P<0.01).Conclusion:1)Daphnetin could reduce the release of pro-inflammatory factor IL-18 in lung adenocarcinoma A549 cells.2)Daphnetin could inhibit the activation of MAPK signal pathway in lung adenocarcinoma A549 cells,and inhibit the activation of STAT3 and NF-κ B pathway mainly through the JNK pathway.3)The proliferation,invasion,migration and clone formation of lung adenocarcinoma A549 cells were inhibited by Daphnetin.