| Background and purposeMetastasis is one of the most life-threatening aspects of malignancy. Effectively controlling of metastasis could obviously improve the survival periods of patients. Clinical therapies for malignancy include surgery, chemotherapy and radiotherapy. Surgery is the best choice for early malignancy. However, most patients were not in early stage when malignancy was discovered, some even with obvious metastasis. Chemotherapy is a common adjuvant therapy which could eradicate residue lesion or metastatic focus. Adjuvant therapy with surgery and chemotherapy could effectively control various kinds of malignant tumors. But chemotherapy drugs induce evident system toxic and side effects as well as adverse reactions of patients. More over, repeated chemotherapy could easily induce tumor cell became drug resistant, which eliminate drug efficiency. So, scientists tried to produce novel anti-tumor reagents without the disadvantages of chemotherapeutics.In 1906, German scientists proposed "targeted therapy" for the first time. The aim was to increase drug concentration in the local lesion, reduce toxic and side effects, decrease dose of drugs applied, making drugs elicit effects safely and effectively. In the present, molecular targeting drugs clinically applied for malignant tumors including:mono antibody families of target drugs (Herceptin), single target of tyrosine kinase inhibiting reagent (imatinib), multiple targets of tyrosine kinase inhibiting reagent (Sorafenib), fusion toxin target drugs(ONTAK).Methods1. Construction, expression and purifying of DT390-TMTP1 and DT390-biTMTPl fusion genes. (1) PCR amplifying gene fragments, product was digested with Ndel and Xhol, purifying and retrieving fragments, which were inserted the pET28a (+) expression vector to get recombinant plasmids of pET28a (+)/DT390-TMTP1 and pET28a (+) /DT390-biTMTP1. (2) Recombinant plasmids were transformed into BL21 (λDE3), which were induced by IPTG to express objective proteins. (3) Inclusion bodies were harvested by sonicating on ice, and purified by 50% Ni-NTA His·Bind slurry. Concentrations and purities of inclusion bodies were determined by SDS-PAGE gels. (4) Purified inclusion bodies were refolded by diluting 10 fold into a redox refolding buffer and concentrated by ultrafiltration. (5) Crystal structure of DT390-TMTP1 and DT390-biTMTPl were analyzed by SWISS-MODEL.2. Toxic effects of DT390-TMTP1 and DT390-biTMTPl fusion proteins for tumor cells and normal cells. (1) PC-3M-1E8, MKN-45 and HEK293 cells were treated with different concentrations (1-6μg/ml) of DT390-TMTP1 and DT390-biTMTP1 fusion proteins for 24 h, and the inhibition rates of the cells growth were measured by MTT assay. (2) Different toxic effects of TMTP1, DT390-TMTP1 and DT390-biTMTP1 fusion proteins for PC-3M-1E8, MKN-45 and HEK293 cells were analyzed by MTT assay. (3) Different toxic effects of TMTP1, DT390-TMTP1 and DT390-biTMTP1 fusion proteins for PC-3M-1E8, MKN-45 and HEK293 cells were analyzed by flow cytometry. (4) Cellular morphology changes of PC-3M-1E8, MKN-45 and HEK293 were observed and photographs were taken under an invert microscope after treated with TMTP1, DT390-TMTP1 and DT390-biTMTP1 fusion proteins. (5) Nuclear morphology changes of PC-3M-1E8, MKN-45 and HEK293 were observed and photographs were taken using a confocal laser scanning microscope after treated with TMTP1,DT390-TMTP1 and DT390-biTMTP1 fusion proteins. 3. Distribution and spread of DT390-biTMTPl and tumor targeting therapy in vivo. (1) Construction of subcutaneous tumor models of prostate cancer and gastric cancer. (2) Model mice were i.p. injected with TMTP1, DT390-TMTP1 and DT390-biTMTP1 fusion proteins, tumor volume and survival rate of mice were detected. (3) Orthotropic gastric cancer model mice were i.p. injected with TMTP1, DT390-TMTP1 and DT390-biTMTP 1 fusion proteins, orthotropic tumor and metastasis were observed. (4) Distribution and spread of DT390-biTMTP1 in model mice were investigated with immunohistochemistry. (5) Expression of proliferation marker (Ki67) and apoptosis markers (Caspase3 and TUNE) in tumors treated with TMTP1, DT390-TMTP1 and DT390-biTMTP1 were detected with immunohistochemistry.Results1. Recombinant plasmids of pET28a (+)/DT390-TMTP1 and pET28a (+)/ DT390-biTMTPl were successfully constructed, which were transformed into BL21 cells and induced with IPTG to produce inclusion bodies. Inclusion bodies were released by sonicating cell pellets on ice in lysing buffer and purified with 50% Ni-NTA His·Bind slurry, after refolded in a redox refolding buffer and concentrated by ultrafiltration, fusion proteins with high purities were obtained. Analysis of SWISS-MODEL showed that crystal structures of catalytic domain and binding domain of diphtheria toxin were the same as wild type diphtherial toxin, indicating our rearrangements didn't result in conformational changes, suggesting the intact toxic activities of recombinant diphtheria toxins.2. Results of MTT assay showed that TMTP1 and DT390-TMTP1 fusion protein couldn't effectively inhibit survival rate of PC-3M-1E8, MKN-45 or HEK293 cells with the concentration of 1-6μg/ml. DT390-biTMTP1 fusion protein significantly killed PC-3M-1E8 and MKN-45 cells, while HEK293 cells were unaffected. Results of flow cytometry showed that TMTP1 and DT390-TMTP1 fusion protein couldn't effectively kill PC-3M-1E8, MKN-45 or HEK293 cells, but DT390-biTMTP1 fusion protein significantly increased the apoptosis rate of PC-3M-1E8 and MKN-45 cells, while HEK293 cells were unaffected. All the three treatments resulted in no morphology changes in HEK293 cells. About 80% MKN-45 and PC-3M-1E8 cells were collapsed after treated by DT390-biTMTP1, while no obvious cellular morphology changes induced by DT390-TMTP1 or TMTP1. All treatments resulted in no nuclear morphology changes in HEK293 cells. DT390-biTMTP1 could induce severely nuclear fragmentation in MKN-45 and PC-3M-1E8 cells, but no changes of nuclear morphology were observed by TMTP1 and DT390-TMTP1.3. Subcutaneous tumor models of prostate cancer and gastric cancer were successfully constructed. Model mice were given i.p. injections of PBS, TMTP1, DT390-TMTP1, and DT390-biTMTP1 for eight times, results showed that DT390-biTMTP1 effectively inhibited PC-3M-1E8 tumor growth and prolonged the survival of mice, but TMTP1 and DT390-TMTP1 failed to exhibit obvious anti tumor effects. Similar results were also obtained in nude mice with MKN-45 subcutaneous tumors, DT390-biTMTP1 significantly slowed down the growth of tumors, while TMTP1 and DT390-TMTP1 didn't inhibit growth of MKN-45 subcutaneous tumors obviously. All MKN-45 orthotropic gastric cancer model mice took place primary tumor, and there were extensive metastasis in liver, spleen, and abdominal wall in TMTP1 and DT390-TMTP1 treated mice, but DT390-biTMTP1 significantly decreased the frequency of metastasis to liver and spleen. Distribution and spread of DT390-biTMTP1 in tumor mice indicate that significantly increased DT390-biTMTP1 was accumulated in the tumor shortly after injection (1 h) and lasted at a high level for 48h, except a transient accumulation in liver and kidney(0.5-1h), there were no amount of DT390-biTMTP1 distributed in heart, spleen, lung and brain when compared with control mice at all time points. DT390-biTMTP1 treated mice had a marked reduction in Ki67 staining, but a distinguished increase in caspase3 staining and tunnel staining, suggesting DT390-biTMTP1 fusion protein elicited toxic effect against subcutaneous tumors via decreasing proliferation, but inducing apoptosis of tumor cell.ConclusionDT390-biTMTP1 fusion protein selectively targets and kills highly metastatic cancer cells and occult metastasis foci, which may be a promising strategy for the treatment of cancer or alternative anticancer agents for tumor metastasis.
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