The Antitumor Activity And Mechanism Studies On Two Leukemia Cell Lines

Objective:Acutemyeloid leukemia?AML?is a heterogeneous cancer,which causes serious harm to human health,and its morbidity and mortality is still rising,has become a very important public health problems.So far,leukemia treatment includes radiotherapy,chemotherapy,immunotherapy,and bone marrow transplantation et al.Chemotherapy is a means of tumor systemic treatment which ccupies an important position in tumor treatment.Among these,the chemotherapy drug arsenic trioxide has been reported with a high remission rate for treatment of acute promyelocytic leukemia,but there are still side effects and drug cytotoxic problems.So,it is urgent to find new more effective chemotherapeutic agents with high biological effect and low toxicity.Polyoxometalates?POMs?are a large family of nanoscale inorganic metaloxide cluster compounds formed by covalent bonding of metal?vanadium,molybdenum,tungsten element,et al?and oxygen.Studies have shown that polyacid compounds have high selectivity inhibition of enzyme function,antitumor activity in vivo and in vitro,broad-spectrum antiviral activity and anti-transcriptase activity.Arsenic is commonly as the hetero atom in polyoxometalates and it can form heteropolyacid compounds with molybdenum,vanadium and tungsten atoms.These compounds may be have the properties of the heteropolyacid and arsenic trioxide and have potential applications in the treatment of acute leukemia.Otherwise,Using the spectroscopy technology to study on the interaction of drugs with biological molecules have important contributed to understand the action mechanism of drugs in vivo,to explain the drug toxicity and pharmacological effects.Polyoxometalates can interact with protein molecules,which are of particular importance for the design of polyacid antitumor drugs.In this paper,arsenomolybdate were synthesized and characterized.the anti-proliferative effect was determined in vitro.In addition,thebinding interactions of compound 1 with biological macromolecules were investigated at physiological conditions using the spectroscopic methods.Methods:The arsenomolybdate was prepared,and then characterized by IR,UV,X-Ray and elemental analysis.The stability of the arsenomolybdate was studied by laser Raman spectroscopy at physiological conditions.Acute leukemia cell lines HL-60 and U937 were selected,and the effect of compound 1 on anti-proliferative effects of acute myeloid leukemia HL-60 and U937 cellswere assessed by MTT assay.The human umbilical vein endothelial cell line HUVEC was selected and the toxicity of the arsenomolybdate to normal cells was tested by MTT.Morphological changes of leukemia cells were detected by confocal laser scanning microscope.The changes of cell cycle and apoptosis of leukemia cells were evaluated by the flow cytometry.The expression of apoptosis-related proteins in leukemia cells was studied by western blot.The binding interactions and apoptosis mechanism of the arsenomolybdate with HSA were investigated at physiological conditions using the methods of UV-Vis absorption spectroscopy,fluorescence and synchronous fluorescence spectroscopy.Results:1.The arsenomolybdate K₂Na[As Mo₆O₂₁?O₂CCH₂NH₃?₃]·6H₂O 1 was synthesized.The structure of the compound is clear and has good stability under physiological conditions.2.The arsenomolybdate can inhibit the proliferation of HL-60 and U937 cells by MTT assay,and the 50% lethal concentration(IC₅₀)value of 8.61 ?M for HL-60 and 14.50 ?M for U937 at 24 h.The inhibitory effect of arsenomolybdate 1 showed dose dependence.Comparing to the positive controls,the anti-leukemia activity of the compound was better than all-trans retinoic acid.The anti-leukemia activity of the compound was similar with arsenic trioxide.3.The toxicity of arsenomolybdate 1 to normal cells HUVEC was lower than that of arsenic trioxide.4.The arsenomolybdate 1 could induce leukemia HL-60 and U937 cell apoptosis.5.The arsenomolybdate 1 could induce G₁ phase cell cycle arrest in HL-60 and U937 cells.6.The arsenomolybdate also induced apoptosis of HL-60 and U937 cells bymodulating cleaved caspase-3 and bcl-2 expression.7.According to the spectroscopic study,the binding of arsenomolybdate and ct DNA was attributable to electrostatic.It can be obtained that the values of 1.4×10⁴and 4.05×10³ M^-1 were obtained for association binding constant of compound 1 at R > 0.625 and R ? 0.625,respectively,where R= [compound 1]/[ct DNA].Arsenomolybdate could interaction with HSA and BSA.The conformation of the HSA and BSA was changed,and the hydrophilicity of the microenvironment increased.The binding constant of compound and HSA was 5.24×103 M^-1,and the binding constant of compound and BSA was 4.90×104M^-1by calculating.Conclusion:1.The arsenomolybdate 1 has a clear structure with an stable performance,and has potential anti-leukemia activity in vitro.2.The arsenomolybdate 1 has low toxicity to normal cells HUVEC;3.The arsenomolybdate 1 could induce cell apoptosis and arrest cell cycle G₁ phase.4.The arsenomolybdate 1 could interaction with ct DNA and serum albumin.