Inhibitory Mechanism Of Dp44mT,Cisplatin And Their Combination On Human Hepatocellular Carcinoma Cell Line HepG2

BackgroundCancer is a systemic wasting disease.Despite decades of studies on it,in developed countries,cancer is still the main cause of death.For most tumors,however,the treatment effect of any single chemotherapic agent is not ideal due to drug resistance,thus a variety of more comprehensive treatment of methods is required.Chemotherapy is an important treatment other than surgery,but its major limitation is unable to distinguish between healthy cells and cancer cells,and will inevitably produce a series of side effects such as nausea,vomiting,loss of appetite,hair loss,liver and kidney damage,bone marrow suppression,and even induce cancer.Therefore,to find the anti-tumor drugs with higher selectivity,low side effects and well tolerated feature is the priorities and hot spots for the clinical works and scientific researches.In recent years,numerous studies have shown that the Di-2-pyridylketone-4,4-Dimethyl-3-thiosemicarbazone(Dp44mT)with broad-spectrum anti-tumor activity.However,the investigation related to the combination of the cisplatin and the Dp44 m T on human hepatoma cell line HepG2,has not yet been reported.In the present study the inhibitiory mechanism of the Dp44mT and cisplatin in individual and combined on human hepatocellular carcinoma cell line HepG2 was prelimetary investigated.PurposeTo investigate the proliferation inhibition of Dp44mT,cisplatin individual and combination on human hepatoma cell line,and the underlying mechanism was also included.MethodsHuman hepatocarcinoma HepG2 cells were cultured in RPMI-1640 medium,and when the cells were in the logarithmic phase,the experiment was carried out,and the experiment was divided into four groups: control group,Dp44mT group,cisplatin group and combination group.1 The morphology of cells was observed by inverted microscope.The growth inhibition rate of tumor cells(HepG2)was measured by the activity of succinate dehydrogenase(MTT)of apoptotic cells,and the inhibition rate of half inhibition concentration(IC50)was obtained from simulated prolifertive curve;2 The ablity of reactive oxygen species(ROS)formation of Dp44mT,cisplatin and their combination in vitro was assessed by fluorescent method;3 The ROS induced by Dp44mT and cisplatin individually and as well as their combination in vivo was detected using similar menthod as above 2.4 Single cell gel electrophoresis(comet tail)was used to evaluated the cellular DNA damage caused by Dp44mT,cisplatin and their combination.5 The effect of the Dp44mT,cisplatin and the combinationits on the growth of human hepatoma cell line HepG2 cell cycle were measured by flow cytometry.Results1 Dp44mT,cisplatin and the combination can significantly inhibit the growth of human hepatoma HepG2 cells,and the inhibition was positively correlated with the concentration of the drugs,compared with the control group,the difference was statistically significant(P <0.05);2 In invitro ROS assessment indicated that Dp44 m T group,cisplatin group and cisplatin combination Dp44mT group,Dp44mT + Vit C group,cisplatin + VitC group,cisplatin + Dp44mT + VitC group had the ability to produce extracellular ROS and among these cisplatin + Dp44mT + VitC group was strongest;3 From ROS measurement in vivo Dp44mT group,cisplatin group,and their combined all induced intracellular reactive oxygen species;4 Single cell gel electrophoresis(comet tail)was used to detect the DNA fragmentation induced by Dp44mT,cisplatin and their combination,the comet tails were appeared in all rested group in a concentration dependent manner,yet combination group produced the longest comet tail;5 Cisplatin lead to the cell cycle of human hepatoma cell line HepG2 arrested in G1 phase,Dp44mT can make the cell cycle of HepG2 arrest in S phase;and their combination can make HepG2 cells cycle arrest in S phase.Dp44 m T blocking effect on cell cycle of HepG2 cells could be enhanced by cisplatin and in a concentration negatively correlated manner,and the lower the concentration of cisplatin,the higher population in S phase caused by Dp44mT.Conclusions1 The proliferation inhibition of the Dp44mT and the cisplatin on HepG2 was a concentration dependent,and the combination of them was showed a synergistic or additive effect.2 The Dp44mT,cisplatin and their combination in vitro or in vivo can all produce the ROS in a concentration dependent manner and the combination of them was more stronger than individaul in ROS generation,accordingly the DNA fragmentation caused by them has similar patern,indicating the proliferation inhibition of them was mediated by ROS3 Cisplatin make the cell cycle of HepG2 arrest in G1 phase;Dp44mT can make the cell cycle of HepG2 arrest in S phase;and two drugs in combination can make HepG2 cells cycle arrest in S phase.Dp44mT blocking effect on cell cycle of HepG2 cells could be enhanced cisplatin,but the enhancement seemed to be more stronger at low concentration than at higher concentration.In conclusion,the proliferation inhibition of the investigated agents involved in ROS mediated DNA damage and cell cycle arrest,implying multiple mechanism contribution to their antitumor activity.