Therapeutic Study Of Tiopronin As Immune Adjuvant On Tumor Of K562Cell In Nude Mice

Objective To investigate the preventive and therapeutic effect of immune adjuvantTiopronin （TIP） combined with interleukin-2（IL-2） on subcutaneous K562cell tumor ofnude mice. Meanwhile to observe their role of removing reactive nitrogen metabolites andenhancing the number and activity of NK cells.Methods The experiment consisted of two stages. The first was prevention stage, fifty-fourmale BALB/C nude mice were randomly divided into three groups，each group containedeighteen nude mice:①I L-2+TIP+CTX group;②IL-2+TIP group;③C TXgroup. Eachgroup was injected the corresponding drugs, IL-2and TIP administered for6days, CTXadministered for3days. Then1×10^7K562cells were subcutaneously inoculated to nudemice, the number of NK cells in peripheral blood was tested by Flow cytometry. Observethe tumor formation in nude mice of each group. The second stage was therapy stage,thirty-three nude mice which was successfully transplanted K562cell tumor wererandomly divided into three groups:①IL-2+TIP group;②IL-2group;③blank controlgroup. Each group contained eleven nude mice,①group was injected IL-2and TIP for14days,②group was injected IL-2for14days,③group was injected normal saline for14days.Tumor volume was measured regularly, and the tumor growth curve was drawn.Nude mice were sacrificed after inoculation for21days. Blood samples and tumor tissuesamples were preserved. The number of NK cells in peripheral blood was tested by Flowcytometry. Serum interferon-gamma （IFN-γ） was detected by ELISA, and serum NaNO₂was detected by the method of nitric acid reductase. Tumor tissue samples were observedby H-E staining.Results1、The stage of prevention:（1）Tumor formation rate: all of nude mice of IL-2+TIPgroup didn’t develop tumors and the tumor formation rate of IL-2+TIP+CTX group andCTX group were17/18（94.4%）、16/18（88.9%） respectively. Tumor formation rate ofIL-2+TIP group was significantly lower than IL-2+TIP+CTX group and CTX group （P＜0.05）; there was no significant difference between IL-2+TIP+CTX group and CTX group（P＞0.05）.（2） NK cells of IL-2+TIP group （0.874±0.235G/L）was significantlyhigher than IL-2+TIP+CTX group （0.215±0.071G/L）and CTX group（0.247±0.126G/L）（P<0.05）, there was no significant difference between IL-2+TIP+CTX group and CTXgroup（P＞0.05）.2、The stage of therapy:（1）The comparisons of tumor volume on the7thday of treatment show that both IL-2+TIP group（0.498±0.146cm³）and IL-2group（0.501±0.070cm³）were significantly smaller than control group（0.920±0.246cm³）（P＜0.01）,there was no significant difference between IL-2+TIP group and IL-2group（P＞0.05）.Thecomparisons of tumor volume on the14thday of treatment show that both IL-2+TIP group（0.935±0.309cm³） and IL-2group （0.879±0.107cm³） were significantly smaller thancontrol group（1.514±0.316cm³）（P＜0.01）, there was no significant difference betweenIL-2+TIP group and IL-2group（P＞0.05）.The comparisons of the difference of tumorvolume after7days of treatment show that both IL-2+TIP group（0.327±0.145cm³） andIL-2group （0.315±0.086cm³） were significantly smaller than control group （0.687±0.262cm³）（P＜0.01）, there was no significant difference between IL-2+TIP group and IL-2group（P＞0.05）.The comparisons of the difference of tumor volume after14days oftreatment show that both IL-2+TIP group （0.764±0.306cm³） and IL-2group （0.693±0.119cm³）were significantly smaller than control group（1.282±0.328cm³）（P＜0.01）, therewas no significant difference between IL-2+TIP group and IL-2group（P＞0.05）.（2）Thecomparisons of number of NK cells after treatment show that both IL-2+TIP group（0.468±0.140G/L）and IL-2group（0.420±0.112G/L）was significantly higher than control group（0.318±0.063G/L）（P＜0.01）, there was no significant difference between IL-2+TIP groupand IL-2group（P＞0.05）.The comparisons of the difference of NK cells before and aftertreatment indicated that both IL-2+TIP group （0.221±0.137G/L） and IL-2group （0.223±0.119G/L）were significantly higher than control group （0.103±0.082G/L）（P＜0.05）,there was no significant difference between IL-2+TIP group and IL-2group（P＞0.05）.（3）There was a significant negative correlation between NK cells number and tumorvolume（P＜0.05）.（4）IFN-γ level of IL-2+TIP group （46.78±16.27umol/L）wassignificantly higher than control group（25.82±10.54umol/L）（P＜0.01）,IL-2group （42.65±19.85umol/L）was significantly higher than control group （25.82±10.54umol/L）（P＜ 0.05）, there was no significant difference between IL-2+TIP group and IL-2group（P＞0.05）.（5）There was a significant positive correlation between IFN-γ level and NKcells（P<0.01）.（6）The comparisons of NaNO₂content level: IL-2group＞IL-2+TIP group＞control group, but there was no significant difference between groups（P＞0.05）.Conclusions1、IL-2can enhance the number and activity of NK cells in nude mice, thusenhance the resistant effect of NK cells to K562cell tumor.2、The above figuredemonstrated that TIP can reduce part of RNM, enhance the level of NK cells and IFN-γ，but the difference was not statistically significant. The adjuvant effect of TIP in tumorimmunotherapy in this study was not significant. The reason may be associated with smallTIP dose and inadequate therapy time.