Radiosensitizing Effect And Mechanism Of Polyoxometalates In HT-29 Cells

Cancer is a major global public health problem and a serious threat to human health.High energy ionizing radiation can kill tumor cells directly by interacting with biomolecules and indirectly by generating free radicals from radiolysis of water molecules in tumor microenvironment.However,due to the special microenvironment of tumor,such as tumor hypoxia,the resistance of tumor cells to radiation is enhanced,and the effect of radiotherapy is not obvious.Therefore,it is of great significance to select highly effective and low-toxicity radiosensitizers to improve the radiosensitivity of tumors.Polyoxometalate（POMs for short）are polyanion clusters formed by high ordinal number metal elements,especially molybdenum,vanadium,and tungsten,connected by oxygen.They are a kind of inorganic clusters with nanoscale structure and particle size generally between 0.5-6nm.It has been shown that metals containing high ordinal number have strong X-ray deposition ability.Therefore,POM is expected to be used as a new sensitizer for radiation therapy of tumor.Objective:POM and POM complex were synthesized,and their composition,structure,morphology,and physiological stability were characterized.Using human colon cancer cell line HT-29 as the model,the radiosensitization effects of POM and POM complex combined with X-ray on POM and POM complex combined with X-ray were clarified,and the mechanism of radiosensitization of POM and POM complex combined with X-ray was elucidated,to provide scientific basis for new radiosensitizer.Methods:1.The POMs Na₆V₁₀O₂₈ and K₃Na₄H₂(Gd W₁₀O₃₆)·2H₂O are synthesized.Using K₃Na₄H₂(Gd W₁₀O₃₆)·2H₂O as the matrix,the dopamine-tungstate gadolinium nanocomplex(Gd W₁₀@PDA)was prepared by hydrothermal method by optimizing the mass ratio of dopamine to tungstate gadolinium and the volume of reaction solvent.A tungstate gadolinium catalase complex(Gd W₁₀@PDA-CAT)was prepared by covalently coupling catalase（CAT）to the surface of Gd W₁₀@PDA using a polydopamine surface functional group.2.Transmission electron microscope（TEM）,ultraviolet spectrophotometer（UV-Vis）,Fourier infrared spectrometer（IR）,Malvern particle size analyzer,thermogravimetric analysis were used to characterize the morphology and composition of POM and POM complex.3.The catalase activity of Gd W₁₀@PDA-CAT was studied by ultraviolet absorption,and the stability of Gd W₁₀@PDA-CAT was evaluated by potassium thiocyanate-titanium trichloride spectrophotometry.4.Human colon cancer cell HT-29 was selected as the cell model to study the effects of X-ray and POM and POM complex on tumor cell proliferation by thiazolam colorimetry（MTT）.The effects of X-ray and POM and POM complex on survival and morphology of HT-29 cells were determined by immunofluorescence method.The effects of X-ray,POM and POM complex on apoptosis of tumor cells were studied by flow cytometry.The inhibitory effect of POM and POM complex combined with X-ray on tumor cell proliferation was studied by clonal colony experiment,and the radiosensitization ratio was calculated.The levels ofγ-H₂AX and ROS in HT-29 cells affected by POM and POM complex combined with X-ray were determined by immunofluorescence method.Results:1.Synthesis of decadadate Na₆V₁₀O₂₈ and tungstate gadolinium complex Gd W₁₀@PDA-CAT,the two substances have clear structure and composition,and have good stability under physiological conditions.2.Decavadate Na₆V₁₀O₂₈ and tungstate gadolinium complex Gd W₁₀@PDA-CAT showed concentration-and dose-dependent inhibition of HT-29 proliferation in human colon cancer cells.The median effective inhibitory concentration(IC₅₀)of Na₆V₁₀O₂₈for 24 hours was 8.994μg/m L,the inhibitory effect of tungstate gadolinium complex on HT-29 cells was low,and the median inhibitory concentration was 81.03μg/m L at 24hours.Gd W₁₀@PDA-CAT had no inhibitory effect on HT-29 cells at the concentration of 50μg/m L,but showed radiosensitization.Hoechst33342/PI staining showed that the tumor cells treated with two POMs combined with X-ray showed strong red fluorescence signal,and the cells showed many apoptosis and necrosis.Hoechst33342/PI staining showed that tumor cells treated with two kinds of POMs combined with X-ray showed strong red fluorescence signal,and many apoptosis and necrosis occurred.Hoechst33342/PI staining showed that tumor cells treated with two kinds of POMs combined with X-ray showed strong red fluorescence signal,and many apoptosis and necrosis occurred in the cells.3.The results of flow cytometry analysis showed that HT-29 cells were incubated with two POMs and then subjected to X-ray irradiation,and the cells in the group were treated with more apoptosis and necrosis,and the difference was statistically significant.4.The results of colony formation experiment showed that both POMs had obvious radiosensitization effect,and the combination treatment with X-ray could significantly inhibit the formation of cell colonies.The radiosensitization ratio of gadolinium ungstate complex was 3.7,and that of decadadate was 8.52.5.The expression level of DNA double-strand break markerγ-H₂AX in tumor cells after different treatments was detected by immunofluorescence method.The results showed that the content of POMs and X-ray combined treatment group was higher than that of other single treatment groups,indicating that the DNA double-strand break occurred significantly,leading to tumor cell apoptosis.6.Reactive oxygen species（ROS）generation in tumor cells in different treatment groups was detected by ROS probes.Tumor cells incubated with POMs and irradiated by X-rays showed strong fluorescence signals,indicating a large amount of ROS generation,thus inducing tumor cell death.Conclusion:1.Successfully synthesized POM and POM complex,the two substances have clear structure and stable properties.2.POM and POM complex can reduce the survival rate of HT-29 cells and have a certain dose-response relationship.3.POM and POM complex containing Gd,W and V high-Z elements can deposit large amounts of radiation in tumor cells,enhance the interaction between X-ray and DNA,lead to a large number of DNA double-strand breaks,increase the level of ROS,and thus enhance the radiosensitivity of tumor cells.