The Effect And Mechanical Study Of Luteolin Combined With Paclitaxel In Esophageal Carcinoma Both In Vivo And In Vitro

Aims1.The first aim was to investigate the in vitro effects and potential mechanisms of luteolin combined with paclitaxel on the cell viability,proliferation,migration and apoptosis of esophageal cancer cells.2.The second aim was to explore the in vivo effect of luteolin and paclitaxel on tumor growth,cells apoptosis and the molecular mechanisms,and to evaluate preliminarily the potential toxicity induced by luteolin and paclitaxel.Methods1.SRB assay was used to detect the effect of luteolin combined with paclitaxel on the cell viability.Colony formation assay was used to detect effect of luteolin combined with paclitaxel on the cell colony formation of esophageal cancer cells.The cell proliferation and tracer detection kit was used to detect the effect of luteolin combined with paclitaxel on the proliferation of esophageal cancer.2.Would healing assay and transwell assay were used to detect the effect of luteolin combined with paclitaxel on the esophageal cancer cell migration.Inverted microscope was used to observe morphology changes of esophageal cancer EC109 and TE-1 cells induced by luteolin combined with paclitaxel.Western Blotting assay was used to detect the expression of SIRT1 and EMT-related proteins.3.The apoptosis assay kit was used to detect the pro-apoptosis effect of luteolin combined with paclitaxel on esophageal cancer cells.The ROS Test Kit was to detect the level of ROS in esophageal cancer cells.Western Blotting assay was used to detect the expression of mitochondrial apoptosis pathway-related proteins and ROS/JNK pathway proteins.4.The esophageal cancer xenograft models were established to explore in vivo anti-tumor effect of luteolin combined with paclitaxel.HE staining and TUNEL staining were used to detect the effect of luteolin combined with paclitaxel on cell apoptosis.The toxicity of the the combination treatment was evaluated by the body weight changes and HE staining of the main organs of nude mice.Western Blotting assay was used to detect the expression of SIRT1,EMT-related proteins,mitochondrial apoptosis pathway-related proteins and ROS/JNK pathway related proteins in nude mice xenografts to explore the in vivo mechanism of luteolin combined with paclitaxel.Results1.luteolin combined with paclitaxel synergistically inhibited the cell viability,proliferation and colony formation of esophageal cancer cells.2.Luteolin and paclitaxel synergistically inhibited cell migration,inhibited the expression of SIRT1 protein,downregulated the expression of epithelial marker proteins(N-Cadherin,?-Catenin,Vimentin)and upregulated expression of epithelial marker proteins(E-Cadherin,Claudin-1,ZO-1).Luteolin combined with paclitaxel could change the morphology of esophageal cancer cells.3.Luteolin combined with paclitaxel synergistically induced cell apoptosis,downregulated the expression of Bcl-2 and Mcl-1 as well as upregulated the expression of Bax,Noxa,Cleaved Caspase-9 and Cleaved Caspase-3 proteins.Luteolin combined with paclitaxel synergistically increased the level of ROS in esophageal cancer cells and upregulated the expression of p-ASK1,p-MKK4,JNK and p-JNK proteins.4.The combination treatment of luteolin and paclitaxel could inhibit the growth of xenografts in nude mice and induce in vivo cell apoptosis;the molecular mechanisms were consistent with the results in vitro;this combination strategy had no significant effect on body weight of nude mice and had no obvious toxicity to heart,liver,spleen,lung and kidney of nude mice.Conclusions1.Luteolin combined with paclitaxel could synergistically regulate the proliferation,migration and apoptosis of esophageal cancer in vitro;the molecular mechanism of inhibiting cell migration is related to the inhibition of SIRT1 expression and EMT process;the mechanism of inducing apoptosis is associated with the activation of ROS/JNK pathway and the mitochondrial apoptotic pathway.2.Luteolin combined with paclitaxel synergistically inhibited tumor growth and promoted cells apoptosis of esophageal cancer xenografts.No obvious toxicity was observed in nude mice during the combined treatment in therapeutic doses.