Small Molecule Tyrosine Kinase Inhibitor ZD6474 On Hepatocellular Cells And Its Mechanism

Background and purposeHepatocellular carcinoma (primary hepatocellular carcinoma, HCC), a high incidence in Africa and southeast Asia, China is a high incidence area, and the low incidence area compared to younger age onset and rapid progression. Liver cancer mortality rate in the nation's No.2 all kinds of tumors and mortality in the last 10 years has been the rise [1]. Current surgical treatment of liver cancer and liver transplantation is a more effective method, but most patients to the hospital when symptoms is already advanced, mass is too large or diffuse, can not be completely removed, the patient has lost 90% of radical surgery possible. Conventional radiotherapy and chemotherapy for unresectable primary liver cancer treatment ineffective. Including the formation of portal vein tumor thrombus, intrahepatic and distant metastasis is poor liver cancer treatment, rapid progression of the major reasons. Therefore, further study on liver cancer, liver cancer and to seek new treatments and drugs is very important. Clinical trial results show that multi-target joint block signal transduction is the development of cancer treatment and drug development[2]. Hepatocellular carcinoma is a tumor rich in blood vessels, tumor blood vessel formation and VEGF-related. Vascular endothelial growth factor (VEGF) is currently the strongest known direct effect on vascular endothelial growth factor receptor in tumor angiogenesis and play a key role in the process of aggression,and but also is an important factor in the promotion of tumor metastasis.ZD6474 (ZactimaTM Vandetanib) is an aniline-based compounds, quinoline yl forest for the potent oral small-molecule VEGFR tyrosine kinase inhibitors, their chemical name (N-4-bromo-2-fluorophenyl)-6 methoxy-7 (1-methyl-piperidine-4)-4 methoxy-quinoline yl Lin-Ammonia. Astrazeneca R & D by pharmaceutical companies, is currently in PhaseⅢclinical trials. The drug can be simultaneously EGF receptor, VEGF receptor and RET tyrosine kinase. In February 2006, FDA approved for the treatment of follicular, medullary, undifferentiated type, as well as local recurrence or metastasis of papillary thyroid carcinoma. ZD6474 can block the VEGF receptor, inhibit tumor angiogenesis directly or indirectly by blocking the EGF receptor,and play an anti-vascular function. Thus, ZD6474 is expected to selectively inhibiting VEGFR2, and EGFR by double-target inhibition of tumor growth.一Small molecule tyrosine kinase inhibitor ZD6474 on liver cancer cell proliferation, cell cycle and apoptosis一materials and methods1. HepG2 cells with 10% calf serum RPMI-1640 medium was 3-5 days a generation of transfer, select the logarithmic growth phase cells as experimental;2. To these groups, cells were observed in size, shape, etc.;3. After detection of ZD6474 effects Hoechst33342/PI HepG2 cell apoptosis;4. To take these groups, with the MTT assay of drugs on human hepatoma HepG2 cell proliferation, according to calculated OD values of cell growth inhibition rate;5. To these groups, cells were collected by flow cytometry cell cycle and apoptosis.二were divided into control group, single drug group. 1. MTT assay of drugs on human hepatoma HepG2 cell proliferation, single-drug group were set to join 0.1,1.0,3.0,10.0,20.0,50.0,100.0μmol/L 8 concentrations group ZD6474, the control group without drug;2. Inverted microscope, Hoechst33342/PI, FCM cell cycle, apoptosis, single-drug groups were added 3 different concentrations of ZD6474 3μmol/L,6μmol/L, 50μmol/L, control group without drug;三,statistical methodsUsing SPSS 13.0 statistical software. Inhibition rate between the three groups were analyzed by repeated measures analysis of variance; cell cycle and apoptosis rate by one-way analysis of variance; multiple comparisons are used LSD method used to analyze the interactive effects of factorial analysis.四,the results1. Inverted microscope, control cells adhered in good condition, showing polygonal cells, larger and plump cytoplasm, cell borders clearly. Experimental cells have different levels of morphological changes, cells became round, size reduced, but the cell membrane structural integrity. Adherent cells were shrunken, rounded off. The concentrations of 3.0μmol/L, morphological changes occur in the cells increased. Abnormal cells with high doses of more common, cells were significantly reduced;2. Hoechst/PI 33342 staining of the ZD6474 group of cells showed significant changes (chromatin condensation, nuclear shrinkage, apoptotic cells with high blue fluorescence) and cell necrosis (high red fluorescence). With the increase of drug concentration, apoptotic cells increased. In high concentrations, even seen some dead cells. ZD6474 can induce apoptosis in HepG2;3. Different concentrations of ZD6474 treatment, HepG2 liver cancer cell growth inhibition rate increased with increasing concentration and the role of administration time and increase, suggesting that ZD6474 on HepG2 cells have a more significant growth inhibition, and this effect significantly time-dependent and dose-dependent. 4. Cell cycle analysis showed G0/G1 cells in treatment group than in the control group of cells had increased, and significant differences (P<0.05); S phase cells decreased compared with the control group, and equally significant difference (P<0.05).5. ZD6474 treated HepG2 cells by flow cytometry showed that, ZD6474 could significantly induce apoptosis in HepG2 cells, and increased with the dose, the apoptosis rate increased.二small molecule tyrosine kinase inhibitor ZD6474 in hepatoma cells the expression of BCL-2 gene I. Methodology Real-time fluorescence quantitative PCR detection of intracellular BCL-2 gene expression. Second, statistical methodsAll data using SPSS 13.0 statistical software for statistical processing. T-test statistical software for statistical processing, taking P<0.05 for the difference was statistically significant. Third, the results of Compared with the control group, Bcl-2 gene expression decreased, and in the control group with significant difference (P <0.05).Conclusion:ZD6474 on the proliferation of HepG2 hepatoma cells suppress the role of a time-and dose-dependent. Treatment group than those in cells in G0/G1 phase cells had an increase in the control group, S-phase cells were decreased compared with the control group, could significantly induce apoptosis in HepG2 cells, and with the dosage increased apoptosis rate increased. Apoptosis of Bcl-2 gene expression decreased, indicating inhibition through the expression of Bcl-2 and promote apoptosis of tumor cells.