Anti-proliferation And Inducing-apoptosis Mechanism Of Wentilactone A In Small Cell Lung Cancer

Cancer statistics in China,2015 demonstrated that lung cancer was the leading cause of cancer deaths in China.According to pathological classification,lung cancer is mainly divided into small-cell lung cancer(SCLC)and non-small cell lung cancer(NSCLC).SCLC,15% of all lung cancer cases,is an aggressive malignancy with a tendency of fast growing rates,early distant metstases,and poor prognosis.Drug resistance leads to high recurrence and high mortality rates with SCLC patients,and the 5-year survival rate of all SCLC patients is no more than 2%.Therefore,it is of great significance to develop SCLC drugs with good therapeutic effect and small side effect.Wentilactone A(WA)is a small molecule marine-derived norditerpenoid compound,which inhibited the proliferation of NCI-H446 by previous preclinical research.In this study,it confirmed the anti-proliferation and inducing-apoptosis effects of WA in SCLC and its mechanism,by using CCK-8 analysis,flow cytometry,cell scratch test,gene chip analysis,gene functional researchs,Western Blot,subcutaneous xenotransplanted tumor models.Part One the Anti-Proliferation and Inducing-Apoptosis Effects of WA in SCLCIn order to study the mechanism of WA anti-SCLC,using CCK-8 analysis,flow cytometry,cell scratch test,Western Blot,subcutaneous xenotransplanted tumor models,CCK-8 analysis confirmed the anti-proliferation effect of WA in SCLC cell lines NCI-H446,NCI-H1688 and LTEP-sm cells.Flow cytometry analysis result showed that WA induced the apoptosis of NCI-H446,NCI-H1688 and LTEP-sm cells in a time-dependent manner.Western Blot test result showed that WA effected the expression of apoptosis-related proteins c-FLIP and c-caspase-3.Cell scratch test result showed that WA inhibited SCLC cells migration.WA inhibited SCLC xenografts growth.In all,WA had anti-proliferation and anti-migration effects and induced the apoptosis of SCLC cells.Part Two the Inducing-Apoptosis Mechanism of WA in SCLC cellsIn order to study the inducing-apoptosis and anti-proliferation mechanism ofWA in SCLC,Affymetrix Human Transcriptome Array chip was used to screening significant changed genes,screening differentially expressed genes in the pathway analysis,and relatively active signaling pathway.Resluts showed that apoptosis pathway,PI3K-AKT pathway related genes significantl changed after WA treatment and the most significant changed gene was AKR1C1 gene,94.74 times down fold.Many researches showed that the expression of AKR1C1 was closely related to the apoptosis of tumor cells.So AKR1C1 was chosed to be deeply studied.Results of Western Blot and RT-PCR analysises confirmed the results of gene chip correctly.In order to study the mechanism of WA inhibiting the expression of AKR1C1 and the mechanism of apoptosis-inducing,overexpression and knock-down of AKR1C1 gene,flow cytometry analysis,Western Blot were used.Results showed that knock-down AKR1C1 gene induced the apoptosis of SCLC cells and depressed the growth of subcutaneous xenotransplanted tumors as same as WA treatment group;overexpression of AKR1C1 inhibited apoptosis of SCLC cells.WA depressed the phosphorylation levels of IGF-1R/IRS-1/PI3K/AKT/Nrf2 and the expression of AKR1C1.The IGF-1R/PI3K/AKT/Nrf2 pathway activator 50 ng/ml IGF-1 promoted the expression of AKR1C1 with lower apoptosis of SCLC cells,IGF-1 reversed the inducing-apoptosis effect of WA.What's more,the result of immunohistochemistry confirmed that WA inhibited the expression of AKR1C1.These results confirmed WA inhibited the expression of AKR1C1 via the IGF-1R/IRS-1/PI3K/AKT/Nrf2 signal pathway and induced apoptosis of SCLC cells.Through these research,we came up with the following conclusions:(1)WA inhibited the proliferation of SCLC cells and induced the apoptosis of SCLC cells;(2)AKR1C1 gene participated in the progress of anti-proliferation and inducing-apoptosis triggered by WA;(3)WA depressed the expression of AKR1C1 via IGF-1R/IRS-1/PI3K/AKT/Nrf2 pathway.