| Lung cancer is one of the most common malignant tumors in clinical, which mortality and morbidity ranks the first cancer. According to the survey of WHO, lung cancer has become the highest malignant tumor morbidity and mortality in wordwide. Ar present, the five year survival rate of less than 15% of patients with lung cancer, the main causes of death form lung cancer is the tumor invasion and metastasis. In recent years, the study found that the epithelial mesenchymal transition (EMT) is an important cause of lung cancer metastasis, its specific mechanism to explore the target drugs for the prevention of tumor recurrence and development of new antitumor therapy has important significance. PTEN signaling pathway play an important role in process of EMT in tumor cells, targeting inhibition of PTEN signaling pathway induced by EMT as a new strategy for the treatment of tumor.In traditional Chinese medicine, qi stagnation and blood stasis is the basic etiological factor and pathogenesis of tumor occurrence, development, which is closely related to the tumor metastasis and malignant. According to syndrome differentiation and treatment, attained perfect legislative principles, promoting blood circulation and removing blood stasis method as the basic method of tumor therapy has been widely used in clinical. The previous studies screened the antitumor activity of the promoting the blood circulation of traditional Chinese medicine, which found that Salvia miltiorrhiza and its effective components has many biological effects on tumor cells. Further studies found that Salvianolic acid A (Sal A), the main water soluble components of Salvia miltiorrhiza, has specific antitumor activity, which could significantly upregulated the phosphorlation level of PTEN protein, however, the regulation of the mRNA level is not obvious. Furthermore, it has safty and low toxicity to human normal cells, which could selectively induce tumor cell apoptosis and inhibit tumor cell invasion and migration. However, the molecular mechanism of its specific antitumor effect remains unknown. Thus, the main objective of this research is to carry out a systematic study of Sal A on the mechanism of inhibiting tumor cell invasion and migration. And put formward the hypothesis:"The mechanism of salvianolic acid A on anti-tumor specificity maybe through targeting PTEN protein, regulating the PTEN signaling pathway and inhibiting the epithelial mesenchymal transition of lung cancer."Part one:Literature reviewBy means of reviewing relevant literatures, the current status of studied on the anti-tumor metastasis of traditional Chinese medicine, Anti lung cancer metastasis research, promoting blood circulation to remove blood stasis of antitumor metastasis and the antitumor metastasis of tumor metastasis research on Salvia miltiorrhiza (Dansen) and sal A were systematic summarized, which lays the foundation for providing practical research programs and determining the study of examined indicators and the starting point on this thesis.Part two:Experimental study1. Elucidated the effects of Sal A on EMT of lung cancer1.1 Investigated the effects of sal A on cell morphology in TGF-β1 induced lung cancer EMT cells. The microscope observation method demonstrated that Sal A treated cells were epithelioid cell phenotype, and Sal A could reverse cells from EMT and increase the adhesion between cells. The results indicated that Sal A could inhibit the cell morphology changes of lung cancer EMT.1.2 Investigated the effects of sal A on cell migration and invasion in TGF-β1 induced lung cancer EMT cells. The cell scratch detection demonstrated that Sal A could significantly inhibit the cell migration of TGF-β1 induced lung ccancer EMT cells (P< 0.05) in a time-dependent manner. The Real-time Cell Analysis (RTCA) showed that Sal A could significantly inhibit the cell invasion of TGF-β1 induced lung ccancer EMT cells(P< 0.05) in a time-dependent manner. The results indicated that Sal A could significantly inhibit the cell migration and invasion of lung cancer EMT cells.1.3 Investigated the effects of sal A on mRNA and protein expression of EMT marker in TGF-β1 induced lung cancer EMT cells. Q-PCR analysis showed that Sal A could significantly increased the mRNA expression of E-cadherin (P< 0.01) and decreased the mRNA expression of Fibronectin and Vimentin (P< 0.01). Immunofluorescence-High content screening (HCS) methods combined with western blot assay demonstrated that Sal A could significantly increased the protein expression of E-cadherin (P< 0.01) and decreased the protein expression of Fibronectin, Vimentin and Snail(P<0.01). The results indicated that Sal A could significantly inhibit the EMT marker in lung cancer EMT cells.1.4 Investigated the effects of sal A on cell skeleton in TGF-β1 induced lung cancer EMT cells. Immunofluorescence-High content screening (HCS) methods showed that Sal A could significantly decrease the number of cell microfilament skeleton (P< 0.01), cytoskeleton area (P< 0.01) and inhibit cytoskeleton rearrangement. The results indicated that Sal A could inhibit the lung cancer cytoskeleton rearrangement, resulting in inhibition of lung cancer EMT.2. Elucidated the molecular mechanism of Sal A on lung cancer EMT through PTEN protein and PTEN signaling pathway.2.1 Investigated the effects of sal A on lung cancer EMT cells through PTEN protein. Immunofluorescence-High content screening (HCS) methods combined with Nanopro analysis demonstrated that Sal A could significantly upregulated the phosphorylation level of PTEN protein (P< 0.01). The results indicated that Sal A could inhibit lung cancer EMT maybe through upregulating the phosphorylation level of PTEN protein.2.2 Investigated the effects of sal A on lung cancer EMT cells through PTEN signaling pathway. Nanopro analysis demonstrated that Sal A could significantly inhibted the PI3K/AKT signaling pathway. However, the inhibition of FAK/P130cas and SHC/MEK/ERK1/2 signaling pathways do not significantly. The results indicated that Sal A could inhibit lung cancer EMT maybe through inhibiting the PI3K./AKT signaling pathway.2.3 Investigated the effects of sal A on cell migration and invasion in lung cancer EMT cells by PTEN inhibitor and siRNA-PTEN interference technology. PTEN inhibitor and s/RNA-PTEN interference technology demonstrated that Sal A could weaken the upregulation of E-cadherin and inhibited cell migration and invasion in lung cancer EMT cells when application of PTEN inhibitor and siRNA-PTEN interference. The results indicated that Sal A could inhibit lung cancer EMT maybe through targeting the PTEN protein and inhibiting the PI3K/AKT signaling pathway.3. Elucidated the interaction of Sal A and PTEN protein.3.1 Investigated the interaction and affinity of Sal A and PTEN protein. The molecules interaction instrument Octect assay demonstrated that Sal A and PTEN protein has interaction and strong sffinity (KD=7.63×10-7). The results indicated that Sal A could inhibit lung cancer EMT maybe through targeting the PTEN protein and interaction with PTEN protein.3.2 Investigated the combined effect after interaction between Sal A and PTEN protein. GENMED PTEN protein phosphatase activity assay kit showed that Sal A could significantly promote the PTEN protein phosphatase activity (P<0.05). The results indicated that Sal A could inhibit lung cancer EMT maybe through targeting the PTEN protein and increasing the PTEN protein phosphatase activity.3.3 Predicted the binding sites of Sal A and PTEN protein. GLIDE reverse docking technology demonstrated that Sal A maybe target the S4 binding pocket of PTEN protein, weaken the inhititory effect of C2 domain on Ptase domain and increase the amount of PTEN generated hydrolysates. The results indicated that Sal A could increase the PTEN protein phosphatase activity maybe through targeting the S4 binding pocket of PTEN protein, weaken the inhititory effect of C2 domain on Ptase domain and increase the amount of PTEN generated hydrolysates. |
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