| Background and ObjectivesHepatocellular carcinoma (HCC) is one of the most frequent tumors with high aggressive and recurrence in China. However, early diagnosis of HCC remains a challenge because most patients have no symptoms in the initial stage. Hence, there is prominent need for oncology imaging modalities or biomarkers capable of identifying early stage tumors, and signs of tumor progression and recurrence of HCC.Toll-like receptors (TLRs) play prominent roles in inflammatory responses against pathogen infection. Current advancement in cancer immunobiology highlights those TLRs as crucial factors involved in tumour growth and progression. For example, there is only an elevated expression of TLR5in gastric carcinoma, colon tumor, prostatic cancer, ovarian cancer and breast carcinoma. Flagellin (FliC) is a specific ligand for TLR5. Studies confirmed that flagellin and flagellin-expressing bacteria (Salmonella) have shown antitumor effects in mouse models of colon and liver metastasis of pancreatic cancer.Occurrence and development of most malignant proliferative diseases were involved in cell cycle regulation mechanism disorders. Cyclin-dependent kinase (CDK) plays a key role in cell cycle regulation, and inhibition of the catalytic activity can play a positive role in the treatment of tumors. Flavopiridol (FLA) was the first CDKs inhibitor to enter clinical trials in American. It inhibits cell proliferation primarily through inhibition of CDK1, CDK2and CDK4kinase activity. However, clinical data suggest that use of these drugs alone cannot achieve the desired therapeutic effect, with the combination of conventional chemotherapeutic drugs show a good therapeutic potential.Radionuclide imaging is able to detect the disease molecularly non-invasively in real time, and also could provide functional changes in the initial stage of disease. Therefore, this study aimed to investigate the effects of different concentrations of flagellin and flavopiridol on proliferation and apoptosis of H22hepatoma cells. We hypothesized that TLR5may be a good biomarker for detection of HCC, so we prepared a radioiodinated anti-TRL5McAb and evaluated its tumor targeting potential using H22hepatocarcinoma-bearing mice model.Methods1. TLR5expression on H22cell line, H22xenograft tumor tissue and normal liver tissue was detected with RT-PCR and immunofluorescence experiments.2. The H22cell line was treated with different concentration of recombinant flagellin or flavopiridol in vitro and then cell proliferation was evaluated by Cell Counting Kit-8assay (CCK-8).3. The rates of apoptosis were calculated on the basis of Annexin V-FITC/PI apoptosis detection by flow cytometry.4. The mice were inoculated subcutaneously on the rear flanks with4x106H22cells in100μl normal saline.5. Anti-TLR5McAb was radioiodinated with Na131I by the Iodogen method, and then the radiotracer was separated from free iodine using Sephadex G-25columns. Radiochemical purity of131I-anti-TLR5McAb was obtained by paper chromatography.6. In-vitro stability of131I-anti-TLR5McAb and its isotype IgG was determined in serum or saline respectively, and evaluated by paper chromatography.7. The uptakes of131I-anti-TLR5McAb by H22cells in vitro were analyzed. 8. Binding affinity of131I-anti-TLR5with TLR5was detected by receptor binding assay in vitro.9. After injection of131I-anti-TLR5McAb or isotype IgG, the biodistribution and tumor targeting imaging in H22tumor-bearing mice were analyzed.Results1. RT-PCR and immunofluorescence experiment showed that TLR5expression of H22cell line and H22-xenografted tumor tissue was higher than that in normal liver tissue.2. CCK-8assay verified that flagellin or flavopiridol had stronger growth inhibiting effect on H22cells in a concentration dependent manner, and flagellin combined with flavopiridol could reach the peak (P<0.05).3. Either flagellin or flavopiridol alone could induce apoptosis of H22cells, but the combined use of them could significantly increase apoptosis (P<0.01).4. Anti-TLR5McAb was successfully labeled with131I. After purified, label rates of131I-anti-TLR5McAb and131I-IgG were both higher than95%. And the specific radioactivities were29.56and25.43GBq/μmoL, respectively. The probes were more stable in serum than normal saline at24h.5. Cellular uptake revealed that uptake of131I-anti-TLR5McAb in H22cells was higher than of Na131I after incubation.6. Receptor binding assay on H22cell line showed that Kd value was (2.81±0.05) nmol/L and Bmax was (4.65±0.09) nmol/L.7. When the tumor diameter reached1cm (2weeks after inoculation), the H22tumor-bearing mice were ready for autoradiography imaging studies.8. Biodistribution study showed that liver and kidney had the higher uptake, tumor uptake was (6.51±0.63)%ID/g at12h post injection,(8.03±0.42)%ID/g at the highest at24h, while the1311-IgG group was (3.83±0.26)%ID/g at24h. As compared to131I-IgG,131I-anti-TLR5McAb displayed rapid H22tumor uptake and better tumor retention.9. Whole body autoradiography showed that the subcutaneous H22tumors were all clearly visible, with good contrast to the background, at all measured time points after injection of131I-anti-TLR5McAb. These findings were in accordance with the high T/NT ratio.ConclusionH22-xenografted tumor tissue exhibited a higher level of TLR5expression, and flagellin can obviously inhibit proliferation of H22cells and induce apoptosis.131I-anti-TLR5McAb was biologically stable with high radiochemical purity. It could target tumor rapidly, and the autoradiography images of tumor were clearly visible. Therefore, it is able to detect lesions in a TLR5-expressing tumor, with high target selectivity, and may offer a promising agent for hepatocarcinoma diagnosis and encourage further investigation. |
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