| BACKGROU:NDLung cancer is the most frequent malignancy all over the world,and the main cancer-related cause of mortality worldwide in both males and females combined[2].In China,there were an nearly 343,991 new cases of lung cancer(19.30%of total cancer incidence cases)and 286,978 lung cancer-related deaths(25.54%of total cancer mortality cases)during 2008-2012,which made lung cancer rapidly became first in the malignant neoplasm for both incidence and mortality[3].Unfortunately,a great many lung cancers were confirmed in its advanced stage and became unresectable.For these patients,chemotherapy became the main treatment.Cisplatin,an effective agent,using worldwide for the treatment of multiple cancers,including lung cancer.To reach the best treatment outcome,it has to be administrated repetitively.However,its therapeutic efficacy is limited due to the development of acquired resistance,and reduced the survival rate of patients.Clinical tumour resistance to chemotherapy could be intrinsic or acquired.Acquired resistance occurs in tumours which are often highly responsive to initial treatment,but on tumour recurrence,exhibit an absolutely different phenotype,and become resistant to both primarily used drugs,and new agents with extremely different structures and mechanisms of action.Clinical resistance to various anticancer agents should not be confused with'multidrug resistance',which in experimental models is related to increased expression of P-glycoprotein(P-gp).Experimental models prove that multidrug resistance can be induced by increased expression of adenosine triphosphate(ATP)-dependent efflux pumps[4].These pumps actively transport chemotherapy drugs out of the cell,The ABCl multi-drug resistance(MDR1)gene generates the transporter P-gp[5],Which is overexpressed in many multidrug resistant cells[6-12].Autophagy is a lysosomal degradation approach that is essential for survival,differentiation,development,and homeostasis.Autophagy primarily serves an adaptive role to protect organisms against various diseases,including infections,cancer,neurodegeneration and heart disease.However,in certain experimental disease models,paradoxically,even the prosurvival functions of autophagy may be detrimental.Excessive activation of autophagy could lead to cell death by destroying major proportions of cytoplasm[13].Thus,autophagic cell death,which would kill cells as an alternative pathway except for apoptosis,represents a new approach to inhibit the growth of apoptosis-resistant cancer cells[14-19].It's believed that diverse Chinese traditional medicine closely related to anti-NSCLC activities,and that even could impair the acquired multiple drug resistance(MDR).What's more,autophagy might be the potential mechanisms which would play a role as a candidate targets of natural active compounds.Recent studies of terpenoids,alkaloid,dietary polyphenols and other active constituents that extracted from various herbs indicates that different natural compounds could either regulate the activity of pro-death autophagy or affect the level of protective autophagy of NSCLC cells,that changing their drug sensitivity and cell viability[20-27].MethodsCell viability was measured by CCK8 assay.Cell proliferation was determined by colony formation assay.Cell invasion and migration ability were measured bywound healing assay and transwell assay.The expression of MDRlgene was valued by qPCR.Protein levels were analyzed by Western-blot analysis.The formation of autophagosome and autolysosome were visualized by electron microscope.Tumor xenograft experiment was established to estimate the effect in vivo.All the experiments were performed three times in triplicates,and results were expressed as meaną SEM.Differences between two groups were assessed by Student t test,and multiple comparisons were conducted using one way ANOVA through SPSS 16 software.Differences were considered statistically significant when P<0.01 or P<0.05.Results1.BBD inhibits the viability of A549 and A549/DDP cells in a dose-time dependent mannerWith the increase of the action time and concentration of Babaodan,the viability and value-adding ability of lung adenocarcinoma A549 and A549/DDP cells gradually weakened.2.BBD suppresses tumour growth via promoting the activity of pro-death autophagyThe increasing concentration of Babaodan does not cause changes in apoptosis-related proteins,but increases the autophagic flow,increases the expression of autophagy-specific protein LC3-?,and increases the number of autophagosomes in the cells to inhibit cells.Growing.And inhibition of autophagy,it can be seen that the inhibition of Babaodan on A549 and A549/DDP cells is also weakened.3.BBD increases cell autophagy through downregulating the PI3K/AKT/mTOR signaling pathwayWith the increase of the concentration of Babaodan,we found that the expression of p-PI3K,p-AKT,p-mTOR protein decreased,LC3-? protein increased;and the combination of PI3K pathway activator IGF-1,Babaodan caused LC3-II The increase was reversed to some extent.Thus,Babaodan increases autophagy by inhibiting the PI3K/AKT/mTOR pathway.4.BBD facilates the antitumour effects of cisplatin Compared with the cisplatin monotherapy group,a certain concentration of Babaodan combined with cisplatin acts on the cells at the same time,which can effectively inhibit the viability and migration invasive ability of A549 and A549/DDP.5.Tumor xenograft experiments demenstrates that BBD inhibits tumour growth and improves cisplatin sensitivityThrough the subcutaneous tumor formation experiment in nude mice,measuring the tumor volume and weight can make Babaodan inhibit tumor cell growth,increase the anti-tumor effect of cisplatin,and the drug process does not produce obvious toxic side effects.ConclusionWe conducted the first study on the role and mechanism of Babaodan:in lung adenocarcinoma by in vivo and in vitro experiments,and demonstrated that it can inhibit the autophagy level of A549 and A549/DDP cells by inhibiting PI3K/AKT/mTOR signaling pathway.Cell growth.At the same time,the expression of the multidrug resistance gene MDR1 and the level of the drug transporter P-gp were reduced,and cisplatin sensitivity was increased.This is of great significance for the exploration of the potential of Babaodan in the application of lung cancer and other tumors. |
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