| Research Purpose:Cinnamaldehyde(CA)is a main chemical component of the essential oil separated from the traditional herb Cinnamomum cassia.Our previous in vitro experiments have proved that CA possesses conspicuous effects incluing apoptosis induction and invasion suppression against various cancer cells,whose underlying mechanism has not been clearly expounded.In addition,we find that epithelial-mesenchymal transition might contribute to the progression and metastasis of tumor.Our present study aims to provide insight into the efficacy of CA on apoptosis and EMT of human non-small cell lung cancer cell lines and the potential molecular mechanism,which might supply further theoretical foundation for Chinese medicine mediated anti-tumor effect against lung cancer.Research Methods:1 MTT assay was conducted to explore the growth prohibitive effect of CA on NSCLC cell lines A549,YTMLC-90 and NCI-H1299.2 Flow cytometry analysis and Hoechst 33258 staining assays were performed to detect the efficiency of CA on apoptosis and nuclear morphology of A549,YTMLC-90 and NCI-H1299.3 An inverted phase contrast microscope was applied to observe the cell morphology variations subsequent to the hypoxia condition simulated by CoCl?.4 Wound-healing assay was used to detect the changes of cell migrative ability.5 Trans well assay was carried out to study the changes of cell invasive capability.6 The influence of CA on the expression of apoptotic,invasive,EMT and Wnt/?-catenin related genes was evaluated by RT-qPCR analysis.7 Western blotting was performed to research the alterations of the expression of apoptotic,invasive,EMT and Wnt/?-catenin related proteins.8 Immunofluorescence staining experiment was conducted to explore the impact of CA on cytoplasmic accumulation as well as nuclear translocation of(3-catenin in A549,YTMLC-90 and NCI-H1299.9 We established xenograft mouse model with A549 cells and CA was intraperitoneally delivered.The suppressive rates of tumor volum and weight were caculated.10 The modifications of the activation of Wnt/?-catenin pathway and apoptotic,invasive,EMT associated genes in harvested tumors were detected by RT-qPCR,Western blotting and immunohistochemisty experiments.Research Results:1 The viability of three types of human NSCLC cell lines were efficiently restrained after treating with CA and the inhibition rate presented both dose-and time-dependent approximately.2 The apoptotic rates of A549,YTMLC-90 and NCI-H1299 cells rose as the concentrations of CA increased.Moreover,Hoechst 33258 staining revealed apparent nuclear condensation,fragmentation and chromatin shrinkage.3 RT-qPCR and Western blotting assays showed that both mRNA and protein levels of Bax increased while those of Bcl-2 and Bcl-xl decreased and both pro-caspase-3 and Pro-PARP get actuated.4 All the three cells converted into spindle-like mesenchymal morphology and lost tight junctions after hypoxia exposure induced by CoCl2 treatment.Besides,both wound-healing and Matrigel-coated Transwell chamber experiments suggested that CoCl2 prompt cell motility.5 E-cadherin was reduced while mesenchymal markers and matrix metalloproteinases(MMPs)were evidently over expressed at both levels which were demonstrated by RT-qPCR and Western blotting assays.6 CA led to not only slower cell movement to the wound district but also reduced number of cells passing through the Matrigel,implying attenuated cell migration and invasion.Moreover,the expression of E-cadherin enhanced while mesenchymal molecules and MMPs were antagonized gradually by CA.7 CoCl2 remarkably promoted ?-catenin and its downstream targets while CA led to prominent decreased expression.Moreover,immunofluorescence staining demonstrated that CoCl2 facilitates cytoplasmic accumulation and nuclear localization of ?-catenin,which get significantly restrained by CA.8 Specific activator LiCl effectively up-regulates ?-catenin and its downstream genes while the facilitation of which resulted from CoCl2 get abrogated by the inhibitor XAV939.In the meantime,cell motility was strengthened by LiCl but compromised when using XAV939.Next,variations of MMPs and EMT proteins caused by LiCl or XAV939 were more convincingly examined.Additionally,CA effectually terminated EMT progression as well as activation of Wnt/p-catenin pathway in agreement with the effect of XAV939 while LiCl not only orchestrated EMT and Wnt/?-catenin transcriptional activities but also partly opposed the inhibitive impact of CA.9 We established xenograft mouse model with A549 cells.Tumor xenografts in both CA groups grew more slowly and the average volume and weight of which were conspicuously lower compared to those in control group.10 After intraperitoneally deliverance of CA into xenograft mouse models,immunohistochemisty of tumor sections exhibited excessiveexpression of Bax,cleaved-PARP,E-cadherin but decreased levels of ?-catenin.Similarly,up-regulation of apoptotic molecules along with down-regulation of EMT related proteins and the components of the Wn tpathway were revealed through RT-qPCR and Western blotting studies.Research Conclusions:1.CA is able to inhibit the proliferation of human non-small cell lung cancer cell lines A549,YTMLC-90 and NCI-H1299 and induce cell apoptosis via improving Bax/Bcl-2 ratio as well as activating Caspase-3 and PARP.2.CA could terminate NSCLC cell motility by way of suppressing epithelial-mesenchymal transition.3.CA reverses epithelial-mesenchymal transition of NSCLC cells through confronting Wnt/?-catenin signaling pathway. |
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