Screening Of Tumor Biomarkers And Application Of Specilic Targeting Liposomes For Malignant Disease

Background: Ovarian cancer is the leading cause of death from all types ofgynecological cancer. Unfortunately, the majority of epithelial ovarian cancers remainclinically undetected until patients have developed late stage disease and the patients’prognosis is very poor, due to lack of effective methods for early diagnosis and neweffective treatment strategies. Tumor biomarkers such as CA-125, CA19-9and CEAthat are currently utilized for the detection of ovarian cancer in clinical practice lackthe sensitivity and specificity needed to detect potentially curable lesions andtherefore not suitable for population screening. Therefore, it is essential to discovernew or supplementary biomarkers to improve diagnosis and treatment of ovariancancer.The stable isotope-based quantitative proteomic approach has opened newapproaches for the discovery of biomarkers. Isobaric tags for relative and absolutequantitation (iTRAQ) analysis followed by liquid chromatography–massspectrometry (LC–MS/MS) is a gel-free mass spectrometric (MS) technique withisobaric amine-specific tags to compare the peptide concentrations between samplesto establish quantitative values for corresponding proteins. iTRAQ method is apowerful tool in which up to eight samples can be analyzed in one experiment.Purpose: To find potential novel biomarkers for detection and prognosis ofovarian cancer, we compared expression levels of proteins between ovarian cancerand normal ovarian tissue by iTRAQ.Method: Quantitative proteomics were used as a screening tool foridentification of differentially expressed proteins as potential biomarkers for cancers.Several of the differentially expressed proteins were validated by real-timequantitative RT-PCR, immunohistochemistry and Western blot analysis. Function ofcandidate proteins were analyzed by gain-of-function and lose-of-function. Results:205proteins were differentially expressed between ovarian cancer andnormal ovarian tissues， these205proteins could be classified into22functionalcategories using the PANTHER classification system.15of the proteins validated byreal-time quantitative RT-PCR analysis, this trend in mRNA level was consistent withdata obtained by the iTRAQ approach. Furthermore, up-regulation of KRT8, PPA1,IDH2, and S100A11were validated by immunohistochemistry and Western blot.Over-expression of legumain correlates with increased cell migration and invasion ofovarian cancer cells. Silencing of S100A11expression suppressed the migration andinvasion properties of ovarian cancer cell in vitro.Conclusion:205proteins were identified differentially expressed betweenovarian cancer and normal ovarian tissues.15of the proteins were validated inmRNA level and protein level. Legumain and S100A11were associated with ovariancancer cell migration and invasion, and they can be used as biomarkers in humanovarian cancer. Background:Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwideand the third leading cause of cancer death. Advances in treatment, imaging, surgicaltechniques and liver transplantation have resulted in considerable improvements intherapy of hepatocellular carcinoma. However, the crucial issues, include inadequatetreatment efficacy due to ineffective targeting of tumor, systemic toxicities and diseasemonitoring, continue to plague the arena of hepatocellular carcinoma therapy.Liposomal nanoparticle function as a well-established delivery tool for drugs or genes and provide a versatile platform for exploring multiple approaches that canpotentially enhance the delivery and targeting of therapies to tumor.Molecular imaging has enabled the noninvasive monitoring of specificmolecular and cellular processes in vivo, including gene expression, progression, andregression of cancer, as well as evaluation of targeted therapy.Hypothesis:We hypothesize that an anti-CD44antibody mediated liposomal nanoparticledelivery system loaded with suicide gene or chemotherapy drugs could specificallytarget the CD44+cells of HCC, and induce their apoptosis. Meanwhile, we employedmolecular imaging with reporter gene methods to probe targeted efficiency and HCCtumor processes.Methods:1. Preparation and characterion of Liposomes. we developed several types ofliposomal nanoparticles, including anti-CD44antibody mediated nanoparticles loadedwith doxorubicin (Lipo-CD44-Dox), CD44targeted TF-liposomes (Lipo-CD44-TF),non targeted TF-liposomes (Lipo-TF), CD44targeted rhodamine B-liposomes(Lipo-CD44-RB) and non targeted rhodamine B-liposomes (Lipo-RB).2. In vitro uptake of CD44targeted liposomes. To test the targeting efficacy anddynamics of CD44antibody conjugated liposomal nanoparticles in vitro, HepG2cellswere cocultured with Lipo-CD44-RB and Lipo-RB. Uptake of RB was analyzed byfluorescence microscopy.3. In situ xenograft tumor model. To track transplanted cells in vivo, HepG2cellswere transduced with a self-inactivating lentiviral vector carrying an ubiquitinpromoter driving firefly luciferase and enhanced green fluorescence protein(Fluc-eGFP) double fusion (DF) reporter gene. HepG2cells (1×106), suspended in10μl of PBS, slowly injected into the upper left lobe of the liver. Then the growthstatus of tumors were monitored by the optical bioluminescence imaging (BLI) ofFLuc.4. Treatment of HCC with CD44targeted liposomes and Bioluminescence imaging of tunor regression. HCC-bearing mice were treated with:1）PBS；2）Lipo-TF/GCV；3）Lipo-CD44-TF/GCV；4） Lipo-CD44-Dox；5）Dox. Then thespecific targeting of the nanoparticles was tracked by BLI of RLuc and the growthstatus of tumor was monitored by BLI of FLuc.5. Immunofluorescence imaging for targeting of the nanoparticles. Furtherstudies of targeting Lipo-CD44-TF was performed by histological analysis of HCCby RFP expression via immunofluorescent microscopic examination. Double stainingof the RFP and CD44showed RFP expression in CD44+HCC cells.6. Apoptosis assay. The possible mechanism of HCC regression by CD44targeted liposomes was explored by TUNEL assays.Results:1. Liposomes were spherical and had the structure of phospholipid bilayersaround the surface as revealed by transmission electron microscopy (TEM), with auniform granularity and average diameter of approximately100nm.2. The uptake of CD44targeted RB-liposome in vitro revealed that RB can bewell taken up by cells at1h and the uptake of RB was saturated at12h byLipo-CD44-RB.3. Rluc from the TF plasmid delivered by Lipo-CD44-TF could be monitoredone week after administration. Dox delivered by Lipo-CD44-Dox can be accumulatedin CD44+HCC and can not be uptaken by other organ.4. The in situ HCC model in NOD/SCID mice was used for this assay. The Flucsignal of the Lipo-CD44-TF/GVC group increased only slightly and showedsignificant differences (P<0.05) when compared to the control and lipo-TF/GCVgroups at d35. Lipo-CD44-Dox also inhibited HCC growth effectively withsignificant improvements in survival, body weight and lower liver toxicity whencompared to the group of Dox.5. Double staining of the RFP and CD44showed RFP expression in CD44+HCCcells, which confirmed that TF could target HCC by the delivery of CD44conjugatedliposomes.6. TUNEL assays revealed that treatment with Lipo-CD44-Dox and Lipo-CD44-TF/GCV resulted in increased apoptosis in tumors when compared to notreatment or non-targeted Lipo-TF/GCV. But apoptosis caused by free Dox fromnormal tissue was more than any other group.Conclusion:The main conclusions are as follows:1. CD44targeted liposomal nanoparticles can be uptaken by HepG2cells invitro.2. CD44targeted liposomal nanoparticles loaded with TF or Dox couldspecifically target HCC expressing CD44, resulted in apoptosis of the targeted cellsand suppression of tumor growth.3. Apoptosis of targeted tumor cells was triggered by CD44targeted liposomalnanoparticles loaded with TF or Dox, and was further amplified because of bystandereffect, and then suppress growth of tumor.