The Proliferation Inhibition And Mechanism Of A Nitrogen Mustard Contained Mannich Base Against Some Tumor Cell Lines

BackgroundMalignant tumor has become one of the main causes of human death,which brings a serious impact on our daily life,mental and physical health.More and more new chemotherapy drugs are springing up,since there is an increasing demand for anti-tumor drugs in the therapy of tumors.Nitrogen mustard is one of the most common anti-tumor drugs in clinical,but haves strong toxic and side effects,therefore many nitrogen mustard derivatives have been synthesized to reduce its toxicity and enhance its selectivity.Kojic acid is an antioxidant,which can effectively scavenge free radicals and protect normal cells.So the organic combination of kojic acid with free radical scavenging ability and DNA alkylate reagent-nitrogen mustard which will form a new nitrogen mustard derivative that may reduce the toxic and side effect on normal cells and improve the anti-tumor effect of nitrogen mustard.We prepared nitrogen mustard containing Mannich base based on the thinking and screen its growth inhibition for some tumor cell lines.Meanwhile,considering the metal-chelating ability of kojic acid in the Mannich base,we also evaluate the cytotoxicity of Mannich base in the presence of copper ion.In view of the excellent anti-tumor activity,the possible mechanism of the Mannich base and its copper complex was investigated preliminarily.AimTo study the inhibition effect and possible mechanism of the Mannich base and its copper on some tumor cells.MethodsHuman hepatocellular carcinoma cells,HepG2 and human colon carcinoma cells,HCT were cultured in vitro.Cells in logarithmic phase were used for the experiment.The growth inhibition rates of a nitrogen mustard contained mannich base on tumor cells(HepG2 and HCT)were measured by detecting the succinate dehydrogenase activity in apoptotic cells(MTT method)in order to get the half inhibition concentration(IC50);The DNA alkylating ability(DNA cross-linking)and generation of the reactive oxygen species(ROS)and DNA cleavag induced by the Mannich base and its copper complex in the Fenton-like reaction were evaluated in vitro;accordingly ROS induction and DNA fragmentation caused by the Mannich base and its copper in vivo were investigated by DCF-AM fluoresence and single cell gel electrophoresis(comet tail);The expriment of melting point of Ct-DNA in the presence or absence of the Mannich base(or its copper complex)was conduced to study the interaction model between the invetigated compound and DNA;The cell cycle analysis was performed by flow cytometry to probe the possible disturbance of cell growth cycle;RT-PCR was used to investigate the effects of the Mannich base and its copper complex on apoptosis related gene expression,exploring the possible molecular mechanism.Results1.Both the Mannich base and its copper complexes showed inhibition effect on HepG2 and HCT cells,and in a concentrations dependent manner.The IC50 of the Mannich base and its copper complexes were(4.5,9)±0.5 ?M for HepG2 cell and 4.5±0.02 ?M for HCT cell,respectively.Accordingly the Mannich base in the presence of copper ion worked better than the Mannich base,and the differences showed statistical significance(P<0.05).2.In vitro ROS assay,it showed that the Mannich base can participate in Fenton-like reaction to generate ROS.And the ROS produced by the Mannich base caused DNA cleavage in a concentration dependent manner.The Mannich base was also shown to have DNA alkylation ability to lead DNA ross-linking based on agarose gel electrophoresis.3.The melting point(MP)experiment showed that the Mannich base and its copper complex could interact with Ct-DNA either via intercaltion or cross-linking for the significant increase of MP(>10?).4.The Mannich base and its copper complex disturbed the cell cycle of HepG2 cells,led to an S phase arrest.5.The results of RT-PCR showed that Mannich base and its copper complex can regulate the expression of Bax,Bcl-2,caspase-3 and caspase-8 in HepG2 cells,a up-regulation of Bax,caspase-3,caspase-8 and down-regulation of Bcl-2 were observed,indicating that apoptosis was involved in their cytotoxicity.Conclusions1.The Mannich base and its copper complex exhibit excellent proliferation inhibition on HepG2 and HCT-11 cell lines,and the inhibition in a concentration dependent manner.2.The Mannich base and its copper complex possed DNA alkylating ability and corss-linking DNA.The DNA fragmentaion induced by the agents may stem from their ROS generation.The interaction between the Mannich base and its copper complex may also involve in intercalation except cross-linking,all of above mentioned contributed their cytotoxicity.3.The Mannich base and its copper complex lead to cell cycle arrest at S phase.4.The cytotoxicity of the Mannich base and its copper complex involved in apoptotic pathway.