A Tumor Targeted Therapy Study Of PcDNA-sTRAIL Combined With¹³¹I-Angiostatin On Tumor-bearing Nude Mice

【objectives】The growth, invasion, and metastasis of tumor are closely related with tumorangiogenesis. Angiogenesis inhibition and tumor apoptosis inducing methods aretwo hot spots in anti-tumor research. In Current clinical therapy, angiostatin （AS）and endostatin are two drugs can effective inhibiting tumor angiogenesis. Therecombine protein of AS has been used in clinical, but couldn’t popularize inclinical therapy because of the disadvantage of high operating cost, high dose oftreatment and long term administration. Study shows that alone or combinetreatment of endostatin has advantage of low toxicity, low immunogenicity andlow drug resistance, but the availability of endostatin still needs to certify in thefuture. Tnf-Related Apoptosis-inducing Ligand （TRAIL） belongs to TNF superfamily and has find out can obviously inducing tumor cell apoptosis.Combined inhibiting tumor angiogenesis, inducing tumor cell apoptosis and radiationtherapy might be a new effective tumor target therapy in the future. In this study,we successfully built, amplified, and purified eukaryotic expression vector ofPcDNA-STRAIL. We succeeded in labeling angiostatin （AS） by usingradioisotope¹³¹I. Finally, we accomplished joint antitumor experiment ontumor-bearing nude mice models by employing both PcDNA-sTRAIL and¹³¹I-AS.【Methods】1. Purify and separate angiostatin （AS） by using affinity chromatography. ObtainAS fragment by limited-digestion using elastase. Use¹³¹I to label angiostatin byIodogen method. Assess the label efficiency, bioactivity, specific activity andin-vivo stability of¹³¹I-AS.2. Total RNA was abstracted with Trizol by one-step way.Use the total RNA astemplate and primer lead to process RT-PCR reaction and obtain the cDNA. DNAstrap was identified by gel electrophoresis.Use PCR amplification one-step waykit obtain objective DNA and then purified it. Establish the T-sTRAIL plasmid byconnecting the DNA strap with PUCm-T vector. Transform T-sTRAIL plasmidinto the competent TGI and screen plasmid with LB medium contain Ampresistant.Exract plasmid by alkaline lysis technique. The objective strap wasreclaimed and purified afterwards. Link PcDNA3.1and T-sTRAIL with T4ligaseafter restriction endonuclease EcoRI and BamH1digest. Transform plasmid intothe competent TGI and screen plasmid with LB medium contain Ampresistant.Establish the PcDNA-sTRAIL eukaryotic vectors, then identify it byprofessional company.Determinate objective protein in293T cell model byWestern blotting method.3. Establish tumor-burdened nude mice models and SPECT imaging.A549cells are cultivated and inoculated subcutaneously to right foreleg in the nude mice toobtain tumor-bearing models. Inject¹³¹I-AS （0.2ml,3.7MBq） intravenously tonude mice models, observe SPECT imaging in2,12,24, and48h.4. Observe combing antitumor effect on tumor-bearing nude mice models.32tumor-bearing nude mice were randomly divided into4groups, adding10%KI in the drinking water for mice3days before experiment. Inject¹³¹I-AS (¹³¹I11.1MBq,AS12.5mg/kg) combining with PcDNA3.1-sTRAIL50μl+Lipofectin50μl（concentration is1μg/μl）,¹³¹I-AS (¹³¹I11.1MBq, AS12.5mg/kg), PcDNA3.1-sTRAIL50μl+Lipofectin50μL（concentration is1μg/μl）and normal saline0.3ml into tumors according to the groups pre-designed, oncea week, for4weeks. Observe GTV （Gross tumor volume） for continuing28days,measure the tumor volume （V=W2×L/2） by vernier caliper and calculateanti-tumor rate. Use HE staining of tumor to observe the morphology changes oftumor tissue.【Results】1. AS was separated and purified by L-LysineSepharose4B affinitychromatography from human blood plasma. Label efficiency of¹³¹I-angiostatinlabeled by Iodogen solid state method was78.2%⁸7.7%, and specific activitywas1.31³.95TBq/g. Observed the stability of the product for7days under-20℃condition. The radiochemical purity of¹³¹I-AS was70.6%. The resultsshowed high specific activity and stablity of¹³¹I-angiostatin labeled by Iodogensolid state method.2. Objective DNA of sTRAIL by gel electrophoresis method find out that the trapat723bp was identified with DNA marker strip.Determinate objective protein ofPcDNA-sTRAIL eukaryotic vector by Western blot method find out that protein successfully express compared with objective protein.3. SPECT imaging found that higher radioactivity in tumor area than other area,which certified the angiostatin was specific absorbed by tumor.4. The average volume of tumor in tumor-burdened nude mice models, thecombined treatment group of PcDNA-sTRAIL and¹³¹I-AS had smaller volume oftumor than the other group. The tumor inhibiting rates of combined treatment ofPcDNA-sTRAIL and¹³¹I-AS increased27%and45%, compared with¹³¹I-ASgroup and TRAIL group respectively. The statistical analysis showed that thetumor volume of combined treatment of PcDNA-sTRAIL and¹³¹I-ASsignificantly different from the other groups in7,14,21,28days respectively.HE staining shows that larger areas necrosis of tumor tissues and inflammatorycell infiltration in combined treatment of¹³¹I-AS-TRAIL group than the othergroups.All the results demonstrated better tumor-inhibiting effect of combing¹³¹I-AS and TRAIL than each alone.【Conclusion】Our research explored combining therapy of PcDNA-sTRAIL and¹³¹I-AS totreat the tumor-burdened nude mice model. The study showed combinationmethod of anti-tumor angiogenesis, inducing tumor apoptosis, and innerradiotherapy in antitumor therapy. The results demonstrated significantanti-tumor effects of the PcDNA-sTRAIL and¹³¹I-AS combining method, andmay provide new and useful information and method in anti-tumor technique.