The Molecular Mechanisms Of Magnolol On Inhibiting Metastasis And Inducing Apoptosis In Human Lung Cancer Cells

Several biological capabilities, including self-sufficiency in growth signals,insensitivity to anti-growth signals, resisting cell death, enabling replicative immortality,inducing angiogenesis, activating invasion and metastasis, immune escape and reprogramming of energy systems, have been acquired by cells during tumorigenesis and cancer metastasis. Although the traditional means of cancer treatment like radiotherapy and chemotherapy have got a certain effect, the efficacy of cancer therapy was disappointing for the specificity and refractoriness of cancer. The anti-cancer components from medical plants, as a supplementary means for radiotherapy and chemotherapy, have intrigued scientists for the high curative effect and low toxicity.Magnolol, a main constituent of the root and stem bark of the Chinese herb Hou pu(Magnolia officinalis), has been reported to have multiple pharmacological effects,including anti-inflammatory, anti-oxidation and antimicrobial effects. As one anti-cancer drug, magnolol could inhibit the proliferation and differentiation of tumor cells, induce tumor cell apoptosis and inhibit tumor metastasis and tumor angiogenesis.In our study, the molecular mechanisms of anti-lung cancer cell metastasis were researched systematically. Meanwhile, the mechanisms of magnolol inducing cell apoptosis in lung cancer also was studied briefly.The main works are as follows:1. The works on anti-metastasis of magnolol in lung cancer cell were as follows:(1) Inhibition rate of H460 and 95 D cells was tested by MTT assay after treatment of 6.25,12.5,25,50,100μM of magnolol for 24 h.(2) The CCK-8 was performed to evaluate the inhibitory effects on adhesion of magnolol in 95 D and H460 cells.(3) The transwell assay was employed to investigate the ability of magnolol on anti-migration and invasion.(4) The transcriptional level of MMP-2 was examined by real time quantitative PCR.(5) The western blot was employed to examine the protein level of MMP-2, Akt,PDK1 and c-Raf in PI3K/Akt signaling pathway.2. The works on inducing lung cancer cell apoptosis of magnolol were as follows:(1) After treated with magnolol(6.25,12.5,25,50,100μM) for different times(12h,24 h,48h,72h), the inhibitory effects of magnolol was shown by MTT assay in A549 cell line.(2) The change of cell shape was observed by fluorescent microscope via AOPI/DAPI stain.(3) The cell apoptosis rate of A549 cells was examined by flow cytometry after PE-Annexin V/7-ADD staining.(4) The transcriptional level of apoptosis related genes was examined by real time quantitative PCR.The results were as follows:1. The results of anti-metastasis study were as follows:(1) Magnolol inhibited the proliferation of 95 D and H460 cells in a dose-dependent manner after treatment for 24 h.(2) After treated with magnolol for 24 h, the results of CCK-8 showed that magnolol could suppress cell adhesion capability of A549 in a dose-dependent manner.(3) After treated with 25μM and 50μM of magnolol for 24 h, the results of Transwell with or without matrigel demonstrated that magnolol inhibited the cell migration and invasion of A549 cells.(4) After treated with 25μM and 50μM of magnolol for 24 h, the results from real time quantitative PCR showed that the MMP-2 expression level was decreased in transcriptional level.(5) The protein level of MMP-2, consistent with real time quantitative PCR, also been decreased. In addition, the results of western blot demonstrated that magnolol also decreased the phosphorylation level of Akt, PDK1 and c-Raf in PI3K/Akt signal pathway.2. The results of inducing cell apoptosis were as follows:(1) After treated with 6.25-100μM of magnolo for 12 h,14h,48 h and 72 h, the MTT assay demonstrated that the inhibition rate of A549 cells increased with increasing ofmagnolol concentration.(2) After dying with AO/PI and DAPI, the early apoptosis and late apoptosis were observed in fluorescent microscope in A549 cells which was treated with 25μM and50μM of magnolol, respectivly.(3) A549 cells labeled by PE-Annexin V/7-ADD exhibited early apoptosis and late apoptosis under treatment of 25μM and 50μM of magnolol.(4) The results of real time quantitative PCR showed that the expression of apoptosis suppressing genes, including Bcl-2 and XIAP, was inhibited on transcriptional level, while the expression level of apoptosis induced genes like Cyto-c and Caspase-3was up-regulated on transcriptional level.In conclusion, the results demonstrated that magnolol could inhibit lung cancer cell invasion and metastasis. In highly metastatic human lung cancer cell lines, magnolol decreased the MMP-2 expression on transcriptional and translational level via modulating the phophorylational level of key regulatory gene in PI3K/Akt signal pathway. Meanwhile, we discussed briefly the effect of inducing cell apoptosis of magnolol in A549 cell line. The results demonsted that magnolol could promote the process of cell apoptosis through mitochandrial pathwy. Therefore, the research of magnolol on the molecular mechanisms of anti-metastasis and inducing apopotosis provieds a scientific basis for its application in the area of anticancer.