Tetrafluorobenzoylethyl Isoleucine Dibutyl Tin Ester (fpldbt,) Induced Non-small Cell Lung Cancer A549 Cells And Their Associated Mechanisms

Background and objective:Lung cancer is the most dangerous cancer throughout the world, and the incidence and death rate of lung cancer has ranked the first in China. According to histological type, lung cancer includes two main types:small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Therefore, it is particularly important for development and research of chemotherapeutic drugs.Chemotherapeutics is the most important treatment for lung adenocarcinoma because it is usually at the later stage when we find this kind of disease in the sufferers, at this moment, NSCLC becomes not suitable for surgery, and not sensitive to radiotherapy. At present, the frequently-used drugs for lung cancer chemotherapy can induce apoptosis of lung cancer cells, but strong toxicity side effect and drug resistance may cause failure of chemotherapy. Therefore, looking for the chemotherapeutic drugs which has high efficiency, minor toxicity, less side effect and ability to overcome multi-drug resistance is one of the urgent problems for researchers to be settled.In summary, A549 cells were selected as experimental cells to investigate in vitro screening of apoptosis-inducing drugs and molecular mechanisms of apoptosis. As well as the synthesis of organotin compounds FPLDBT anti-tumor activity in vitro study, and then, to explore the growth inhibition by FPLDBT and the molecular mechanisms of FPLDBT-induced apoptosis in A549 cells in vitro. We hoped that these studies could be in the treatment of lung cancer at the cellular level and the development of new chemotherapeutic agents for the treatment of lung cancer to provide a more complete theoretical basis, and make a positive contribution.Methods:1.MTT assay on cell viability, by which the FPLDBT was screened initially.2.Detection of cell apoptosis:-Observation of cell morphological changes by Phase Contrast Microscope.-Observation of nuclear fragmentation by Hoechst 33258 staining combined with Fluorescence Microscopy.-Observation of morphological changes of apoptosis by Acridine orange (AO) fluorescence staining combined with Fluorescence Microscopy.-Detection of the activity of ROS in A549, the content of ROS was determined by colorimetric assay.-Detection of apoptosis rates by Nucleosome ELISA assay kits.3.Detection of changes in cell cycle distribution:-Flow cytometry was used to detect the changes about the cell cycle of A549 after treated with FPLDBT. 4. Methods for determining whether caspase-3,-4 were activated by FPLDBT, as follows:-Analyzing caspase-3,-4 activities by caspase-3,-4 colorimetric assay kits.-Identification the influences of caspase-3,-4 on apoptosis induced by FPLDBT with caspase inhibitors.Results:1.The test results of anti-tumor activity of FPLDBT (the results of MTT assay on cells livability)-Influences of FPLDBT on viability of A549 cell: The results showed that:Within 48 hours, FPLDBT significantly reduced the survival rate of the A549 cell and significantly inhibited the growth of the A549 cell in a dosage dependent manner.2. Results of FPLDBT-induced apoptosis in A549 cells-Observation of morphological changes by Fluorescent inverted phase contrast microscope: The results showed that:After A549 cells were exposed to 0.8μM/L FPLDBT for 48 hours, cell morphological changes by Phase Contrast Microscope showed that cell shrinkage, vacuolization evident, apoptotic bodies occurred, and many cells detached from the dishes, showing a typical morphological characteristics of apoptosis; While treated with 0.1% DMSO, cell morphology had no obvious changes.-Results of detection of nuclear fragmentation by Hoechst 33258 staining combined with fluorescence microscopy: The results showed that:After A549 cells were exposed to 0.8μM/L FPLDBT for 48 hours, cell morphological changes by fluorescence microscopy revealed a uniform light blue nuclei, chromatin condensation, apoptotic bodies clearly visible, showing the typical morphological characteristics of apoptosis; While the DMSO group and the control group had no obvious change in cell nuclear morphology.-Results of detection of cell apoptosis by Acridine orange (AO) staining combined with fluorescence microscopy: The results showed that:After A549 cells were exposed to 0.8μM/L FPLDBT for 48 hours, cell morphological changes by fluorescence microscopy showed that pehromatin condensation and marginalization, emerging nuclei fragmentation, apoptotic bodies clearly visible, showing the typical morphological characteristics of apoptosis; While the DMSO group and the control group had no significant change in cell morphology.-The results of the activity test of ROS: The results showed that:When A549 cells were treated with 0.8μM/L FPLDBT for 48 hours, compared with the control group, the activity of ROS increased a lot.-Results of Nucleosome ELISA assay: The results showed that:When A549 cells were treated with 0.8μM/L FPLDBT for 48 hours, compared with the control group, FPLDBT could effectively induce apoptosis in A549 cells, and showed a dose-time-dependent manner.3.Influence of FPLDBT on the cell cycle of A549-Influence of FPLDBT on A549 cells cycle distribution: The results showed that:Flow cytometry analysis demonstrated that cells, after being treated with FPLDBT (0.8μM/L) for 48 hours, compared with the control group, most were blocked in G1 phase, could not enter S phase, in a time-dependent manner.4. Research findings of FPLDBT-induced apoptosis in A549 cells and its related molecular-Assay results of activities of caspases: The results showed that:In the apoptotic process, FPLDBT activated caspase-3 and-4, in which the activation of caspase-3 level was weaker than caspase-4.-Detection influences of caspases inhibitors on FPLDBT-induced apoptosis of A549 cells: Compared to 0.8μM/L FPLDBT treatment group, caspase-4 inhibitor (Z-LEVD-FMK) significantly reversed the FPLDBT-induced apoptosis in A549 cells. And caspase-3 inhibitor (Z-DEVD-FMK) reversed partially the FPLDBT-induced apoptosis.Conclusion:1. FPLDBT could significantly inhibit the proliferation of A549 cells in a concentration-dependent manner.2. FPLDBT induced A549 cells arrest at G1.3. FPLDBT could effectively induce A549 cells apoptosis in a dose-time-dependent manner. ROS and caspases might participate in this process of apoptosis.4. According to the effect of caspases in the process of apoptosis induced by FPLDBT in A549 cells, we speculated that the endoplasmic reticulumial pathway might play a major role in this process of apoptosis.