Influence Of Arsenic Trioxide On Excretion Process Of Mouse Hepatocarcinoma Cell H₂₂ Derived-Exosome And Its Combined Therapeutic Application With Exosome

Exosomes are small membrane vesicles from multi-vesicular endosomes.Exosomes are involved in many physiological and pathological processes.Primary investigation results showed exosome is promising to be a novel technique in biomedicine and application field.Yet due to the following aspects,such as nano-grade volume,low density and yield, complicated extraction process,restricted the reseach on exosomes.Some studies indicated that the procedure of exosome excretion was related to the change of endochylema calcium ion.For example,monensin,a Na⁺/H⁺ exchanger,inducing a calcium ion inflow,may increase the production of exosome obviously in K562 cells,which can be suppressed by calcium antagonist BAPTA-AM.Arsenic trioxide,essential component of arsenicum sublimatum, proved to cure APL significantly.Other researches indicated that arsenic trioxide could induce apoptosis of multi-tumour cells and promote intracellular free calcium ion.In view of above characters,we select arsenic trioxide as an intervention agent to approach the exosome excretion changes of mouse hepatoma carcinoma cell H₂₂.Membrane fusion mechanism-SNARE(soluble NSF attachment protein receptor),is one of mechanisms to explain the exocytosis,influenced by intracellular free calcium ion,among the neuroendocrine cells.The exosomal origin and cyclic process was similar to the exocytosis/endocytosis process of neuroendocrine cells,moreover, exosomes and neuroendocrine vesicles were of same morphology and size. In our study,we detected the expression of SNARE associated protein and its expression changes.These were experimental works for further exploring exosomal mechanism.In addition,we observed therapeutic effect of exosome combined with arsenic trioxide on mouse model of subcutaneous hepatoma inoculated in vivo.These provide some enlightenments for further research on exosome and function as the experiment basis and the rationale of exosome in the tumor clinical immunotherapy.Methods:1.Influence of arsenic trioxide on exosomal excretion of mouse hepatoma carcinoma cell H₂₂:The growth curve of tumour cells and arsenic trioxide concentration was drawn by MTT method and be sure that cells grow in the log phase and be extracted when most of cells(90%) are alive. Exosomes were extracted and purified by fractional centrifugation and sucrose density gradient centrifugation.The total protein of exosome was determined by Bradford method.The calcium concentration changes in viable cells were determined by the fluorescence probe Fura-2 and inverted fluorescence microscope detection system for calcium.The expression level of syntaxin was detected by Western-Blot method.2.Therapeutic investigation of exosome combined arsenic trioxide on mouse model inoculated subcutaneouslly with H₂₂ cells:The concentration of cells was adjusted into 5×10⁵ cells per milliliter and 0.2 ml of them were transplanted subcutaneouly into the anterior limb armpits of the mice,to generate mouse solid tumor model.Some of mice were administered with arsenic trioxide in the abdominal cavity since the second day after tumour cells inoculation,once a day for consecutive 10 days.Exosomes were administered subcutaneously in the medial part of right posterior limbs of other mice on the fourth day after inoculation,once per 4 days,consecutive for three times.Calculate the tumour inhibition ratio;observe and measure the tumor growth;semi-quantitate the infiltration of CD4⁺,CD8⁺T lymphocytes in tumor tissue;calculate the mice survival rate.Results:1.Influence of arsenic trioxide on exosomal excretion of mouse hepatoma carcinoma cell H₂₂:1.1 The log phase of 5×10⁴/ml Hepato-carcinoma H₂₂ cells were from 24 hours to 48 hours.Compared with the control,when H₂₂ cells were treated with 1.25μmol/L arsenic trioxide for 48 hours after passage,the cell-inhibition-ratio was 10 percent approximately.The 5×10⁴/ml H₂₂ cells suspension in the log phase were passed into normal culture flasks of two groups(control group,experimental group) respectively.The final concentration of arsenic trioxide in the experimental group was 1.25μmol/L. 48 hours later,200 ml H₂₂ cell suspension were obtained and underwent a series of ultraspeed fractional centrifugation and sucrose density gradient centrifugation.2 ml of purified exosomes were gathered and were determined for the total proteins in the two groups above via Bradford method.The results were(0.85±0.13) mg/ml and(0.96±0.10) mg/ml respectively,and significantly elevated in the arsenic trioxide group as comparing with the control group(P＜0.05).1.2 The results of intracellular free Ca²⁺ concentration showed an obvious increase after treating with a low concentration of arsenic trioxide (1.25μmol/L),and that a peak was achieved in 24 h(P＜0.05),and afterward it decreased and returned to the basic level 72 h later.1.3 Western-Blot indicated,when treated with the low concentration arsenic trioxide(1.25μmol/L).the expression of synatxin protein increased significantly in hepatocarcinoma H₂₂ cells.2 Therapeutic investigation of exosome combined arsenic trioxide on mouse model inoculated subcutaneouslly with H₂₂ cells:2.1 The growth of tumor H₂₂ inoculated in mouse subcutaneously:The difference arose since 16^th day after inoculation.The tumor average volumes in the two combined groups and the arsenic trioxide alone treatment group are smaller than the one in control group at the same day(P＜0.05) and no difference was found between the combined groups and arsenic trioxide alone group(P＞0.05).The tumor average volume in the combined group since 20^th day after inoculation was smaller than those of the arsenic trioxide alone group(P＜0.05) and the control group(P＜0.05).No differences were found among the two EXO alone treatment groups and the control group all along(P＞0.05).2.2 Mice survival rate in different groups:On the 25^th day after tumor inoculation the mice survival rates in control group,arsenic trioxide group,arsenic trioxide combined with EXOn group,arsenic trioxide combined with EXOa group,EXOn group,EXOa group were 30%,50%,70%,80%,30%and 30%,respectively.No significant difference in statistics was concluded from above data,which might be due to the small number of samples.2.3 Mouse tumor(H₂₂) inhibition ratio:The tumor inhibition ratios in arsenic trioxide group,arsenic trioxide combined with EXOn group,arsenic trioxide combined with EXOa group, EXOn group,EXOa group were 44.58%,66.27%,65.06%,4.82%,and 6.03%,respectively.2.4 CD4⁺,CD8⁺ T lymphocytes infiltration in tumor(H₂₂) tissue: The average optical(AO) values of CD4⁺T lymphocytes in control group, arsenic trioxide group,arsenic trioxide combined with EXOn group, arsenic trioxide combined with EXOa group,EXOn group,EXOa group were 0.12±0.02,0.18±0.03,0.32±0.08,0.34±0.06,0.13±0.05 and 0.14±0.04,respectively and the AO values of CD8⁺ T lymphocytes in control group,arsenic trioxide group,arsenic trioxide combined with EXOn group,arsenic trioxide combined with EXOa group,EXOn group, EXOa group were 0.11±0.01,0.15±0.02,0.18±0.04,0.20±0.03,0.13±0.02 and 0.12±0.03,respectively.Compared with the control group, there were no differences in exosome alone treatment groups(EXOn, EXOa)(P＞0.05).A significant difference was established in the combined treatment groups.Comparing with the arsenic trioxide alone treatment group,there were significant difference in the two combined groups(P＜0.05).No differences were found between two combined treatment groups (P＞0.05),and between the two EXO groups(P＞0.05).Conclusion:1.Low concentration of arsenic trioxide(1.25μmol/L) may promote the intracellular free calcium concentration,enhance the expression of syntaxin in mice hepatocarcinoma cells(H₂₂),and the excreting of exosomes in mice hepatocarcinoma cells(H₂₂).These demonstrated,that the exosomal excretion process of mice hepatocarcinoma cells is matching the exocytosis mechanism of the SNARE compounds hypothesis partially.2.Exosomes has a synergic therapeutic effect on transplanted subcutaneous mice tumor from hepatocarcinoma cells H₂₂.which indicated, exosome may function as a biological agent in the anti-tumour aspect.