Anti-tumor Mechanism Of Isoquinoline Alkaloids And VEGFR2 Inhibitor Screening Study

With the increasing in coal industry and a large number of vehicle exhaust emissions caused serious air pollution.About the last 20 years,the incidence of lung cancer showed an increasing trend in our country.Today male lung cancer patients in China has occupied the first place in the male malignancies,but also the rapid increase in the incidence of women,and it is likely to beyond the ovarian cancer as women's first high-risk caner in the future.Lung cancer has clearly become a health hazard to human life in a vicious disease.Therefore,the development of drugs to treat lung cancer is particularly urgent.Isoquinoline alkaloids is widely distributed in the biosphere,and it has a good biological activity in anti-inflammatory,antibacterial,immune system,regulate cardiovascular disease,some types of isoquinoline alkaloids also has anti-tumor effects.We chose the self-synthetic isoquinoline alkaloids imine B4,B13,B20 using for the anti-tumor activity screening in lung cancer A549 cells.It was found that isoquinoline alkaloids imide B4,B13,B20 exhibits good proliferation inhibition on A549 cell in vitro.Our research found that isoquinoline alkaloids imide B20(take 24?M treatment 3,6,12h)can significantly increase the intracellular ROS level in A549 cells,and induce mitochondrial membrane potential decreased in A549 cells.Isoquinoline alkaloids imide B20 treatment of A549 cells(take 2,6,12,24?M concentrations 24h)Western Blot results showed that isoquinoline alkaloids imide B20 can enhance the expression of p53,HSP70,HSP90,GRP78,cleaved caspase-3,cleaced PARP and cleaved caspase-9,and significantly increased the ratio of Bax/Bcl2,while reducing mitochondrial cytochrome c and cytochrome c in the cytoplasm increases.After the joint NAC(oxidative damage inhibitors)inhibit intracellular ROS,reversing PARP,Caspase-3,Caspase-9 cleavage caused by B20 treatment with A549 cells.At the same time,using Annexin-V-PI method to detect apoptosis(B20 take 24?M treatment 24h),apoptosis was found in the control group was 6.6%,B20 dosing group apoptosis was 31.3%.Accordingly,isoquinoline alkaloids imine B20 can induce human lung cancer A549 cells produce ROS and induced endogenously apoptosis in vitro,and the apoptosis is dependent on the production of ROS.Further study found that isoquinoline alkaloids imide B20 at 24?M treatment of lung cancer A549 will produce a large vacuole membrane structure after 48 hours later;Western Blot results showed that the isoquinoline alkaloid imide B20 can induce autophagy-related protein expression LC-3 ? amount increased;fluorescence microscope observation showed that,B20 can induce transfection GFP-LC-3 plasmid A549 cells showed a large number of GFP-LC-3 spots gather,and within 24h with B20(take 2,6,12,24?M concentration)treatment of A549,the proportion of green GFP-LC-3 punctate intracellular accumulation occurred increased from3.7%to 34.3%;In conjunction with flow cytometry using Acridine orange staining found in acidic autophagic dissolved proteasome with B20 concentration increased;after use autophagy inhibitors Baf A1 and B20 treatment of lung cancer cells A549,there was significantly enhanced in A549 cells apoptosis.The results show that isoquinoline alkaloids imide B20 A549 cells can be induced autophagy and autophagy as a protective effect.Our study also found that,B20(take 2,6,12,24?M)treatment of A549 cells to increase the JNK phosphorylation levels,suggesting that the Western blot test results found:There is a clear expression of P-JNK when B20(at 24 ?M concentration)treatment of A549.When using the NAC and B20 combined treatment A549,then JNK phosphorylation level was inhibited.After the joint use of JNK inhibitors sp600125(10?M)and Sh JNK interference A549,B20 induced apoptosis and autophagy generated was significantly inhibited.In summary,this part of the study found that isoquinoline alkaloids imide B4,B13,B20 can significantly inhibit the value of A549 cells;isoquinoline alkaloids imide B20 can induce lung cancer A549 cells to produce ROS,and A549 cells stably expressing wild-type P53,and the induction of the mitochondrial pathway of apoptosis occurs in vitro;while B20 Induced A549 autophagy and activated JNK.B20 anti-tumor effect on A549 cells is induced apoptosis,and this effect is dependent on the generation of ROS and JNK activation.The formation of tumor blood vessels in tumor growth and metastasis and evolution plays a very important role.Growth of solid tumors is dependent on continuous and extensive growth of blood vessels.Therefore,the inhibition of tumor angiogenesis,can effectively inhibit tumor growth.In the process of tumor angiogenesis,-vascular endothelial growth factor and vascular endothelial growth factor receptor interaction mediates its signal transduction pathway.Wherein,VEGF/VEGFR is the main control factor in regulating angiogenesis.There are already a variety of targeting VEGF/VEGFR signaling pathway specific tyrosine kinase inhibitor approved to medicine market and successfully used in the clinical treatment of tumors.Development of tumor angiogenesis mechanism for tyrosine kinase inhibitor,is currently the main method of anti-angiogenic strategy.For the purpose to develop the independent property anti-tumor angiogenesis tyrosine kinase inhibitors,our autonomous synthetic YQY,ZJQ series of compounds were cellular and animal models of levels of anti-angiogenic activity screening.We found ZJQ-24 is an active compounds.And we studied the anti-angiogenesis function and the mechanism of ZJQ-24.First,we use chorioallantoic membrane model and MTT assay for the synthesis of compounds(targeting the receptor tyrosine kinases)were screened,and we screened several compounds that can inhibit chorioallantoic membrane angiogenesis.Then we found that ZJQ-24 inhibited VEGF-induced HUVEC proliferation and the IC50 of ZJQ-24 is 9.02±0.3?M(+VEGF)37.5±0.2?M(-VEGF).Subsequently,scratches experiment found(taken within 24h 2.5?M concentrations HUVEC)ZJQ-24(51.4±0.6)%inhibition of migration of endothelial cells.At the same time,ZJQ-24 at 5?M make(48.5±1.2)%inhibition of microtubule formation HUVEC.Further studies showed that ZJQ-24 can inhibit the level of VEGFR2 phosphorylation,and leading to the inhibition of down-stream signaling by the receptors like AKT,ERK,FAK,Src and other signaling molecules,and have a concentration-dependent manner.ZJQ-24 inhibits the proliferation of HCT116,SW480 and HEPG-2 cells(HCT116 IC50=8.10±1.8?M,SW480 IC50-7.90±1.3?M,HEPG-2 IC50=8.3±2.2?M),but not significantly inhibits the proliferation of normal embryonic lung cells MRC-5(IC50>100).ZJQ-24(within 24h take 1,2.5,5,10,20 ?M)can induce HEPG-2 cells showed PARP and Caspase-3 cleavage,suggesting ZJQ-24 can induce apoptosis in HEPG-2 tumor cells.In summary,ZJQ-24 by inhibiting VEGFR2 and its downstream signaling molecules produced significant anti-angiogenic effect.Meanwhile ZJQ-24 have good anti-tumor activity and can induce tumor cell HEPG-2 produced PARP and Caspase-3 cleavage to produce apoptosis.Thus,we synthesized compound ZJQ-24 have significant anti-angiogenic effects and anti-tumor effect.