The Effect And Molecular Mechanism Of Ailanthone Inhibiting The Growth Of Non-small Cell Lung Cancer Through Repressing DNA Replication Via Downregulating RPA1

【Background and Objective】Lung cancer is the first in the incidence and mortality of malignant tumors in China,and non-small cell lung cancer accounts for 80%-85% of the total number of lung cancers.With the in-depth study of traditional Chinese medicine,it has become a focus on against cancer.RPA1(replicating protein A1)has been shown to be highly expressed in a variety of tumors,but it has not been studied in non-small cell lung cancer.In the previous work,it was found that Ailanthone,a main active compound of the stem barks of Ailanthus altissima,can inhibit the growth of non-small cell lung cancer.Therefore,This study focused on the mechanism of Ailanthone inhibiting the growth of non-small cell lung cancer,Comparison of pharmacodynamics between Ailanthone and clinical first-line treatment drugs,expression of RPA1 and its role in non-small cell lung cancer.【Methods】Firstly,the expression of DNA damage marker γ-H2 AX was detected by immunofluorescence;Detection of differentially expressed genes and signaling pathways in Ailanthone on non-small cell lung cancer by c DNA microarray;Brd U incorporation and fluorochrome-labelled antibody detection were applied to detect the DNA replication by Ailanthone;detection of changes in m RNA levels of RPA1,PCNA,POLE2 and LIG1 in the DNA replication pathway by real time PCR;detection of changes in RPA1 and PCNA protein levels by western blot;Confirmation of antitumor effect of skunk ketone by establishing RPA1 overexpressing cell line;the anti-tumor effect of Ailanthone in vivo was detected by subcutaneous xenograft tumour models and orthotopic lung tumour models and bioluminescent imaging.Secondly,Analysis of expression and survival analysis of RPA1 in lung adenocarcinoma by TCGA database;Detection of RPA1 m RNA and protein levels in 8 non-small cell lung cancer tissues and adjacent normal tissues by real time PCR and western blot;The effect of RPA1 on the activity of non-small cell lung cancer by establishing RPA1 knockout cell line.Finally,the anti-tumor effect of Ailanthone and clinical first-line chemotherapy drug cisplatin was compared in vitro and in vivo by MTT activity assay and A549 subcutaneous tumor-bearing mouse model.【Results】1.Ailanthone induced DNA damage in H1299 and H1975 cells but not in A549 cells.2.c DNA microarray showed that Ailathone significantly changed 1222 genes,involving 21 signaling pathways in A549、H1299 and H1975.3.Ailanthone can inhibit DNA replication in non-small cell lung cancer.4.Ailanthone can down-regulate the expression of RPA1 and PCNA and inhibit DNA replication mainly by down-regulating RPA1 protein.5.The inhibitory effect of Ailanthone on RPA1 overexpressing non-small cell lung cancer was reduced.6.Ailanthone inhibits tumor growth in a variety of non-small cell lung cancer models.7.RPA1 is highly expressed in non-small cell lung cancer,which is closely related to poor prognosis in lung adenocarcinoma.8.The activity of RPA1 knockout non-small cell lung cancer was significantly inhibited.9.The results of MTT assay showed that Ailanthone inhibited non-small cell lung cancer with higher effect than cisplatin in vitro and in vivo.【Conclusion】1.Ailanthone inhibits the growth of non-small cell lung cancer mainly by down-regulating RPA1 which inhibition of DNA replication in vitro.2.Ailanthone can inhibit the growth of subcutaneous xenograft tumour models and orthotopic lung tumour models and prolongthe survival of mice.3.RPA1 is highly expressed in non-small cell lung cancer and the cell activity of RPA1 knockout is significantly decreased.4.The inhibitory effect of Ailanthone on the growth of non-small cell lung cancer in vitro and in vivo is stronger than cisplatin.