| ObjectiveTo verify the effect of Yiqi Chutan formula on intervening the TGF-β signaling pathways, reversing epithelial to mesenchymal transition, inhibitting the action of the invasion and metastasis of lung cancer cells by culture A549 cells with the TGF-β1 to build the research platform, in vivo and vitro.Methods1:To establish research platform of EMT A549 lung cancer cell line (A549-M) by stimulating the A549 lung adenocarcinoma cancer cell line with the TGF-β1.2:In vivo, establish tumor-burdened mice model by subcutaneously inoculating A549 cells and A549-M cells in nude mice, respectively. Each group was separately treated with Yiqi Chutan formula(YQCTF) and normal saline, marked YQCTF groups and control groups. After 21 days of lavage, the mice were subjected to euthanasia. The volume and weigh of the tumors were recorded, and tumor inhibitory rates were calculated. The expression of EMT-related proteins were detected by western-blot.3:In vitro, MTT method for determination the effect of different concentration of serum containing on cell proliferation;four groups were established separately treated with blank serum, drug-contained serum TGF-31 (10 ng/mL)+blank·serum, SB-431542+TGF-β1 (10 ng/mL)+blank serum and TGF-β1 (10 ng/mL)+drug-contained serum. Morphological changes were observed under phase contrast microscope; cell invasion and metastasis ability was tested by the scratches experiment and Transwell experiment;the expression of the EMT-related proteins and Smad2/3 protein were detected by western-blot.Results1:Typical forms of EMT appeared in A549 cells after 24 hours of TGF-β1(10ng/mL) stimulation. Obvious mesenchymal cells forms were showed after long-term of TGF-β1 incubation and were to maintain 5 days after the removal of TGF-β1.2:At the end of the experiment in vivo, the weight of the tumors in A549 YQCTF group and A549-M YQCTF group were obviously reduced compared with A549 control group and A549-M control group, respectively. The tumor inhibitory rate in A549 YQCTF group and A549-M YQCTF group were 31.3% and 29.08%, respectively. The volume of the tumors in four groups were on the increase after the establishment of the mice model. On the 1st,3rd and 6th day, no significant statistical difference was showed between the four groups. The volume of the tumors in A549 YQCTF group were significantly reduced compared with A549 control group on 15th,18th and 21st day, separately, while the tumors’ volume of A549-M YQCTF group were reduced compared with A-549 control group on 18th and 21st day. However, significant difference were neither showed between the A549 control group and the A549-M control group nor between the A549 YQCTF group and the A549-M YQCTF group(P>0.05). Results showed that the YQCTF could increase the weight of nude mice, but not statistically significant compared with control group. Western-blot results indicated that the expression of E-cadherin in A549-M control group was lower and the expression of Vimentin is higher compared with A549 control group. The expression of E-cadherin was lower and the expression of Vimentin was higher in A549-M YQCTF group and A549 YQCTF group compared with A549-M control group and A549 control group, separately. The expression of Smad2/3 in A549-M control group and A549-M YQCTF group was higher compared with A549 control group and A549 YQCTF group, and was higher in A549-M control group compared with A549-M YQCTF group.3:Experiment in vitro showed that normal A549 cells appeared to be the paving stone sample cubic shape under microscope, and after 24 hours of stimulation of TGF-β1 (10ng/mL) the shape of the A549 cells was transformed from cuboidal epithelium form to long and thin spindle form. After treated with 20% drug-contained serum, cell apoptosis was observed and epithelial-mesenchymal transition was reduced compared with blank serum group. TGF-β1-induced A549 cells with SB-431542 showed reduced epithelial-mesenchymal transition compared with group without SB-431542. MTT results showed that after 24 hours of intervention, cell growth inhibitory rates in 20% drug-contained serum group,10% drug-contained serum group,5% drug-contained serum group and 2.5% drug-contained serum group were lower compared with 30% drug-contained serum group (P<0.01), while no significant difference was showed between each two groups of the four groups. After 36 hours of intervention, cell growth inhibitory rates in 10% drug-contained serum group,5% drug-contained serum group and 2.5% drug-contained serum group were lower compared with 20% drug-contained serum group and 30% drug-contained serum group (P<0.01), while no significant difference was showed between each two groups of the first three groups. After 48 hours of intervention, cell growth inhibitory rates in 10% drug-contained serum group,5% drug-contained serum group and 2.5% drug-contained serum group were lower compared with 30% drug-contained serum group (P<0.01), and 2.5% drug-contained serum group lower compared with 20% drug-contained serum group. No significant difference was showed between each two groups of 20% drug-contained serum group,10% drug-contained serum group and 5% drug-contained serum group. After 72 hours of intervention, cell growth inhibitory rates in 10% drug-contained serum group,5% drug-contained serum group and 2.5% drug-contained serum group were lower compared with 20% drug-contained serum group and 30% drug-contained serum group (P<0.01-0.05), and no significant difference was observed between each two groups of 10% drug-contained serum group,5% drug-contained serum group and 2.5% drug-contained serum group. When treated with 30% drug-contained serum, cell growth inhibitory rates were higher when treated after 24 hours compared with 48 hours or 72 hours treatment (P<0.01). When treated with 20% drug-contained serum, cell growth inhibitory rates were higher when treated after 36 hours,48 hours and 72 hours compared with 24 hours treatment (P<0.01); When treated with 10% drug-contained serum, cell growth inhibitory rates were higher when treated after 72 hours compared with 24 hours or 36 hours treatment (P<0.05); When treated with 5% and 2.5% drug-contained serum, there was no significant difference between evey group. Drug containing serum inhibits the cell proliferation rate was correlated with the concentration and the effect of the length of time. Scratch experiment results showed that the nick spacing in blank serum group is broader than in TGF-β1+blank serum group and TGF-β1+drug-contained serum group (P<0.01～0.05), Transwll experiment results showed that transmenbrane cell number in blank serum group was significantly different from TGF-β1+blank serum group (P=0.000). Transmenbrane cell number in blank serum group was significantly different from TGF-β1+drug-contained serum group (P=0.003). Transmenbrane cell number in TGF-β1+blank serum group was significantly different from TGF-β1+drug-contained serum group (P=0.000)Western-blot results showed that TGF-β1 could reduce the expression of E-cadherin, while SB-431542 and YQCTF could suppress this progress, and YQCTF drug-contained serum could up-regulate the expression of E-cadherin. TGF-β1 could up-regulate the expression of Vimentin, while SB-431542 and YQCTF could reduce this process, and YQCTF drug-contained serum could down-regulate the expression of Vimentin. TGF-β1 could stimulate the expression of the Smad2/3 pathway, while SB-431542 and YQCTF could reduce the expression of this pathway.Conclusion1:TGF-β1 could induce the transformation from epithelium cells to mesenchymal cells of human lung adenocarcinoma cell line A549.2:YQCTF could possibly reverse the cell transformation from epithelial to mesenchymal and suppress the growth, invasion and matastasis of lung cancer cells through inhibiting the expression of Smad2/3 pathway, up-regulating the expression of E-cadherin and down-regulating the expression of Vimentin. |
PDF Full Text Request