Studies On Anti-tumor Activities And Mechanisms Of DN3,a Natural Ent-kaurane Diterpenoid Analog

Object:To explore the antitumor activity and molecular mechanism of DN3,a new natural ent-kaurene diterpenoid,in vitro.Method1.We evaluated the antitumor activity of DN3 by cytotoxicity test,in vitro.The tumor cells included esophageal cancer cell line EC-109,lung cancer cell line PC-9,prostate cancer cell line PC-3,gastric cancer cell lines HGC-27,MGC-803,BGC-823,gastric epithelial cell line GES-1.We found that gastric cancer cells were sensitive to the DN3.MGC-803,HGC-27 were selected from the gastric cancer cells as the test cell lines,GES-1 as normal cell.DAPI staining was used to detected the changes of cell nucleus by fluorescence microscope;the flow cytometry was used to quantitatively analyze the apoptosis of tumor cells by DN3.The effect of DN3 on the mitochondrial membrane potential was analyzed by fluorescence microscopy and flow cytometry.The changes of ATP content in unit protein cells were detected by fluorescence microplate reader.The expressions of p53,caspase-3 associated mitochondrial pathway protein were detected by western blot.2.The effect on clone formation and migration were induced by DN3 to tumor cells.Cell clone formation was detected by cloning method.Scratch-healing assay and transwell assay were used to explore cell migration.Western blot was performed to detect E-cadherin,?-catenin and N-cadherin which were related to EMT signaling pathway.3.Reactive oxygen scavenger NAC can reverse the antitumor activity of DN3 or not.After adding NAC to preincubate cells,cytotoxicity test,apoptosis detection test,JC-1 staining test and western blot were used to detect the reverse effect of NAC on the anti-tumor activity induced by DN3.Results1.DN3 which treated cells 24 h had a significant proliferation inhibitory on gastric tumor cells.The cytotoxicity of HGC-27 and MGC-803 were 4.72±1.37?M,7.36±0.86?M,respectively,which were sensitive relatively and showed a concentration dependence to the compound.However,GES-1 was 22.54 ± 1.65 ?M,so we can say that the DN3 had a certain selectivity on tumor cells.Further studies found that DN3 made nucleus shrinking,cracking,formation of apoptotic bodies.It also had significant apoptosis induction,decreased the MMP within a short time,reduced ATP level.In addition,western blot analysis showed that DN3 induced up-regulation of p53,activated caspase-3.Cytochrome c was released from the mitochondria into the cytoplasm and hexokinase-2 was stripped from the mitochondrial membrane to the cytoplasmic matrix.2.Below the lower concentration of induced apoptosis,DN3 significantly inhibited the number and size of clones of gastric cancer cells as for MGC-803 and HGC-27.It significantly reduced the ability of cell healing and migration,also up-regulated?-catenin and N-cadherin but down-regulated E-cadherin protein expression.3.After preincubation of NAC,the cytotoxicity of DN3 was significantly reduced.The effect on inducing apoptosis and membrane potential reduction induced by DN3 were also obviously reversed.More interestingly,NAC can also block the regulation of DN3 on Bcl-2 family associated proteins(Bim,Bad etc.)Conclusion DN3 can inhibit the proliferation of various tumor cells and has obvious tumor cell selectivity.It is the first time that DN3 can induce a series of molecular signals to induce cell apoptosis and migration by inducing the rise of ROS level,which provides a material and theoretical basis for the development of new antitumor drugs.