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Objective:To investigate targeted therapy of ovarian cancer with new fusion proteins that were produced by fusing the first 390 amino acids of diphtheria toxin(DT390)to the TMTP1 peptide.Methods:The cisplatin-resistant cell line,C13*,and cisplatin-sensi-tive cell line,OV2008,were selected as models and divided into control,TMTP1,DT390-TMTP1,DT390-biTMTP1,and DT390-triTMTP1 groups.Laser scanning confocal microscopy was used to observe nuclear morphology.3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide(MTT)and flow cytometry assays were used to detect cell survival and apoptosis,respectively.The formation of subcu-taneous tumors in nude mice following injection of C13*cells was used to observe the formation and growth of ovarian cancer.Apop-tosis of cells in the subcutaneous tumor tissue was detected by the terminal deoxynucleotidyl transferase dUTP nick-end labeling(TU-NEL)assay.Results:Laser scanning confocal microscopy showed that DT390-biTMTP1 and DT390-triTMTP1 induced nuclear shrinkage and fragmentation.The MTT assay showed that cell survival was obviously reduced with increasing concentrations of DT390-biTMTP1 and DT390-triTMTP1. Flow cytometry revealed that DT390-biTMTP1 and DT390-triTMTP1 significantly increased cell apoptosis (P<0.05).The apoptosis rates of the DT390-biTMTP1 and DT390-triTMTP1 groups were 66.0%±12.0% and 72.9%±4.6%,respectively.These were higher than the 55.5%±8.9% and 65.1%±9.8% obvserved in OV2008 cells.DT390-biTMTP1 and DT390-triTMTP1 significantly in-hibited the tumor formation (P<0.01) and growth (P<0.05), and increased apoptosis (P<0.05) of subcutaneous tumors. However, DT390-TMTP1 had insignificant effects on C13*and OV2008 cells.Conclusions:DT390-biTMTP1 and DT390-triTMTP1 preferentially tar-geted and inhibited ovarian cancer cells.These fusion proteins may be a promising strategy for clinical therapy of ovarian cancer.
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Objective To synthesize 18F-AlF-NOTA-G-TMTP1 and evaluate its potential for PET imaging on nude mice bearing high-metastatic potential hepatoma cells.Methods NOTA-G-TMTP1 was synthesized by the standard Fmoc-solid phase synthetic protocols and radiolabeled with 18F using NOTA-AlF chelation method.The nude mice models bearing low-metastatic potential HCC97L and high-metastatic potential HCCLM3 xenografts were established separately.The tumor-targeting characteristics of 18F-AlF-NOTA-G-TMTP1 were assessed by microPET/CT and biodistribution assay.Results NOTA-G-TMTP1 was labeled with 18F in one step with (25±6)％ labeling yield (n=5).The radiochemical purity of 18F-AlF-NOTA-G-TMTP1 was more than 95％ with a specific activity more than 11.1 GBq/μmol.The octanol/water partition coefficient (logP) for 18F-AlF-NOTA-G-TMTP1 was-3.166±0.022.The tumor to muscle ratios were 1.8± 0.4 and 4.7±0.2 at 35 min post injection for HCC97L and HCCLM3,respectively.The uptake of 18F-AlF-NOTA-G-TMTP1 in HCCLM3 tumor was inhibited (61.4％) by unlabeled G-TMTP1.Conclusion 18F-AlF-NOTA-G-TMTP1 has been successfully synthesized.It shows specific uptake by tumor induced by the high-metastatic potential hepatoma cells.
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TMTP1,a 5-amino acid peptide NVVRQ,obtained by using the flagella peptide library screening in our previous studies,can be used for the labeling of malignant in situ and metastatic lesions,and even micro-metastases.In this study,TMTP1 was assessed for its ability to specifically target the malignant hematopoietic cells and metastatic lesions of hematological malignancies.FITC-TMTP 1 was chemically synthesized.Immunofluorescence assay and competitive test were carried out to determine the specific binding capacity of TMTPI to hematological malignant cell lines,including HL60,k562,SHI-1,Jurkat,Raji,El-4 and umbilical cord blood mononuclear cells.Mononuclear cells were isolated from the bone marrow of healthy subjects and patients with chronic myeloid leukemia.Then the cells were co-clutured with TMTP1 or scrambled peptides and the binding and affinity of TMTP1 peptide to the primary cells of hematological malignancies were flow cytometrically analyzed.The binding specificity of TMTP 1 to target hematological malignancies was measured in vivo by intravenous injection of FITC-conjugated TMTP1 into El-4 lymphoma-bearing mice.The results showed that TMTP1 specifically bound to the cells of a series of hematological malignancies,including HL60,k562,Jurkat,Raji,El-4 and chronic myeloid leukemia primary cells but not to bone marrow mononuclear cells from healthy subjects.By contrast,TMTP1 could bind to the metastatic foci of lymphoma originating from the EL-4 cell line while the scrambled peptide failed to do so.Moreover,the occult metastases could be identified,with high specificity,by detecting FITC-TMTP1.We are led to conclude that TMTP1,as a novel tumor-homing peptide,can serve as a marker for primary malignant and metastatic lesions for the early diagnosis of hematological malignances and a carrier of anticancer drugs for cancer treatment.