| Background:Glioma, accounts for about 45～55% of brain tumors, is one of the most common intracranial malignant tumor which can be divided into four grades by WHO, grade Ⅰ, Ⅱ for lower malignant degree, and Ⅲ, Ⅳ malignant degree for higher malignant degree. At present, the treatment for glioma includes surgery, radiation therapy, chemotherapy, targeted therapy and comprehensive therapy. Although novel treatments have sprung up in recent years, the prognosis of glioma hasn’t been improved significantly due to the invasive growth, no obvious demarcation from adjacent tissue and easy relapse of glioma. Median survival time after comprehensive treatment of patients with lower malignant grade(WHO grade Ⅰ-Ⅱ), WHO gradeⅢ and grade Ⅳ are 8-10 years,3-4 years and 14.6-17 months, respectively. Less than 1% of the patients receiving radiotherapy alone can survive for five years. Therefore, it is very important to find new targets and treatments to improve the efficiency of glioma therapy.Autophagy is an important process of intracellular substance flow which is conservative during eukaryotic evolution. After wrapping by double- membrane autophagic vesicles, damaged proteins and organelles were transferred into lysosomes (animals) or vacuole (yeast and plants) to degrade and recycle. This process is of great importance in providing energy, remove abnormal organelles, prevent chromosomal abnormalities and maintain genomic stability. Recent studies showed that tumor cell itself is characterized by high metabolism and demand more nutrients and energy than normal cell during progression, but can be hardly satisfied by tumor microenvironment. For example, occasions like the period between oncogenesis and tumor angiogenesis, when tumor grows up and vascular collapse occurs or when tumor cells detaches from the primary focal region will cause nutritional deficiencies or supply disruptions, which induce tumor cells to get autophagy survival advantage through autophagy and survive under harsh environment. There are also evidence indicated that after chemotherapy or radiotherapy, cancer cells will produce a lot of by-products like damaged organelles and proteins. Activation of autophagy can remove these harmful substances and provide substrate and energy that enable repair of damaged DNA. Certain levels of autophagy may improve the effect of chemotherapy, suggesting that increasing the activation of autophagy may also directly lead to autophagic death of tumor cells. Autophagy is also known as type II programmed cell death and mechanism for antitumor drugs to directly or indirectly induce cell death. Treating various tumors through regulating autophagic signaling pathways has been gradually accepted by clinical practice, thus autophagy as a novel therapeutic method becomes topical research field for researchers.ZD6474 (ZactimaTM Vandetanib), also named (N-4-2-fluorine bromine-phenyl)-6 methoxy-7 (1-methyl piperidine-4) methoxy sinensis quinzoline-4-ammonia, is a anilino quinzoline compounds and potent oral small molecule tyrosine kinase inhibitors (TKI) which can simultaneously act on epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR) and RET tyrosine kinase in tumor cells. Developed by Astrazeneca pharmaceutical company, ZD6474 has been approved by FDA in February 2006 as a drug for follicular, medulla, undifferentiated type and locally advanced or metastatic papillary thyroid carcinoma is currently applied in Ⅲ phase clinical trials for other solid tumors. Its possible pharmacological mechanism is to inhibit tyrosine kinase phosphorylation and signal transduction in tumor cells by acting on EGFR, VEGFR and RET tyrosine kinase, thus suppress damage repair of tumor cells, block cell division in G1 phase, induce or maintain cellular apoptosis and decrease formation of new blood vessels, etc.AimTo investigate the effect of tyrosine kinase inhibitors ZD6474 on cell autophagy in glioma cells, detect gene expression of autophagy-relative signaling pathway induced by tyrosine kinase inhibitors ZD6474 and provide theoretical basis for developing novel antitumor drugs targeting autophagy.Chapter 1 Small molecule tyrosine kinase inhibitors ZD6474 can promote autophagy in glioma cellsMethod1. Glioma cells U251 and U87MG were maintained in DMEM medium containing 10% fetal bovine serum, passaged regularly and chosen for experiment at exponential growth phase;2. CCK8 method was used to detect inhibition of cell proliferation by drug administration in brain glioma cells;3. U87MG and U251 cells were respectively divided into control group and single drug administration group and been observed for autophagy by electron microscope after treating with ZD6474;4. Using method for detection of GFP-LC3 to observe autophagy of U87MG and U251 with fluorescence microscope after ZD6474 treatment;5. Detect protein expression of LC31 Ⅰ/Ⅱ in brain glioma cells U87MG and U251 by western blot in above groups.Content and process1.Using CCK8 method to determine the inhibition on growth of U87MG and U251 after drug administration, single drug administration group were treated with ZD6474 of 0,1.0,2.0,4.0,8.0,16.0,32.0μmol/L for 72 h and control group were treated without drugs;2. Human brain glioma cells U87MG and U251 were divided into single drug administration group with ZD6474 treatment in three different concentrations at 2μmol/L,4μmol/L and 8μmol/L and control group without treatment. In 4μmol/L ZD6474 single administration group, human brain glioma cells U87MG and U251 were treated for 6,12,24h, respectively while control group were treated at the same time but without drugs, Western blot was used to detect expression of autophagy-related protein LC31 Ⅰ/Ⅱ;3. Human brain glioma cells U87MG and U251 in single drug administration group were treated with 4μmol/LZD6474 while control group was treat without drugs, after 24h, autophagic vacuole or autophagic lysosome was observed with electron microscope cells;4. Established cell line transfected with autophagy marker GFP-LC3, after treating with drugs of different concentration for 48h,. Laser confocal microscopy was used to observe cell autophagy;Three different methods were used to detect cell autophagy after drug administration; combined use of a variety of detection methods can ensure the reliability of experimental results.Statistical methodAll data were statistically processed with average value, expressed in average value+/-standard deviation (mean+/-SD) and analyzed with single factor analysis of variance (One-Way ANOVA) or two independent sample t test (Independent-samples t test) using spssl6.0 software, P<0.05 was considered statistically significant.Resultsl.The small molecule tyrosine kinase inhibitors ZD6474 can significantly inhibit proliferation of U87MG and U251 in vitro in an obvious dose-effect pattern;2. Conversion from symbolic protein for autophagy LC3 Ⅰ to LC3Ⅱ enhanced as ZD6474 concentration and action time increased;3. Under transmission electron microscopy, intracellular ultrastructure, mitochondria, endoplasmic reticulum, nuclear chromatin, nucleus, cell membranes and Golgi complexes were clearly visible but no autophagy-lysosome in control group. After being treated with small molecule tyrosine kinase inhibitors ZD6474, autophagy and autophagy-lysosome with some visible double membrane structure as well as mitochondria swelling in the cytoplasm were clearly visible in glioma cells U87MG and U251;4. Confocal laser observation of glioma cells U251 and U87MG expressing GFP-LC3 showed that green fluorescence positive cells with a large dot plaque increased significantly which is significantly different from control group, indicating that small molecule tyrosine kinase inhibitors ZD6474 may induce the autophagy of U87MG and U251.Conclusion1. In this part, we provide evidence that small molecule tyrosine kinase inhibitors ZD6474 can remarkably inhibit proliferation of glioma cells U87MG and U251;2. Small molecule tyrosine kinase inhibitors ZD6474 can induce aotuphagy of U87MG and U251 in a time and concentration-dependent pattern; Autophagy can be enhanced with increasing of concentration of the drug.Chapter 2 Mechanism underlying autophagy induction in U87MG and U251 by small molecule tyrosine kinase inhibitors ZD6474MethodUsing Western Blot to detect the expression of autophagy-related signaling pathway proteins like AKT, P-AKT, S6 and 4EBP1, as well as autophagy-related protein Atg7 and Beclinl to explore whether or not the drug induce autophagy through PI3K/AKT/mTOR pathway.Content and processOur findings validate the assumption that small molecule tyrosine kinase inhibitors ZD6474 induced autophagy occurrence in glioma cells U87MG and U251. Previous studies indicated that autophagy is related to mTOR pathways. To investigate whether or not small molecule tyrosine kinase inhibitors ZD6474 induced autophagy by adjusting the PI3K/AKT/mTOR signaling pathway inU87MG and U251 glioma cells, we further explore the mechanism underlying.1. Single drug administration group in human brain glioma cells U87MG and U251 was treated with 4uM ZD6474 for 0,6,12h, respectively. Western Blot was used to determine expression of downstream protein in mTORCl pathway like S6, p-S6,4EBP1, p-4EBP1 and AKT, p-AKT as well as autophagy-related protein Atg7 and Beclinl;2. In order to further confirm the action of drug in mTOR pathway, we use insulin to activate mTOR in cells.4μmol/L ZD6474 was administrated in glioma cells before detection of p-S6, p-AKT and LC3 Ⅰ/Ⅱ protein expressions.Statistical methodAll data were statistically processed with average value, expressed in average value+/-standard deviation (mean+/-SD) and analyzed with single factor analysis of variance (One-Way ANOVA) or two independent sample t test (Independent-samples t test) using spss16.0 software, P<0.05 was considered statistically significant.Results1. Western blot showed that after 72h treatment in single drug administration group,PI3K/AKT/mTOR pathway related signal protein S6,4ebp1 and phosphorylated AKT increase in the control group as comparing to experimental group, but autophagy related protein expression was not obviously changed;2. After activation of mTOR pathway by insulin in U251, S6 and phosphorylated AKT levels increased, LC3 Ⅰ/Ⅱ protein levels also had remarkable changes.ConclusionAbove results suggested that small molecule tyrosine kinase inhibitors ZD6474 can induce autophagy in U87MG and U251 by regulating PI3K/AKT/mTOR signaling pathways.Chapter 3 Inhibition of autophagy induced by small molecule tyrosine kinase inhibitor ZD6474 can promote apoptosis in U87MG and U251Method1. Knockdown autophagy-related genes in U87MG and U251 with siRNA Atg7 and detect autophagy-related proteins by Western blot;2. Measure apoptosis of U251 and U87MG after SiRNA Atg7 knockdown or ZD6474 treatment with with Annexin V kit.3. Measure apoptosis of U87MG and U251 with Annexin V kit after combined administration autophagy inhibitor chloroquine and 4uM ZD6474;4. Detect activity of Caspase-9 and Caspase-3 in U87MG and U251 using Caspase kit.Content and process1. First, we knocked down autophagy-related genes with siRNA Atg7 in U87MG and U251 and detected expression of autophagy-related protein LC3 Ⅰ/Ⅱ to determine cell apoptosis after siRNA knockdown of autophagy-related gene Atg7;2. Then we detected the apoptosis of U87MG and U251 with with Annexin Ⅴ kit. after SiRNA Atg7 knockdown and with or without ZD6474 administration of 0μmol/L,2μmol/L,4μmol/L,8μmol/L;3. In order to further confirm that inhibition on autophagy would increase apoptosis, we use 5μmol/L autophagy inhibitor chloroquine combined with 4μmol/L ZD6474 in drug administration group of U87MG and U251 cells and no drugs in control group before detection of apoptosis with Annexin V kit;4. To further testify which apoptotic pathway participate in suppression of autophagy, we detected activity of Caspase-9 and Caspase-3 with Caspase kit in groups as follows 25μmol/L autophagy inhibitor in chloroquine treatment group, 4μmol/L ZD6474 in single-agent treatment group, ZD6474 (4μmol/L x24h) plus chloroquine (25μmol/L×4 hours) in treatment group, and control group without drugs.Statistical methodAll data were statistically processed with average value, expressed in average value+/-standard deviation (mean+/-SD) and analyzed with single factor analysis of variance (One-Way ANOVA) or two independent sample t test (Independent-samples t test) using spss16.0 software, P<0.05 was considered statistically significant.Results1. First, we use the siRNA Atg7 to silence U87MG and U251 cell autophagy genes, cut its protein expression level, detect autophagy proteins LC3 Ⅰ/Ⅱ levels Then we further study of siRNA silence Atg7 autophagy genes protein after cell apoptosis;2. Subsequent tests with CCK8 to detect U87MG and U251 cell proliferation. U87MG and U251 cells can be divided into four groups, control group, SiRNA Atg7 silence, ZD6474 single drug treatment group and SiRNA Atg7 silence+ZD6474 group;3. In order to further confirm increased inhibition of autophagy and apoptosis, we divided U87MG and U251 cells into four groups:control group, single chloroquine 25μmol/L group, ZD6474 4μmol/L group and chloroquine 25μmol/L joint ZD6474 4μmol/L group. detected the apoptosis of U87MG and U251 with with Annexin V kit;4. Further testing cell autophagy inhibition are implemented through the apoptosis pathway, we use the Caspase activity kit to detect autophagy inhibitor chloroquine 25umol/L treatment group, ZD6474 single-agent 4μmol/L, ZD6474 mu (4μmol/L×24 hours) and chloroquine (25μ/L×24 hours), control group without medicine, and the changes of Caspase-3 and Caspase-9 in U87MG and U251 cells.ConclusionSmall molecule tyrosine kinase inhibitors can induce autophagy of brain glioma cells U87MG and U251; Inhibition of autophagy can promote apoptosis of brain glioma cells;... |
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