The Mechanism Of Dehydronorcantharidin Silver Coordination Polymer Induced Apoptosis In Lung Cancer Cells A549

Background and ObjectiveSilver complexes have been shown to possess antimicrobial and anticancer properties. Ag-SP-DNC, a novel silver and singly protonated dehydronorcantharidin complex, was synthesized in our previous study. In this study, we offer evidence that Ag-SP-DNC elicits a reactive oxygen species (ROS)-mediated mitochondrial apoptosis in lung cancer cells. Ag-SP-DNC inhibited the growth of A549cells by inducing G2/M phase cell cycle arrest and apoptosis. Ag-SP-DNC induced apoptosis was associated with the levels of intracellular ROS. The further study revealed that Ag-SP-DNC disrupted the mitochondrial membrane potential, induced the caspase-3activation and led to the translocation of apoptosis inducing factor and endonucleaseG to the nucleus. Our findings have important implications for the development of silver complexes for anticancer applications.Methods1. Culture A549cell line routinely to logarithmic phase.2. Using MTT assay to detect the cell viability.3. Using SRB assay to detect the cell viability.4. Using PI staining to detect the cell cycle by flow cytometry. 5.GSH was measured by5,5’-dithiobis (2-nitrobenzoic acid)DTNB.6.The cellular morphology was observed under an inverted phase contrast microscope.7. The microfilament of A549cells were detected by using TRITC-phalloidin and analyzed by confocal laser scanning microscopy.8. Using Annexin V-FITC and propidumiodide(PI) to measure the cell apoptosis rate by flow cytometry.9. Using JC-1staining to measure the mitochondrial membrane potential in A549cells by flow cytometry.10. Using Fluorescent probe DCFH-DA to detect the level of reactive oxygen species in A549cells.11. Using caspase-3(Asp175) antibody (Alexa fluor488conjugate) to measure the active level of caspase.12. Using Fluo-3/AM staining to measure the level of Ca2+.13. Western blot to detect the level of AIF, EndoG, Bcl-2and P53.14. The statistical analysis was done using Student’s t test with the SPSS16.0statistical program, p<0.05was accepted as significant, and p<0.01was regarded as highly significant.Results1. Different concentrations of Ag-SP-DNC can cause evident inhibition of the proliferation of A549cells.2.10μmol·L-1Ag-SP-DNC induces G2/M phase arrest in A549cells.3.10μmol·L-1Ag-SP-DNC can reduce the intracellular GSH expression.4. Compared with normal control group, cell morphology can obviously change by using10μmol·L-1Ag-SP-DNC treated., The cell body shrinkage round.5.10μmol·L-1Ag-SP-DNC has obviously influence to the microfilament structure of A549cell, the microfilament structure of A549cell is obvious destruction. 6.10μmol·L-1Ag-SP-DNC has obviously increase the apoptosis rate of A549cell, cell apoptosis rate increased significantly; If add external of GSH at the same time, apoptosis rate recovery to the normal level of control group.7. Compared with the normal control group,10μmo·L-1Ag-SP-DNC has obviously influence to the A549cell mitochondrial membrane potential, cell mitochondrial membrane potential occur obviously damage (green/red ratio increased); If add external of GSH at the same time, cell mitochondrial membrane potential recovery to the level of normal control group.8. Compared with normal control group,10μmol·L-1Ag-SP-DNC has obviously increase the level of reactive oxygen species, the level of ROS occur obviously increase; If add external of GSH at the same time, the level of ROS recovery to the level of normal control group.9.10μmol·L-1Ag-SP-DNC can obviously increase the caspase-3activity level in the A549cell.10.10μmol·L-1Ag-SP-DNC can obviously increase the intracellular free Ca2+concentration level of A549cell.11.10μmol·L-1Ag-SP-DNC can obviously increase the intracellular AIF, EndoG, P53protein expression and reduce the Bcl-2protein expression.ConclusionsIn conclusion, our study revealed that Ag-SP-DNC induces ROS-mediated mitochondrial dependent apoptosis in lung cancer cells. Ag-SP-DNC treatment induced ROS generation, decreased the expression of Bcl-2and increased the expression of p53in A549cells. The ROS elevation and the disequilibrium expression of anti-apoptotic and pro-apoptosis proteins led to mitochondrial membrane potential disruption and calcium overload. Then the caspase-3was activated, and the AIF and EndoG translocated from mitochondria to the nucleus. Both caspase-dependent and caspase-independent apoptosis were induced by Ag-SP-DNC in lung cancer cells.