The Effect Of¹⁸F-FDG On The Growth Of Lewis Lung Cancer Xenografts In C57bl/6Mice

Today,“Century molecular”¹⁸F-2-Deoxy-2-Fluoro-D-Glucose (¹⁸F-FDG) is one of the mostimaging agents clinically used to detect tumor metabolism and widely applied to PET/CT imaging.Recent studies indicate that imaging agents can inhibit tumor initiation, promotion and progress todisplay antitumor efficacy.¹⁸F intaked by human or animal tissues decay to emit β+rays with0.635Mev of average energy, β+and γ rays from annihilation radiation will internally irradiatetissues and impair cells.¹⁸F-FDG is glucose analogs, it can not be decomposed into CO₂and H₂Oafter entering cells like ordinary glucose and will concentrate in cells, the concentration of¹⁸F-FDGin body is positively correlated with the levels of cellular metabolism. The metabolism levels ofmalignant tumor cells are higher, which cause the higher¹⁸F-FDG concentration in cells, the higherconcentration, the larger internal irradiation dose, the stronger radiation effects, and the bettertherapeutic effects to suppress and destruct tumor.Objectives: Lewis lung cancer transplant model in C57BL/6mice was established and used forthe in vivo experiment to explore the possibility for positron radiophar-maceuticals. The inhibitionof¹⁸F-FDG on the growth of Lewis lung cancer transplant and effects of¹⁸F-FDG on the prolifera-tion and apoptosis of tumor cell and microvessel count （MVC） were observed，thereby providing areliable evidence for the possibility of its clinical application.Methods：（1） Cell cuture Lewis lung cancer cells were growed in RPMI1640medium andcultured at37℃in5%CO2and satured humidity;（2） Cell subculture Lewis lung cancer cellswere injected subcutaneously into groin at the right in C57BL/6mice, subcultured per two weeks,the third generation cells subcultured with the means were used in following tests;（3）Establishment of transplantation tumor model The third generation subculture mice wereexecuted by cervical vertebrae dislocation, tumors were dissociated and sterilized with75%ethanoland grinded with dismembyator and filtered with200mesh sieve, the concentration of tumor cells were adjusted as１×107/L,0.2ml of cells were subcutaneously injected in groin at the right in eachmouse and Lewis lung cancer subcutaneous transplantation tumor models in¹⁸ C57BL/6mice wereestablished, and divided as high dose¹⁸F-FDG treatment group, low dose¹⁸F-FDG treatment groupand control by using random number approach,6mice in each group. and divided as high dose¹⁸F-FDG treatment group,low dose¹⁸F-FDG treatment group and control by using random numberapproach,6mice in each group.¹⁸.5×107Bq and9.25×107Bq of¹⁸F-FDG and0.2mL equal volumeof physiological saline were once administrated intraperitoneally, respectively, on the7th day aftertransplantation.（4） Monitoring of tumor volume The size of subcutaneous tumor nodules inmice were measured by using slide gauge with the accuracy of0.1mm and recorded, respectively,on the7th,10th,13th,16th,19th and22th day after transplantation;（5） Determination ofinhibition rate of tumor growth The mice were executed by cervical vertebrae dislocation on the22th day after tumor transplantation, subcutaneous tumors were quickly dissociated and weighed byequiarm balance, inhibition rate of tumor growth （IRTG） was computed according to the formula:IRTG=(average tumor weight in control-average tumor weight in¹⁸F-FDG treated group)/（average tumor weight in control）×100%;（6） Determination of expressions of proteins Theexpressions of Bcl-2、Survivin、PCNA、VEDF and CD34were immunohistochemically assayedwith SP means;（7） Measurement of tumor cell apotosis by flow cytometry （FCM） The micewere executed by cervical vertebrae dislocation on the22th day after tumor transplantation,subcutaneous tumors were quickly dissociated. Small bulk of tumor tissues was thrice washed withphosphate buffered saline （PBS） and trypsinized with0.25%trypsin and blowed into single cellsuspension, the concentration of cells was adjusted as1×106/ml,1ml cell suspension was taken andfixed with70%ethanol precooled at-20℃and placed at4℃for4h and centrifuged and fixationsolutions were discarded; cells were washed with PBS and filtered with sieve and treated at37℃with5mg/ml RNase for30minutes and stained in the dark with50μg/ml PI for15minutes andapoptotic cells were detected by using FCM, the results were analysed and processed with WinMDI2.9software. Results:（1） Dynamic variation of tumor volume Tumor appears in all model mice on the7thday after transplantation, the tumor nodules with the diameter to exceed0.5cm weresubcutaneously touched in all model mice. Dynamic variation of tumor volume may be seen in table1. The tumor growth in¹⁸F-FDG-treated mice gradually slowed down，the tumor growth in highdose¹⁸F-FDG group was notably suppressed，but that in control was fast, the rise of growth curvewas obvious and seen from figure1. In the later phase of the experiment, differences of mean tumorvolume between different two group mice weresinificant（P <0.05）.（2） The inhibition of tumorgrowth The inhibition rates of tumor growth in high and low dose¹⁸F-FDG groups were56.37%and20.19%, respectively. The inhibition rate of tumor growth in high dose group was more thanthat in low dose group （P<0.05）.（3） Apoptosis The apoptosis rates in high and low dose¹⁸F-FDGgroups and control were （31.87±14.05）%,（15.08±7.29）%☆, and （3.65±1.44）%, respectively. Theapoptosis rates in the irradiated groups were more than that in control（P<0.05）, that in high dose¹⁸F-FDG group was more significant（P<0.01）.（4） Expressions of Bcl-2and Survivin proteinsThe expressions of Bcl-2proteins in high and low dose groups and control were1.63±10.95,29.75±15.00and57.38±5.95, respectively; The expressions of Survivin proteins in high and lowdose groups and control were8.64±3.2,13.29±3.02and20.57±5.03, respectively. The expressionsof Bcl-2and Survivin proteins in irradiated mice tumors were strikingly less than those in control（P<0.05）, compared with those in low dose group, differences between high dose treated mice andcontrol were more significant（P<0.01）.（5）Expressions of PCNA protein The expressions ofPCNA in high and low dose groups and control were46.80±6.77,36.22±4.85and32.96±6.07，respectively，differences between¹⁸F-FDG-treated mice and control were significant（P<0.05）.（6）Expressions of CD34and VEGF proteins The expressions of CD34proteins in high andlow dose groups and control were30.51±6.63,37.95±7.92and43.65±1.76, respectively; theexpressions of VEGF proteins in high and low dose groups and control were3.146±0.085,3.504±0.1¹⁸ and4.749±0.792, respectively; the values of MVD in high and low dose groups andcontrol were6.35±2.11,7.64±2.45and9.00±2.15, respectively.The expressions of CD34and VEGF proteins in irradiated mice were less than those in control（P<0.05）. Those suggest that¹⁸F-FDGmay predominantly inhibit tumor growth, the mechanisms may be associated with thedownregulation of Bcl-2and Survivin protein expressions to promote apoptosis of tumor cells, thedecrease of PCNA protein expression to restrain the proliferation of tumor cells and the reduction ofVEGF and CD34proteins expressions to suppress tumor vessel growth.Conclusions:¹⁸F-FDG may make the mice with transplanted Lewis lung cancer internallyexposed to positrons and γ rays from positron-electron annihilation radiation and destruct moleculesin Lewis lung cancer cells and down-regulate the expressions of Survivin, Bcl-2and PCNA toinduce cell apoptosis and inhibit the proliferation of cells, simultaneously,¹⁸F-FDG can make MVDreduced to inhibit the expression of VEGF to affect angiogenesis in tumor and finally preventtransplants in mice from growing.