| BackgroundGastric cancer(GC)is one of the most common malignancies,ranked second in cancer mortality worldwide.Although chemotherapeutic drugs are play important role in cancer treatment,the side effects and the drug resistance force people to find new therapeutic strategies.Iron chelation is a hot topic in the research of antitumor drugs.Transition metals,such as copper and iron,play an important role in cell growth and proliferation.Dithiocarbamate is a kind of metal chelating agent which has strong complexation ability to transition metals.It is involved in regulating cell apoptosis and oxidative stress.But Dithiocarbamate inactivate the enzyme because of its affinity to metals.The enzyme is an important biological macromolecule in the body of life,and most of the enzymes rely on metal ions.Therefore,the derivatives of dthiocarbamate are prepared by structural modification.The derivative of the disulfide has strong antitumor activity on liver cancer,and the dipyridylhydrazone dithioc arbamte butyric acid ester(Dpdtb A)is also one of derivatives of disulfide carbamate,and its inhibitory effect on GC and the mechanisms are merit to explore.ObjectiveTo explore the effect of Dpdtb A on the growth of MGC-803 cells and SGC-7901 cells in human gastric cancer,and to reveal the mechanism of its influence on the growth of gastric cancer.Methods1.Dpdtb A was obtained by chemical synthesis,and the structure was confirmed by magnetic resonance and mass spectrometry.DMSO is used as a solvent to prepare the solution.Routine-fostering with human gastric MGC-803 and SGC-7901 cells.2.The cell proliferation effect of Dpdtb A was assessed by MTT assay.When detecting the effect of Dpdtb A on cell growth,cells were divided into 8 experimental groups and 1 DMSO group.When detecting whether ROS is related to the effect of Dpdtb A on cell growth,we has NAC acted on cells for 2h,then divided them into 8 experimental groups and 1 DMSO group.The final concentration of Dpdtb A in the experimental group was 0.15 ?M?0.3 ?M?0.6 ?M?1.25 ?M?2.5 ?M?5 ?M?10 ?M and 20 ?M,the DMSO group only added DMSO.Observed and recorded the cell morphology changes after 48 hours of Dpdtb A treated with inverted microscope.3.The effect of Dpdtb A on reactive oxygen species was detected with DCFH-DA by flow cytomeay(FCM).Cells were divided into 3 experimental groups,the DMSO group and the blank control group.The final concentration of Dpdtb A in the experimental group was 2.5 ?M?5 ?M and 10 ?M,the blank control group did not deal with it.4.Annexin V-FITC and PI staining were used to detect the effect of Dpdtb A on cell apoptosis by FCM,The groups and intervention were as same as cell reactive oxygen species(ROS).5.Rhodamine 123(Rh 123)was used to detect the effect of Dpdtb A on mitochondrial membrane potential by FCM.The groups and intervention were as same as cell ROS.6.The changes of apoptosis related proteins(casepase 8?p53? Cyt C? Bax?Bcl-2),Cath D,LC3-I and LC3-II were measured by Western Blot,And the Bax/Bcl-2 ratio was calculated.To detect the effect of Dpdtb A on apoptosis associated protein,cells were divided into 3 experimental groups and DMSO group,The final concentration of Dpdtb A in the experimental group were 1.25 ?M?2.5 ?M and 5 u M.To detect the effect of Dpdtb A on Cath D protein in MGC-803 cells: cells were divided into 2 experimental groups and DMSO group,The final concentration of Dpdtb A in the experimental group were 2.5 ?M and 5 u M.To detect whether ROS plays role in the apoptosis which induced by Dpdtb A: cells were divided into Dpdtb A+NAC group?Dpdtb A group and DMSO group.To detect whether p53 play role in the apoptosis which induced by Dpdtb A: cells were divided into Dpdtb A+PFT-? group?DMSO+PFT-? group?Dpdtb A group and DMSO group.To detect whether ROS play role in autophagy in MGC-803 cells which induced by Dpdtb A: cells were divided into Dpdtb A+NAC group? Dpdtb A+3-MA group? Dpdtb A group?NAC group? 3-MA group and DMSO group.All the above groups were named according to the intervention drugs.7.The change of lysosomal membrane permeability of MGC-803 cells was observed by Lyso-Tracker Red staining and fluorescence microscope.Cells were divided into 2 experimental groups and the DMSO group.The final concentration of Dpdtb A in the experimental group was 2.5 ?M and 5 ?M.8.The effect of Dpdtb A on Cath D of in MGC-803 cells was detected by immunofluorescence.Cells were divided into 1 experimental group and the DMSO group.The final concentration of Dpdtb A in the experimental group was 2.5 ?M.9.Acridine orange staining was used to detect the effect of Dpdtb A on cell autophagy by fluorescence microscope.Cells were divided into 2 experimental groups and 1 DMSO group;whether autophagy inhibitor 3-MA had effect on cell autophagy: After 3-MA treated on cells for 2h,cells were divided into 2 experimental group and 1 DMSO group.The final concentration of Dpdtb A in the experimental group was 2.5 ?M and 5 ?M.10.PI staining was used to detect the effect of Dpdtb A on cell cycle by FCM.Cells were divided into 2 experimental group,DMSO group and blank control group.The final concentration of Dpdtb A in the experimental group was 2.5 ?M and 5 ?M,and the blank control group did not deal with it.11.SPSS17.0 statistical software was used for statistical analysis.The data were expressed by mean±standard deviation.The Mean was compared by analysis of varian and the level was ?=0.05Results1.Dpdtb A inhibits the growth of human gastric cancer cells.The IC50 of Dpdtb A on MGC-803 cells and SGC-7901 cells were 3.80 ± 0.40 ?M and 4.20 ± 0.52 ?M respectively.2.Dpdtb A induced more ROS production to inhibite the growth of MGC-803 and SGC-7901 cells.Cells were treated with Dpdtb A for 1.5 h,the fluorescence of DCFH-DA enhanced,indicating that the Dpdtb A induced ROS production of GC cells in a concentration dependent manner.NAC is ROS scavenger,the inhibitory effect of Dpdtb A against both cell lines in the presence of NAC was significantly attenuated.For MGC-803 cell,the IC50 was increased by 3 folds in the presence of NAC;similarly for SGC-7901 cell,Dpdtb A led to 30% growth inhibition at higher concentration(20 ?M)in the presence of NAC.3.Dpdtb A induced apoptosis in MGC-803 and SGC-7901 cells.Treated MGC-803 and SGC-7901 with Dpdtb A for 24 h cells,the Dpdtb A induced early apoptosis and later apoptosis of GC cells in a concentration dependent manner,while the early apoptosis populations changed significantly;And the the fluorescence of Rh123 was enhanced with increasing of Dpdtb A.4.Dpdtb A can affect the expression of apoptosis related proteins.Treated MGC-803 and SGC-7901 with Dpdtb A for 24 h,the expression of p53,Cyt C,Caspase 8 and Bax increased in a concentration dependent manner,the relative ratio of Bax/Bcl-2 was increased and higher than DMSO group.5.Dpdtb A affects the expression of apoptosis related proteins closely related to ROS.Treated MGC-803 and SGC-7901 with NAC for 2h and add Dpdtb A,the expression of p53,Cyt C,Caspase 8 and Bax in Dpdtb A plus NAC group were lower than that of Dpdtb A group but higher than that of DMSO group;The ratio of Bax/Bcl-2 in Dpdtb A plus NAC group was lower than Dpdtb A group but higher than DMSO group too.6.p53 regulates Dpdtb A induced apoptosis.After p53 inhibitor,PFT-? acts on cells for 2h then add Dpdtb A,the expression of p53 in Dpdtb A+PFT-? group is lower than that in Dpdtb A group,and DMSO plus PFT-? group is also lower than that of DMSO group,suggesting that PFT-? inhibits the expression of p53 protein.With the decrease of p53,the expression of Cyt C,Bax and caspase 8 decreased,and the expression of Bcl-2 increased.The ratio of Bax/Bcl-2 ranked from Dpdtb A group,Dpdtb A plus PFT-? group,DMSO group to DMSO plus PFT-? group(P<0.05).7.Dpdtb A changed lysosome membrane permeability and induced the release of enzyme in MGC-803 cells.Treated MGC-803 and SGC-7901 with Dpdtb A for 24 h,the red fluorescence enhanced in a concentration dependent manner,compared with the DMSO group.The Cath D of DMSO group cells was granular aggregated,but it was diffuse after Dpdtb A treatment,and the amount of Cath D increased in a concentration dependent manner.8.Dpdtb A induced autophagy in MGC-803 cells.The orange red fluorescence increased in a concentration dependent manner,indicating that the autophagesome increased.While,Pretreated cells with autophagic inhibitor 3-MA for 2 h then add Dpdtb A,the orange red fluorescence decreased.9.ROS was associated the autophagy in MGC-803 cells induced by Dpdtb A.The expression of LC3-I changed little.The expression of LC3-II highest in Dpdtb A group,higher in Dpdtb A plus NAC group,and lowest in Dpdtb A plus 3-MA group,NAC group,3-MA group and DMSO group.10.Dpdtb A affect on cell cycle.Treated MGC-803 and SGC-7901 with Dpdtb A for 24 h.induce cell cycle blocked in a concentration dependent manner,Dpdtb A blocked the cells in the G1 phase.Conclusions:1.Dpdtb A can inhibit the growth of gastric cancer cells.2.The inhibition may stem from induction of ROS generation,subsequently provoking p53 response,triggering apoptosis,autophagy,lysosomal cell death and cell cycle arrest. |
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