Study The Mechanism Of Alocasia Rhizome In Treatment Of Non-small Cell Lung Carcinoma(NSCLC)

ObjectiveTo study the mechanism of Alocasia Rhizome in treatment of non-small cell lung carcinoma(NSCLC)both in silico and in vitro.Methods1.To study the mechanism of Alocasia Rhizome in silico(1)Collecting all compounds of Alocasia Rhizome for the target prediction and analysis of the KEGG and GO enrichment via PTS database and DAVID database;(2)All biomarkers targets of non-small cell lung cancer(NSCLC)are collected from the CTD database and compared with the predicted targets above;(3)Randomly selected 3 non-small cell lung cancer related protein targets from the common-targets data set,and pick out the small molecular compounds from Alocasia Rhizome by ranking score of molecular docking,which shows they have potential therapeutic effect of treating NSCLC.2.To study the mechanism of Alocasia Rhizome in vitro(1)To study the effects of different concentrations of candidates on A549 cell proliferation activity by the cell growth inhibition test(MTT);(2)To detect the effects of the apoptosis of A549 cells of different concentrations of candidates by using Flow cytometry after Annexin V-APC/7-AAD;(3)Flow cytometry was used to detect the influence of different concentration of candidates on the cell cycle of A549 cells;(4)Detection of expression of apoptosis related protein Bcl-2,Bax and Cleaved caspase-3 with Western Blot.Results1.28 compounds of Alocasia Rhizome were obtained successfully from large-scale text data,and the predicted 639 potential targets of these 28 compounds were collected through PTS database.The 639 potential targets were analyzed via KEGG pathway and GO enrichment analysis and found that there are 136 signaling pathways involved,including apoptosis pathway and multiple cancer associated signaling pathways,for example,the prostate cancer pathway,the non-small cell lung carcinoma signaling pathway and the small cell lung carcinoma pathway,etc.;in addition,the results of GO analysis suggested that the main function of these molecules associated with protein serine/threonine kinase activity,protein tyrosine kinase activity,etc.A total of 106 biomarkers of NSCLC were obtained from the CTD database.By comparison and analysis,there are 21 common targets between the 639 predicted targets and the 106 biomarkers.3 non-small cell lung carcinoma related protein targets were randomly selected(EML4-ALK、EGFR、KRAS).The small molecular compounds(Daucosterol and Grossamide)from Alocasia Rhizome were finally pick out based on ranking score of molecular docking,which indicated that they have potential therapeutic effect of treating NSCLC.2.The MTT assay results show that both Daucosterol and Grossamide could inhibit A549 cell proliferation with different concentration after 24 h,and the cell survival rates were significantly lower and showed a dose effect relationship between concentration(P <0.01),compared to the control group(0μ M);The apoptosis rate of A549 cells of different concentrations of Daucosterol and Grossamide were detected by FCM,and the results indicate the apoptosis ratio increased significantly(P <0.01),compared to the control group(0μ M);and the percentage of G1 phase cells of different concentration increased significantly after 24h(P<0.01).On the other hand,the cells of percentage in G2 and S phases decreased significantly(P <0.01),compared to the control group(0μ M);finally,the cell apoptosis-related protein indicators were detected by Western blot,and the results show that the different concentrations of Daucosterol and Grossamide significantly increased the expression of Bax and down-regulation of Bcl-2 protein expression(P <0.01),compared to the control group(0μ M),then the ratio of bcl-2/bax decreased;besides,with the increase of drug concentration,the expression of cleaved caspase-3 also increased significantly(P <0.01).ConclusionIn this study,Alocasia Rhizome has been discovered that it has multi-targets and involved in multi-layer signaling pathway through the bioinformatics analysis,suggesting that it can act on multi-targets and regulate multiple signaling pathways which play a significant role in the treatment of NSCLC.In addition,it has also been proved that chemicals of Alocasia Rhizome can influence the growth of tumor cells through arresting cell cycle and inducing apoptosis in vitro experiments.With the combination of doing research both in silico and in vitro,Alocasia Rhizome is regarding as a natural antitumor drug with development value as it has the characteristics of multi-elements and multi-targets of overall adjustment in the treatment of NSCLC.