Apatinib Inhibits Angiogenesis in Intrahepatic Cholangiocarcinoma by Regulating the Vascular Endothelial Growth Factor Receptor-2/Signal Transducer and Activator of Transcription Factor 3/Hypoxia Inducible Factor 1 Subunit Alpha Signaling Axis.

INTRODUCTION: Intrahepatic cholangiocarcinoma (ICC), which is difficult to diagnose and is usually fatal due to its late clinical presentation and a lack of effective treatment, has risen over the past decades but without much improvement in prognosis. OBJECTIVE: The study aimed to investigate the role of apatinib that targets vascular endothelial growth factor receptor-2 (VEGFR2) in ICC. METHODS: MTT assays, cell scratch assays, and tube formation assays were used to assess the effect of apatinib on human ICC cell line (HuCCT-1) and RBE cells proliferation, migration, and angiogenic capacity, respectively. Expression of vascular endothelial growth factor (VEGF), VEGFR2, signal transducer and activator of transcription factor 3 (STAT3), pSTAT3, and hypoxia inducible factor 1 subunit alpha (HIF-1α) pathway proteins was assessed using Western blotting and mRNA expression analysis in HuCCT-1 was performed using RT-qPCR assays. The pcDNA 3.1(-)-VEGFR2 and pcDNA 3.1(-)-HIF-1α were transfected into HuCCT-1 and RBE cells using Lipofectamine 2,000 to obtain overexpressed HuCCT-1 and RBE cells. RESULTS: We found that apatinib-inhibited proliferation, migration, and angiogenesis of HuCCT-1 and RBE cells in vitro in a dose-dependent manner. We also proved that apatinib effectively inhibits angiogenesis in tumor cells by blocking the expression of VEGF and VEGFR2 in these cells. In addition, we demonstrated that apatinib regulates the expression of STAT3 phosphorylation by inhibiting VEGFR2. Finally, we showed that apatinib regulates ICC angiogenesis and HIF-1α/VEGF expression via STAT3. CONCLUSIONS: Based on the above findings, we conclude that apatinib inhibits HuCCT-1 and RBE cell proliferation, migration, and tumor angiogenesis by inhibiting the VEGFR2/STAT3/HIF-1α axis signaling pathway. Apatinib can be a promising drug for ICC-targeted molecular therapy.

New approach for development of sensitive and environmentally friendly immunoassay for mycotoxin fumonisin B(1) based on using peptide-MBP fusion protein as substitute for coating antigen.

Here, on the basis of mimotope of small analytes, we demonstrated a new approach for development of sensitive and environmentally friendly immunoassay for toxic small analytes based on the peptide-MBP fusion protein. In this work, using mycotoxin fumonisin B1 (FB1) as a model hapten, phage displayed peptide (mimotope) that binds to the anti-FB1 antibody were selected by biopanning from a 12-mer peptide library. The DNA coding for the sequence of peptide was cloned into Escherichia coli ER2738 as a fusion protein with a maltose binding protein (MBP). The prepared peptide-MBP fusion protein are "clonable" homogeneous and FB1-free products and can be used as a coating antigen in the immunoassay. The half inhibition concentration of the quantitative immunoassay setup with fusion protein (F1-MBP and F15-MBP) was 2.15 ± 0.13 ng/mL and 1.26 ± 0.08 ng/mL, respectively. The fusion protein (F1-MBP) was also used to develop a qualitative Elispot assay with a cutoff level of 2.5 ng/mL, which was 10-fold more sensitive than that measured for chemically synthesized FB1-BSA conjugates based Elispot immunoassay. The peptide-MBP fusion protein not only can be prepared reproducibly as homogeneous and FB1-free products in a large-scale but also can contribute to the development of a highly sensitive immunoassay for analyzing FB1. Furthermore, the novel concept might provide potential applications to a general method for the immunoassay of various toxic small molecules.

Application of mimotope peptides of fumonisin b1 in Peptide ELISA.

Anti-fumonisin B(1) (FB(1)) McAb 1D11 was used as the target for biopanning from a phage random loop-constrained heptapeptide library. After three cycles of panning, seven phages with three mimotope peptides were selected to mimic the binding of FB(1) to 1D11. After the identification of phage ELISA, the phage clone that showed the best linear range of detection was chosen for further research. One peptide with the inserted peptide sequence of the phage was synthetized, named CT-452. An indirect competitive ELISA (peptide ELISA) for detecting FB(1) was established using the CT-452-bovine serum albumin conjugate as coating antigen. The linear range of the inhibition curve was 1.77-20.73 ng/mL. The half inhibitory concentration (IC50) was 6.06 ng/mL, and the limit of detection was 1.18 ng/mL. This method was compared with conventional indirect ELISA (commercial ELISA kit) and high-performance liquid chromatography (HPLC), and the results showed the reliability of the peptide ELISA for the determination of FB(1) in cereal samples. The relationship between the CT-452 and FB(1) standard concentrations in peptide ELISA was evaluated. The results indicated that synthetic peptide CT-452 can replace the FB(1) standard to establish an immunoassay free of FB(1).

Association between insulin-like growth factor-2 expression and prognosis after transcatheter arterial chemoembolization and octreotide in patients with hepatocellular carcinoma.

OBJECTIVE: To investigate the association between the change of IGF-2 level in serum after transcatheter arterial chemoembolization (TACE) and hepatocellular carcinoma (HCC) progression, especially in relation to metastasis. METHODS: IGF-2 in serum was measured by quantitative sandwich enzyme-linked immunosorbent assay before, 3 days and 4 weeks after TACE in 60 patients with HCC. The occurrence of HCC metastasis was also evaluated, 3 months after TACE. RESULTS: (1) The average serum level of IGF-2 in the 60 patients with HCC was 136.5 ± 87.3 pg/ml; (2) A tendency for increase was observed with heterogenous uptake of octreotide and portal vein thrombosis. Metastatic foci were found in 37/38 patients in the group with IGF-2 increasing (97.0%), in contrast to 3/22 (13.6%) patients with IGF-2 decrease. CONCLUSION: The increase of IGF-2 level in serum appears to be associated with the occurrence of metastatic HCC after TACE and chemotherapy.

[Transhepatic arterial chemoembolization with gemcitabine and carboplatin for the treatment of stage III hepatocellular carcinoma].

OBJECTIVE: To evaluate the efficiency and safety of transhepatic arterial chemoembolization (TACE) with gemcitabine and carboplatin for the treatment of stage III hepatocellular carcinoma (HCC). METHODS: Sixty-one HCC patients were treated by TACE. During TACE, At first, intra-arterial infusion of carboplatin 300 mg/m2, then gemcitabine 1000 mg/m2 with 5-30 ml of ultra-lipoidal iodide oil emulsion was used for arterial embolization. The toxicity and hepatic damage were observed according to WHO anticancer drug toxicity criteria and Child-Pugh classification criteria, respectively. The survival time was also observed during follow-up. RESULTS: The blood toxicity was bone marrow suppression presented as grade I leucopenia in 39.3%, grade II in 29.5%, grade III-IV in 18.0%. Grade II-III nausea and vomiting developed in 96.8% of the patients. Hepatic function damage became aggravated in 16 patients from A to B class, in 2 from A to C class, and in 6 from B to C class according to Child-Pugh classification criteria. The median survival time was 20 months with a range of 5 to 3 5 months. CONCLUSION: Transhepatic arterial chemoembolization using carboplatin and mixture of gemcitabine with ultra-lipoidal iodide oil emulsion is safe and effective in the management of stage III hepatocellular carcinoma. This regimen can also improve their quality of life.

Association between vascular endothelial growth factor and metastasis after transcatheter arterial chemoembolization in patients with hepatocellular carcinoma.

BACKGROUND: Hypoxia up-regulates vascular endothelial growth factor (VEGF) and stimulates the growth of hepatocellular carcinoma (HCC) cells. This study was designed to investigate the association between changes in plasma VEGF levels after transcatheter arterial chemoembolization (TACE) and HCC progression, especially in relation to metastasis. METHODS: Plasma VEGF levels were measured by quantitative sandwich enzyme-linked immunosorbent assay (ELISA R&D system). Plasma VEGF levels were measured before, 3 days and 4 weeks after TACE in 30 patients with HCC. The development of metastasis was evaluated at the end of the third month after TACE. RESULTS: The plasma VEGF levels of the 30 patients with HCC were 154.47+/-90.17 pg/ml. The total plasma VEGF levels after TACE increased compared with their basal levels (P<0.05), and the plasma VEGF levels had a tendency to increase in patients with heterogeneous uptake of iodizdoil and portal vein thrombosis. Follow-up for six months showed metastatic foci in 20 patients (74%) with increased plasma VEGF, but none of the patients with decreased plasma VEGF developed metastasis. CONCLUSION: Increased plasma VEGF expression is associated with the development of metastasis in HCC after TACE.

[Relation between vascular endothelial growth factor and reoccurence-metastasis after transcatheter arterial chemoembolization in hepatocellar carcinoma].

OBJECTIVE: To investigate the relation between changes in serum vascular endothelial growth factor (VEGF) level after transcatheter arterial chemoembolization (TACE) and hepatocellular carcinoma (HCC) progression, especially in relation to metastasis. METHODS: Serum VEGF expression level, measured by quatitative sandwich enzyme-linked immunosorbent assay (ELISA, R&D system), was measured before, 3 days and 4 weeks after TACE in 30 patients with HCC. The development of metastasis was evaluated at the end of the third month after TACE. RESULTS: 1. The serum VEGF level in 30 patients was 154.47 +/- 90.17 pg/ml, 2. Post-TACE total serum VEGF level increased as compared with their basal level in 30 patients (P < 0.05) and serum VEGF level had a tendency to increase in patients with heterogeneous uptake of iodized oil and portal vein thrombosis. During the follow-up of 1 - 2 years, metastatic foci were found in 74% (20) patients with SVEGF increase, while none of the patients showing SVEGF decrease developed metastasis. CONCLUSION: Serum VEGF expression increase is associated with the development of metastasis in hepatocellular carcinoma after TACE.