?-Elemene Enhances The Effect Of Afatinib On Non-small Cell Lung Cancer By Inhibiting OCT4 Expression

In the past decades,non-small cell lung cancer(NSCLC)has an increasing incidence and poor prognosis.Although the current first-line medicine,afatinib, promotes patient survival,acquired resistance to afatinib was also observed in many patients which due to stem-like cells.Thus,overcoming afatinib resistance to stem cell-like properties is a critical component of the treatment of NSCLC.?-Elemene(bELE)is a compound from a Chinese herb which has been found inhibit glioma stem-like cells(GSLCs)growth.CD133~+lung cancer cells can significantly inhibit the immunotherapeutic effect of lung cancer.CD133,also known as prominin-1,has been widely used as a dry marker in non-small cell lung cancer.OCT-4is a member of the transcription factor family of POU domain and is usually expressed in pluripotent stem cells.Studies have shown that OCT4 plays an important role in maintaining the self-renewal of CD133~+lung cancer cells and the characteristics of cancer stem cells.Some studies suggest that?-Elemene can inhibit the activation of Notch1 and inhibit the proliferation of cancer stem cells from gastric cancer.At the same time,?-Elemene can inhibit the proliferation of cancer stem cells from gastric cancer,promote the differentiation of cancer stem cells into normal cancer cells,and enhance the sensitivity of cancer stem cells to temozolomide.However,in lung cancer(non-small cell lung cancer),whether beta-elemene inhibits the proliferation of cancer stem cells has not been reported.In this study,A549 non-small cell lung cancer cells were cultured in vitro.After the intervention of beta-elemene and alfatinib,the changes of cell proliferation,spherogenesis,self-renewal,expression of OCT4 and drug sensitivity were studied by means of molecular biology and cell biology.So we investigated whether bELE could enhance the effect of afatinib on NSCLC.We hope to provide new targets and directions for better treatment of lung cancer in clinic through this study.In this study,we found that bELE could inhibit the growth of cancer stem cells by down-regulating OCT4 expression,thereby enhancing the killing effect of afatinib on NSCLC.Objective:1.To study the effect of?-Elemene on the proliferation of A549 cells inhibited by afatinib and on apoptosis of A549 cells induced by afatinib.2.To study the effect of?-Elemene combined with afatinib on the self-renewal ability of A549 cells.3.To study the effect of?-Elemene combined with afatinib on the Proliferation of A549 Cells with Stem Cell-like Features and the expression of OCT4 in lung cancer A549 cells treated with the Combination of bELE and Afatinib.Methods:1.CCK-8 was used to detect the cell viability and proliferation of lung cancer A549 cells after elemene,alfatinib and elemene combined with alfatinib at different concentrations.2.The morphological changes of cells in each group were observed under the microscope after the treatment of elemene,alfatinib and elemene at different concentrations combined with alfatinib.3.MTT was used to detect drug sensitivity.4.Cells in each group were labeled with FITC-CD133 and PE-OCT4 respectively.The percentage of CSCs in A549 cells was detected by flow cytometry.5.The self-renewal ability of lung cancer cells in each group was tested by tumour spherogenesis experiment.6.Cells were treated by grouping,stained by flow cytometry,and then observed under the microscope.The distribution of apoptotic cells in each stage was observed,and the apoptotic cells were detected and analyzed.7.Western blot was used to detect the expression of apoptotic proteins in cells treated with different conditions.8.The expression of OCT-4 was detected by qRT-PCR and Western blot after elemene,alfatinib and elemene combined with alfatinib at different concentrations.Results:1.The IC50 of lung cancer A549 cells treated with?-Elemene for 24 hours was 25ug/ml.The IC50 of lung cancer A549 cells treated with afatinib for 24 hours was 5?M.Compared with the group without pre-treatment with?-Elemene,the inhibitory effect of afatinib on A549 cell proliferation was significantly enhanced in the group with pre- treatment with?-Elemene.2.After Hoechst 33258 staining,the number of labeled living cells in the group of?-Elemene combined with afatinib was significantly less than that in the other groups,and the cell survival rate was decreased. 3.The proportion of OCT-4 positive cells in?-Elemene and afatinib group was decreased separately.Compared with single component,their combined application could significantly reduce the proportion of OCT-4 positive cells.4.Alfatinib inhibited the growth of A549 cells treated with beta-elemene for 6hours,and the inhibitory effect increased with the increase of the concentration of alfatinib.5.The combination of?-Elemene and afatinib inhibited the spherical formation of tumor cells.6.When A549 cells were treated with?-Elemene and/or afatinib for 24 hours,the combined effect of them could induce apoptosis more effectively than that of?- Elemene and afatinib alone(P<0.05).7.The combination of beta-elemene and alfatinib could increase the expression of apoptotic protein,while the expression of anti-apoptotic protein was inhibited.8.Compared with the single use of?-Elemene and afatinib,their combination significantly reduced the expression of OCT-4.The combined use of?-Elemene and afatinib also significantly reduced the expression of OCT-4 at the level of mRNA compared with the single application.Conclusion:1.Proliferation Inhibition Effect of afatinib Is Enhanced Synergistically by bELE in A549 Cells,Induction of Apoptosis by afatinib Is also Enhanced Synergistically by bELE in A549 Cells.2.The self-renewal ability of A549 cells was weakened by bELE combined with afatinib.3.The Combination of bELE and Afatinib reduced Inhibited the Proliferation of A549 Cells with Stem Cell-like Features.And the Combination of bELE and Afatinib Downregulated the Expression of OCT-4.