Synthesis Of TH1082and Mechanism Of Apoptosis In Human Melanoma A375Cells Induced By TH1082

Background：The wide application of indole in life sciences has stimulated the development ofa number of methods for its derivative synthesis. Replacement at the C4, C5, C6orC7position of indole by a sp2-hybridized nitrogen provides a skeleton containing ahydrogen-bond donor and acceptor in a rigid three-atom arrangement, respectively.This class of compounds can be considered as the bioisosteres of indole or purine,which show a variety of medicinal relevance and potential biological activities, suchas antitumor, antiproliferative, antiviral, antibacterial, antiangiogenesis,protein-kinases inhibition and so on. Studies of preparations of7-azaindolederivatives have attracted much attention due to their important role in designingpotential drug candidates. We designed and synthesized a new7-azaindole derivativeTH1082, which could inhibit proliferations of many human cancer cell lines via MTTassay and it could inhibit A375cells most effectively in a time and dose dependentmanner. But the mechanism of antiproliferation in A375cells was not clear, it isnecessary to detect furthermore.Object：To investigate the mechanism of apoptosis in human melanoma A375cellsinduced by TH1082.Methods：1. To detect the effects of anti-proliferation and IC50value of24h by MTT assay.2. To observe the morphological changes of A375cells treated with differentconcentrations of TH1082for24h by fluorescence microscope after AO/EB,Hoechst33342/PI and DAPI staining. And to count the apoptotic ratio of A375cells.3. To measure the mitochondrial transmembrane potential in A375cellstreated with different concentrations of TH1082for24h by fluorescence microscopeor flow cytometry after JC-1staining.4. To detect the expressions of Bcl-2and Bax in A375cells treated withdifferent concentrations of TH1082for24h by Western Blot and QPCR.5. To detect the expressions of Cyt c, Smac and AIF in A375cells treated withdifferent concentrations of different concentrations of TH1082for24h by WesternBlot.6. To detect the activities of caspase-3, caspase-8and caspase-9in A375cellstreated with different concentrations of TH1082for24h.7. To detect the expressions of caspase-3, caspase-9and PARP in A375cellstreated with different concentrations of TH1082for24h.8. To detect the effects of TH1082of Bcl-2family and apoptotic rate on A375cells treated with Z-VAD-FMK.9. To detect the expressions of proteins of Death receptor signaling pathwayin A375cells treated with different concentrations of TH1082for24h by WesternBlot.10. To detect the cell cycle of A375cells treated with different concentrationsof TH1082for24h by PI staining. To detect the expressions of c-myc, CDK4,cyclin D1, p53and p27kip1in A375cells treated with different concentrations ofTH1082for24h by Western Blot.11. To detect the expressions of proteins of PI3K/Akt signaling pathway inA375cells treated with different concentrations of TH1082for24h by Western Blot.12. To detect the expressions of proteins of MAPK signaling pathway in A375cells treated with different concentrations of TH1082for24h by Western Blot. 13. To detect the expressions of p-ERK1/2, c-myc, CDK4, cyclin D1, p53,p27kip1, Bcl-2and Bax by Western blot. To detect the apoptotic ratio and cell cycle ofA375cells.Results：1. Effects of TH1082on proliferation of A375cellsTo examine the effects of TH1082on the proliferations of A375, A549,HCT116, HeLa, HepG2, K562, MCF-7, SGC, SHSY5Y and SMMC cells by MTTassays. TH1082could inhibit the proliferations of these cell lines in a time-anddose-dependent manner. TH1082significantly inhibited the proliferation of A375cells and the IC50was25.38±1.32μg/mL for24h.2. Effects of TH1082on morphological changes of A375cells2.1Morphological changes in A375cellsAfter the treatments with different concentrations of TH1082(0,5,10and20μg/mL) for24h in A375cells, the cells were observed by fluorescencemicroscope. We could see more and more A375cells floating in PRIM-1640, andfewer adherent cells were at the bottom of6-well-plate. The proliferation of A375cells was inhibited by TH1082in a dose dependent manner.2.2AO/EB staining and apoptotic rateAfter the treatments with different concentrations of TH1082(0,5,10and20μg/mL) for24h in A375cells, the cells were observed by fluorescencemicroscope. More and more orange dots could be seen. It indicated that TH1082could induce the apoptosis of A375cells by a dose dependent manner. The apoptoticrates were3.1±0.82%,9.5±2.09%,18.9±2.25%and39.5±2.02%, respectively.2.3Hoechst33342/PI stainingAfter the treatments with different concentrations of TH1082(0,5,10and20μg/mL) for24h in A375cells and Hoechst33342/PI staining, the cells wereobserved by fluorescence microscope. More and more red dots could be seen. Itindicated that TH1082could induce the apoptosis of A375cells by a dose dependentmanner. 2.4DAPI stainingAfter the treatments with different concentrations of TH1082(0,5,10and20μg/mL) for24h in A375cells and DAPI staining, the cells were observed byfluorescence microscope. The result indicated that TH1082could induce theapoptosis of A375cells by a dose dependent manner.3. Study on mechanism of apoptosis in A375cells induced by TH10823.1Effects of TH1082on mitochondrial transmembrane potential in A375cellsA375cells were treated with different concentrations of TH1082(0,5,10and20μg/mL) for24h, then A375cells were stained by JC-1. The mitochondrialtransmembrane potential significantly decreased in a dose-dependent manner onA375cells and they were14.05±0.57,11.83±0.62,6.79±0.84and3.29±0.27,respectively.3.2Effects of TH1082on Bcl-2and Bax by Western Blot and QPCRA375cells were treated with different concentrations of TH1082(0,5,10and20μg/mL) for24h, and then the expressions of Bcl-2and Bax were detected byWestern blot and QPCR assays. TH1082could depress the expression of Bcl-2andincrease the expression of Bax both in Western blot and QPCR assays.3.3Effects of TH1082on the expressions of Cyt c, Smac and AIFA375cells were treated with different concentrations of TH1082(0,5,10and20μg/mL) for24h, and then the expressions of Cyt c, Smac and AIF were detectedby western blot assay. TH1082could up-regulate the expression of Cyt c, Smac andAIF in A375cells.3.4Effects of TH1082on caspase-3and caspase-9in A375cellsA375cells were treated with different concentrations of TH1082(0,5,10and20μg/mL) for24h, the activities of caspase-3(3.58±0.47,5.07±0.81and12.10±1.51folds vs control group) and caspase-9(1.92±0.16,2.42±0.22and5.08±0.65folds vs control group) were significantly increased by TH1082inA375cells. TH1082could up-regulate the expressions of caspase-3, caspase-9and PARP. And the activities of caspase-3(8.01±1.40fold vs control group), caspase-8(8.08±1.10fold vs control group) and caspase-9(3.58±0.27fold vs control group)were significantly decreased by TH1082after treated with Z-VAD-FMK in A375cells, the apoptotic rate decreased from39.8±1.20%to21±1.94%.3.5Effects of TH1082on Death receptor signaling pathwayA375cells were treated with different concentrations of TH1082(0,5,10and20μg/mL) for24h, the activity of caspase-8(2.10±0.19,5.19±0.70and11.59±1.74folds vs control group) was significantly increased by TH1082in A375cells. Theexpressions of caspase-8, Fas and FasL were detected by Western blot. TH1082could up-regulate the expressions of caspase-8, Fas and FasL. And the activity ofcaspase-8(8.08±1.10fold vs control group) was significantly decreased by TH1082after treated with Z-VAD-FMK in A375cells.3.6Effects of TH1082on cell cycle in A375cellsA375cells were treated with different concentrations of TH1082(0,5,10and20μg/mL) for24h, and detected by PI staining. TH1082could induce A375cellsG0/G1arrest (61.68±1.80%,76.16±2.24%,80.90±1.16%and87.09±1.31%) in adose-dependent manner.3.7Effects of TH1082on PI3K/Akt signaling pathwayA375cells were treated with different concentrations of TH1082(0,5,10and20μg/mL) for24h, and detected by western blot assay. TH1082could up-regulatethe expressions of p-PTEN and GSK-3β, and down-regulate the expressions ofPTEN, p-Akt(Thr308) and p-Akt(Ser473).3.8Effects of TH1082on MAPK signalingA375cells were treated with different concentrations of TH1082(0,5,10and20μg/mL) for24h, and detected by western blot assay. TH1082could up-regulatethe expressions of p-ERK1/2, and there were no changes in ERK1/2, p38MAPK,p-p38MAPK or JNK. There was no expression of p-JNK. After A375cells treatedwith PD98059, TH1082could decrease the expressions of p-ERK1/2, p53, p27kip1and Bax, and increase the expressions of c-myc, CDK4, cyclin D1and Bcl-2. And G0/G1phase arrest decreased from87.09±1.31%to78.6±0.65%, meanwhileapoptotic rate decreased from39.8±1.20%to16.1±1.64%. TH1082coulddown-regulate the expressions of Ras and c-Raf. These results indicated that TH1082could induce apoptosis in A375cells through ERK1/2signaling pathway.Conclusion：1. TH1082could inhibit significantly the proliferation of A375cells in a time-and dose-dependent manner.2. TH1082could induce apoptosis in A375cells.3. TH1082induced apoptosis in A375cells through both Mitochondrailpathway and Death receptor pathway.4. TH1082induced G0/G1phase arrest of A375cells by regulating ERK1/2signaling pathway, and Ras/c-Raf could regulate this effect.5. TH1082induced apoptosis in A375cells by PI3K/Akt pathway.