AIM: To establish clone cells with different metastatic potential for the study of metastasis-related mechanisms. METHODS: Cloning procedure was performed on parental hepatocellular carcinoma (HCC) cell line MHCC97, a...AIM: To establish clone cells with different metastatic potential for the study of metastasis-related mechanisms. METHODS: Cloning procedure was performed on parental hepatocellular carcinoma (HCC) cell line MHCC97, and biological characteristics of the target clones selected by in vivo screening were studied. RESULTS: Two clones with high (MHCC97-H) and low (MHCC97-L) metastatic potential were isolated from the parent cell line. Compared with MHCC97-L, MHCC97-H had smaller cell size (average cell diameter 43 microm vs 50 microm) and faster in vitro and in vivo growth rate (tumor cell doubling time was 34.2h vs 60.0h). The main ranges of chromosomes were 55-58 in MHCC97-H and 57-62 in MHCC97-L. Boyden chamber in vitro invasion assay demonstrated that the number of penetrating cells through the artificial basement membrane was (37.5 +/- 11.0) cells/field for MHCC97-H vs (17.7 +/- 6.3)/field for MHCC97-L. The proportions of cells in G0-G1 phase, S phase, and G2-M phase for MHCC97-H/MHCC97-L were 0.56/0.65, 0.28/0.25 and 0.16/0.10, respectively, as measured by flow cytometry. The serum AFP levels in nude mice 5wk after orthotopic implantation of tumor tissue were (246 +/- 66) microg.L(-1) for MHCC97-H and (91 +/- 66) microg.L(-1) for MHCC97-L. The pulmonary metastatic rate was 100% (10/10) vs 40% (4/10). CONCLUSION: Two clones of the same genetic background but with different biological behaviors were established, which could be valuable models for investigation on HCC metastasis.展开更多
AIM: To characterize the anticancer function of cytokine-induced killer cells (CIK) and develop an adoptive immunotherapy for the patients with primary hepatocellular carcinoma (HCC), we evaluated the proliferation ra...AIM: To characterize the anticancer function of cytokine-induced killer cells (CIK) and develop an adoptive immunotherapy for the patients with primary hepatocellular carcinoma (HCC), we evaluated the proliferation rate, phenotype and the antitumor activity of human CIK cells from healthy donors and HCC patients in vitro and in vivo. METHODS: Peripheral blood mononuclear cells (PBMC) from healthy donors and patients with primary HCC were incubated in vitro and induced into CIK cells in the presence of various cytokines such as interferon-gamma (IFN-gamma), interleukin-1 (IL-1), IL-2 and monoclonal antibody (mAb) against CD3. The phenotype and characterization of CIK cells were identified by flow cytometric analysis. The cytotoxicity of CIK cells was determined by (51)Cr release assay. RESULTS: The CIK cells were shown to be a heterogeneous population with different cellular phenotypes. The percentage of CD3+/CD56+ positive cells, the dominant effector cells, in total CIK cells from healthy donors and HCC patients, significantly increased from 0.1-0.13% at day 0 to 19.0-20.5% at day 21 incubation, which suggested that the CD3+ CD56+ positive cells proliferated faster than other cell populations of CIK cells in the protocol used in this study. After 28 day in vitro incubation, the CIK cells from patients with HCC and healthy donors increased by more than 300-fold and 500-fold in proliferation cell number, respectively. CIK cells originated from HCC patients possessed a higher in vitro antitumor cytotoxic activity on autologous HCC cells than the autologous lymphokine-activated killer (LAK) cells and PBMC cells. In in vivo animal experiment, CIK cells had stronger effects on the inhibition of tumor growth in Balb/c nude mice bearing BEL-7402-producing tumor than LAK cells (mean inhibitory rate, 84.7% vs 52.8%, P【0.05) or PBMC (mean inhibitory rate, 84.7% vs 37.1%, P【0.01). CONCLUSION: Autologous CIK cells are of highly efficient cytotoxic effector cells against primary hepatocellular carcinoma cells and might 展开更多
AIM: To establish the role of vascular endothelial growth factor (VEGF) in the oncogenesis of human gastric carcinoma more directly. METHODS: The expression of VEGF and its receptor kinase-domain insert containing rec...AIM: To establish the role of vascular endothelial growth factor (VEGF) in the oncogenesis of human gastric carcinoma more directly. METHODS: The expression of VEGF and its receptor kinase-domain insert containing receptor (KDR) in human gastric cancer tissue were observed by immunohistochemical staining. VEGF levels were manipulated in human gastric cancer cell using eukaryotic expression constructs designed to express the complete VEGF(165) complimentary DNA in either the sense or antisense orientation. The biological changes of the cells were observed in which VEGF was up-regulated or down-regulated. RESULTS: VEGF-positive rate was 50%, and VEGF was mainly localized in the cytoplasm and membrane of the tumor cells, while KDR was mainly located in the membrane of vascular endothelial cells in gastric cancer tissues and peri-cancerous tissue. In 2 cases of 50 specimens, the gastric cancer cells expressed KDR, localized in both the cytoplasm and membrane. Introduction of VEGF(165) antisense into human gastric cancer cells (SGC-7901, immunofluorescence intensity, 31.6%)) resulted in a significant reduction in VEGF-specific messenger RNA and total and cell surface VEGF protein (immunofluorescence intensity, 8.9%) (P【0.05). Conversely, stable integration of VEGF(165) in the sense orientation resulted in an increase in cellular and cell surface VEGF (immunofluorescence intensity, 75.4%) (P【0.05). Lowered VEGF levels were associated with a marked decrease in the growth of nude mouse xenografted tumor (at 33 days postimplantation, tumor volume: 345.40 +/- 136.31 mm3)(P【0.05 vs control SGC-7901 group: 1534.40 +/- 362.88 mm3), whereas up-regulation of VEGF resulted in increased xenografted tumor size (at 33 days postimplantation, tumor volume: 2350.50 +/- 637.70 mm3) (P【0.05 vs control SGC-7901 group). CONCLUSION: This study provides direct evidence that VEGF plays an important role in the oncogenesis of human gastric cancer.展开更多
AIM: To test the hypothesis to block VEGF expression of SMMC-7721 hepatoma cells may inhibit tumor growth using the rat hepatoma model. METHODS: Amplify the 200 VEGF cDNA fragment and insert it into human U6 gene cass...AIM: To test the hypothesis to block VEGF expression of SMMC-7721 hepatoma cells may inhibit tumor growth using the rat hepatoma model. METHODS: Amplify the 200 VEGF cDNA fragment and insert it into human U6 gene cassette in the reverse orientation transcribing small antisense RNA which could specifically interact with VEGF165, and VEGF121 mRNA. Construct the retroviral vector containing this antisense VEGF U6 cassette and package the replication-deficient recombinant retrovirus. SMMC-7721 cells were transduced with these virus and positive clones were selected with G418. PCR and Southern blot analysis were performed to determine if U6 cassette integrated into the genomic DNA of positive clone. Transfected tumor cells were evaluated for RNA expression by ribonuclease protection assays. The VEGF protein in the supernatant of parental tumor cells and genetically modified tumor cells was determined with ELISA. In vitro and in vivo growth properties of antisense VEGF cell clone in nude mice were analyzed. RESULTS: Restriction enzyme digestion and PCR sequencing verified that the antisense VEGF RNA retroviral vector was successfully constructed.After G418 selection, resistant SMMC-7721 cell clone was picked up. PCR and Southern blot analysis suggested that U6 cassette was integrated into the cell genomic DNA. Stable SMMC-7721 cell clone transduced with U6 antisense RNA cassette could express 200 bp small antisense VEGF RNA and secrete reduced levels of VEGF in culture condition. Production of VEGF by antisense transgene-expressing cells was 65+/-10 ng/L per 10(6) cells, 42045 ng/L per 10(6) cells in sense group and 485+/-30 ng/L per 10(6) cells in the negative control group, (P【 0.05). The antisense-VEGF cell clone appeared phenotypically indistinguishable from SMMC-7721 cells and SMMC-7721 cells transfected sense VEGF. The growth rate of the antisense-VEGF cell clone was the same as the control cells. When S.C. was implanted into nude mice, growth of antisense-VEGF cell lines was greatly inhibited compared with co展开更多
Metastatic human HCC model is needed for the studies on mechanism and intervention of metastatic recurrence. By using orthotopic implantation of histologically intact tissues of 30 surgical specimens, a patient-like m...Metastatic human HCC model is needed for the studies on mechanism and intervention of metastatic recurrence. By using orthotopic implantation of histologically intact tissues of 30 surgical specimens, a patient-like metastatic model of human HCC in nude mice (LCI-D20) and a low metastatic model of human HCC in nude mice (LCI-D35) have been established. All mice with transplanted LCI-D20 tumors exhibited extremely high metastatic ability including spontaneous metastasis to liver, lungs, lymph nodes and peritoneal seeding. Remarkable difference was also found in expression of some of the invasiveness related genes and growth factors between the LCI-D20 and LCI-D35 tumors. PAI-1 increased gradually following tumor progression in LCI-D20 model, and correlated with tumor size and AFP level. Phasic expression of tissue intercellular adhesion molecule-1 in this model was also observed. Using corneal micropocket model, it was demonstrated that the vascular response induced by LCI-D20 tumor was stronger than that induced by LCI-D35 tumor. Similar report on metastatic human HCC model in nude mice and human HCC cell line with metastatic potential was rarely found in the literature. This LCI-D20 model has been widely used for the studies on intervention of metastasis, including anti-angiogenesis,antisense approach, metalloproteinase inhibitor, differentiation inducer, etc. It is concluded that the establishment of metastatic human HCC model in nude mice and human HCC cell line with metastatic potential will provide important models for the in vitro and in vitro study of HCC invasiveness, angiogenesis as well as intervention of HCC recurrence.展开更多
AIM: To investigate anti-tumor activities and apoptosis-regulated mechanisms of bufalin in the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice.METHODS: BEL-7402 cells of human hep...AIM: To investigate anti-tumor activities and apoptosis-regulated mechanisms of bufalin in the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice.METHODS: BEL-7402 cells of human hepatocellular carcinoma were inoculated to form subcutaneous tumors, and were implanted into the liver to establish orthotopic transplantation tumor models of human hepatocellular carcinoma in nude mice. Seventy-five animals were randomized divided into five groups (n = 15). Bufalin was injected intraperitoneally into three groups at doses of 1.5 mg/kg (BF1), 1 mg/kg (BF2) and 0.5 mg/kg (BF3) for d 15-24, respectively. The NS group was injected an equal volume of saline as above and adriamycin was injected intraperitoneally into the ADM group at a dose of 8.0 mg/kg for d 15. Ten mice in each group were killed at d 25 and the survival time in each group was calculated. We also observed the morphologic alterations in the myocardium, brain, liver, kidney and tumor tissues by pathology and electron microscopy, measured the apoptotic rate by TUNEL staining method, and detected the expression of apoptosis-regulated genes bcl-2 and bax by immunohistochemical staining and RT-PCR in tumor tissues. RESULTS: The tumor volumes in each group of bufalin were reduced significantly (35.21 ± 12.51 vs 170.39 ± 25.29; 49.83 ± 11.46 vs 170.39 ± 25.29; 83.99 ± 24.63 vs 170.39 ± 25.29, P < 0.01, respectively), and the survival times were prolonged in group BF1-2 (31.8 ± 4.2 vs 23.4 ± 2.1 and 29.4 ± 3.4 vs 23.4 ± 2.1, P < 0.05, respectively), and necrosis was mainly in severe or moderate degree in group BF1-2. No morphologicalchanges were detected in the myocardium, brain, liver and kidney tissues. Apoptotic characteristics could be seen in group BF1-2. The positive rates of bcl-2 and bax protein expression of each group by immunohistochemical staining were 10.0%, 10.0%, 20.0%, 10.0% and 20.0%; 90.0%, 80.0%, 80.0%, 40.0% and 30.0%, respectively. Loss of expression of bcl-2 mRNA in each group was to be found and the展开更多
Background The earthworm fibrinolytic enzyme （EFE） is a complex protein enzyme that is widely distributed in the earthworm＇s digestive cavity. Possessing strong protein hydrolysis activity, EFE not only has a direc...Background The earthworm fibrinolytic enzyme （EFE） is a complex protein enzyme that is widely distributed in the earthworm＇s digestive cavity. Possessing strong protein hydrolysis activity, EFE not only has a direct effect on fibrin, but also can activate plasminogen. Its therapeutic and preventative effects on thrombosis-related disease have been confirmed clinically. Recently, there has been increased interest in the anti-tumor activity of EFE. In this study, the anti-tumor activity of EFE, isolated from Eisenia foetida, on human hepatoma cells was evaluated in vitro and in vivo. The potential mechanisms involved were also studied. Methods In vitro experiments were performed in four human hepatoma cell lines： HLE, Huh7, PLC/PRF/5 and HepG2. After treatment with EFE in various concentrations, the inhibition of the rate of cell proliferation was measured. For the in vivo studies, tumor-bearing models xenografted with Huh7 cells were developed in nude mice, and then the mice were fed with EFE once a day for 4 weeks, and the control group received only saline. An inhibitory effect on tumor growth was observed. Also, apoptosis was observed with flow cytometric assay and fluorescent dye staining with acridine orange and ethidium bromide （AO/EB）. The expression of matrix metalloproteinase 2 （MMP-2） were detected by Western blotting assay. Results After treatment with various concentrations of EFE, the proliferation of all hepatoma cell lines was suppressed to varying degrees in vitro. The IC50 for HLE, Huh7, PLC/PCF/5 and HepG2 were 2.11, 5.87, 25.29 and 17.30 uku/ml, respectively. After administration of EFE orally for 4 weeks, the growth of tumor xenograft of Huh7 cells in nude mice was significantly inhibited in vivo. The tumor inhibitory rates in the EFE 500 uku/（kg·d） and 1000 uku/（kg·d） groups were 46.08% （compared with control group, P=0.026） and 57.52% （compared with control group, P=0.002） respectively. Meanwhile, the average weight of body, spleen or thymus did not show any 展开更多
AIM: The transcription factor EGR-1 (early growth response gene-1) plays an important role in cell growth, differentiation and development. It has identified that EGR-1 has significant transformation suppression activ...AIM: The transcription factor EGR-1 (early growth response gene-1) plays an important role in cell growth, differentiation and development. It has identified that EGR-1 has significant transformation suppression activity in some neoplasms, such as fibrosarcoma, breast carcinoma. This experiment was designed to investigate the role of egr-1 in the cancerous process of hepatocellular carcinoma (HCC) and esophageal carcinoma (EC), and then to appraise the effects of EGR-1 on the growth of these tumor cells. METHODS: Firstly, the transcription and expression of egr-1 in HCC and EC, paracancerous tissues and their normal counterpart parts were detected by in situ hybridization and immunohistochemistry, with normal human breast and mouse brain tissues as positive controls. Egr-1 gene was then transfected into HCC (HHCC, SMMC7721) and EC (ECa109) cell lines in which no egr-1 transcription and expression were present. The cell growth speed, FCM cell cycle, plate clone formation and tumorigenicity in nude mice were observed and the controls were the cell lines transfected with vector only. RESULTS: Little or no egr-1 transcription and expression were detected in HCC, EC and normal liver tissues. The expression of egr-1 were found higher in hepatocellular paracancerous tissue (transcription level P=0.000; expression level P=0.143, probably because fewer in number of cases) and dysplastic tissue of esophageal cancer (transcription level P=0.000; expression level P=0.001). The growth rate of egr-1-transfected HHCC (HCC cell line) cells and ECa109 (EC cell line) cells was much slower than that of the controls. The proportion of S phase cell, clone formation and tumorigenicity were significantly lower than these of the controls' (decreased 45.5% in HHCC cells and 34.1% in ECa109 cells; 46.6% and 41.8%; 80.4% and 72.6% respectively). There were no obvious differences between SMMC7721 (HCC) egr-1-transfected cells and the controls with regard to the above items. CONCLUSION: The decreased expression of egr-1 might play a role in 展开更多
AIM: To evaluate antihepatoma effect of antisense phosphorothioate oligodeoxyribonucleotides (S-ODNs) targeted to alpha-fetoprotein (AFP) genes in vitro and in nude mice. METHODS: AFP gene expression was examined by i...AIM: To evaluate antihepatoma effect of antisense phosphorothioate oligodeoxyribonucleotides (S-ODNs) targeted to alpha-fetoprotein (AFP) genes in vitro and in nude mice. METHODS: AFP gene expression was examined by immunocytochemical method or enzyme-linked immunosorbent assay. Effect of S-ODNs on SMMC-7721 human hepatoma cell growth in vitro was determined using microculture tetrazolium assay. In vitro antitumor activities of S-ODNs were monitored by measuring tumor weight differences in treated and control mice bearing SMMC-7721 xenografts. Induction of cell apoptosis was evaluated by fluorescence-activated cell sorter (FACS) analysis. RESULTS: Antisense S-ODN treatment led to reduced AFP gene expression. Specific antisense S-ODNs, but not control S-ODNs, inhibited the growth of hepatoma cells in vitro. In vitro, only antisense S-ODNs exhibited obvious antitumor activities. FACS analysis revealed that the growth inhibition by antisense S-ODNs was associated with their cell apoptosis induction. CONCLUSION: Antisense S-ODNs targeted to AFP genes inhibit the growth of human hepatoma cells and solid hepatoma, which is related to their cell apoptosis induction.展开更多
AIM: To establish a relevant animal model of human gastrointestinal cancer, which can be used for repetitive investigations, so as to improve our understanding and management of carcinogenesis and cancer metastasis. M...AIM: To establish a relevant animal model of human gastrointestinal cancer, which can be used for repetitive investigations, so as to improve our understanding and management of carcinogenesis and cancer metastasis. METHODS: Intact tissues of human colorectal and pancreatic cancers were transplanted in nude mice. The biological characteristics of the original and the corresponding transplanted tumors were investigated by HE staining, PAS staining and immunostaining. The metastases in the livers and lungs of nude mice were investigated by immunostaining with biotinylated mab KL-1 and by RT-PCR using CK20 specific primers. RESULTS: There were totally 9 of 16 surgical specimens growing in nude mice subcutaneously and/or orthotopically (4 of 6 colorectal and 5 of 10 pancreatic cancer). Tumor cell content of the specimens and freezing of tissue specimens are important factors influencing the growth of transplanted tumor. In the group of fresh tumor tissues with greater than 50% tumor cell content, the success rate of the transplantation was 100% (3 cases of pancreatic cancer and 3 cases of colorectal cancer). The orthotopically trans-planted tumors resemble the original tumor morphologically and biologically, including TAA expression such as CEA by immunohistochemistry, and CEA level in the serum of mice. Ki-67 labeling index and the expression of TAA especially K-ras, 17-1A and RA-96, are associated with the potential of tumor growth in nude mice. Micrometastases in the lungs and livers of tumor bearing mice can be detected by immunostaining with biotinylated mab KL-1 and CK20-specific RT-PCR. CONCLUSION: An orthotopic transplantation model for human colon and pancreatic cancer in nude mice has been set up. We have also established sensitive detection methods with CK-immunohistochemistry and CK20-RT-PCR to study xenotransplanted human cancer and its metastatic cancer cells in the liver and lung of nude mice. This study may be helpful in understanding the mechanism of cancer metastasis and in developing new diagnostic展开更多
AIM To investigate the effects of taxol onSMMC-7721 human hepatoma and itsmechanisms.METHODS In vitro cell growth was assessedby trypan blue exclusion method.Experimentalhepatoma model was established by seedingSMMC-7...AIM To investigate the effects of taxol onSMMC-7721 human hepatoma and itsmechanisms.METHODS In vitro cell growth was assessedby trypan blue exclusion method.Experimentalhepatoma model was established by seedingSMMC-7721 cells subcutaneously into Balb/c(nu/nu)nude mice.In vivo tumor growth wasdetermined by measurement of tumor diameterwith Vernier calipers.The syntheses of DNA,RNA and protein were analyzed by incorporationof ~3H-thymidine,~3H-uridine and ~3H-leucinerespectively.Using light and electronmicroscopes to observe the morphologicalchanges of cells including mitosis andapoptosis.RESULTS Taxol was effective against SMMC-7721 human hepatoma cell growth in the rangesof 2.5 nmol/L-10 nmol/L with mitotic arrestand apoptosis in vitro.DNA,RNA and proteinsyntheses in cells were also obviouslysuppressed by in vitro treatment of taxol for72 h.Taxol at 2.5 nmol/L reduced ~3H-thymidineuptake to about 34% of the control value(P【0.05).Increasing the dose of taxol to20 nmol/L resulted in a greater decrease in ~3H-thymidine incorporation to 60% of the controlvalue(P【0.01).At a concentration of 20 nmol/L,the ~3H-uridine and ~3H-leucine uptakeswere reduced to 52%(P【0.05)and 63%(P【0.01),respectively.In vivo,taxolsignificantly inhibited SMMC-7721 tumor growthat 10 mg/kg,i.p.,once daily for 10 d.A morethan 90% decrease in tumor volume wasobserved by day 11(P【0.01)similarly withmitotic arrest and cell apoptosis.CONCLUSION Taxol has a marked anticanceractivity in SMMC-7721 human hepatoma both invitro and in nude mice.Its mechanisms might beassociated with mitotic arrest,subsequently,apoptosis of the hepatoma cells.No obvioustoxicity was observed with in vivoadministration of taxoi.展开更多
基金Supportod ty the State Key Basic Research Program Grant G1998051211 the Fund for Leading Specialty of Shanghai Metropolitan Bureau of Public Health.
文摘AIM: To establish clone cells with different metastatic potential for the study of metastasis-related mechanisms. METHODS: Cloning procedure was performed on parental hepatocellular carcinoma (HCC) cell line MHCC97, and biological characteristics of the target clones selected by in vivo screening were studied. RESULTS: Two clones with high (MHCC97-H) and low (MHCC97-L) metastatic potential were isolated from the parent cell line. Compared with MHCC97-L, MHCC97-H had smaller cell size (average cell diameter 43 microm vs 50 microm) and faster in vitro and in vivo growth rate (tumor cell doubling time was 34.2h vs 60.0h). The main ranges of chromosomes were 55-58 in MHCC97-H and 57-62 in MHCC97-L. Boyden chamber in vitro invasion assay demonstrated that the number of penetrating cells through the artificial basement membrane was (37.5 +/- 11.0) cells/field for MHCC97-H vs (17.7 +/- 6.3)/field for MHCC97-L. The proportions of cells in G0-G1 phase, S phase, and G2-M phase for MHCC97-H/MHCC97-L were 0.56/0.65, 0.28/0.25 and 0.16/0.10, respectively, as measured by flow cytometry. The serum AFP levels in nude mice 5wk after orthotopic implantation of tumor tissue were (246 +/- 66) microg.L(-1) for MHCC97-H and (91 +/- 66) microg.L(-1) for MHCC97-L. The pulmonary metastatic rate was 100% (10/10) vs 40% (4/10). CONCLUSION: Two clones of the same genetic background but with different biological behaviors were established, which could be valuable models for investigation on HCC metastasis.
基金Science and Technology Development Foundation of Beijing Institute of Infectious Diseases,No.01 Z094
文摘AIM: To characterize the anticancer function of cytokine-induced killer cells (CIK) and develop an adoptive immunotherapy for the patients with primary hepatocellular carcinoma (HCC), we evaluated the proliferation rate, phenotype and the antitumor activity of human CIK cells from healthy donors and HCC patients in vitro and in vivo. METHODS: Peripheral blood mononuclear cells (PBMC) from healthy donors and patients with primary HCC were incubated in vitro and induced into CIK cells in the presence of various cytokines such as interferon-gamma (IFN-gamma), interleukin-1 (IL-1), IL-2 and monoclonal antibody (mAb) against CD3. The phenotype and characterization of CIK cells were identified by flow cytometric analysis. The cytotoxicity of CIK cells was determined by (51)Cr release assay. RESULTS: The CIK cells were shown to be a heterogeneous population with different cellular phenotypes. The percentage of CD3+/CD56+ positive cells, the dominant effector cells, in total CIK cells from healthy donors and HCC patients, significantly increased from 0.1-0.13% at day 0 to 19.0-20.5% at day 21 incubation, which suggested that the CD3+ CD56+ positive cells proliferated faster than other cell populations of CIK cells in the protocol used in this study. After 28 day in vitro incubation, the CIK cells from patients with HCC and healthy donors increased by more than 300-fold and 500-fold in proliferation cell number, respectively. CIK cells originated from HCC patients possessed a higher in vitro antitumor cytotoxic activity on autologous HCC cells than the autologous lymphokine-activated killer (LAK) cells and PBMC cells. In in vivo animal experiment, CIK cells had stronger effects on the inhibition of tumor growth in Balb/c nude mice bearing BEL-7402-producing tumor than LAK cells (mean inhibitory rate, 84.7% vs 52.8%, P【0.05) or PBMC (mean inhibitory rate, 84.7% vs 37.1%, P【0.01). CONCLUSION: Autologous CIK cells are of highly efficient cytotoxic effector cells against primary hepatocellular carcinoma cells and might
文摘AIM: To establish the role of vascular endothelial growth factor (VEGF) in the oncogenesis of human gastric carcinoma more directly. METHODS: The expression of VEGF and its receptor kinase-domain insert containing receptor (KDR) in human gastric cancer tissue were observed by immunohistochemical staining. VEGF levels were manipulated in human gastric cancer cell using eukaryotic expression constructs designed to express the complete VEGF(165) complimentary DNA in either the sense or antisense orientation. The biological changes of the cells were observed in which VEGF was up-regulated or down-regulated. RESULTS: VEGF-positive rate was 50%, and VEGF was mainly localized in the cytoplasm and membrane of the tumor cells, while KDR was mainly located in the membrane of vascular endothelial cells in gastric cancer tissues and peri-cancerous tissue. In 2 cases of 50 specimens, the gastric cancer cells expressed KDR, localized in both the cytoplasm and membrane. Introduction of VEGF(165) antisense into human gastric cancer cells (SGC-7901, immunofluorescence intensity, 31.6%)) resulted in a significant reduction in VEGF-specific messenger RNA and total and cell surface VEGF protein (immunofluorescence intensity, 8.9%) (P【0.05). Conversely, stable integration of VEGF(165) in the sense orientation resulted in an increase in cellular and cell surface VEGF (immunofluorescence intensity, 75.4%) (P【0.05). Lowered VEGF levels were associated with a marked decrease in the growth of nude mouse xenografted tumor (at 33 days postimplantation, tumor volume: 345.40 +/- 136.31 mm3)(P【0.05 vs control SGC-7901 group: 1534.40 +/- 362.88 mm3), whereas up-regulation of VEGF resulted in increased xenografted tumor size (at 33 days postimplantation, tumor volume: 2350.50 +/- 637.70 mm3) (P【0.05 vs control SGC-7901 group). CONCLUSION: This study provides direct evidence that VEGF plays an important role in the oncogenesis of human gastric cancer.
基金Project supported by National Natural Science Foundation of China,No.863 Z2001-04
文摘AIM: To test the hypothesis to block VEGF expression of SMMC-7721 hepatoma cells may inhibit tumor growth using the rat hepatoma model. METHODS: Amplify the 200 VEGF cDNA fragment and insert it into human U6 gene cassette in the reverse orientation transcribing small antisense RNA which could specifically interact with VEGF165, and VEGF121 mRNA. Construct the retroviral vector containing this antisense VEGF U6 cassette and package the replication-deficient recombinant retrovirus. SMMC-7721 cells were transduced with these virus and positive clones were selected with G418. PCR and Southern blot analysis were performed to determine if U6 cassette integrated into the genomic DNA of positive clone. Transfected tumor cells were evaluated for RNA expression by ribonuclease protection assays. The VEGF protein in the supernatant of parental tumor cells and genetically modified tumor cells was determined with ELISA. In vitro and in vivo growth properties of antisense VEGF cell clone in nude mice were analyzed. RESULTS: Restriction enzyme digestion and PCR sequencing verified that the antisense VEGF RNA retroviral vector was successfully constructed.After G418 selection, resistant SMMC-7721 cell clone was picked up. PCR and Southern blot analysis suggested that U6 cassette was integrated into the cell genomic DNA. Stable SMMC-7721 cell clone transduced with U6 antisense RNA cassette could express 200 bp small antisense VEGF RNA and secrete reduced levels of VEGF in culture condition. Production of VEGF by antisense transgene-expressing cells was 65+/-10 ng/L per 10(6) cells, 42045 ng/L per 10(6) cells in sense group and 485+/-30 ng/L per 10(6) cells in the negative control group, (P【 0.05). The antisense-VEGF cell clone appeared phenotypically indistinguishable from SMMC-7721 cells and SMMC-7721 cells transfected sense VEGF. The growth rate of the antisense-VEGF cell clone was the same as the control cells. When S.C. was implanted into nude mice, growth of antisense-VEGF cell lines was greatly inhibited compared with co
基金Partly supporled by the State Key Basic Research Program Grant of China(G1998051211)Leading Speciality Grant of Shanghai Health Bureau.
文摘Metastatic human HCC model is needed for the studies on mechanism and intervention of metastatic recurrence. By using orthotopic implantation of histologically intact tissues of 30 surgical specimens, a patient-like metastatic model of human HCC in nude mice (LCI-D20) and a low metastatic model of human HCC in nude mice (LCI-D35) have been established. All mice with transplanted LCI-D20 tumors exhibited extremely high metastatic ability including spontaneous metastasis to liver, lungs, lymph nodes and peritoneal seeding. Remarkable difference was also found in expression of some of the invasiveness related genes and growth factors between the LCI-D20 and LCI-D35 tumors. PAI-1 increased gradually following tumor progression in LCI-D20 model, and correlated with tumor size and AFP level. Phasic expression of tissue intercellular adhesion molecule-1 in this model was also observed. Using corneal micropocket model, it was demonstrated that the vascular response induced by LCI-D20 tumor was stronger than that induced by LCI-D35 tumor. Similar report on metastatic human HCC model in nude mice and human HCC cell line with metastatic potential was rarely found in the literature. This LCI-D20 model has been widely used for the studies on intervention of metastasis, including anti-angiogenesis,antisense approach, metalloproteinase inhibitor, differentiation inducer, etc. It is concluded that the establishment of metastatic human HCC model in nude mice and human HCC cell line with metastatic potential will provide important models for the in vitro and in vitro study of HCC invasiveness, angiogenesis as well as intervention of HCC recurrence.
文摘AIM: To investigate anti-tumor activities and apoptosis-regulated mechanisms of bufalin in the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice.METHODS: BEL-7402 cells of human hepatocellular carcinoma were inoculated to form subcutaneous tumors, and were implanted into the liver to establish orthotopic transplantation tumor models of human hepatocellular carcinoma in nude mice. Seventy-five animals were randomized divided into five groups (n = 15). Bufalin was injected intraperitoneally into three groups at doses of 1.5 mg/kg (BF1), 1 mg/kg (BF2) and 0.5 mg/kg (BF3) for d 15-24, respectively. The NS group was injected an equal volume of saline as above and adriamycin was injected intraperitoneally into the ADM group at a dose of 8.0 mg/kg for d 15. Ten mice in each group were killed at d 25 and the survival time in each group was calculated. We also observed the morphologic alterations in the myocardium, brain, liver, kidney and tumor tissues by pathology and electron microscopy, measured the apoptotic rate by TUNEL staining method, and detected the expression of apoptosis-regulated genes bcl-2 and bax by immunohistochemical staining and RT-PCR in tumor tissues. RESULTS: The tumor volumes in each group of bufalin were reduced significantly (35.21 ± 12.51 vs 170.39 ± 25.29; 49.83 ± 11.46 vs 170.39 ± 25.29; 83.99 ± 24.63 vs 170.39 ± 25.29, P < 0.01, respectively), and the survival times were prolonged in group BF1-2 (31.8 ± 4.2 vs 23.4 ± 2.1 and 29.4 ± 3.4 vs 23.4 ± 2.1, P < 0.05, respectively), and necrosis was mainly in severe or moderate degree in group BF1-2. No morphologicalchanges were detected in the myocardium, brain, liver and kidney tissues. Apoptotic characteristics could be seen in group BF1-2. The positive rates of bcl-2 and bax protein expression of each group by immunohistochemical staining were 10.0%, 10.0%, 20.0%, 10.0% and 20.0%; 90.0%, 80.0%, 80.0%, 40.0% and 30.0%, respectively. Loss of expression of bcl-2 mRNA in each group was to be found and the
文摘Background The earthworm fibrinolytic enzyme （EFE） is a complex protein enzyme that is widely distributed in the earthworm＇s digestive cavity. Possessing strong protein hydrolysis activity, EFE not only has a direct effect on fibrin, but also can activate plasminogen. Its therapeutic and preventative effects on thrombosis-related disease have been confirmed clinically. Recently, there has been increased interest in the anti-tumor activity of EFE. In this study, the anti-tumor activity of EFE, isolated from Eisenia foetida, on human hepatoma cells was evaluated in vitro and in vivo. The potential mechanisms involved were also studied. Methods In vitro experiments were performed in four human hepatoma cell lines： HLE, Huh7, PLC/PRF/5 and HepG2. After treatment with EFE in various concentrations, the inhibition of the rate of cell proliferation was measured. For the in vivo studies, tumor-bearing models xenografted with Huh7 cells were developed in nude mice, and then the mice were fed with EFE once a day for 4 weeks, and the control group received only saline. An inhibitory effect on tumor growth was observed. Also, apoptosis was observed with flow cytometric assay and fluorescent dye staining with acridine orange and ethidium bromide （AO/EB）. The expression of matrix metalloproteinase 2 （MMP-2） were detected by Western blotting assay. Results After treatment with various concentrations of EFE, the proliferation of all hepatoma cell lines was suppressed to varying degrees in vitro. The IC50 for HLE, Huh7, PLC/PCF/5 and HepG2 were 2.11, 5.87, 25.29 and 17.30 uku/ml, respectively. After administration of EFE orally for 4 weeks, the growth of tumor xenograft of Huh7 cells in nude mice was significantly inhibited in vivo. The tumor inhibitory rates in the EFE 500 uku/（kg·d） and 1000 uku/（kg·d） groups were 46.08% （compared with control group, P=0.026） and 57.52% （compared with control group, P=0.002） respectively. Meanwhile, the average weight of body, spleen or thymus did not show any
基金the National Natural Scientific Foundation of China,No.39670298
文摘AIM: The transcription factor EGR-1 (early growth response gene-1) plays an important role in cell growth, differentiation and development. It has identified that EGR-1 has significant transformation suppression activity in some neoplasms, such as fibrosarcoma, breast carcinoma. This experiment was designed to investigate the role of egr-1 in the cancerous process of hepatocellular carcinoma (HCC) and esophageal carcinoma (EC), and then to appraise the effects of EGR-1 on the growth of these tumor cells. METHODS: Firstly, the transcription and expression of egr-1 in HCC and EC, paracancerous tissues and their normal counterpart parts were detected by in situ hybridization and immunohistochemistry, with normal human breast and mouse brain tissues as positive controls. Egr-1 gene was then transfected into HCC (HHCC, SMMC7721) and EC (ECa109) cell lines in which no egr-1 transcription and expression were present. The cell growth speed, FCM cell cycle, plate clone formation and tumorigenicity in nude mice were observed and the controls were the cell lines transfected with vector only. RESULTS: Little or no egr-1 transcription and expression were detected in HCC, EC and normal liver tissues. The expression of egr-1 were found higher in hepatocellular paracancerous tissue (transcription level P=0.000; expression level P=0.143, probably because fewer in number of cases) and dysplastic tissue of esophageal cancer (transcription level P=0.000; expression level P=0.001). The growth rate of egr-1-transfected HHCC (HCC cell line) cells and ECa109 (EC cell line) cells was much slower than that of the controls. The proportion of S phase cell, clone formation and tumorigenicity were significantly lower than these of the controls' (decreased 45.5% in HHCC cells and 34.1% in ECa109 cells; 46.6% and 41.8%; 80.4% and 72.6% respectively). There were no obvious differences between SMMC7721 (HCC) egr-1-transfected cells and the controls with regard to the above items. CONCLUSION: The decreased expression of egr-1 might play a role in
基金Supported by the National Postdoctoral Science Foundation of China,No.199711.
文摘AIM: To evaluate antihepatoma effect of antisense phosphorothioate oligodeoxyribonucleotides (S-ODNs) targeted to alpha-fetoprotein (AFP) genes in vitro and in nude mice. METHODS: AFP gene expression was examined by immunocytochemical method or enzyme-linked immunosorbent assay. Effect of S-ODNs on SMMC-7721 human hepatoma cell growth in vitro was determined using microculture tetrazolium assay. In vitro antitumor activities of S-ODNs were monitored by measuring tumor weight differences in treated and control mice bearing SMMC-7721 xenografts. Induction of cell apoptosis was evaluated by fluorescence-activated cell sorter (FACS) analysis. RESULTS: Antisense S-ODN treatment led to reduced AFP gene expression. Specific antisense S-ODNs, but not control S-ODNs, inhibited the growth of hepatoma cells in vitro. In vitro, only antisense S-ODNs exhibited obvious antitumor activities. FACS analysis revealed that the growth inhibition by antisense S-ODNs was associated with their cell apoptosis induction. CONCLUSION: Antisense S-ODNs targeted to AFP genes inhibit the growth of human hepatoma cells and solid hepatoma, which is related to their cell apoptosis induction.
基金Supported by the German Foundation"Hensel-Stiftung"and Foundation of Health Ministry of China,No.D39901
文摘AIM: To establish a relevant animal model of human gastrointestinal cancer, which can be used for repetitive investigations, so as to improve our understanding and management of carcinogenesis and cancer metastasis. METHODS: Intact tissues of human colorectal and pancreatic cancers were transplanted in nude mice. The biological characteristics of the original and the corresponding transplanted tumors were investigated by HE staining, PAS staining and immunostaining. The metastases in the livers and lungs of nude mice were investigated by immunostaining with biotinylated mab KL-1 and by RT-PCR using CK20 specific primers. RESULTS: There were totally 9 of 16 surgical specimens growing in nude mice subcutaneously and/or orthotopically (4 of 6 colorectal and 5 of 10 pancreatic cancer). Tumor cell content of the specimens and freezing of tissue specimens are important factors influencing the growth of transplanted tumor. In the group of fresh tumor tissues with greater than 50% tumor cell content, the success rate of the transplantation was 100% (3 cases of pancreatic cancer and 3 cases of colorectal cancer). The orthotopically trans-planted tumors resemble the original tumor morphologically and biologically, including TAA expression such as CEA by immunohistochemistry, and CEA level in the serum of mice. Ki-67 labeling index and the expression of TAA especially K-ras, 17-1A and RA-96, are associated with the potential of tumor growth in nude mice. Micrometastases in the lungs and livers of tumor bearing mice can be detected by immunostaining with biotinylated mab KL-1 and CK20-specific RT-PCR. CONCLUSION: An orthotopic transplantation model for human colon and pancreatic cancer in nude mice has been set up. We have also established sensitive detection methods with CK-immunohistochemistry and CK20-RT-PCR to study xenotransplanted human cancer and its metastatic cancer cells in the liver and lung of nude mice. This study may be helpful in understanding the mechanism of cancer metastasis and in developing new diagnostic
文摘AIM To investigate the effects of taxol onSMMC-7721 human hepatoma and itsmechanisms.METHODS In vitro cell growth was assessedby trypan blue exclusion method.Experimentalhepatoma model was established by seedingSMMC-7721 cells subcutaneously into Balb/c(nu/nu)nude mice.In vivo tumor growth wasdetermined by measurement of tumor diameterwith Vernier calipers.The syntheses of DNA,RNA and protein were analyzed by incorporationof ~3H-thymidine,~3H-uridine and ~3H-leucinerespectively.Using light and electronmicroscopes to observe the morphologicalchanges of cells including mitosis andapoptosis.RESULTS Taxol was effective against SMMC-7721 human hepatoma cell growth in the rangesof 2.5 nmol/L-10 nmol/L with mitotic arrestand apoptosis in vitro.DNA,RNA and proteinsyntheses in cells were also obviouslysuppressed by in vitro treatment of taxol for72 h.Taxol at 2.5 nmol/L reduced ~3H-thymidineuptake to about 34% of the control value(P【0.05).Increasing the dose of taxol to20 nmol/L resulted in a greater decrease in ~3H-thymidine incorporation to 60% of the controlvalue(P【0.01).At a concentration of 20 nmol/L,the ~3H-uridine and ~3H-leucine uptakeswere reduced to 52%(P【0.05)and 63%(P【0.01),respectively.In vivo,taxolsignificantly inhibited SMMC-7721 tumor growthat 10 mg/kg,i.p.,once daily for 10 d.A morethan 90% decrease in tumor volume wasobserved by day 11(P【0.01)similarly withmitotic arrest and cell apoptosis.CONCLUSION Taxol has a marked anticanceractivity in SMMC-7721 human hepatoma both invitro and in nude mice.Its mechanisms might beassociated with mitotic arrest,subsequently,apoptosis of the hepatoma cells.No obvioustoxicity was observed with in vivoadministration of taxoi.
