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Of tetrafluorobenzoylethyl isoleucine induced by dibutyltin ester (FPLDBT) in non-small cell lung cancer A549 cell apoptosis and its related mechanism

Author: HuZunLi
Tutor: ZhaoYunFeng
School: Qufu Normal University
Course: Biochemistry and Molecular Biology
Keywords: Lung cancer A549 cells Apoptosis Organic tin small molecule compounds caspase
CLC: R734.2
Type: Master's thesis
Year: 2010
Downloads: 28
Quote: 0
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Abstract


Background and Objective: Lung cancer is today the world's first cancer killer, topped the list has become a serious threat to human health and life diseases in China, the morbidity and mortality. The histological type of lung cancer is divided into small cell lung cancer (small cell lung cancer, SCLC) and non-small cell lung cancer (non-small cell lung cancer, NSCLC) two categories. Non-small cell lung cancer (NSCLC) accounts for more than 80% of lung cancer, but treatment Ⅲ ~ Ⅳ lost opportunities of surgery, surgery patients, recurrence, and the transfer rate of up to 50% or more, and is very sensitive to radiotherapy. Therefore, systemic chemotherapy is an important means for the treatment of non-small cell lung cancer. Currently used chemotherapy drugs in the chemotherapy early effect, can effectively induce apoptosis in lung cancer cells, but serious side effects and easy to produce drug resistance, resulting in the failure of chemotherapy. Therefore, to find efficient and toxicity, new chemotherapy drugs to overcome the resistance to become one of the main problems solved in the current lung cancer treatment. In summary, the purpose of this research project is to study: non-small cell lung cancer A549 cells, the study of synthetic the organic tin small molecule compounds FPLDBT vitro anti-tumor activity, then explore on cultured non-small cell lung cancer The molecular mechanisms of growth inhibition and apoptosis in A549 cells, the hope that these studies can provide some theoretical basis for the treatment of lung cancer and the development of new lung cancer chemotherapy drugs, and make a positive contribution. Research Methods: the MTT assay cell viability, FPLDBT, conducted the initial screening. 2. Detection of apoptosis situations: - inverted phase contrast microscope Morphological changes were observed;-Hoechst 33258 staining and combined fluorescence microscope nucleus fragmentation; - acridine orange (AO) staining and fluorescence microscopy further testing cells apoptosis; - the level of reactive oxygen species (ROS) changes in the content of intracellular reactive oxygen species detected with a fluorescence spectrophotometer; - Nucleosome ELISA kit to detect apoptosis rate. Affect the detection of the cell cycle: - flow cytometry FPLDBT on the cell cycle. 4.FPLDBT of caspase-3 and -4 activation of the detection method is as follows: - use of caspase-3, -4 enzyme activity assay kit analyze the activity changes of caspase-3, -4; - the caspases inhibitor detection caspase- 3 of 4 on FPLDBT cell growth. Research results: 1.FPLDBT anti-apoptotic activity of test results (MTT assay of cell viability results)-FPLDBT A549 cell viability: The results show that: within 48 hours, FPLDBT significantly reduced the survival rate of A549 cells, A549 have significantly inhibited the growth and significant dose effect. Induction of apoptosis of A549 cells 2.FPLDBT research results - inverted phase contrast microscope Morphological changes were observed: The results showed that: 0.8μM / L of FPLDBT treatment of A549 cells 48 hours after the inverted phase contrast microscope, cell shrinkage, vacuolization obvious apoptotic bodies produce death and detached from the bottom of the dishes, the cells showed typical apoptotic morphological features; 0.1% DMSO solvent control group compared with the control group, no significant changes in cell morphology. -Hoechst 33258 staining and fluorescence microscopy nucleus fragmentation Results: The results showed that: 0.8μM / L of FPLDBT treatment of A549 cells 48 hours after observed under a fluorescence microscope found that the nucleus was blue, the phenomenon of nuclear fragmentation, apoptotic bodies clearly visible, showing typical apoptosis morphological characteristics; DMSO solvent control group compared with the control group, the nuclei were uniformly bright blue, no significant morphological changes. - Acridine orange (AO) staining and fluorescence microscopy to detect chromatin condensation results: The results showed that: 0.8μM / L of FPLDBT treatment of A549 cells after 48 hours under a fluorescence microscope observed chromatin condensation and marginalization, and apoptosis The bodies appeared, showing typical apoptotic morphological features; DMSO solvent control group compared with the control group, no significant changes in chromatin. Determination of intracellular reactive oxygen species-A549: The results showed that: 0.8μM / L of FPLDBT treatment of A549 cells 48 hours after detection of intracellular reactive oxygen content and fluorescence spectrophotometer found that, compared with control cells, intracellular The active oxygen content increased significantly. -Nucleosome ELISA detection level of nucleosome changes Results: The results showed that treatment of A549 cells for 48 hours: 0.8μM / L of FPLDBT Nucleosome ELISA kit detected, compared with the control group of cells, cell kernel level increased significantly. Of 3.FPLDBT on the A549 cell cycle - flow cytometry cell cycle distribution Results: The results showed that: the cell cycle distribution by flow cytometry and found that, compared with the control group, 0.8μM / L of FPLDBT 48 hours after The vast majority of A549 cell cycle arrest in G1 phase, can not enter S phase. -Caspases activity detection of molecular mechanisms 4.FPLDBT induced apoptosis of A549 cells results: The results show that: apoptotic process, FPLDBT also activated caspase-3, -4, where the degree of activation of caspase-4 in caspase-3. -Caspases inhibitor of FPLDBT-induced A549 cell apoptosis: caspases inhibitor, apoptosis ELISA kit to detect apoptosis rate found: compared to cells treated with FPLDBT inhibitor of caspase-4 (the Z-LEVD- FMK) significantly block the FPLDBT induced apoptosis, caspase-3 inhibitor (Z-DEVD-FMK) also inhibited the FPLDBT induced apoptosis, but to a lesser extent. Study conclusions: 1.FPLDBT a concentration-dependent manner significantly reduces the survival rate of A549 cells, inhibit the proliferation of A549 cells. 2.FPLDBT allows cell cycle arrest in G1 phase. 3.FPLDBT can effectively induce apoptosis in A549 cells, to participate in this process of apoptosis by ROS and caspases. 4 according to the role played by caspases in FPLDBT-induced apoptosis of A549 cells, we speculate that this process of apoptosis within the endoplasmic reticulum apoptosis pathway-based.

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CLC: > Medicine, health > Oncology > Respiratory system tumors > Lung tumors
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