Experimental Studies Of Effects Of As₂O₃on C57BL/6Mice Lewis Lung Cancer Pleural Xenograft Malignant Pleural Effusion

【Background and Objective】Lung cancer is the leading cause of malignant death all over the world. Malignantpleural effusion is a frequent complication of lung cancerï¼›its presence diminishes thepatient’s ability to perform routine daily activities and is suggestive of end stage disease withvery short life expectancy. Management of MPE is considered palliative because it does notimprove survival. An efficiency， low toxicity， and economic drug is urgent. Angiogenesis,pleural vascular hyperpermeability, and inflammation are considered central to thepathogenesis of MPE. MPE’s central pathogenesis are angiogenesis， pleural vascularhyperpermeability，and inflammation. Recently，researches on treatment of MPE focus on itspathogenesis，such as VEGF-A antibody，NF-κB antibody. But they are so espensive that，akind of effective，harmfulless，cheaper drug is of required urgently.As₂O₃，the traditional Chinese medicine，was firstly used to treatAPL efficiently，withlow toxicity. In our clinical practice，local injection of As₂O₃into pleural cavity gained goodclinical effect. The color of pleural effusion turned red into yellow and the volume of pleuraleffusion decreased gradually without apparent chest pain. Miller reported that arsenic trioxidecan bind to the catalytic residues（Cys-179） in the activation loop of the IKK catalyticsubunits. As a result NF-ΚB activation was blocked. We hypothesized that As₂O₃could beused for treating MPE，with Possible mechanisms，inhibiting NF-κB activation，reduceing theexpression of VEGF-A indirectly.In this study，we firstly set up the pleural xenograftmodel of Lewis lung cancer cell. Then we observe the effects of As₂O₃on MPE and therelated factors. Make sure the mechanisms of As₂O₃as a treatment of MPE.【Contents and Method】Part1Set up C57BL/6mice Lewis lung cancer pleural xenograft malignantpleural effusion modelLewis lung cancer cells were injected into C57BL/6mice pleural cavity. The inoculationdensity of group A was5.0×106cells/ml，0.2ml/mouse. CT was given to mice14days afterintrapleural LLC cell.Part2Experimental studies of effects of As₂O₃on C57BL/6mice Lewis lung cancer pleural xenograft malignant pleural effusion80C57BL/6mice Lewis lung cancer pleural xenograft malignant pleural effusion modelwere divided into five groups randomly，with16/group.Group A（NS）：NS was intrapleural to mice，0.01ml/10gï¹’dï¼›Group B（As₂O₃2.5mg/kgï¹’d）：As₂O₃was intrapleural to mice，2.5mg/kgï¹’dï¼›Group C（As₂O₃5mg/kgï¹’d）：As₂O₃was intrapleural to mice，5mg/kgï¹’dï¼›Group D（Bleomycin15mg/kgï¹’d）：Bleomycin was intrapleural to mice，15mg/kgï¹’dï¼›Group E（Bortezomib15mg/kgï¹’d）：Bortezomib was intrapleural to mice，0.1mg/kgï¹’dï¼›Intervent7days Continuously.1.8mice from every group were used to determine pleural vascular permeability inmice bearing MPE. Each mouse received200μl of50mg/ml Evans’ blue solution （total dose10mg）intravenously and were killed1h later. Pleural fluid and serum Evans’blueconcentration were determined by measuring absorbance at a wave length of630nm incomparison to standards of known Evans’ blue concentrations.2.Other mice was used to test MPE volume，VEGF-A、TNF-α mRNA and their proteinexpressions in MPE and tumor nodes，activity of NF-κB of tumor nodes，vascular density，Pleural vascular permeability.3.The arithmetic mean and standard deviation（s.d.） were calculated for the data, andstatistically evaluated using ANVOA. P<0.05was considered statistically significant.【Results】1.The success rate of setting up models in this study is100%.2. MPE volumes of experimental groups（EGs） are more than control group（CG）（0.906ml）remarkably（P<0.05）. MPE volume of As₂O₃5mg/kg（0.089ml） is the lest，being notablelyless than groups of As₂O₃2.5mg/kg（0.579ml），Bortezomib（0.579ml） and bleomycins（0.600ml）（P<0.05）.There is no significant difference betweenAs₂O₃2.5mg/kg，Bortezomiband bleomycins（P>0.05）.3. MPE Evans’blue concentration of EGs are lower remarkably than CG（1.844mg/ml）（P<0.05）. The concentration ofAs₂O₃5mg/kg（0.344mg/ml） is the lest，being notablelyless than groups of As₂O₃2.5mg/kg（1.002mg/ml），Bortezomib （0.655mg/ml）and bleomycins （1.201mg/ml）（P<0.05）.There is no significant differences betweenAs₂O₃2.5mg/kg，Bortezomib and bleomycins（P>0.05）.4.IHC CD31、CD34：microvessel density of EGs are lower remarkably than CG（CD31/CD34：200±12/191±9）（P<0.05）.CD31：MVD of As₂O₃2.5mg/kg，As₂O₃2.5mg/kg andbleomycins are（92±7）、（45±2）and（113±5）respectively.The differences between eachother are not significant（P>0.05）.CD34：MVD of As₂O₃2.5mg/kg，As₂O₃2.5mg/kg andbleomycins are（60±5），（30±3）and（115±7）respectively. The differences between eachother are not significant（P>0.05）.5. MPE VEGF expression level of EGs are lower remarkably than CG（2990.94pg/ml）（P<0.05）. MPE VEGF expression level ofAs₂O₃5mg/kg（110.28pg/ml） is the lest.Thereis no significant difference between As₂O₃2.5mg/kg（535.16pg/ml），As₂O₃5mg/kg，Bortezomib（555.92pg/ml）and bleomycin（s1603.17pg/ml（）P<0.05）.There is no significantdifferences between As₂O₃2.5mg/kg，Bortezomib and bleomycins（P>0.05）.6. MPE TNF-α expression level of EGs are lower remarkably than CG（39.51pg/ml）（P<0.05）. MPE TNF-α expression level ofAs₂O₃5mg/kg（1.62pg/ml） is the lest.Thereis no significant difference between As₂O₃2.5mg/kg（8.69pg/ml），As₂O₃5mg/kg，Bortezomib（6.59pg/ml） and bleomycins（20.19pg/ml）（P<0.05）.There is no significant differencesbetween As₂O₃2.5mg/kg，Bortezomib and bleomycins（P>0.05）.7. Pleural tumor nodules VEGF-A、TNF-αmRNA expression level of EGs are lowerremarkably than CG（P<0.05），There is no significant differences between each EG（P>0.05）.8. Pleural tumor nodules VEGF-A、TNF-αprotein expression level of EGs are lowerremarkably than CG.9.EMSA：Pleural tumor nodules NF-κB activity of each EG is lower than CG，withAs₂O₃5mg/kg being the lowest.The results suggest that: As₂O₃inhibits transcription and expression of TNF-α byreducing the activity of NF-κB，thereby reducing the transcription and expression of VEGF-Ato reduce the pleural tumor angiogenesis and reduce pleural vascular permeability. This effectis of concentration-dependent enhancement. 【Conclusion】1、Development of a mouse model of pleural xenograft tumor by the intrapleural injection ofLewis lung cancer cells has a high success rate，with the obvious formation of pleuraleffusion.2、The intrapleural injection of arsenic trioxide is effective against malignantpleural effusion.3、 The effect of arsenic trioxide is similar with NF-κB inhibitors， and it isconcentration-dependent.