Anti-cancer Study Of Fused Polypeptide Aimed At CXCR4

Malignancy has become the top one disease of threat to humanhealth, and breast cancer and lung cancer are the ranked first malignancyto women’s and men’s health respectively. The stage Ⅲ and Ⅳ breastcancer still have a high relapse rate and mortality owing to the metastasisand drug-resistant. Prevention of metastasis has become an importantpoint of therapy of breast cancer. Meanwhile, the five-year survival rateof lung cancer has not markedly improved since1970’s. The therapeuticstrategy against malignancy has gradually changed from "finding anddestroying tumor" to "targeting and controlling tumor" as the time ofmolecular oncology coming. The targeting therapy has become theimportant study direction for prevention and therapy of tumor.The peptide drug is a development direction against tumor targetingtherapy, which has some advantages, such as low toxicity and low tissueaccumulation, high biological activity and high specificity. It can be usedto treat cancer that selecting the role-specific small-peptide drugs aimedat tumor-specific regulatory factors and blocking their active sites.Chemokine receptor4(CXCR4) and its ligand stromal-derived factor-1(SDF-1, or CXCL12) are known to play an important role in regulation of metastasis in a variety of solid tumors. In our previous research, a fusedpolypeptide TAT-DV3-BH3was designed, which aims at CXCR4overexpressing in many tumors，and some antitumor researches had beendone in vitro and in vivo. The results indicated that the fused polypeptidemight retain all functions of their components. They include that TAT(HIV-1trans-activator, TAT) carries TAT-DV3-BH3into cells efficientlyas protein transduction peptides; synthetic DV3is a targeting sequencebinding CXCR4, endues TAT-DV3-BH3target differentially many tumorcells or tumor tissues with high expression of CXCR4; BH3is the onlydomain of P53up-regulated modulator of apoptosis (PUMA) and makesTAT-DV3-BH3be able to induce the tumor cells apoptosis and inhibit thegrowth of tumors. However, targeting and inhibiting tumor is not enoughto therapy of tumor because metastasis is a key factor of tumor therapyand prognosis.Consequently it is a worth study direction that designeddual-function fused polypeptide with inhibition the growth, migration andmetastasis of tumor. Based on DV1could bind and inhibit the CXCR4,the present study will i) design and synthesize dual-function fusedpolypeptide TAT-DV1-BH3and explore its effects on the growth,migration and metastasis of tumor cells and their mechanisms; ii)synthesize the fused polypeptide TAT-DV1-DEF used TAT-DV1as anefficient targeting vector for tumor and study the anti-tumor effects of DEF, which is an unknown peptide obtained from our previous study.Meantime, evaluate the feasibility of TAT-DV1used as an efficienttargeting vector for tumor and offer some experimental data for thefurther study of anti-tumor targeting peptide drugs.PART IEXPERIMENTAL STUDY OF ANTI-BREAST CANCERAGAINST CXCR4Objective:1. Design and synthesize dual-function fused polypeptideTAT-DV1-BH3, and control polypeptides TAT-DV1and DV1-BH3.2. Validate the targeting specificity of TAT-DV1-BH3; and analyzeits effects on the growth of high metastasis human breast cancer cell lineMDA-MB-231and low metastasis human breast cancer cell line MCF-7.3. Analyze the effects of TAT-DV1-BH3on the migration andmetastasis of high metastasis human breast cancer cell lineMDA-MB-231.4. Evaluate the feasibility of TAT-DV1used as efficient targetingvector for tumor.Methods:1. Screening the various cell lines used cytotoxic test with CCK-8and analyzing the effects of fused polypeptides on the growth of these cell lines.2. Observing the distribution of fused polypeptide TAT-DV1-BH3incells with the laser confocal microscopy.3. Analyzing the efficiency of polypeptides entering cells with flowcytometry.4. Observing the change of nucleus morphology under thefluorescence microscope after the cells treated with fused polypeptideTAT-DV1-BH3and stained with DAPI.5. Analyzing the effects of fused polypeptide TAT-DV1-BH3on themigration of cell line MDA-MB-231with wound healing experiment.6. Evaluating the effects of fused polypeptide TAT-DV1-BH3on themetastasis of MDA-MB-231with transwell test.7. Analyzing the apoptosis signal pathway of breast cancer cell lineMDA-MB-231treated by TAT-DV1-BH3through detecting theexpression of caspase-3,8and9with Western blot test.8. Validating the mechanisms of migration and metastasis of cell lineMDA-MB-231treated with TAT-DV1-BH3by Western blot test.Results:1. The experimental cell lines were screened by cytotoxic test withCCK-8, including human embryonic kidney cell line HEK-293, humanhigh metastasis breast cancer line MDA-MB-231and human lowmetastasis breast cancer line MCF-7. 2. The results from cytotoxic test with CCK-8indicated that fusedpolypeptide TAT-DV1-BH3could differentially inhibit the growth oftumor cells and not do non-tumor cells HEK-293. TAT-DV1-BH3coulddose-dependently depress the growth of human breast cancer cell linesMDA-MB-231and MCF-7.3. The results from flow cytometry and laser confocal microscopedisplayed that TAT-DV1-BH3could enter cells quickly and efficiently (＞90%) after they were co-cultured with TAT-DV1-BH3for1h, anddistributed in cytoplasm and mainly co-located in mitochondria. ButDV1-BH3without TAT entered cells more difficultly (＜1.7%).4. The morphological changes of nuclear were observed underfluorescence microscope. After the cells treated with fused polypeptideTAT-DV1-BH3for72h, cell line MDA-MB-231exhibited remarkableapoptosis characters such as membrane break, chromatin condensationand fragmentation. The results from Western blot validated that fusedpolypeptide TAT-DV1-BH3induced breast cancer cells apoptosis throughmitochondria pathway same as PUMA (BH3).5. The results from wound healing test and transwell test indicatedthat the fused polypeptide TAT-DV1-BH3could blocked migration andmetastasis of human high metastasis breast cancer cell lineMDA-MB-231significantly. The inhibition was validated to depress theexpression level of PI3K and MMP-9through blocked CXCR4activity. Conclusion:The study has succeeded in designing dual-function fusedpolypeptide TAT-DV1-BH3. In vitro, TAT-DV1-BH3could enter cellsmore quickly and efficiently and execute the function of DV1and BH3.TAT-DV1-BH3could inhibit the growth of human breast cancer cell linesdose-dependently and differentially through inducing cells apoptosis viathe mitochondria pathway same as PUMA (BH3). Through blocking thefunction of CXCR4, fused polypeptide TAT-DV1-BH3could inhibit theexpression of PI3k and MMP-9and depress significantly the migrationand metastasis of human high metastasis breast cancer cell lineMDA-MB-231. All of these indicate that the strategy of dual-andmulti-function fused polypeptides is feasible and TAT-DV1might be atargeting and efficient vector for studies of anti-tumor drugs. It not onlyoffered important experimental data for further study of anti-tumorfunction of fused polypeptides but also widened the design for fusedpolypeptides. PART IIANTI-LUNG CANCER STUDY OF FUSED POLYPEPTIDEWITH DEFObjective:1. Use TAT-DV1as targeting and efficient vector to study thefunction of an unknown peptide DEF based on our previous studies.2. Further evaluate the feasibility of TAT-DV1as targeting andefficient vector for tumor therapy.Methods:1. Designing and synthesizing fused polypeptide TAT-DV1-DEF,and control polypeptides TAT-DV1and DEF.2. Analyzing the effects of the fused polypeptide TAT-DV1-DEF onthe growth of human lung adenocarcinoma cell lines GLC-82withCCK-8test.3. Observing the distribution of fused polypeptide TAT-DV1-DEF incells with the laser confocal microscopy.4. Analyzing the efficiency of polypeptides entering cells and theapoptosis rate with flow cytometry.5. Observing the changes of nucleus morphology under thefluorescence microscope after the cells treated with fused polypeptideTAT-DV1-DEF and stained with DAPI.6. Detecting cell apoptosis related protein caspase-3after treated with fused polypeptide TAT-DV1-DEF for72h.Results:1. The results from cytotoxic test with CCK-8indicated that fusedpolypeptide TAT-DV1-DEF could specifically inhibit the growth ofhuman lung adenocarcinoma cell line GLC-82and not do non-tumor cellsHEK-293.2. The results of flow cytometry and laser confocal microscopydisplayed that TAT-DV1-DEF cells co-cultured with TAT-DV1-DEF for1h could enter cells more quickly and efficiently (about100%), anddistributed in cytoplasm and mainly co-located in mitochondria. But DEFwithout TAT entered cells more difficultly (60.58%).3. The morphological changes of nuclear were observed underfluorescence microscope. After the cells treated with fused polypeptideTAT-DV1-DEF for72h, cell line GLC-82exhibited typical apoptosischaracters. The rate of apoptosis increases with increasing ofconcentration of TAT-DV1-DEF with flow cytometry.4. The results from Western blot indicated that human lungadenocarcinoma cell line GLC-82was induced apoptosis after treatedwith fused polypeptide TAT-DV1-DEF for72h.Conclusion:The present study has tries to use TAT-DV1as an efficient tumortargeting vector for exploring the function of an unknown peptide DEF. The results of preliminary experiments show that the fused polypeptideTAT-DV1-DEF has anti-tumor activity and may lay the basis for thefurther in-depth study of DEF. Secondly, TAT-DV1can be used as thespecific and efficient transmission vectors for anti-tumor polypeptidedrugs, which helps the further study of fused polypeptide.