| Objective:To explore the effect of 10H:≤3,6-diazophene thiazide on the biological behavior of hepatocellular carcinoma cell line HepG2 and its mechanism,and to provide a new idea for the comprehensive treatment of liver cancer.Methods:In this study,the samples were divided into three groups,namely,the untreated group,the positive control group received the standard control IC50 concentration,and the treatment group receiving the IC50 concentration of the test compound 1 OH-3,6-diazophene thiazide(PTZ).The synthesis of PTZ compounds was synthesized with reference to the information obtained in the publication.Liver cancer cell line HepG2.was selected.The proliferation function was measured by MTT assay,the morphology of apoptosis was evaluated by(AO lPI)double staining,the apoptosis was quantitatively determined by membrane protein V-FITC staining,and the invasive ability of cells was verified by Transwell assay.The mechanism of anti-liver cancer action of PTZ was explained by measuring intracellular reactive oxygen species(ROS),mitochondrial cytochrome c,cystine asparaginase activity and mitochondrial thioredoxin reducase activity.Results:The samples were divided into three groups,namely,the untreated group,the positive control group received the standard control IC50 concentration,and the treatment group receiving the IC50 concentration of the test compound PTZ.HepG2 cancer cells were inoculated in 96 well flat plate with a density of 1 × 105 cells per hole.The compounds were dissolved in DMSO to the reserve concentration of 80mM and further diluted to 2.5,10,40,80μM,0μM as the untreated control,and incubated in CO2 incubator for 24 hours.PTZ test exhibited that PTZ showed inhibitory effect on the growth of HepG2 HCC cells in each concentration range.After 24 hours of treatment,the inhibitory concentration of PTZ on 50%maximum response(IC50)of HepG2 HCC cells was 37.03μM,and the IC50 value of positive control(DDP)was 109.23μM.AO/PI double staining(Acridine orange/propidium iodide)was used to qualitatively observe the apoptosis of HepG2 cancer cells treated with PTZ.The cancer cells formed fragment nucleus,condensed nucleus,released DNA content and formed membrane blistering.At the same time,it was noted that PTZ also induced the necrosis of liver cancer cells,and the hepatocytes stained the whole cell red.The apoptosis of HepG2 cells was quantitatively detected by membrane associated protein V-FITC.Flow cytometry showed that the inhibitory effect of PTZ on HepG2 cells was three times higher than that of positive control.Further detection of cell cycle checkpoint showed that the number of untreated HCC cells was the highest in GO/G 1 phase(80.93%),while most of the HCC cells treated with DDP and PTZIC50 were in S phase activation state and S phase stagnation state(74.90%in platinum group and 81.91%in PTZ group).At the same time,Transwell assay showed that the inhibition rate of PTZ on the invasion of HepG2 cancer cells was 43.64%.The results of intracellular reactive oxygen species(ROS)showed that the activity of ROS increased to 218.62%when HepG2 cells were treated with PTZ at the concentration of IC50,and the activity of ROS increased to 158.16%compared with the group treated with DDP(the activity of ROS in the untreated group was standardized to 100%).At the same time,flow cytometry showed significant amount of mitogen c in DDP and PTZ treatment groups.In the process of studying the mechanism of apoptosis induced by PTZ,the activities of four kinds of cytidine asparaginase(Caspase-3/7,8,9,10)in Caspase-3/7,8,9,10 were quantitatively determined.the results showed that the activities of cytidase(caspase-3/7,caspase-85caspase-9 and caspase-10)in HepG2 treated with PTZ were higher than those in control group.Cysteinase activity).The up-regulated cystine asparaginase activity suggests that PTZ can induce apoptosis of HepG2 cancer cells through internal and external pathways.Finally,the somal thioredoxin reductase was measured.The values of the untreated control were standardized to 100%and the normal TrxR enzyme activity was hypothesized.The results showed that the TrxR enzyme activity of the DDP treatment group was inhibited to 19.52%,while the TrxR enzyme activity of the PTZ treatment group was inhibited by 21.43%.Conclusion:The high mortality rate in patients with liver cancer is caused by the resistance of cancer cells to chemotherapeutic drugs.In this study,the inhibitory effect of PTZ on HepG2 cells was 3 times higher than that of DDP at 1 OH-3,6-diazophene thiazide(PTZ),compared with Cisplatin.in the study of the mechanism of PTZ inhibiting HCC cells,it was found that PTZ could activate S phase cell cycle checkpoint and inhibit the replication process of DNA,thus inhibiting cell division.At the same time,it was further analyzed that the inhibitory effect of PTZ existed after the proliferation-electrostatic effect began.Therefore,the genomic damaged cancer cells were finally removed from the tumor cell population.In addition,the increase of the cell concentration of reactive oxygen species(ROS)and the inhibition of the activity of mitothioredoxin reducase(TrxR)showed that PTZ selectively attacked the mitochondria of cancer cells,thus limiting the cell proliferation and increasing its sensitivity to apoptosis signal. |
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