[Effects of Rapamycin on angiogenesis and tumor progression in human hepatocellular carcinoma implantation mice].
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To study the effects of Rapamycin (RPM) on angiogenesis and tumor progression in human hepatocellular carcinoma (HCC) implantation mice.Tumor tissues of HCC were implanted into the liver of nude mice. Then, nude mice were treated with RPM and cyclosporine A (CsA). Real-time PCR was used to detect the mRNA expression of vascular endothelial growth factor (VEGF). Immunohistochemical stain and image analysis were used to detect the protein expression of VEGF and proliferating cell nuclear antigen (PCNA) and microvessel density (MVD) was counted by endothelial cells immunostained by anti-CD34 antibody. The concentration of VEGF in the peripheral blood was detected by ELISA.(1) The tumor weights were (372 +/- 35) mg, (769 +/- 39) mg and (751 +/- 42) mg in RPM, CsA and control group respectively. The tumor weight was significantly decreased in RPM group and no difference in CsA group compared with control group. (2) The expression of VEGF mRNA, VEGF and PCNA protein in tumor tissues and concentration of VEGF in the peripheral blood were remarkably down-regulated in RPM group compared with control group (P < 0.05) and were not remarkably different in CsA group from in control (P > 0.05).(3) Comparing with the control, the tumor MVD was remarkably decreased in RPM group (P < 0.05), and no difference in CsA group (P > 0.05).RPM can inhibit angiogenesis and tumor progression of HCC by down-regulated the gene and protein expression of VEGF and inhibited the growth of tumor.Keywords:
Tumor progression
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Objective To observe the growth and angiogenesis of human colon cancer HCT116 implanted in nude mice after silencing VEGF gene expression with RNAi technique. Methods The sub-cloned recombinant plasmid shRNA (pAVU6+27-VEGF-siRNA) and empty plasmid (pAVU6+27) were transfected into human colon carcinoma cell line HCT116 with DOTAP method, respectively named as group C and group B. The positive transfected cell clones were screened with G418. The cells receiving no plasmid transfection served as control (group A). Tumorigenicity and tumor growth of transfected cells were studied in a mouse model. VEGF expression and microvessel density (MVD) in tumor tissue were measured by immunohistochemistry. Results The mean volume and weight of the implanted tumor in group C [(0.169±0.009) cm3, (0.192±0.022) g] were significantly lower than group A [(0.256±0.016) cm3, (0.324±0.026) g] and group B [(0.248±0.014) cm3, (0.316±0.015) g] (P0.05). The protein expressions of VEGF in group C were significantly decreased as compared with those in group A and group B (P0.05), but no significant difference between group A and group B (P0.05). Moreover, MVD in group C was also decreased as compared with that in group A and group B (P0.05), but no significant difference between group A and group B (P0.05). There was a positive correlation between VEGF and MVD (r=0.810, P0.01). Conclusion Silencing VEGF gene can decrease VEGF gene expressions, suppress tumor angiogenesis and inhibit tumor growth in nude mice bearing human colon cancer cell line HCT116.
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Aim To observe the regulatory effect of LZBZY on the growth of H22 tumor and the expression of vascular endothelial growth factor(VEGF) in transplanted H22 tumor of mice,and explore whether the inhibition of tumor is related to the inhibition of vascular growth.Methods The mice were vaccinated with H22 cells suspension(1×107/ml) 0.2 ml each one.The observation was made on the weight of the transplanted tumor of the mice,then the inhibitory rate was calculated.The expression of VEGF was determined by the immunohistochemical method.Results The LZBZY group had significant effect on inhibiting the tumor growth(P0.05,P0.01),when compared with the model group,the pathological detective results showed that the positive rate of expression of VEGF was 100% in each group with the degree difference.Moreover,the expression of VEGF was decreased obviously in LZBZY group(P0.05,P0.01).Conclusion The LZBZY can inhibit the growth of the transplanted H22 tumor of mice and decrease the expression of the VEGF.
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To examine the effect of celecoxib on tumor growth and angiogenesis in an orthotopic implantation tumor model of colon cancer.Human colorectal adenocarcinoma HT-29 cells were implanted subcutaneously in nude mice. Four groups of animals received different doses of celecoxib after tumor implantation. After 42 days, all animals were evaluated for changes in body weight, the volume and weight of colorectal tumors, and tumor growth inhibition. The content of prostaglandin E(2) (PGE(2)) in the tumor tissue homogenate was estimated by radioimmunoassay (RIA). Cyclooxygenase-2 (COX-2) and CD34 expression in tumor tissue was assessed by immunohistochemistry, and the microvessel density (MVD) of tumor tissue was determined. Apoptosis of the tumor cells was detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). The expression of vascular endothelial growth factor (VEGF) mRNA and matrix metalloproteinase-2 (MMP-2) mRNA extracted from the tumor tissue was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR).There was no statistically significant change in the animals' body weight between the treatment groups. However, with increasing doses of celecoxib, the volume and weight of the tumor decreased. The rates of tumor growth inhibition for the L (low), M (medium) and H (high) groups were 25.30%, 38.80%, and 76.92%, respectively, which were significant compared to the C (control) group. There were significant differences in COX-2 expression in the tumor tissue between all groups, except between the L and M groups. Celecoxib exposure also reduced PGE2 levels in the tumor tissue homogenates. The level of PGE(2) correlated to the weight of tumor (r=0.8814, P < 0.05) and to COX-2 expression (r=0.8249, Puse < 0.05). Compared to the control group, the tumor cells from celecoxib-treated mice had a significantly higher apoptotic index. Celecoxib also decreased CD34(+) expression in tumors from treated mice. There were significant differences in the MVD between all groups except between groups H and M. Celecoxib significantly reduced the expression of VEGF and MMP-2 mRNA in the group H but not in L and M groups. The MVD in tumor tissue was closely related to the PGE2 levels, as well as the expression of VEGF and MMP-2 mRNA (r = 0.9006, r = 0.8573 and r = 0.6427, respectively; P < 0.05).By inhibiting COX-2, PGE(2) synthesis, and VEGF and MMP-2 mRNA expression in tumor tissue, celecoxib enhances tumor cell apoptosis, thereby inhibiting the growth and angiogenesis of orthotopically implanted tumors in a mouse model of human colorectal cancer.
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Objective To study the inhibitory effects of Protopanaxidiol(Ppd) on vascular endothelial growth factor(VEGF) and its mRNA expression of liver cancer.Methods Liver cancer model animals were divided into control group,positive drug group,cyclophosphamide group,low,medium and high dosage of Ppd group(25,50,100 mg/kg),10 ones in each group.After two weeks,the animals were killed to make histological section for immunohistochemical and hybridization in situ stain.Results Control group had increased tumor interstitial vessel density,the enhanced VEGF and mRNA expression positively relating to vessel density,while Ppd treatment group had decreased above indexes(P0.01);and medium and high dosage of Ppd had stronger inhibitory effect than cyclophosphamide(P0.01).Conclusions Ppd inhibits VEGF and its mRNA expression in tumor tissue and tumor angiogenesis,thus inhibits tumor growth.
Liver Cancer
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Objective To study the effects of glycogen synthase kinase-3β (GSK-3β) knock-down on the growth and the angiogenesis of human pancreatic cancer xenografts in nude mice.Methods Panc-1 cells were inoculated subcutaneously in athymic nude mice to establish xenograft models.The mice were divided into negative control group,vector control group and experimental group (n =8 each).After 8 weeks,the mice were killed.Tumor volume and weight were measured in nude mice beating xenografts.The inhibitory rate was calculated according to the weights of xenografts.The expression of proliferating cell nuclear antigen (PCNA) and CD31 proteins was assessed by using immunohistochemitry.Proliferation index (SPF) and microvessel density (MVD) were respectively counted according to PCNA and CD31 staining.The expression level of vascular endothelial growth factor (VEGF) was detected by using real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting.Results As compared with control group,the growth rate of human pancreatic cancer xenografts of nude mice in experimental group was decreased notably (P ＜0.05) and the inhibitory rate was 35.17％.In experimental group,the SPF index [(69.55 ± 2.64) ％] was significantly higher than in the negative control group [(83.26 ±2.83) ％] and the vector control group [(83.65 ± 2.65) ％] (P ＜ 0.05).In experimental group,the MVD index [(17.2 ± 2.9)] was higher than in the negative control group [(27.2 ± 3.1)] and the vector control group [(27.2 ± 3.1)] (P ＜ 0.05).The expression levels of VEGF mRNA and protein [(0.089 38 ±0.008 84 and 0.450 6 ±0.014 6) respectively] were significandy higher in xenografts tissues than in the negative control group (0.139 06 ± 0.003 35 and 0.675 8 ± 0.026 2 respectively) and the vector control group (0.138 89 ± 0.001 75 and 0.664 5 ± 0.010 5 respectively),but there was no significant difference between the negative control group and the vector control group (P ＞ 0.05).Conclusion In vivo GSK-3β knock-down can inhibit the growth and the angiogenesis of human pancreatic cancer xenografts in nude mice,which may be related to the down-regulation of VEGF.
Key words:
Pancreatic cancer; Glycogen synthase kinase-3β ; Microvessel density ; Vascular endothelial growth factor
CD31
Nude mouse
Proliferation index
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Objective To observe the effects of mutant p27 (p27mt) gene on the angiogenesis of SMMC-7721 cells in nude mice.Methods A mouse model of human hepatocellular carcinoma (SMMC-7721 cell line) was established by subcutaneous transplantation in BALB/c mice.The 30 mice were then divided into three groups,which were injected with PBS,Ad-LacZ and Ad-p27mt respectively,and the angiogenesis of the transplanted liver tumor was observed.The expression of p27 was detected by Western blotting,the expression levelss of vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-9 were immunohistochemically determined,and Ⅷ factor was examined through immunofluorescence.Results As compared with blank control and the Ad-Lac-Z groups,p27 protein expression in Ad-p27mt group was obviously increased,but the expression of VEGF and MMP-9 in Ad-p27mt group was significantly decreased (P ＜0.05).The average absorbence (AA) of Ⅷ factor in Ad-p27 group was 0.201 ±0.103,and that in the blank control and the Ad-Lac-Z groups was 0.346 ± 0.069 and 0.351 ± 0.045 respectiyely,with difference being significant between the former and the latter.Conclusion p27mt could express p27 protein in hepatocellular carcinoma,and the p27 protein could significantly inhibit the angiogenesis of tumor through down-regulating the expression of VEGF and MMP-9.
Key words:
Mutant p27 gene; Angiogenesis; Carcinoma, hepatocellular
Nude mouse
Immunofluorescence
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Objective To explore rapamycin's inhibitory effect on proliferation of H22 hepatic cancer in mice.Methods In vitro study: H22 hepatic cancer cell lines were cultured with rapamycin,CsA,FK506,and 5-FU,respectively,for 48 h.The different drugs' inhibitory effect on proliferation was determined through MTT.The influences of different agents on the H22 hepatic cancer cell cycle were observed by flow cytometry.The vascular endothelial growth factor(VEGF) concentration of the supernatant fluid of the cultured H22 hepatic cancer cell was detected by ELISA.In vivo study: C57BL/6 to Balb/c mice allogenic skin transplant was established,and the H22 hepatic cancer cell was implanted under skin.Rapamycin,CsA,FK506 and 5-FU were fed to the mice,respectively.The effect of different immunosuppressors on the survival of skin graft was observed while the proliferation of the transplant tumor was investigated.VEGF concentration of treated mice serum was examined by ELISA.The microvessel density of the transplanted tumor was observed through immunohistochemistry staining of CD34.Results The proliferation of the H22 hepatic cancer cells was inhibited by rapamycin at the concentration of 0.01-1 mg/L.When the H22 hepatic cancer cells were cultured with different dose of rapamycin,the VEGF concentration of the supernatant fluid decreased significantly(P0.05).The number of S phase cells decreased significantly compared to that of other agents(P0.05).When the mice in different groups were fed with 1.5 mg/(kg·d) and 4.5 mg/(kg·d) rapamycin,the lengthened survival time of the skin grafts was similar to that in CsA and FK506 groups.But the tumor volume was smaller than that in CsA and FK506 groups(P0.05).Compared to that in the control group,the VEGF concentration of mice serum decreased in rapamycin group (P0.05),and the microvessel density of the transplant tumor was reduced greatly(P0.05).Conclusion Rapamycin,as an immunosuppressor,significantly resists immunologic rejection and inhibits the proliferation of H22 hepatic cancer,thus having its advantage in treating malignant hepatic cancer with liver transplantation.
Liver Cancer
MTT assay
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OBJECTIVE:To establish the nude mouse model with breast cancer MCF-7 cells in which VEGF-C was interfered with Lentivirus-Mediated Small Interfering RNA(VEGF-C/siRNA),and investigate the relationship between VEGF-C expression and microvessel density(MVD) and lymphatic microvessel density(LVD).METHODS: VEGF-C/siRNA vectors were constructed and transducted into MCF-7 breast cancer cells,and rea1-time PCR was performed to detect the mRNA expression of VEGF-C.The MCF-7 cells which transfected with siRNA Vectors were injected into the nude mouse,and the volume of tumor was measured.The VEGF-C expression,MVD and LVD in transplant tumors were detected by immunohistochemistry.RESULTS: VEGF-C gene expression was down-regulated significantly in VEGF-C/siRNA interfered MCF-7 cells by 49.2%(P=0.017).The transplant tumor could grow in 3 groups of nude mice,6 in KD group,7 in NC group,8 in CON group.Cell number of transplant tumor in KD group decreased,and apoptosis and necrosis were observed in many cancer cells with HE staining.The immunohistochemical results showed that VEGF-C protein expression in KD group was decreased significantly(CON group:1+,1++,4+++;NC group:1++,5+++;KD group:5+,1++,P=0.02).LVD were 5.33±1.63,8.17±1.83,and 8.50±1.05 in KD group,NC group and CON group,respectively.LVD in KD group was lower significantly(P=0.005).There was not difference of MVD in KD group(8.83±1.72),NC group(11.17±2.48) and CON group(11.02±1.41,P=0.097).CONCLUSION: The VEGF-C expression can be successfully knocked down with VEGF-C/siRNA Lentivirus,VEGF-C can stimulate tumor lymphangiogenesis and have no effect on tumor angiogenesis.
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To investigate the effect of vascular endothelial growth factor (VEGF) on tumor angiogenesis in gallbladder carcinoma.Fifty one patients with gallbladder carcinoma were enrolled as observation group. Thirty healthy people were included as control group. Chemically synthesized siRNA sequences targeting VEGF was transfected with VEGF-siRNA. A blank group (group B), a negative control group (transfected with independent sequence, group C), and an inhibition group (transfected with VEGF siRNA, group D) were established. Physiological saline was set as group A. The expression of VEGF was detected by qRT-PCR. The expression of VEGF protein was detected by Western blot. MVD was used to measure microvessel density. CCK-8, Transwell and flow cytometry were used to detect cell proliferation, invasion and apoptosis.The tumor volume of nude mice and VEGF mRNA expression in group D was significantly smaller than that in group B and C (P<0.05). The MVD density in group B and C was significantly higher than that in group D (P<0.01). The proliferation of cells was detected from the 3rd day, and the proliferation of cells in the blank and negative control groups was faster than that of the inhibition group (P<0.05). The apoptosis rate of the blank group and the negative control group was lower than that of the inhibition group (P<0.001).VEGF is highly expressed in serum of patients with cholangiocarcinoma, it promotes angiogenesis, proliferation and invasion of gallbladder cancer cells, and inhibits apoptosis of tumor cells.
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OBJECTIVE To investigate the effects of rapamycin (RPM) in inhibiting the growth and metastasis of hepatocellular carcinoma (HCC). METHODS Human HCC cells of the line MHCC97H with a high potential of metastasis were divided into 3 groups to be cultured with cyclosporine A (CsA) 100 ng/ml, RPM 10 ng/ml, or CsA + RPM for 48 hours. Flow cytometry was used to examine the apoptosis and cell cycle MTT method was used to examine the effect of RMP on the proliferation of the MHCC97H cells. RT-PCR was used to detect the mRNA expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hypoxia-inducible factor-1alpha (HIF-1alpha), and transforming growth factor b (TGFb). Another MHCC97H cells were cultured in complete medium without RPM for 48 hours, then the protein expression of VEGF in the supernatant was detected by ELISA. Twenty-eight nude LCI-D20 mice were inoculated with human HCC cells and then divided into 4 groups to be fed with CsA (25 mg/kg), RPM (2 mg/kg), CsA + RPM, and normal saline (0.2 ml, as control group) for 35 days. Then the mice were killed to take the weight of inoculated tumor, measure the blood drug concentration, calculate the lung metastasis rate and number of metastatic foci, and observe pathology of the lung. RESULTS CsA showed no effect on the cycle of the MHCC97H cells. The MHCC97H cells of the RPM and CsA + RPM groups arrested at the stage G(0)/G(1) (both P = 0.000). MMT method also showed that the proliferation of the MHCC97H cells in the RPM and CsA + RPM groups were blocked (P = 0.003 and P = 0.002). However, CsA did not influence the proliferation of the MHCC97H cells. Flow cytometry showed that RPM did not promote the apoptosis of the MHCC97H cells. RT-PCR showed that RPM down-regulated the mRNA expression of VEGF and HIF-1alpha (both P < 0.05), however, did not influence the mRNA expression of bFGF, TGFb, and TGFb. The VEGF protein level in the supernatant of the culture fluid of MHCC97H cells of the RPM group was (890.3 +/- 25.1) pg/ml, significantly lower than that of the control group, (1583.7 +/- 17.3) pg/ml (P = 0.000). The tumor inhibiting rate of the RPM group was 63.7%, not significantly different from that of the RPM + CsA group (80.9%, P = 1.000). The metastatic rate of the CsA and control groups were both 100% with a higher number of metastatic tumors in the CsA group (P = 0.046). CONCLUSION RPM significantly inhibits the growth and metastasis of HCC. RPM-based immunosuppressive regimen may be of value in HCC patients receiving liver transplantation.
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