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
AIM: To study the effects of sense and antisense KAI1 genes on the growth and invasion of human hepatocellular carcinoma (HCC) cell line MHCC97-H.METHODS: KAI1 sense and antisense eukaryotic expression plasmids were c...AIM: To study the effects of sense and antisense KAI1 genes on the growth and invasion of human hepatocellular carcinoma (HCC) cell line MHCC97-H.METHODS: KAI1 sense and antisense eukaryotic expression plasmids were constructed using subclone technique and transfected into MHCC97-H cells respectively by DOTAP liposome. After successful transfection was confirmed, in vitro growth curve, cell cycles, plate clone formation efficiency, invasive ability in Boyden Chamber assay and ultrastructural morphology were studied.RESULTS: KAI1 sense and antisense genes had no significant effects on the cell growth curve and cell cycles.After transfection with sense KAI1 gene, decreased invasive ability in Boyden Chamber assay and decreased amount of mitochondria, but no significant changes of plate clone formation efficiency were observed in MHCC97-H-S cells.The plate clone formation efficiency and invasive ability in Boyden Chamber assay were significantly increased in MHCC97-H-AS cells, after transfection with antisense KAI1 gene. Furthermore, increased amount of mitochondria,rough endoplasmic reticulum, Golgi apparatus and expanded endoplasmic reticulum were also noted in MHCC97-H-AS cells.CONCLUSION: Changes of KAI1 expression in HCC cells may alter their invasive and metastasis ability of the tumor.展开更多
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 figure out the effect of somatostatin analogue Octreotide on proliferation and invasion of human hepatocellular carcinoma cell MHCC97-H and the underlying mechanism in vitro and in vivo. Methods: MHCC97-...Objective: To figure out the effect of somatostatin analogue Octreotide on proliferation and invasion of human hepatocellular carcinoma cell MHCC97-H and the underlying mechanism in vitro and in vivo. Methods: MHCC97-H cells were treated with Octreotide at the concentration of 0.2 ug/mL in vitro, proliferation related to time was evaluated. After treated with Octreotide at the concentration of 0.2 ug/mL for 48 h, MHCC97-H cells were observed by transmission electron microscope. Cell proliferation was detected by MTT assay after MHCC97-H cells were treated with Octreotide at different concentrations including 0.05, 0.1, 0.2, 0.4, 0.6 and 0.8 ug/mL for 36 h in vitro. 27 nude mice, in which MHCC97-H tumor mass was planted orthotopically, were divided into 3 groups randomly including control group (intraperitoneal injection with equal volume normal saline; n=8), low dose treated group (intraperitoneal injection with Octreotide at 50 ug/kg?d; n=9) and large dose treated group (intraperitoneal injection with Octreotide at 200 ug/kg?d; n=10). All mice were raised for 35 d and sacrificed. The information about survival time, the weight at death point and the pathology change of liver and lung was collected. The expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinases-2 (MMP-2) in mouse HCC tissues were detected by immunohistochemistry finally. Results: MTT assays showed that Octreotide inhibited the proliferation of MHCC97-H cells significantly. Apoptosis cells were found by transmission electron microscope after treatment with Octreotide at 0.2 ug/mL for 48 h in vitro. The proliferation was inhibited significantly by Octreotide in a dose-dependant manner (r=0.86, P<0.01). Compared with control group, the treated group had the heavier weight at death point and lower intrahepatic metastasis ratio (P<0.05), meanwhile, there was not significant difference in treated groups (P>0.05). The positive expression ratios of VEGF and MMP-2 in treated groups were lower than those in control group (P<0.05), while there was no apparent difference in treated groups (P>0.05). Conclusion: Octreotide could inhibit the proliferation of MHCC97-H cells in vitro via inducing apoptosis and the inhibitory function acts in a dose-dependant manner. Octreotide could improve survival of mice with MHCC97-H cells and inhibit the metastasis of MHCC97-H cells in vivo. Regulation of VEGF and MMP-2 expression by Octreotide would be involved in its inhibition in vivo.展开更多
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.
基金Supported by the National Natural Science Foundation of China,No.30070348
文摘AIM: To study the effects of sense and antisense KAI1 genes on the growth and invasion of human hepatocellular carcinoma (HCC) cell line MHCC97-H.METHODS: KAI1 sense and antisense eukaryotic expression plasmids were constructed using subclone technique and transfected into MHCC97-H cells respectively by DOTAP liposome. After successful transfection was confirmed, in vitro growth curve, cell cycles, plate clone formation efficiency, invasive ability in Boyden Chamber assay and ultrastructural morphology were studied.RESULTS: KAI1 sense and antisense genes had no significant effects on the cell growth curve and cell cycles.After transfection with sense KAI1 gene, decreased invasive ability in Boyden Chamber assay and decreased amount of mitochondria, but no significant changes of plate clone formation efficiency were observed in MHCC97-H-S cells.The plate clone formation efficiency and invasive ability in Boyden Chamber assay were significantly increased in MHCC97-H-AS cells, after transfection with antisense KAI1 gene. Furthermore, increased amount of mitochondria,rough endoplasmic reticulum, Golgi apparatus and expanded endoplasmic reticulum were also noted in MHCC97-H-AS cells.CONCLUSION: Changes of KAI1 expression in HCC cells may alter their invasive and metastasis ability of the tumor.
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.
文摘Objective: To figure out the effect of somatostatin analogue Octreotide on proliferation and invasion of human hepatocellular carcinoma cell MHCC97-H and the underlying mechanism in vitro and in vivo. Methods: MHCC97-H cells were treated with Octreotide at the concentration of 0.2 ug/mL in vitro, proliferation related to time was evaluated. After treated with Octreotide at the concentration of 0.2 ug/mL for 48 h, MHCC97-H cells were observed by transmission electron microscope. Cell proliferation was detected by MTT assay after MHCC97-H cells were treated with Octreotide at different concentrations including 0.05, 0.1, 0.2, 0.4, 0.6 and 0.8 ug/mL for 36 h in vitro. 27 nude mice, in which MHCC97-H tumor mass was planted orthotopically, were divided into 3 groups randomly including control group (intraperitoneal injection with equal volume normal saline; n=8), low dose treated group (intraperitoneal injection with Octreotide at 50 ug/kg?d; n=9) and large dose treated group (intraperitoneal injection with Octreotide at 200 ug/kg?d; n=10). All mice were raised for 35 d and sacrificed. The information about survival time, the weight at death point and the pathology change of liver and lung was collected. The expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinases-2 (MMP-2) in mouse HCC tissues were detected by immunohistochemistry finally. Results: MTT assays showed that Octreotide inhibited the proliferation of MHCC97-H cells significantly. Apoptosis cells were found by transmission electron microscope after treatment with Octreotide at 0.2 ug/mL for 48 h in vitro. The proliferation was inhibited significantly by Octreotide in a dose-dependant manner (r=0.86, P<0.01). Compared with control group, the treated group had the heavier weight at death point and lower intrahepatic metastasis ratio (P<0.05), meanwhile, there was not significant difference in treated groups (P>0.05). The positive expression ratios of VEGF and MMP-2 in treated groups were lower than those in control group (P<0.05), while there was no apparent difference in treated groups (P>0.05). Conclusion: Octreotide could inhibit the proliferation of MHCC97-H cells in vitro via inducing apoptosis and the inhibitory function acts in a dose-dependant manner. Octreotide could improve survival of mice with MHCC97-H cells and inhibit the metastasis of MHCC97-H cells in vivo. Regulation of VEGF and MMP-2 expression by Octreotide would be involved in its inhibition in vivo.
