The Anti-tumor Mechanism Of Benzaldehyde Nitrogen Mustard-2-Pyridine Carboxylic Acid Hydrazone And Its Copper Complex

BackgroundCancer has been a common disease which threatens human life.Usually the localized cancer can be removed by surgery,but the metastatic cancer have to rely on the chemotherapy.Nitrogen mustard is widely used in clinic,but the side effects and the drug resistance of these drugs limit their use.The anti-tumor characteristics of iron chelating agent and its ability to inhibit invasion and metastasis have shown good prospects for clinical application.Multiple targets drugs can significantly reduce the side effects and enhance the therapeutic effect.we combined the iron chelating agent into the designation of derivatives of nitrogen mustard to form a novel multifunction antitumor drug.ObjectiveIn order to exploit a highly efficient and low toxic antitumor drugs,In this paper,we condensated Pyridine Carboxylic Acid Hydrazine?iron chelator?with Benzaldehyde Nitrogen Mustard to form a multifunational antitumor drug.In view of the biological function of copper ions,the Copper Complex of the Nitrogen Mustard derivative was also investigated in proliferation inhibition.Their antitumor mechanism was preliminarily evalucidated.Methods1 The Benzaldehyde Nitrogen Mustard-2-Myridine Carboxyl Acid Hydrazone and its Copper Complex was prepared based on literature.2 MTT assay was used to detect the cytotoxicity of Benzaldehyde NitrogenMustard-2-Myridine Carboxyl Acid Hydrazone and its Copper Complex on different tumor cell,and their median inhibitory concentrations?IC50?were calculated.3 Fluorescence Spectrophotometer method was used to assess the ability of ROS production with Benzaldehyde Nitrogen Mustard-2-Myridine Carboxyl Acid Hydrazone and its Copper Complex in vitro and in vivo.4 Single Cell Electrophoresis Method was used to detect the celluar DNA damage under the Benzaldehyde Nitrogen Mustard-2-Myridine Carboxyl Acid Hydrazone and its Copper Complex,The melting point method was used to detect the combination action of the drugs and DNA.5 RT-PCR and Western Blot were used to detect the apoptosis related genes and their protein expression of the Benzaldehyde Nitrogen Mustard-2-Myridine Carboxyl Acid Hydrazone and its Copper Complex.6 The Flow Cytometry is used to observe the drug effect on cell cycle on HepG₂.7 Acridine Orange Staining method and lysosome red fluorescent probe staining method was used to detect the Benzaldehyde Nitrogen Mustard-2-Myridine Carboxyl Acid Hydrazone and Its Copper Complex of autophagy and lysosomal permeability and mitochondrial permeability.Results1 The Benzaldehyde Nitrogen Mustard-2-Myridine Carboxyl Acid Hydrazone and its Copper Complex was characterized as described in literature.Both of them can be dissolved in 75% DMSO.2 The Benzaldehyde Nitrogen Mustard-2-Myridine Carboxyl Acid Hydrazone and Its Copper Complex have growth inhibitory effect on HepG₂ and HCT-116 cells?for HepG₂:IC50=31.6±1.7?mol/L;for HCT-116: IC50=44.1±1.5?mol/L?,especially the Copper Complex showed stronger growth inhibition on tumor cell,10 times stronger than the Benzaldehyde Nitrogen Mustard-2-Myridine Carboxyl Acid Hydrazone?forHepG₂ :IC50=3.5±0.8?mol/L;for HCT-116:IC50 = 4.5 ± 0.5 ?mol/L?.3 Fluorescence method and Active Oxygen detection reagent kit show the Benzaldehyde Nitrogen Mustard-2-Myridine Carboxyl Acid Hydrazone and its Copper Complex can promote the formation of ROS in vivo and in vitro.The Copper complex can produce stronger ROS in low concentration,consistent with its stronger cytotoxicity.4 The Single Cell Electrophoresis Assay showed the Benzaldehyde Nitrogen Mustard-2-Myridine Carboxyl Acid Hydrazone and Its Copper Complex can cause DNA breaks in HepG₂ cells and its Copper Complex can make most of the DNA fragmentation in low concentration.Melting Point Method showed the Benzaldehyde Nitrogen Mustard-2-Myridine Carboxyl Acid Hydrazone and Its Copper Complex mainly interact by trench or electrostatic with from complexes.5 RT-PCR and Western Blot results showed that the apoptosis related gene and protein of Bcl-2 family was activated,the expression of Bax and Caspase-8 was increased,Bcl-2decreased,protein Cyclin D related to cycle was changed.6 Both Benzaldehyde Nitrogen Mustard-2-Pyridine Carboxyl Acid Hydrazone and its Copper Complex caused an arrest in the S phase and in a dose dependent relationship.7 The proliferation inhibition of the Benzaldehyde Nitrogen Mustard-2-Myridine Carboxyl Acid Hydrazone and its Copper Complex may involve autophagy.This was preliminarily confirmed by evaluation of the AVOs in HepG₂ cells.Conclusion1.The antitumor activity of Benzaldehyde Nitrogen Mustard-2-Pyridine Carboxyl Acid Hydrazone and its Copper Complex are related to the production of ROS.Both of them can cause the DNA fragmentation and induce the apoptosis of tumor cells.2.Both they can cause S phase arrest,which is related to their DNA alkylation.3.Copper Complex showed a stronger inhibitory effect,which is related to its stronger ability of ROS production.In addition,the Benzaldehyde Nitrogen Mustard-2-Pyridine Carboxyl Acid Hydrazone and its Copper Complex can induce tumorcell death through autophagy.