The Study On Anti-Tumor Mechanisms Of Dithiocarbamate Derivatives With α,β-unsaturated Ketone Scaffold

In recent years,dithiocarbamates have become the focus of research in the field of pharmacy due to the unique molecular structure.In addition,it has a wide array of biological activities,including anti-fungal,anti-inflammatory,anti-tumor,antioxidation,inhibition of carbonic anhydrase activities and so on.Combining dithiocarbamate with other different anti-tumor scaffold is an important research field of dithiocarbamate chemical modification.In our previous studies,the dithiocarbamate has always been used as a linkage to combine different antiproliferative active scaffolds to design new chemical entities.Our group have successfully reported a series of dithiocarbamates derivatives as potential antitumor agents.In natural and synthetic drugs,α,β-unsaturated ketones are a very important pharmacophore due to its unique chemical structure and it could alkylate with sulfhydryl groups to bind with tumor cells and kill them.Therefore,it has been deeply studied in the development of antitumor drugs.Based on the this,our group utilized the principles of drug design,which led to the molecular hybridization of dithiocarbamate and α,β-unsaturated ketone scafford to integrate them in one molecular platform to generate a new hybrid.Additionally,we evaluated the antiproliferative activity of the derivatives in vitro.By screening,the compound with high biological activity is selected for further antitumor research.Looking forward to contributing the development of novel antitumor drug candidates and the discovery of efficient,low-toxic antitumor compounds.The main contents of this research are as follows:1.MTT assay was used to assess the anti-tumor activity of dithiocarbamate derivatives with α,β-unsaturated ketone scafford in vitro.The five kinds of tumor cell lines were selected for antitumor activity evaluation.The experimental results showed the synthetic derivatives induced cell inhibition in selected cancer cells,especially exhibited a stronger inhibitory effect better than 5-FU on PC3 cells.Compound ZL-8 showed the better anti-tumor activity.Therefore,ZL-8 was selected as the target compound for further mechanism study.2.We evaluated the effect of compound ZL-8 on PC3 cells proliferation.The study showed that compound ZL-8 inhibited PC3 cells proliferation in a concentrationdependent manner and decreased the clone formation dramatically.The results of comet assay and flow cytometry showed that compound ZL-8 could cause DNA damage,induce G2 / M phase arrest and affect the expression of related proteins at the G2 / M phase in PC3 cells.These results revealed that compound ZL-8 can inhibit the proliferation,causes DNA damage and induce cycle arrest in PC3 cells.3.The apoptotic pathway is one of the major ways of cell death,so we explored whether compound ZL-8 induced apoptosis in PC3 cells.The results showed that ZL-8 can significantly induce apoptosis in PC3 cells and the apoptosis rate was significantly increased with increases in their concentrations.Morphological changes of PC3 cells were determined using DAPI staining,a large number of cells appeared that small and shrinking of the nucleus,chromatin condensation,deepening,nuclear fragmentation with the same characteristics of apoptotic cells.By JC-1 staining assay to detect mitochondrial membrane potential and Western blot assay,we found that compound ZL-8 can significantly reduce the mitochondrial membrane potential,regulate the expression of Bcl-2 and IAP family proteins and upregulate the expression of death receptor DR5 and related FADD proteins,led to an increased activation of Caspase8 / Caspase3 to induce caspase-dependent apoptosis death.These results suggest that compound ZL-1 exerted anti-tumor effects by activating the mitochondrial pathway and death receptor DR5-mediated pathway to induce PC3 cells apoptosis.4.Numerous evidence has shown that overproduction of ROS plays an important role in anti-tumor.We used flow cytometry to detect intracellular ROS levels,the results showed that compound ZL-8 caused an increase of ROS level in PC3 cells.To further investigate the internal factors that causing increased intracellular ROS levels,we found that ZL-8 inhibits the activity of superoxide dismutase(SOD)and catalase in PC3 cells through the SOD activity assay kit and the catalase(CAT)activity assay kit.The docking result shows that the inhibition activity of compound ZL-8 maybe derive from its interferance to the balance between the extened and folded conformations of catalase.In addition,exogenous antioxidant NAC,an inhibitor of ROS production,blocked the effects of compound ZL-8 on the inhibition of cell proliferation,DNA damage,apoptosis induction,mitochondrial membrane potential reduction and apoptosis related proteins regulation in PC3 cells.Taken together,these results indicate that the elevated level of intracellular reactive oxygen species plays an essential role in the anti-tumor effect of compound ZL-8.5.The ability to metastasize distantly is the most important biological characteristic of malignant tumors,and it is also the major cause of death in patients with cancer.We used Wound-Healing assay and Transwell assay to evaluate the antimetastatic ability of compound ZL-8 in PC3 cells.Our founding showed that compound ZL-8 inhibited the rate of "wound" healing and decreased the number of cells that penetrate transwell chamber.In addition,Western blot assay results showed that compound ZL-8 increased the expression of E-cadherin,while decreased the expression of N-cadherin,Vimentin,and reduced the activation of matrix metalloproteinases MMP2,MMP9.Therefore,Compound ZL-8 inhibited the metastasis of PC3 cells,and the decreased migration ability of PC3 cells was related with its inhibition of EMT.Above all,we evaluated the anti-tumor activity of dithiocarbamate derivatives with α,β-unsaturated ketone scafford,and selected compound ZL-8 as the target compound.We found that compound ZL-8 can inhibit cell proliferation,induce apoptosis and reduce cell migration in PC3 cells.In addition,compound ZL-8 induced the increase of intracellular ROS level,which was the main role of its anti-tumor effect.Significantly,it may provide a theoretical basis and an experimental basis for the development of promising anti-cancer drugs.