Jiao Glutamyl Proline The Cyhexatin Ester (ppc) Induced Human Lung Adenocarcinoma Cells And Its Mechanism

Backgroud:Lung cancer is one kind of disease which jeopardize people' s health and lives seriously, and the incidence and death rate of this disease has ranked the first in our country. Moreover, among all types of lung cancer, lung adenocarcinoma is the most common one and its incident rate is the highest. However, no effective treatments for this kind of disease could be available now, because the molecular mechanisms underlying lung adenocarcinoma cells' apoptosis have not been clarified. Therefore, the clarifying of this mechanism would play an important role on the treatments for lung cancer.Chemotherapeutics is the most common treatment for lung adenocarcinoma for the reason that it is usually at the later stage when we found this kind of disease in the sufferers. Therefore, looking for the potent chemotherapeutic agents and sensitizing agents are the main tasks for researchers studied on lung cancer. Based on the backgrounds mentioned above, the molecular mechanisms underlying A549 cell apoptosis were investigated through the method of the induced apoptosis of this kind of cell by organotin small molecular compound.Objective: To investigate the growth inhibition by organotin small molecular compound and the molecular mechanisms of apoptosis in A549 cell lines in vitro and provide theoretical basis at the cell-level for the therapy of lung cancer.Methods: In the experiment to investigate the in vitro effects of organotin compound on the growth of cultured cells, A549 cell were exposed to escalating doses of PPC in the culture medium. MTT assay was used to test the proliferation of the cells treated by PPC, by which small molecules were screened initially; at the same time, cell morphological changes were observed by Phase Contrast Microscope; changes in cell nucleus were detected using H33258 staining through which we could observe the typical phenomena of apoptosis; Terminal deoxynucleotidyl Using DeadEndm Colorimetric TUNEL System assay was used to show DNA fragmentation; LDH assay to determine whether cells treated by the small molecules underwent necrosis. The changes of p53 protein level were analyzed by Western blot assay; Flow cytometry was used to detect the changes about the cell cycle of A549; the combination of fluorescent probe with the technical of Confocal laser scanning microscope were used to measure the concentration change of Ca²⁺ in A549after treated in different time in vitro.Results: The results of MTT assay on cells livability within 48 hours showed that PPC induced apoptosis in A549 cells with a dosage dependent manner, and the initiative concentration is 0.1μM. After being treated A549 cells Observation of cell morphological changes by Phase Contrast Microscope showed that cells become longer , membrane blebbing and apoptotic bodies occurred. Ultimately, many cells detached from the dishes and underwent death.; Using Hoechest33258 staining, the nuclear fragmentation, chromatin condensation and apoptotic body were observed in A549 cells through fluorescence microscope; Using DeadEndTM Colorimetric TUNEL System, nuclear fragmentation and intranucleosomal DNA fragmentation were detected in A549 cells after the treatment with PPC(0.5μM) .LDH assay showed that when cells were treated by 0.25μM PPC for 48h ,there was no significant difference in LDH activity present in the mediums between the control and the test group,However,the difference is rather obvious when the concentration of PPC increased to 0.5μM, and the difference became significant (P<0.01, n=3). Western blot assay confirmed that when cells were exposed to 0.5μM PPC forl2,24,48h ,in comparison with the control group,p53 protein level in the test group increased .Flow cytometry analysis demonstrated that cells, after being treated with PPC (0.5μM ) for 12,24,48 hours, most were blocked in G0/G1 phase in a time-dependent manner with no obvious M phase changes; The test of Ca²⁺ fluorescent probe showed that PPC (0.5μM) can increase [Ca²⁺] in A549 cells evidently (P<0.05or P<0.01, n=3) in an apparent time-dependent manner.Conclusions:1. PPC can apparently inhibit the growth of A549 cells in a dose and time dependent manner.2. In A549 cells, the form of cell death was mainly apoptosis.3. Effects of PPC in cell cycling were time related, and A549 cell was arrested in G1 phase.4. PPC might induce apoptosis in A549 cells through up-regulating p53 and [Ca²⁺].