| Objective:In the past two decades,the number of deaths caused by lung cancer in China has exceeded 20% of the total number of cancer deaths,and the morbidity and mortality have increased rapidly.The number of patients with non-small cell lung cancer accounts for about 85% of lung cancer patients.Although surgical treatment can remove the lesion area,there is still a high recurrence rate in the later stage,and the prognosis is not satisfactory.Corosolic acid is a natural pentacyclic triterpenoid compound and has a very broad application prospect in anti-tumor.Anti-tumor drugs have a variety of pathways to induce tumor cell apoptosis,in which oxidative stress-induced reactive oxygen species is closely related to tumor cell apoptosis,and corosolic acid is a traditional Chinese medicine monomer that effectively inhibits tumor growth.The effect may be to induce apoptosis of tumor cells through oxidative stress.Therefore,this subject is based on non-small cell lung cancer,with oxidative stress as the research focus,looking for the upstream and downstream of its signaling pathway,revealing the mechanism of corosolic acid inhibition of non-small cell lung cancer growth.Method:1.MTT assay was used to detect the growth of different lung cancer cell lines H1650,SPC-A-1,A549 and H460 by different concentrations of corosolic acid(1,2.5,5,10,25,50,100,250 μmol/L).2.Different concentrations of corosolic acid(0,5,10,15,20,25 μmol/L)were mixed with non-small cell lung cancer H1650 cells.MTT assay was used to detect the effect of corosolic acid on the growth of H1650 cells.3.The effect of different concentrations of corosolic acid on ROS in H1650 cells was detected by DCFH-DA probe.4.Corosolic acid was combined with oxidative stress/signal pathway-related inhibitors,and MTT assay was used to detect the growth of H1650 cells.The DCFH-DA probe was used to detect the effect of corosolic acid in combination with oxidative stress/signal pathway-related inhibitors on intracellular ROS.5.The effect of different concentrations of corosolic acid on the protein expression of H1650 cells was detected by Western Blot.Corosolic acid was used in combination with oxidative stress/related signaling pathway inhibitors to detect the effects of key proteins and phosphorylation levels in Smad,p38,JNK1/2,and ERK1/2 signaling pathways in H1650 cells.6.The nude mouse solid tumor model was constructed by using non-small cell lung cancer H1650 cells,and divided into blank group,control group,positive drug group(cisplatin)and different doses of corosolic acid.Continuous intragastric administration for 14 days,the tumor volume was measured and the nude mice were weighed every day.After the animal treatment,the tumor tissue volume and weight were counted.Western Blot was used to detect the protein levels of different concentrations of corosolic acid on tumor tissues,such as Smad2,p38,JNK1/2,ERK1/2,cleaved-Caspase3,Caspase3.Results:1.Corosolic acid can inhibit the growth of different types of lung cancer cell lines H1650,SPC-A-1,A549,and H460.2.Corosolic acid inhibited the growth of non-small cell lung cancer H1650 cells in a concentration-dependent manner;corosolic acid had no significant effect on the proliferation of wi-38 cells at concentrations below 20 μmol/L.3.DCFH-DA probe was used to detect the changes of ROS in H1650 cells.The results showed that the concentration of RFU increased in a concentration-dependent manner after treatment with different concentrations of corosolic acid,indicating that corosolic acid can induce ROS formation in H1650 cells.4.Antioxidant pretreatment was combined with corosolic acid.Compared with the corosolic acid treatment group,the RFU values of the NAC,Catalase,and Apocynin pretreatment groups were significantly decreased,indicating that the antioxidant can inhibit the ROS formation of H1650 cells induced by corosolic acid.5.The combination of antioxidants and corosolic acid can promote the growth of H1650 cells,such as the combination with Tocopherol,NAC,Catalase,and Apocynin,and there is a significant difference compared with the group treated with corosolic acid alone.6.Western Blot assay showed that the phosphorylation level of Smad2 in cytoplasmic protein increased gradually with the increase of corosolic acid concentration in a dose-dependent manner.At the same time,the phosphorylation levels of p38 and JNK1/2 protein were also gradually increased.The levels of Smad2 and Smad3 proteins in nuclear proteins were also gradually increased,indicating that corosolic acid can promote nuclear translocation of Smad2 and Smad3 proteins.7.The combination of an oxidative stress-related pathway inhibitor and the corosolic acid group can promote the growth of H1650 cells,such as SB525334,PD98059,SB203580,and there is a significant difference compared with the corosolic acid alone group.8.Animal experiments showed that the 2.5mg/kg-40mg/kg corosolic acid group can effectively inhibit the growth of H1650 tumor cells.Western Blot assay showed that the expression of Smad2 phosphorylation level was up-regulated and the expression of p38 phosphorylation level was down-regulated in the corosolic acid group(2.5mg/kg-40mg/kg)compared with the control group,with statistical significance.Corosed acid group(5mg/kg,10mg/kg,20mg/kg)cleaved-Caspase3 protein expression was up-regulated,with statistical significance.Conclusion:1.Corosolic acid can effectively inhibit the growth of different lung cancer cell lines,including non-small cell lung cancer.2.Corosolic acid induces apoptosis in non-small cell lung cancer through oxidative stress.3.Corosolic acid mainly regulates the phosphorylation of Smad protein in Smad signaling pathway and promotes nuclear translocation,and activates Caspase signaling pathway to induce apoptosis of non-small cell lung cancer by regulating p38 signaling pathway in MAPK. |
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