| Bovine lactoferrin(bLF),as a natural glycoprotein,is an important component of the host innate immune system.In recent years,with the advancement of pharmaceutical research,bLF has been shown to exhibit excellent activities in antibacterial,antiviral,animal intestinal function regulation and immunomodulation.In addition,bLF can inhibit the proliferation of many malignant tumors and their cells,such as liver,lung,colon and esophageal cancers,and has broad-spectrum anti-tumor effects.It has been shown that bLF can significantly inhibit the proliferation of lung cancer,but there are still many aspects of its anti-lung cancer mechanism of action that have not been elucidated.In order to investigate the growth inhibitory effect of bLF on lung cancer A549 cells and its potential mechanism of action,this study intends to investigate the effect of bLF on the proliferation and migration of A549 cells by CCK-8,colony formation,scratch assay and flow cytometry;then to investigate the effect of bLF on A549 cells by Hoechst 33342 staining,flow cytometry and fluorescence probe combined with fluorescence microscopy;finally to investigate the effect of bLF on A549 cells by Hoechst 33342 staining,flow cytometry and fluorescence probe combined with fluorescence microscopy.The effect of bLF on apoptosis was then investigated by Hoechst 33342 staining,flow cytometry and fluorescence probe combined with fluorescence microscopy.The following results were obtained from this study:1.bLF was clearly shown to effectively inhibit the proliferation of A549 cells.The results of CCK-8 method showed that the cell viability of A549 cells treated with different concentrations of bLF(0,0.5,1.0,1.5,and 2.0 mg/m L)for 12 h,24 h and 48 h respectively were significantly reduced in a time-and drug concentration-dependent manner,and in addition,the results of colony formation showed that,compared with the blank control group,with increasing drug concentration,the number of colonies formed by A549 cells The number of colonies formed by A549 cells decreased gradually with increasing drug concentration compared with the blank control.2.bLF induced S-phase block in A549 cells and its molecular mechanism was elucidated.In this process,bLF downregulated the m RNA level of Cyclin A1 in cancer cells and inhibited the expression of P21,CDK2 and Cyclin D1 proteins,resulting in a reduction of the Cyclin A1-CDK2 complex,which caused S-phase block in cancer cells.3.bLF was found to have an inhibitory effect on the migration of A549 cells.The results of the scratch assay showed that bLF inhibited cell migration in a drug concentration-dependent manner.Moreover,Western blot showed that bLF down-regulated MMP-2 protein and up-regulated E-cadherin protein levels,indicating that bLF inhibited the migration of A549 cells by regulating some migration-related factors.4.The molecular mechanism by which bLF triggers endo-apoptosis in A549 cells was first investigated.We used Hoechst33342 staining,Annexin V-FITC,PI double staining and mitochondrial membrane potential and apoptosis staining to demonstrate that bLF induced apoptosis in A549 cells and caused disruption of mitochondrial membrane potential and intracellular ROS production.It was further demonstrated by Western blot that bLF caused downregulation of Bcl-2 and upregulation of Bax,Caspase-3 and P53,indicating that bLF mediated apoptosis in A549 cells through the mitochondrial pathway.In conclusion,bLF inhibited the proliferation,cell cycle progression and migration of A549 cells in vitro,and also induced intracellular reactive oxygen species production,which led to disturbance of mitochondrial membrane potential and induced apoptosis through the endogenous mitochondrial pathway.This suggests that bLF has a strong anti-lung cancer function and its potential in the treatment of various types of cancers in humans and animals can be further explored. |
PDF Full Text Request