Objective： To assess the anti-invasive effect of DDB and its possible active mechanism in human hepatocellular carcinoma MHCC97-H with high metastasis potential. Methods： MTT assay was used to evaluate the cytotoxic...Objective： To assess the anti-invasive effect of DDB and its possible active mechanism in human hepatocellular carcinoma MHCC97-H with high metastasis potential. Methods： MTT assay was used to evaluate the cytotoxicity of DDB to MHCC97-H cells and the anti-adhesion of DDB on MHCC97-H cells to laminin （LN） and fibronectin （FN）. The anti-invasive effect of DDB was detected by the transwell chamber experiment. VEGF, nm23-H1 and uPAR mRNA transcriptions were determined by RT-PCR assay. The secretion and expression of a-fetal protein （AFP） were analyzed by ELISA and flow cytometry, respectively. Results： DDB at non-cytotoxic concentrations （10, 50 and 100 μmol/L） obviously inhibited the adhesion of MHCC97-H on LN and FN. In the transwell chamber experiment, the inhibition rates of the invasion of DDB 50 and 100 μmol/L on MHCC97-H cells were 25.8% and 32.3%, respectively. By RT-PCR assay, DDB 50 and 100 μmol/L decreased VEGF, nm23-H1 and uPAR mRNA expressions in MHCC97-H cells. The ELISA assay showed that 50, 100 and 200 μmol/L DDB decreased the AFP secretion of MHCC97-H cells, the inhibitory rates were 16.5%, 17.5% and 48.5%, respectively. DDB also decreased the expression of AFP in MHCC97-H cells by flow cytometry assay. Conclusion： DDB, an anti-hepatitis drug, at non-cytotoxic concentrations showed significant anti-invasion effect in human hepatocellular carcinoma MHCC97-H cells, and the inhibition of VEGF, nm23-H1 and uPAR expression should contribute to the anti-invasion property of DDB.展开更多
Objective： To study the anti-angiogenesis effect of melittin on human hepatoma Mock/MHCC97-H cells by regulatingthe expression of cathepsin S （CatS） in vivo. Methods： Models of in situ transplantation tumor of Moc...Objective： To study the anti-angiogenesis effect of melittin on human hepatoma Mock/MHCC97-H cells by regulatingthe expression of cathepsin S （CatS） in vivo. Methods： Models of in situ transplantation tumor of Mock/MHCC97-Hcells and silencing cathepsin shRNA-CatS/ MHCC97-H cells in nude mice were established. The model mice wererandomly divided into four groups. In the A1 group, the mice were inoculated with shRNA-CatS/MHCC97-H cells andtreated with melittin. In the A2 group, the mice were inoculated with shRNA-CatS/MHCC97-H cells and treated withsaline. In the B1 group, the mice were inoculated with Mock/MHCC97-H cells and treated with melittin. In the B2 group,the mice were inoculated with Mock/MHCC97-H cells and treated with saline. The A1 and B1 group were injected withmelittin （80 mg/kg） intraperitoneally every day. The A2 and B2 group were injected with 0.2 mL normal salineintraperitoneally every day. After administration for 25 days, the animals were sacrificed. The tumor size and weight innude mice in each group were recorded. The expression of CD34 protein in the xenograft tumor tissues was detected byimmunohistochemistry. The expression of Cat S, VEGF-A, p-VEGFR2, Ras, Raf, p-Raf, MEK1, p-MEK1, ERK1/2 andp-ERK1/2 proteins were detected by western blot. Results： The B1 group had significantly smaller tumor volumes andlower tumor weights than the B2 group （both P 〈 0.001）. There was no significant difference between the A1 group andA2 group in tumor volumes and weights. The number of CD34-positive microvessels in the B2 group was significantlyhigher than that in the A2 group （P 〈 0.001）. The number of CD34-positive microvessels in the B1 group wassignificantly lesser than that in the A1 group （P 〈 0.001）. Most strikingly, in the model featuring inoculation ofMock/MHCC97-H cells, CatS, VEGF-A, p-VEGFR2, Ras, Raf, p-Raf, MEK1, p-MEK1, ERK1/2 and p-ERK1/2expression were inhibited when treated with melittin. However, in the model featuring the inoculation ofshRNA-CatS/MHCC97-H cells, no such effects were observed with similar treatments. Conclusion： Melittin can inhibitthe growth of tumors and angiogenesis by blocking the CatS-VEGf-A signaling pathway.展开更多
文摘Objective： To assess the anti-invasive effect of DDB and its possible active mechanism in human hepatocellular carcinoma MHCC97-H with high metastasis potential. Methods： MTT assay was used to evaluate the cytotoxicity of DDB to MHCC97-H cells and the anti-adhesion of DDB on MHCC97-H cells to laminin （LN） and fibronectin （FN）. The anti-invasive effect of DDB was detected by the transwell chamber experiment. VEGF, nm23-H1 and uPAR mRNA transcriptions were determined by RT-PCR assay. The secretion and expression of a-fetal protein （AFP） were analyzed by ELISA and flow cytometry, respectively. Results： DDB at non-cytotoxic concentrations （10, 50 and 100 μmol/L） obviously inhibited the adhesion of MHCC97-H on LN and FN. In the transwell chamber experiment, the inhibition rates of the invasion of DDB 50 and 100 μmol/L on MHCC97-H cells were 25.8% and 32.3%, respectively. By RT-PCR assay, DDB 50 and 100 μmol/L decreased VEGF, nm23-H1 and uPAR mRNA expressions in MHCC97-H cells. The ELISA assay showed that 50, 100 and 200 μmol/L DDB decreased the AFP secretion of MHCC97-H cells, the inhibitory rates were 16.5%, 17.5% and 48.5%, respectively. DDB also decreased the expression of AFP in MHCC97-H cells by flow cytometry assay. Conclusion： DDB, an anti-hepatitis drug, at non-cytotoxic concentrations showed significant anti-invasion effect in human hepatocellular carcinoma MHCC97-H cells, and the inhibition of VEGF, nm23-H1 and uPAR expression should contribute to the anti-invasion property of DDB.
基金This work was supported by grants from the National Science Foundation of China （No. 81360372）.
文摘Objective： To study the anti-angiogenesis effect of melittin on human hepatoma Mock/MHCC97-H cells by regulatingthe expression of cathepsin S （CatS） in vivo. Methods： Models of in situ transplantation tumor of Mock/MHCC97-Hcells and silencing cathepsin shRNA-CatS/ MHCC97-H cells in nude mice were established. The model mice wererandomly divided into four groups. In the A1 group, the mice were inoculated with shRNA-CatS/MHCC97-H cells andtreated with melittin. In the A2 group, the mice were inoculated with shRNA-CatS/MHCC97-H cells and treated withsaline. In the B1 group, the mice were inoculated with Mock/MHCC97-H cells and treated with melittin. In the B2 group,the mice were inoculated with Mock/MHCC97-H cells and treated with saline. The A1 and B1 group were injected withmelittin （80 mg/kg） intraperitoneally every day. The A2 and B2 group were injected with 0.2 mL normal salineintraperitoneally every day. After administration for 25 days, the animals were sacrificed. The tumor size and weight innude mice in each group were recorded. The expression of CD34 protein in the xenograft tumor tissues was detected byimmunohistochemistry. The expression of Cat S, VEGF-A, p-VEGFR2, Ras, Raf, p-Raf, MEK1, p-MEK1, ERK1/2 andp-ERK1/2 proteins were detected by western blot. Results： The B1 group had significantly smaller tumor volumes andlower tumor weights than the B2 group （both P 〈 0.001）. There was no significant difference between the A1 group andA2 group in tumor volumes and weights. The number of CD34-positive microvessels in the B2 group was significantlyhigher than that in the A2 group （P 〈 0.001）. The number of CD34-positive microvessels in the B1 group wassignificantly lesser than that in the A1 group （P 〈 0.001）. Most strikingly, in the model featuring inoculation ofMock/MHCC97-H cells, CatS, VEGF-A, p-VEGFR2, Ras, Raf, p-Raf, MEK1, p-MEK1, ERK1/2 and p-ERK1/2expression were inhibited when treated with melittin. However, in the model featuring the inoculation ofshRNA-CatS/MHCC97-H cells, no such effects were observed with similar treatments. Conclusion： Melittin can inhibitthe growth of tumors and angiogenesis by blocking the CatS-VEGf-A signaling pathway.
