| [Background]In recent years,the incidence and mortality of lung cancer are increasing year by year,which is the leading cause of death of malignant tumors.Traditional treatments for lung cancer are mainly surgery,radiotherapy and chemotherapy,although these therapies had brought significant effects,it also brought a great side effects,increasing the suffering of patients and reducing life quality of patients.Compared with traditional therapy,molecular targeted therapy has high selectivity and low toxicity,which can prolong the survival time of patients and improve the quality of life.Tumor molecular targeted therapy is a therapy which is based on tumor cell biology,using specific tumor tissue or cells with a(or relatively specific)molecular structure as a target,combined with the specific target of these molecules with antibody or ligand to achieve a direct treatment or treatment oriented therapy purpose.It is known that epidermal growth factor receptor(EGFR)is one of the main molecular targets of lung cancer treatment.EGFR tyrosine kinase inhibitor(TKIs)is used in clinical EGFR mutant non-small cell lung cancer treatment has achieved remarkable achievements.The growth factor receptor binding protein 2(Grb2)plays an important role in the signal transduction process as adapter protein of EGFR.It was reported that alpha-lipoic acid(?-LA)plays an important role in inhibiting the growth of many tumor cells and inducing apoptosis.In this study,we found that A549 cells proliferation was inhibited by ?-LA,the results showed that the level of Grb2 m RNA was significantly decreased in A549 cells treated with ?-LA.Therefore,we speculate that Grb2-EGFR may mediate ?-LA to inhibit Non-small cell lung cancer cells proliferation.[Methods]1.CCK-8 detected ?-LA for A549 and NCI-H1975 cell proliferation,The concentration of ?-LA was 1.0m M and 2.0m M respectively,the detection time was 0h,24 h,48h,72 h,96h,120 h.Determine the alpha-LA working concentration.2.Differential expression analysis of transcriptome gene of A549 cells treated with 2 m M ?-LA by RNA-Seq technique.3.The expression of Grb2 m RNA and protein in A549 and NCI-H1975 cells treated with 2.0m M ?-LA was detected by RT-PCR and Western blot.Determining how long the drug treats cells.4.A549 and NCI-H1975 cells were transfected with Grb2 si RNA and p ENTER-Grb2 plasmids respectively to silence or overexpress Grb2 respectively.Western blot was used to validate the transfection effect.CCK-8 method was used to detect the changes of cell proliferation after Grb2 silenced and overexpressed.5.The effect of 2.0m M ?-LA on EGFR and ERK protein levels in A549 and NCI-H1975 cells before and after activation of EGFR-ERK pathway by 50 ng / m L EGF was detected by Western blot.6.Silencing and overexpression of Grb2,the expression of EGFR and ERK protein were detected by Western blot in A549 and NCI-H1975 cells respectively;after silencing Grb2,50 ng / m L EGF activated EGFR-ERK pathway,the expression of EGFR and ERK protein in the above two kinds of cells detected by Western blot.7.Grb2 was silenced or overexpressed firstly,and then treated with 2 m M ?-LA,EGFR and ERK protein levels were detected by Western blot in A549 and NCI-H1975 cells.[Results]1.1.0m M and 2.0m M ?-LA inhibited the proliferation of A549 and NCI-H1975 cells.2.A549 cells were treated with 2.0m M ?-LA,the expression of differentially expressed genes in the transcription group showed a significant decrease in Grb2 m RNA levels.3.The levels of Grb2 m RNA and protein were decreased at 12 h and 24 h after treated with 2.0m M ?-LA in A549 and NCI-H1975 cells.4.Silencing Grb2,the proliferation of A549 and NCI-H1975 cells was inhibited,and the proliferation of A549 and NCI-H1975 cells was not changed after overexpression of Grb2.5.The levels of P-EGFR and P-ERK protein in A549 and NCI-H1975 cells treated with 2.0m M ?-LA decreased,and the total protein levels of EGFR and ERK did not change.2.0m M ?-LA inhibited upregulation of 50 ng / ml of EGF-induced p-EGFR and p-ERK protein levels.6.The level of p-EGFR protein decreased and the level of P-ERK protein did not change significantly after silencing Grb2,at the same time,silencing Grb2 could decrease the level of EGF-induced p-EGFR and p-ERK protein,and the total protein level of EGFR and ERK did not change in A549 and NCI-H1975 cells,.7.Silencing Grb2,A549 and NCI-H1975 cells were treated with 2 m M ?-LA,and the levels of p-EGFR and p-ERK protein were decreased;after overexpression of Grb2,the levels of p-EGFR and p-ERK protein were not significantly changed in A549 and NCI-H1975 cells treated with 2 m M ?-LA.[Conclusions]1.2.0m M ?-LA on A549 and NCI-H1975 cells in the inhibition of proliferation began at 24 h,A549 cell proliferation was significantly increased until the end of 120 h test;NCI-H1975 cell proliferation inhibition in 96 h the most obvious.2.2.0m M ?-LA reduced m RNA and protein levels of Grb2 in A549 and NCI-H1975 cells3.2.0m M ?-LA inhibits EGFR and ERK phosphorylation in EGFR-ERK pathway in A549 and NCI-H1975 cells.4.Silencing Grb2,P-EGFR protein levels were decreased and P-ERK protein levels did not change significantly in A549 and NCI-H1975 cells.In summary,Grb2-EGFR mediated ?-LA to inhibit NSCLC cells proliferation. |
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