Antitumor Effect And Mechanism Of Thymoquinone In Lung Adenocarcinoma Cells

BackgroundAt the beginning of the 20th century,lung cancer is still a rare tumor in the world.in recent years,the incidence of lung cancer is on the rise,which poses a great threat to human life and health.Lung cancer is divided into small cell lung cancer and non-small cell lung cancer,which account for about 85%of lung cancer.epidemiological findings in recent years show that the incidence of lung adenocarcinoma has exceeded that of lung squamous cell carcinoma in both smokers and non-smokers.It has become the most common pathological type of lung cancer.Multidisciplinary comprehensive treatment is the main treatment of lung cancer at present,in which chemotherapy is still an important treatment.Thyraquinone is the main active component extracted from the medicinal plant black seed or black algae in southwestern Asia.it has antioxidant,anti-atherosclerotic,anti-inflammatory,anti-apoptosis,anti-tumor and other biological activities,but its mechanism of action is not in-depth study.P53 gene is one of the important tumor suppressor factors in cells.it plays an active role in tumor microenvironment and immunomodulation.it has a variety of biological functions,such as inducing cell growth arrest and apoptosis.Cell differentiation and cell energy metabolism.P53 gene mutation is very common in patients with non-small cell lung cancer(NSCLC),the frequency of p53 gene mutation is as high as 50%.Lung cancer models in vitro and in vivo show that p53 mutation plays a key role in malignant transformation,histological progression,invasion and metastasis of lung cancer,but the role of p53 mutation in apoptosis of lung adenocarcinoma cells induced by thyraquinone and the anti-tumor mechanism of thyraquinone have not been reported..ObjectiveTo observe the effect of thyraquinone on proliferation and apoptosis of lung adenocarcinoma cells and its relationship with p53 gene,and to explore the mechanism of thyraquinone against lung adenocarcinoma cells.Materials and MethodsHuman lung adenocarcinoma cell lines A549(p53+/+)and H1299(p53-/-)were purchased from Shanghai Institute of Cell Biology,Chinese Academy of Sciences.the cells were treated with different concentrations of thyraquinone to examine the antitumor effect of thyraquinone.The morphological changes and necrosis rate of the two kinds of cells were detected by fluorescence microscope,and the effects of thyraquinone on the proliferation of the two kinds of cells were detected by MTT assay.The effects of thyraquinone on apoptosis and reactive oxygen species(Ros)production of two kinds of cells were detected by flow cytometry,and the effects of thyraquinone pretreatment with ROS scavenger NAC on the proliferation,apoptosis and reactive oxygen species(Ros)production of the two kinds of cells were detected.RT-PCR technique was used to detect the effect of thyraquinone on the expression of three key enzymes in glycolysis pathway in A549 cells,and RT-qPCR technique was used to detect the expression of SAT1,PTGS2 and GPX4 related to ferroptosis in A549 cells.Detection of protein expression after thyloquinone treatment by Western blot.ResultsAfter treated with thyraquinone on A549 cells and H1299 cells,the morphology of A549 cells and H1299 cells changed significantly and the necrosis rate increased in a dose-dependent manner under inverted fluorescence microscope.A549 cells were more sensitive to thyloquinone and had higher necrosis rate than H1299 cells,and the results of MTT assay showed that thyraquinone inhibited the proliferation of A549 cells and H1299 cells,and the results of MTT assay showed that thyraquinone inhibited the proliferation of A549 cells and H1299 cells,and the results of MTT assay showed that thyraquinone could inhibit the proliferation of A549 cells and H1299 cells.However,the inhibitory effect on the proliferation of A549 cells was more obvious,which was consistent with the results observed under microscope,and the results of apoptosis detection showed that thyraquinone could promote the apoptosis of the two kinds of cells in a dose-dependent manner,and the inhibition of the proliferation of A549 cells was more obvious.The results of Ros detection showed that the content of Ros in A549 cells and H1299 cells pretreated with ROS scavenger NAC increased in a dose-dependent manner.The content of Ros and apoptosis rate of two kinds of cells pretreated with NAC were significantly lower than those treated with thyraquinone at the same concentration.The results of MTT were the same as those of apoptosis.The results of RT-PCR showed that thyraquinone had no significant effect on the expression of three rate-limiting enzymes in glycolysis pathway in A549 cells,but the results of RT-qPCR showed that thyraquinone had no significant effect on the expression of three rate-limiting enzymes in A549 cells.The expression of mRNA of GPX4 related to ferroptosis decreased,while the expression of mRNA of SAT1 and PTGS2 increased.the results of;Western blot showed that the expression of p53 and SAT1 protein increased in A549 cells in a dose-dependent manner.Conclusion1.In vitro,thyraquinone could inhibit the proliferation of A549 cells and H1299 cells,induce apoptosis,and showed p53 dependence.2.Thyraquinone promotes apoptosis of A549 cells and H1299 cells by promoting the production of Ros,but this process is not related to glycolysis pathway.3.Thyraquinone induces ferroptosis in lung adenocarcinoma cells by down-regulating the expression of GPX4.