| Aminopeptidase N (APN/CD13), a zinc-dependent exopeptidase, is highlyexpressed on the surface of cancer cells and is thought to be involved in thedegradation of extracellular matrix barriers and angiogenesis and promotes cancergrowth and metastasis. Asparagine-glycine–arginine (NGR) is a tumor-homingtripeptide, which can selectively combine with APN/CD13that is overexpressed onthe surface of some tumor cells.13F-1, a novel5-fluorouracil (5-FU) prodrug, isformed by a tripeptide NGR(NO2) which was synthesized and conjugated with5-fluorouracil. We examined the effect of13F-1on human colonic carcinomagrowth by both in vitro and in vivo studies. Its efficacy and mechanisms in micewere then compared with those of5-FU.OBJECTIVEWe evaluated the efficacy and the mechanism of13F-1as a novel peptidomimeticscompound for treatment of cancers by targeting APN/CD13. METHODS1．Effects of13F-1on Colo205cells in vitro⑴MTT and the clonogenic assay: The Colo205cells were randomlydivided into several groups: The negative control group: the Colo205cells werecultured for24h,48h and72h in serum-free RPMI-1640medium. The treatment of13F-1group: the final concentration of13F-1were1,5,10,50,100μM and werecultured for24h,48h and72h. MTT and the clonogenic assay were used to evaluatethe growth inhibition of Colo205cells.⑵The Colo205cells were randomly divided into five groups: The negativecontrol group: the Colo205cells were cultured for24h in serum-free RPMI-1640medium. The group of5-FU: the final concentration of5-FU were100μM and thenwere cultured for24h. The treatment of13F-1group: the final concentration of13F-1were1,5,10μM and were cultured for24h. Annexin-V/PI staining was usedto quantify the percentages of apoptosis in the total cell population. Hoechst33342staining was used to observe the apoptosis morphology of Colo205cells. Thefluorescent, lipophilic and cationic probe, JC-1, was used to measure themitochondrial membrane potential (ΔΨm) of cancer cells. Immunofluorescence flowcytometry and Western blotting were used to measure the expression of APN.2．Effects of13F-1on human colon cancer Colo205nude mice invivo⑴Establish the human colon cancer Colo205xenografts: Thirty of femaleBALB/C-nu mice, establish nude mice model with subcutaneous inoculation thecolo205cells in the left forefoot. The Colo205cells were randomly divided into fivegroups: The negative control group,5-FU(50mg/kg), high dose13F-1(100mg/kg),model group13F-1(50mg/kg), low dose13F-1(25mg/kg)(n=6). Every group wasinjected for3weeks through tail vein after inoculation24h.⑵Mice were sacrificed after inoculation for22days, and the xenografts wereremoved for weighting and further analysis. After plucking the eyeball and fetchingthe blood, then detected the changes of peripheral blood elements by the automatedhematology analyzer.Immunohistochemical staining and Western blotting were used to detect the APN expression levels. We carried out the experiments of terminaldeoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining todetermine apoptotic cells in human colonic carcinoma Colo205xenografts.Apoptosis-related protein expression were detected by Western blotting. The levelsof SOD and ROS in Colo205cells and xenografts were examined in the Colo205cells and the supernatant of tissue homogenate using the kits of ROS and SOD.RT-PCR was used to determine the mRNA levels of MnSOD and NADPH oxidase-1(Nox-1).RESULTS1．Effects of13F-1on Colo205cells in vitro⑴MTT and the clonogenic assay:13F-1at1to50μM for up to72hproduced a dose-and time-dependent inhibition on cell proliferation (1to5μM for24h, and1μM for48h, P>0.05,10μM for24h, P <0.05, vs. the vehicle control).Statistical analysis indicated that the median inhibitory concentration (IC50) in48hexposure time was32.80±1.13μM. The results of clonogenic assay were showedthat13F-1significantly inhibited Colo205clone formation in a dose-andtime-dependent manner (1μM, p <0.05;5-100μM, p <0.01vs. the vehicle control).13F-1at50and100μM for72h exposure inhibited clone formation by100%.5-FUat50μM for48and72h also completely inhibited clone formation.⑵The apoptosis rates were detected by the Annexin V-FITC/PI staining,there was few cells occurred apoptosis in the negative control group. The apoptosisrates were significantly increased in the13F-1at final concentration of1,5and10μM after24h(P<0.05). Compared with the control group, there was no significantdifference of the apoptosis rates of10μM5-FU. The apoptosis morphology ofColo205cells after Hoechst33342staining were observed by fluorescence microscopy,the nucleus of nomal Colo205cells were round and lighe blue. Colo205cells occurredapoptosis and the nucleus of apoptosis cells turned into bright blue after enrichmentand were lobulated, fragmented and margination.13F-1at6,30and150μM significantly decreased the Δψmof Colo205cells(P<0.05),5-FU at10μM showed nosignificant decrease of Δψmand the apoptosis. Immunofluorescence flow cytometryand Western blotting analysis showed that13F-1significantly reduced the expressionof APN, compared with the negative control group, the changes of APN expression of10μM5-Fu showed no significant.2．Effects of13F-1on human colon cancer Colo205nude mice invivo⑴During the experiments, weight loss were not appear in the negativecontrol group and13F-1groups, they were generally in good condition, but aftertreatment of5-FU, the weight of nude mice significantly decreased(P<0.05). In the5-FU-treated mice, significant reduces were seen in total WBC, neutrophil,lymphocyte and platelet counts, and there were no significant drops in blood countsin the13F-1-treated mice (p>0.05). The toxicity of13F-1was significantly reducedcompared to the group of5-FU.⑵Three weeks after tail vein injection, the tumor growth inhibition of5-FU(50mg/kg),13F-1at25mg/kg,50mg/kg and100mg/kg groups were56.3%,36.1%,50.4%and63.9%(P<0.05), there was significant difference between5-FU at50mg/kg and13F-1at150mg/kg(P<0.05). After treatment of13F-1, the expressionof APN was significantly decreased(P<0.05), and the expression of5-FU showed nosignificant difference(P>0.05). Compared to the control, the apoptosis rates showeda significant difference of13F-1groups(P<0.05), but the group of5-FU showed nosignificant difference(P>0.05). At the same time, western blotting results revealedthat the levels of apoptosis-related protein of cleaved-9, cleaved-3and cleaved PARPand the ratio of Bax/Bcl-2increased(P<0.05), but, the levels of cleaved-9, cleaved-3,cleaved PARP and the ratio of Bax/Bcl-2showed no significant changes of5-FUgroup. In addition, compared to the control, the levels of ROS increase and the levelsof SOD decreased after treatment of13F-1. The expression of NOX-1mRNA insignificantly increased, while of MnSOD mRNA in expression significantly with decreased (P <0.05).CONCLUSION13F-1is a peptidomimetics compound with a inhibition effect on the activity. Thepossible mechanisms was that13F-1could inhibited the expression of APN, and theninhibited human colon cancer Colo205xenografts growth by inducingROS-dependent mitochondrial apoptosis pathway. |
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