| Objective: To investigate 6,8-ditrifuoromethyl-7-acetoxychrysin(dFMAChR) induces human lung carcinoma cell line (A549) cells growth inhibition and apoptosis in vitro,and evaluate anti-lung cancer effect of dFMAChR in vivo, and explore the mechanism of dFMAChR to induce lung cancer cell apoptosis by activating PPARγ,upgrading PTEN, inhibitting p-Akt, and activating Caspase-3, for provide a laboratory and theoretic evidence on searching for a new potent and promising chemical entity for lung cancer chemotherapy.Methods: Human lung carcinoma cell line (A549) cells were cultured in vitro. The proliferative inhibitory effect of dFMAChR on A549 cells were examined by MTT assay. Plate-colon and Soft ager-colon assay was used to test colon formation inhibitory effect of A549 by dFMAChR. DNA agarose electrophoresis was used to detect the fragment of DNA of A549 cell. Hochest staining was used to observe apoptosis induced function of dFMAChR to A549 cells . PI stain flow cytometry (FCM) was used to analyse the apoptosis after treatment with dFMAChR. Human lung carcinoma xenograft in nude mouse model was established to investigate the anti-lung cancer effect of dFMAChR in vivo.To assess the effect of dFMAChR on tumor angiogenesis, immunohistochemistry for VEGF was used. Effect of dFMAChR on the expression of PPARγ, PTEN,p-Akt and caspase-3 protein in A549 cells was detected by western blotting, to explore molecular mechanism of the anti-lung cancer action of. dFMAChR.Results: MTT assay showed inh dFMAChR ihibited proliferation and growh of A549 cells in a dose-dependent. the proliferation inhibitory rate was 11.26%, 18.60%, 39.46% ,48.11% ,69.99% and 53.85% respectively after treatment with dFMAChR at various concentration of 0.3,1.0,3.0,10.0,30.0μM and Chrysin at 30.0μM for 48h. The proliferation inhibition rate(69.9%)of dFMAChR at 30.0μM is higher than that of the lead compound Chrysin (53.85%) at same concentration. Plate-colon assay showed that the colon inhibitory rate was 25.0%,50.0%,75.6% and 17.3% after treatment with dFMAChR at of various concentration 3.0,10.0,30.0μM and 30.0μM ChR for 8d respectively. the colon inhibitory rate of dFMAChR (75.6%) at 30.0μM is higher than that of the lead compound Chrysin (17.3%) at corresponding concentration. Clone formation in soft agar showed that the colon inhibitory rate was 43.67%,58.50%,84.03% and 26.67% after treatment with dFMAChR at various concentration 3.0,10.0,30.0μM of and 30.0μM ChR for 8d respectively. the colon inhibitory rate(84.03%) of dFMAChR at 30.0μM is higher than that of the lead compound Chrysin (26.67%) at corresponding concentration. The potency of dFMAChR was stronger than that of lead compound ChR. DNA agarose gel electrophoresis shown that DNA ladder bands could appear after treatment with dFMAChR at 10.0,30.0μM for 48h. dFMAChR significantly induced apoptosis. The results of Hochest staining showed that the apoptosis of A549 cells can be induced by dFMAChR(3.0,10.0,30.0μM),We could find the number of apoptosis cells increased accompaniment by the increasement of concentration ,showed the apoptosis cells nucleus condensation. The potency of dFMAChR was stronger than that of lead compound ChR. PI staining flow cytometry(FCM) analysis indicated that treatment after with dFMAChR for 48h in the concentration of 3.0,10.0 ,30.0μM and the same concentration ChR for 48h, the rate of apoptosis was 12.01%,33.9%,43.5%and 5.80%,8.4%,25.3% respectively ,and the rate of apoptosis of positive control drugs DDP and TAX was 8.3%,12.5% which revealed that dFMAChR significantly induced apoptosis. Therapeutic trial of human lung carcinoma xenograft in nude mouse model indicated that dFMAChR significantly inhibited the growth of human lung carcinoma xenografts in Balb/c-nu mice. The tumor weight inhibitory rate of dFMAChR at 10.0 mg/kg,20.0 mg/kg,40.0mg/kg was 56.7%,76.1% and 82.8% respectively. Immunohistochemistry for VEGF was used . dFMAChR significantly inhibited the VEGF expression of human lung carcinoma cell xenografts in Balb/c-nu mice. Treatment at various concentration dFMAChR and ChR for 48h, the expression of PPAR,PTEN and Caspase-3 was enhanced expression of p-Akt was decreased in a time and concentration-dependent. Western-blotting analysis indicated that after exposure to dFMAChR at 0.3,3.0,30.0μM for 24h the protein level of PPARγ,PTEN and Caspase-3 was up-regulated by 11.2%,28.6%,43.5% , 15.4%,27.3%,35.6%,12.8%,26.9%,42.5%,the protein expression of p-Akt was down-regulated by 6.8%,27.9%,44.1% respectively in comparision with the control group. After A549 cells were treated with dFMAChR at 10.0μM for 6,12 and 24h, the protein expression of PPARγ,PTEN and Caspase-3 was up-regulated 6.3%,29.5%,46.7%,35.3%,43.1%,57.5%,25.3%,38.8%,56.5%,the protein expression of p-Akt was down-regulated by 34.9%,47.2%,55.1%,respectively in comparision with the control group.Conclusion:1) 6,8-ditrifluoromethyl-7-acetoxychrysin posses the inhibitory effect of the proliferation and growth of A549 cells in a dose-dependent manner.2) 6,8-ditrifluoromethyl-7-acetoxychrysin can significantly induce apoptosis of A549 cells.3) 6,8-ditrifluoromethyl-7-acetoxychrysin can significantly inhibit the growth of human lung carcinoma xenograft in nude mice in dose-dependent manner.4) The inhibition of growth and induction of apoptosis of A549 cells by 6, 8-ditrifluoromethyl-7-acetoxychrysin are associated with upregulation of PPARγ,PTEN and Caspase3 protein level expression and downregulation of PAkt protein level expression.
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