Artemisinin, also termed qinghaosu, is extracted from the traditional Chinese medicine ar- temesia annua L. (the blue-green herb) in the early 1970s, which has been confirmed for effectively treating malaria, Additi...Artemisinin, also termed qinghaosu, is extracted from the traditional Chinese medicine ar- temesia annua L. (the blue-green herb) in the early 1970s, which has been confirmed for effectively treating malaria, Additionally, emerging data prove that artemisinin exhibits anti-cancer effects against many types of cancers such as leukemia, melanoma, etc. Artemisinin becomes cytotoxic in the presence of ferrous iron. Since iron influx is high in cancer cells, artemisinin and its analogs selectively kill can- cer cells with increased intracellular iron concentrations. This study is aimed to investigate the selective inhibitory effects of artemisinin on SMMC-7721 cells in vitro and determine the effect of holotransfer- fin, which increases the concentration of ferrous iron in cancer cells, combined with artemisinin on the anticancer activity. MTT assay was used for assessing the proliferation of SMMC-7721 cells treated with artemisinin. The induction of apoptosis and inhibition of colony formation in SMMC-7721 cells treated with artemisinin were determined by TdT-mediated dUTP nick end labeling (TUNEL) and col- ony formation assay, respectively. The results showed that artemisinin at various concentrations signifi- cantly inhibited growth, colony formation and cell viability of SMMC-7721 cells (P〈0.05), likely due to induction of apoptosis of SMMC-7721 cells. Of interest, it was found that incubation of artemisinin combined with holotransferrin sensitized the growth inhibitory effect of artemisinin on SMMC-7721 cells (P〈0.01). Our data suggest that treatment with artemisinin leads to inhibition of viability and pro- liferation, and apoptosis of SMMC-7721 ceils. Furthermore, we observed that holotransferrin signifi- cantly enhanced the anti-cancer activity of artemisinin. This study may provide a potential therapeutic choice for liver cancer.展开更多
Objective: To explore the effects of matrine on HCCR1 and HCCR2 expression in cultural human hepatocellular carcinomas (HCC) cells at the level of gene and protein. Methods: Three methods, representational differe...Objective: To explore the effects of matrine on HCCR1 and HCCR2 expression in cultural human hepatocellular carcinomas (HCC) cells at the level of gene and protein. Methods: Three methods, representational difference analysis (RDA) of cDNA, microarrays and fluorescence in situ hybridization (FISH) were used to detect levels of mRNA and protein expression of HCCR1 and HCCR-2 before and after treatment of matrine. Results: Matrine had inhibitory effect on the mRNA and protein expression of HCCR1 and HCCR2 in cultural HCC cells. Conclusion: Matrine has inhibitory effect on gene transcription, protein expression of HCCR 1 and HCCR2 in cultural HCC cells.展开更多
To investigate the effect of nano-apatite on the expression of the telomerase gene of human hepatocellular carcinoma cell lines and further explore the mechanism of the nano-apatite inhibiting cancer cells. Using the...To investigate the effect of nano-apatite on the expression of the telomerase gene of human hepatocellular carcinoma cell lines and further explore the mechanism of the nano-apatite inhibiting cancer cells. Using the hybridization in situ method to detect the expression of the telomerase gene of human hepatocellular carcinoma cells treated with the nano-apatite for 4 h at 37 ℃ . The hybridization in situ showed that the cytoplasm of the positive cells was stained in nigger- brown. The positive cell rate of the control group was 88.49% , the cisplatin group was 25.6% , the nano-apatite group was 63.6% . The activity of telomerase gene was both obviously dedined comparing with the control group and the difference had significance ( p 〈 0. 05, p 〈 0.01 ). The nanoapatite obviously inhabit the expression of the telomerase gene of human hepatocellular carcinoma cells.展开更多
AIM: To characterize the anticancer function of cytokine-induced killer cells (CIK) and develop an adoptiveimmunotherapy for the patients with primary hepatocellularcarcinoma (HCC), we evaluated the proliferation rate...AIM: To characterize the anticancer function of cytokine-induced killer cells (CIK) and develop an adoptiveimmunotherapy for the patients with primary hepatocellularcarcinoma (HCC), we evaluated the proliferation rate,phenotype and the antitumor activity of human CIK cellsfrom healthy donors and HCC patients in vitro and in vivo.METHODS: Peripheral blood mononuclear cells (PBMC) fronhealthy donors and patients with primary HCC were incubatedin vitro and induced into ClK cells in the presence of variouscytokines such as interferon-gamma (IFN-γ), interleukin-1(IL-1), IL-2, and monoclonal antibody (mAb) against CD3.The phenotype and characterization of CIK cells wereidentified by flow cytometric analysis. The cytotoxicity of CIKcells was determined by 51 Cr release assay.RESULTS: The CIK cells were shown to be a heterogeneouspopulation with different cellular phenotypes. Thepercentage of CD3+/CD56+ positive cells, the dominanteffector cells, in total CIK cells from healthy donors andHCC patients, significantly increased from 0.1-0.13 % at day0 to 19.0-20.5 % at day 21 incubation, which suggested thatthe CD3+ CD56+ positive cells proliferated faster than othercell populations of CIK cells in the protocol used in thisstudy. After 28 day in vitro incubation, the ClK cells frompatients with HCC and healthy donors increased by morethan 300-fold and 500-fold in proliferation cell number,respectively. CIK cells originated from HCC patientspossessed a higher in vitro antitumor cytotoxic activity onautologous HCC cells than the autologous lymphokine-activated killer (LAK) cells and PBMC cells. In in vivoanimal experiment, CIK cells had stronger effects on theinhibition 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 inhibitoryrate, 84.7% vs37.1%, P<0.01).CONCLUSION: Autologous CIK cells are of highly efficientcytotoxic effector cells against primary hepatocellularcarcinoma cells and might serve as an alternative adoptivetherapeutic strategy for HCC patients.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 cytotoxicit...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 α-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 adhe- sion 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
AIM: To investigate the density of mast cells (MCs) in human hepatocellular carcinoma (HCC), and to determine whether the MCs density has any correlations with histopathological grading, staging or some baseline patie...AIM: To investigate the density of mast cells (MCs) in human hepatocellular carcinoma (HCC), and to determine whether the MCs density has any correlations with histopathological grading, staging or some baseline patient characteristics.METHODS: Tissue sections of 22 primary HCCs were histochemically stained with toluidine blue, in order to be able to quantify the MCs in and around the neoplasm using a computer-assisted image analysis system. HCC was staged and graded by two independent pathologists. To identify the sinusoidal capillarisation of each specimen 3μm thick sections were histochemically stained with sirius red, and semi-quantitatively evaluated by two independent observers. The data were statistically analysed using Spearman′s correlation and Student′s t-test when appropriate.RESULTS: MCs density did not correlate with the age or sex of the patients, the serum alanine aminotransferase (ALT) or aspartate aminotransferase (AST) levels, or the stage or grade of the HCC. No significant differences were found between the MCs density of the patients with and without hepatitis C virus infection, but they were significantly higher in the specimens showing marked sinusoidal capillarisation.CONCLUSION: The lack of any significant correlation between MCs density and the stage or grade of the neoplastic lesions suggests that there is no causal relationship between MCs recruitment and HCC. However, as capillarisation proceeds concurrently with arterial blood supply during hepatocarcinogenesis, MCs may be considered of primary importance in the transition from sinusoidal to capillary-type endothelial cells and the HCC growth.展开更多
We report the cloning and functional characterization of human cyclin L2, a novel member of the cyclin family. Human cyclin L2 shares significant homology to cyclin L1, K, T1, T2, and C, which are involved in transcri...We report the cloning and functional characterization of human cyclin L2, a novel member of the cyclin family. Human cyclin L2 shares significant homology to cyclin L1, K, T1, T2, and C, which are involved in transcriptional regulation via phosphorylation of the C-terminal domain of RNA polymerase Ⅱ. The cyclin L2 protein contains an N-terminal "cyclin box" and C-terminal dipeptide repeats of alternating arginines and serines, a hallmark of the SR family of splicing factors. A new isoform and the mouse homologue of human cyclin L2 have also been cloned in this study. Human cyclin L2 is expressed ubiquitously in normal human tissues and tumor cells. We show here that cyclin L2 co-localizes with splicing factors SC-35 and 9G8 within nuclear speckles and that it associates with hyperphosphorylated, but not hypophosphorylated, RNA polymerase Ⅱ and CDK p110 PITSLRE kinase via its N-terminal cyclin domains. It can also associate with the SC-35 and 9G8 through its RS repeat region. Recombinant cyclin L2 protein can stimulate in vitro mRNA splicing. Overexpression of human cyclin L2 suppresses the growth of human hepatocellular carcinoma SMMC 7721 cells both in vitro and in vivo, inducing cellular apoptosis. This process involves up-regulation of p53 and Bax and decreased expression of Bcl-2. The data suggest that cyclin L2 represents a new member of the cyclin family, which might regulate the transcription and RNA processing of certain apoptosis-related factors, resulting in tumor cell growth inhibition and apoptosis.展开更多
AIM: To investigate the effects of suberoylanilide hydroxamic acid(SAHA) on proliferation and apoptosis of a human hepatocellular carcinoma cell line(HepG2.2.15) and hepatitis B virus(HBV) replication.METHODS: HepG2.2...AIM: To investigate the effects of suberoylanilide hydroxamic acid(SAHA) on proliferation and apoptosis of a human hepatocellular carcinoma cell line(HepG2.2.15) and hepatitis B virus(HBV) replication.METHODS: HepG2.2.15 cells were treated with different concentrations of SAHA.Cell morphology was examined by confocal laser scanning microscopy,and cell proliferation was determined using a MTT colorimetric assay.Flow cytometry was used to detect apoptosis and determine cell cycle phase,while hepatitis B surface antigen and hepatitis B e antigen content were measured using chemiluminescence.Reverse transcription polymerase chain reaction was performed to measure HBV DNA in cell lysate.RESULTS: Cell proliferation rates were significantly reduced by the addition of SAHA.The inhibitory effect of SAHA on cell proliferation was both time-and dosedependent.After 24 h of treatment with SAHA,the early cell apoptotic rate increased from 3.25% to 21.02%(P = 0.041).The proportion of G0 /G1 phase cells increased from 50.3% to 65.3%(P = 0.039),while that of S phase cells decreased from 34.9% to 20.6%(P = 0.049).After 48 h of treatment,hepatitis B surface antigen and hepatitis B e antigen content increased from 12.33 ± 0.62 to 25.42 ± 2.67(P = 0.020) and 28.92 ± 1.24 to 50.48 ± 1.85(P = 0.026),respectively.Furthermore,HBV DNA content increased from 4.54 ± 0.46 to 8.34 ± 0.59(P = 0.029).CONCLUSION: SAHA inhibits HepG2.2.15 cell proliferation,promotes apoptosis,and stimulates HBV replication.In combination with anti-HBV drugs,SAHA may potentially be used cautiously for treatment of hepatocellular carcinoma.展开更多
AIM: To study the in vitro and in vivo inhibitory effects of genistein on invasive potential of Bel 7402 hepatocellular carcinoma (HCC) cells and to explore the underlying mechanism.METHODS: Bel 7402 HCC cells were ex...AIM: To study the in vitro and in vivo inhibitory effects of genistein on invasive potential of Bel 7402 hepatocellular carcinoma (HCC) cells and to explore the underlying mechanism.METHODS: Bel 7402 HCC cells were exposed to genistein. The invasive activity of tumor cells was assayed in transwell cell culture chamber. p125FAK expression and cell cycle were evaluated by a functional assay. Cell apoptosis analysis was performed with TUNEL method. In addition, bilateral subrenal capsule xenograft transplantation of HCC was performed in 10 nude mice.Genistein was injected and the invasion of HCC into the renal parenchyma was observed. Microvessels with immunohistochemical staining were detected.RESULTS: Genistein significantly inhibited the growth of Bel 7402 cells, the inhibitory rate of tumor cells was 26 -42%. The invasive potential of Bel 7402 cells in vitro was significantly inhibited, the inhibitory rate was 11-28%. Genistein caused G2/M cell cycle arrest, S phase decreased significantly. The occurrence of apoptosis in genistein group increased significantly. The expression of p125FAK in 5 μg/mL genistein group (15.26±0.16%)and 10 μg/mL genistein group (12.89±0.36%) was significantly lower than that in the control group (19.75± 1.12%,P<0.05). Tumor growth in genistein-treated nude mice was significantly retarded in comparison to control mice, the inhibitory rate of tumor growth was about 20%. Genistein also significantly inhibited the invasion of Bel 7402 cells into the renal parenchyma of nude mice with xenograft transplant. The positive unit value of microvessels in genistein-treated group (10.422±0.807)was significantly lower than that in control group (22.330 ± 5.696, P< 0.01).CONCLUSION: Genistein can effectively inhibit the invasive potential of Bel 7402 HCC cells by altering cell cycle, apoptosis and angiogenesis, inhibition of focal adhesion kinase may play a significant role in this process.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 Iow 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-Iincreased gradually following tumor progression in LCID20 model, and correlated with tumor size and AFP level,Phasic expression of tissue intercellular adhesion molecule-I 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 antiangiogenesis, 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 vivo and in vitro study of HCC invasiveness, angiogenesis as well as intervention of HCC recurrence.展开更多
AIM: To study the viscoelastic properties of humanhepatocytss and helatocellulsr carcinoma (HCC) cellsunder cytoslelstal perturbation, and to further to study theviscoelastic properties and the adhesive properties of ...AIM: To study the viscoelastic properties of humanhepatocytss and helatocellulsr carcinoma (HCC) cellsunder cytoslelstal perturbation, and to further to study theviscoelastic properties and the adhesive properties of mousehepatorna cells (HTC) in different cell cycls.METHODS: Micropipette aspiration technique was adopted tomeasure viscoelastic coefficients and adhesion force tocollagen coated surface ofthe cells. Three kinds ofcytoskeleton perturbing agents, colchiclnes (Col),cytochalssin D (CD) and vinblastine (VBL), were used totreat HCC cells and hepatocytes and the effects of thesetreatent on cell viscoelastic coefficients were investigated.The experimental results were analyzed with a thres-elsmentstandard linear solid. Further, the viscoelastic properties ofHTC cells and the adhesion force of different cycle HTC cellswere also investigated. The synchronous G1 and S phasecells were achieved through thymine-2-desoryriboside andcolchicines sequential blockage method and thymine-2-desoryriboside blockage method respectively.RESULTS: The elastic coefficients, but not viscouscoefficient of HCC cells (k1 = 103.6± 12.6N.m-2, k2 =42.5±10.4N. m-2, μ = 4.5 ± 1.9Pa. s), were significantly higherthan the corresponding value for hepatocytes (K1 = 87.5 ±12.1N.m-2, k2 =33.3± 10.3N.m-2, μ=5.9±3.0Pa. s, P<0.01). Upon treatment with CD, the viscoelastic coefficients ofboth hepotocytes and HCC cells decreased consistently,with magnitudes for the decrease in elastic coefficients ofHCC cells (k1: 68.7 N.m-2 to 81.7N.m-2, 66.3 % to 78.9 %;k2: 34.5 N.m-2 to37.1N.m-2, 81.2% to 87.3 %, P<0.001)larger than those for normal hepatocytes (k1: 42.6N. m-2 to49.8N.nt-2, 48.7% to56.9 %; k2: 17.2N.m-2 to 20.4N.m-2,51.7 % to 61.3 %, P< 0.001). There was a little decrease inthe vlscous coefficient of HCC cells (2.0 to 3.4Pa. s, 44.4 to75.6 %, P<0.001) than that for hepatocytes (3.0 to 3.gPa.s, 50.8to 66.1% P<0.001). Upon trastment with Col andVBL, the elastic coefficients of hepatocytes generallyincreased or tended to increase while those of HCC cellsdecreased. HTC cells with 72.1% ofG1 phase and 98. 9 % ofS phase were achieved and high K1, k2 value and low μvalue were the general characlteristics of HTC cells. G1phase cells had higher K1 value and lower tμ value than Sphase cells had, and G1 phase HTC cells had strongeradhesive forces [(275.9±232.8) x 10-10N] than S phase cells[(161.2± 120.4) x l0-10N, P<0.001).CONCLUSION: The difference in both the pattern and themagnitude of the effect of cytoskeletal perturbing agent onthe viscoelastic properties between HCC cells andhepatocytes may reflect differencss in the state of thecytusieleton structure and function and in the sensitivity toperturbing agent treatment between trinse two types of cells.Change in the viscoelastic properties of cancer cells mayaffect significantly tumor cell invasion and metastasis as wellas interactions between tumor cells and their micro-mechanical environments.展开更多
To investigate the modulating effects of survivn antisense oligonucletode (ASODN) on the cell cycle and apoptosis of human hepatocellular carcinoma (HCC) cell line SMMC-7721 and explore its mechanism.Methods Survivin ...To investigate the modulating effects of survivn antisense oligonucletode (ASODN) on the cell cycle and apoptosis of human hepatocellular carcinoma (HCC) cell line SMMC-7721 and explore its mechanism.Methods Survivin ASODN was transfected into SMMC-7721 cells mediated by DOTAP liposomal reagent.Electron microscopy,flow cytometry and RT-PCR were used to detect the changes in cell ultrastructure,apoptosis,cell cycle and the expression of cyclinB1 mRNA,respectively.Results After transfection of survivin ASODN,the expression of cyclinB1 mRNA in the cells significantly increased and increase in G2-M arrest and apoptosis appeared.Meanwhile,the cell ultrastructure had apoptotic changes such as chromatin condensation and apoptotic body formation.Conclusion Survivin ASODN can induce the expression of cyclinB1 that may result in G2-M arrest.Consequently,apoptosis is triggered.Survivin ASODN transfection might be an improtant new treatment for HCC.14 refs,2 figs,1 tab.展开更多
AIM: To evaluate the effects of sulindac in inducing growth inhibition and apoptosis of human gastric cancer cells in comparison with human hepatocellular carcinoma (HCC)cells.METHODS: The human gastric cancer cell li...AIM: To evaluate the effects of sulindac in inducing growth inhibition and apoptosis of human gastric cancer cells in comparison with human hepatocellular carcinoma (HCC)cells.METHODS: The human gastric cancer cell lines MKN45 and MKN28 and human hepatocellular carcinoma cell lines HepG2and SMMC7721 were used for the study. Anti-proliferative effect was measured by MTT assay, and apoptosis was determined by Hoechst-33258 staining, electronography and DNA fragmentation. The protein of cyclooxygenase-2 (COX(2) and Bcl-2 were detected by Westem dot blotting.RESULTS: Sulindac could initiate growth inhibition and apoptosis of MKN45, MKN28, HepG2 and SMMC7721 cells in a dose-and time-dependent manner. Growth inhibitory activity and apoptosis were more sensitive in HepG2 cells than in SMMC7721 cells, MKN45 and MKN28 cells. After 24hours incubation with sulindac at 2mmol. L-1 and 4mmol.L-1, the level of COX-2 and Bcl-2 protein were lowered in MKN45, SMMC7721 and HepG2 cells but not in MKN28 cells.CONCLUSION: Sulindac could inhibit the growth of gastric cancer cells and HCC cells effectively in vitro by apoptosis induction, which was associated with regression of COX-2and Bcl-2 expression. The growth inhibition and apoptosis of HCC cells were greater then that of human gastric cancer cells. The different effects of apoptosis in gastric cancer cells may be related to the differentiation of the cells.展开更多
The prognosis of hepatocellular carcinoma (HCC) stillremains dismal, although many advances in its clinicalstudy have been made. It is important for tumor control toidentity the factors that predispose patients to dea...The prognosis of hepatocellular carcinoma (HCC) stillremains dismal, although many advances in its clinicalstudy have been made. It is important for tumor control toidentity the factors that predispose patients to death. Withnew discoveries in cancer biology, the pathological andbiological prognostic factors of HCC have been studied quiteextensively. Analyzing molecular markers (biomarkers) withprognostic significance is a complementary method. A largenumber of molecular factors have been shown to associatewith the invasiveness of HCC, and have potential prognosticsignificance. One important aspect is the analysis ofmolecular markers for the cellular malignancy phenotypeThese include alterations in DNA ploidy, cellularproliferation markers (PCNA, Ki-67, Mcm2, MIB1, MIA, andCSE1L/CAS protein), nuclear morphology, the p53 geneand its related molecule MDM2, other cell cycle regulators(cyclin A, cyclin D, cyclin E, cdc2, p27, p73), oncogenesand their receptors (such as ras, c-myc, c-fms, HGF, c-met, and erb-B receptor family members ), apoptosisrelated factors (Fas and FasL), as well as telomeraseactivity. Another important aspect is the analysis ofmolecular markers involved in the process of cancerinvasion and metastasis. Adhesion molecules (E-cadherin,catenins, serum intercellular adhesion molecule-1, CD44variants), proteinases involved in the clegradation ofextracellular matrix (MMP-2, MMP-9, uPA, uPAR, PAl), aswell as other molecules have been regarded as biomarkersfor the malignant phenotype of HCC, and are related toprognosis and therapeutic outcomes. Tumor angiogenesisis critical to both the growth and metastasis of cancersincluding HCC, and has drawn much attention in recentyears. Many angiogenesis-related markers, such as vascularendothelial growth factor (VEGF), basic fibroblast growthfactor (bFGF), platelet-derived endothelial cell growth factor( PD-ECGF ), thrombospondin ( TSP ), angiogenin,pleiotrophin, and endostatin (ES) levels, as well asinratumor microvessel density (MVD) have been evaluatedand found to be of prognostic significance. Body fluid(particularly blood and urinary) testing for biomarkers iseasily accessible and useful in clinical patients. Theprognostic significance of circulating DNA in plasma orserum, and its genetic alterations in HCC are otherimportant trends. More attention should be paid to thesetwo areas in future. As the progress of the human genomeproject advances, so does a clearer understanding of tumorbiology, and more and more new prognostic markers withhigh sensitivity and specificity will be found and used inclinical assays. However, the combination of some items, i.e., the pathological features and some biomarkersmentioned above, seems to be more practical for now.展开更多
AIM: To evaluate the receptor protein which can specifically bind to β2GPⅠon the membrane of hepatocellular carcinoma (HCC) cell line SMMC-7721, and to study the biological function of the receptor.METHODS: Through ...AIM: To evaluate the receptor protein which can specifically bind to β2GPⅠon the membrane of hepatocellular carcinoma (HCC) cell line SMMC-7721, and to study the biological function of the receptor.METHODS: Through β2GPⅠ-affinity chromatography column, the peptid-polysome-mRNA complex, which can specially bind to β2GPⅠ, stayed with the column and was separated from the whole polysome of liver cells, and then eluted and collected. Using cDNA synthesis kit and cDNA PCR kit, the corresponding cDNA was obtained and sequenced. RT-PCR was used to amplify annexinⅡ, and flow cytometry was used to study the competitive binding of annexinⅡ with β2GPⅠto SMMC-7721.RESULTS: A total of 1.1 kb of the cDNA fragment of the specific binding protein of β2GPⅠon liver cell membrane was obtained. The sequence of cDNA shared high homology with human annexinⅡ (98%). AnnexinⅡ was expressed on the membrane of SMMC-7721, and could compete with β2GPⅠfor combining with SMMC-7721.CONCLUSION: The receptor for β2GPⅠon membrane of SMMC-7721 cells is annexinⅡ, which might bridge HBV to infect hepatocytes.展开更多
AIM: Cyclooxygenase-2 (COX-2) has been suggested to beassociated with carcinogenesis. We sought to investigatethe effect of the selective COX-2 inhibitor, Nimesulide onproliferation and apoptosis of SMMC-7721 human he...AIM: Cyclooxygenase-2 (COX-2) has been suggested to beassociated with carcinogenesis. We sought to investigatethe effect of the selective COX-2 inhibitor, Nimesulide onproliferation and apoptosis of SMMC-7721 human hepatomacells.METHODS: This study was carried out on the culture ofhepatic carcinoma SMMC-7721 cell line. Variousconcentrations of Nimesulide (0、200 μmol/L、300 μmol/L、400μmol/L) were added and incubated. Cell proliferation wasdetected with MTT colorimetric assay, cell apoptosis byelectron microscopy, flow cytometry and TUNEL.RESULTS: Nimesulide could significantly inhibit SMMC-7721cells proliferation dose-dependent and in a dependentmanner compared with that of the control group. Theduration lowest inhibition rate produced by Nimesulide inSMMC-7721 cells was 19.06 %, the highest inhibition ratewas 58.49 %. After incubation with Nimesulide for 72 h, themost highest apoptosis rate and apoptosis index of SMMC-7721 cells comparing with those of the control were 21. 20 %+1.62% vs2.24% +0.26% and21.23+ 1.78 vs2.01+0.23(p<0.05).CONCLUSION: The selective COX-2 inhibitor, Nimesulide caninhibit the proliferation of SMMC-7721 cells and increaseapoptssis rate and apoptosis index of SMMC-7721 cells. Theapoptoois rate and the apoptosis index are dose-dependent.Under electron microscope SMMC-7721 cells incubated with 300μmol and 400 μmol Nimesulide show apoptotic characteristics.With the clarification of the mechanism of selective COX-2inhibitors, These COX-2 selective inhibitors can become thechoice of prevention and treatment of cancers.展开更多
AIM To demonstrate the relationship betweenH-ras oncogene and hepatocellular carcinoma(HCC) metastasis.METHODS Activated H-ras oncogene wastransfected into SMMC 7721, a cell line derivedfrom human HCC, by calcium phos...AIM To demonstrate the relationship betweenH-ras oncogene and hepatocellular carcinoma(HCC) metastasis.METHODS Activated H-ras oncogene wastransfected into SMMC 7721, a cell line derivedfrom human HCC, by calcium phosphatetransfection method. Some metastasis-relatedparameters were detected in vitro, includingadhesion assay, migration assay, expression ofcollagenase ⅣV (c ⅣV ase) and epidermal growthfactor receptor (EGFR).RESULTS The abilities of H-ras-transfected cellclones in adhesion to laminin (LN) or fibronectin(FN), migration, c Ⅳ ase secretion increasedmarkedly, and the expression of EGFR elevatedmoderately. More importantly, these alterationswere consistent positively with the expressionof p21, the protein product of H-ras oncogene.CONCLUSION H-ras oncogene could inducethe metastatic phenotype of HCC cell in vitro toraise its metastatic potential.展开更多
AIM: The goal of this study was to characterize the AlPreceptor, its possible signal transduction pathway and itsproliferative functions in human hepatoma cell line Bel 7402.METHODS: Cell proliferation enhanced by AFl...AIM: The goal of this study was to characterize the AlPreceptor, its possible signal transduction pathway and itsproliferative functions in human hepatoma cell line Bel 7402.METHODS: Cell proliferation enhanced by AFlP was detectedby MTT assay, 3H-thymidine incorporation and S-stsgepercentage of cell cycle analysis. With radioactive labeled 125 I-AFP for receptor binding assay; cAMP acctmuation, ProteinKinase A activity were detected by radioactive immunosorbentassay and the change of intracellular free calcium ([Ca2+ ], )was monitored by scanning fluorescence intensity under TCS-NT confocal microscope. The expression of oncxgenes N- ras,p53, and p21ras in the cultured cells in vitro were detected byNorthem blotting and Western blotting respectively.RESULTS: It was demonstrated that AFP enhanced theproliferation of human hepatoma Bel 7402 cell in a dosedependlent fashion asshown in MTT assay, 3H-thymidineincorporation and S-phase percentage up to 2-fold. Twosubtypes of AFP receptors were identified in the cells withKds of 1.3 x 10-9 mol. L-1 and 9.9 x 10-8 mol. L-1 respectively.Pretreatnent of cells with AFP resulted-in a significantincrease (625 %) in cAMP accumulation. The activity ofprotein kinase A activity were increased up to 37.5, 122.6,73.7 and 61.2 % at treatment time point 2, 6, 12 and 24hours. The level of intracellular calcium were elevated afterthe treatment of alpha-fetoprotsin and achieved to 204 % at 4min. The results also showed that AFP (20 mg. L-1 ) couldupregulate the expression of N-ras oncogenes and p53 andp21ras in Bel 7402 cells. In the later case, the alteration ware 81.1%(12 h) and 97.3 %(12 h) respectively compared with control.CONCLUSION: These results demonstrate that AFP is apotential growth factor to promote the proliferation of humanhepatoma Bel 7402 cells. Its growth-regulatory effects aremediated by its specific plasma membrane receptorscoupled with its transmembrane signaling transductionthrough the pathway of cAMP-PKA and intracellular calciumto regulate the expression of oncogenes.展开更多
AIM: To explore the role of SF/HGF-Met autocrine and parscrine in metastasis of hepatocellular carcinoma (HCC).METHODS: SF/HGF and c-met transcription and protein expression in HCC were examined by RT-PCR and Western ...AIM: To explore the role of SF/HGF-Met autocrine and parscrine in metastasis of hepatocellular carcinoma (HCC).METHODS: SF/HGF and c-met transcription and protein expression in HCC were examined by RT-PCR and Western Blot in 4 HCC cell lines, including HepG2, Hep3B,SMMC7721 and MHCC-1, the last cell line had a higher potential of metastasis. Sf/hgf cDNA was transfected by the method of Lipofectin into SMMC7721. SF/HGF and c-met antibody were used to stimulate and block SF/HGF-c-met signal transduction. Cell morphology, mobility, and proliferation were respectively compared by microscopic observation, wound healing assay and cell growth curve.RESULTS: HCC malignancy appeared to be relative to its met-SF/HGF expression. In MHCC-1, c-met expression was much stronger than that in other cell lines with lower potential of metastasis and only SF/HGF autocrine existed in MHCC-1. After sf/hgf cDNA transfection or conditioned medium of MHCC-1 stimulation, SMMC7721 changed into elongated morphology, and the abilities of proliferation ( P < 0.05) and mobility increased. Such bio-activity could he blocked by c-met antibody ( P< 0.05).CONCLUSION: The system of SF/HGF-c-met autocrine and paracrine played an important role in development and metastasis potential of HCC. Inhibition of SF/HGF-c-met signal transduction system may reduce the growth and metastasis of HCC.展开更多
INTRODUCTIONHepatocellular carcinoma (HCC) is one of the mostcommon human malignancies worldwide[1,2], and isclosely associated with infection of HBV and HCVand contamination of aflatoxin B1[3-6]. Althoughthe molecula...INTRODUCTIONHepatocellular carcinoma (HCC) is one of the mostcommon human malignancies worldwide[1,2], and isclosely associated with infection of HBV and HCVand contamination of aflatoxin B1[3-6]. Althoughthe molecular mechanisms of hepatocarcinogenesisremain poorly understood, an increasing number ofgenetic abnormalities have been recognized[7-10],for example, the p16 gene[11,12] the p53gene[13-18], the E-cadherin gene[19], and the c-mycgene[20].展开更多
文摘Artemisinin, also termed qinghaosu, is extracted from the traditional Chinese medicine ar- temesia annua L. (the blue-green herb) in the early 1970s, which has been confirmed for effectively treating malaria, Additionally, emerging data prove that artemisinin exhibits anti-cancer effects against many types of cancers such as leukemia, melanoma, etc. Artemisinin becomes cytotoxic in the presence of ferrous iron. Since iron influx is high in cancer cells, artemisinin and its analogs selectively kill can- cer cells with increased intracellular iron concentrations. This study is aimed to investigate the selective inhibitory effects of artemisinin on SMMC-7721 cells in vitro and determine the effect of holotransfer- fin, which increases the concentration of ferrous iron in cancer cells, combined with artemisinin on the anticancer activity. MTT assay was used for assessing the proliferation of SMMC-7721 cells treated with artemisinin. The induction of apoptosis and inhibition of colony formation in SMMC-7721 cells treated with artemisinin were determined by TdT-mediated dUTP nick end labeling (TUNEL) and col- ony formation assay, respectively. The results showed that artemisinin at various concentrations signifi- cantly inhibited growth, colony formation and cell viability of SMMC-7721 cells (P〈0.05), likely due to induction of apoptosis of SMMC-7721 cells. Of interest, it was found that incubation of artemisinin combined with holotransferrin sensitized the growth inhibitory effect of artemisinin on SMMC-7721 cells (P〈0.01). Our data suggest that treatment with artemisinin leads to inhibition of viability and pro- liferation, and apoptosis of SMMC-7721 ceils. Furthermore, we observed that holotransferrin signifi- cantly enhanced the anti-cancer activity of artemisinin. This study may provide a potential therapeutic choice for liver cancer.
文摘Objective: To explore the effects of matrine on HCCR1 and HCCR2 expression in cultural human hepatocellular carcinomas (HCC) cells at the level of gene and protein. Methods: Three methods, representational difference analysis (RDA) of cDNA, microarrays and fluorescence in situ hybridization (FISH) were used to detect levels of mRNA and protein expression of HCCR1 and HCCR-2 before and after treatment of matrine. Results: Matrine had inhibitory effect on the mRNA and protein expression of HCCR1 and HCCR2 in cultural HCC cells. Conclusion: Matrine has inhibitory effect on gene transcription, protein expression of HCCR 1 and HCCR2 in cultural HCC cells.
文摘To investigate the effect of nano-apatite on the expression of the telomerase gene of human hepatocellular carcinoma cell lines and further explore the mechanism of the nano-apatite inhibiting cancer cells. Using the hybridization in situ method to detect the expression of the telomerase gene of human hepatocellular carcinoma cells treated with the nano-apatite for 4 h at 37 ℃ . The hybridization in situ showed that the cytoplasm of the positive cells was stained in nigger- brown. The positive cell rate of the control group was 88.49% , the cisplatin group was 25.6% , the nano-apatite group was 63.6% . The activity of telomerase gene was both obviously dedined comparing with the control group and the difference had significance ( p 〈 0. 05, p 〈 0.01 ). The nanoapatite obviously inhabit the expression of the telomerase gene of human hepatocellular carcinoma cells.
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 adoptiveimmunotherapy for the patients with primary hepatocellularcarcinoma (HCC), we evaluated the proliferation rate,phenotype and the antitumor activity of human CIK cellsfrom healthy donors and HCC patients in vitro and in vivo.METHODS: Peripheral blood mononuclear cells (PBMC) fronhealthy donors and patients with primary HCC were incubatedin vitro and induced into ClK cells in the presence of variouscytokines such as interferon-gamma (IFN-γ), interleukin-1(IL-1), IL-2, and monoclonal antibody (mAb) against CD3.The phenotype and characterization of CIK cells wereidentified by flow cytometric analysis. The cytotoxicity of CIKcells was determined by 51 Cr release assay.RESULTS: The CIK cells were shown to be a heterogeneouspopulation with different cellular phenotypes. Thepercentage of CD3+/CD56+ positive cells, the dominanteffector cells, in total CIK cells from healthy donors andHCC patients, significantly increased from 0.1-0.13 % at day0 to 19.0-20.5 % at day 21 incubation, which suggested thatthe CD3+ CD56+ positive cells proliferated faster than othercell populations of CIK cells in the protocol used in thisstudy. After 28 day in vitro incubation, the ClK cells frompatients with HCC and healthy donors increased by morethan 300-fold and 500-fold in proliferation cell number,respectively. CIK cells originated from HCC patientspossessed a higher in vitro antitumor cytotoxic activity onautologous HCC cells than the autologous lymphokine-activated killer (LAK) cells and PBMC cells. In in vivoanimal experiment, CIK cells had stronger effects on theinhibition 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 inhibitoryrate, 84.7% vs37.1%, P<0.01).CONCLUSION: Autologous CIK cells are of highly efficientcytotoxic effector cells against primary hepatocellularcarcinoma cells and might serve as an alternative adoptivetherapeutic strategy for HCC patients.
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 α-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 adhe- sion 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the grants from the Foundation"Michele Rodriguez".Istituto Scientifico per le Misure Quantitative in Medicina,Milan,Italy
文摘AIM: To investigate the density of mast cells (MCs) in human hepatocellular carcinoma (HCC), and to determine whether the MCs density has any correlations with histopathological grading, staging or some baseline patient characteristics.METHODS: Tissue sections of 22 primary HCCs were histochemically stained with toluidine blue, in order to be able to quantify the MCs in and around the neoplasm using a computer-assisted image analysis system. HCC was staged and graded by two independent pathologists. To identify the sinusoidal capillarisation of each specimen 3μm thick sections were histochemically stained with sirius red, and semi-quantitatively evaluated by two independent observers. The data were statistically analysed using Spearman′s correlation and Student′s t-test when appropriate.RESULTS: MCs density did not correlate with the age or sex of the patients, the serum alanine aminotransferase (ALT) or aspartate aminotransferase (AST) levels, or the stage or grade of the HCC. No significant differences were found between the MCs density of the patients with and without hepatitis C virus infection, but they were significantly higher in the specimens showing marked sinusoidal capillarisation.CONCLUSION: The lack of any significant correlation between MCs density and the stage or grade of the neoplastic lesions suggests that there is no causal relationship between MCs recruitment and HCC. However, as capillarisation proceeds concurrently with arterial blood supply during hepatocarcinogenesis, MCs may be considered of primary importance in the transition from sinusoidal to capillary-type endothelial cells and the HCC growth.
文摘We report the cloning and functional characterization of human cyclin L2, a novel member of the cyclin family. Human cyclin L2 shares significant homology to cyclin L1, K, T1, T2, and C, which are involved in transcriptional regulation via phosphorylation of the C-terminal domain of RNA polymerase Ⅱ. The cyclin L2 protein contains an N-terminal "cyclin box" and C-terminal dipeptide repeats of alternating arginines and serines, a hallmark of the SR family of splicing factors. A new isoform and the mouse homologue of human cyclin L2 have also been cloned in this study. Human cyclin L2 is expressed ubiquitously in normal human tissues and tumor cells. We show here that cyclin L2 co-localizes with splicing factors SC-35 and 9G8 within nuclear speckles and that it associates with hyperphosphorylated, but not hypophosphorylated, RNA polymerase Ⅱ and CDK p110 PITSLRE kinase via its N-terminal cyclin domains. It can also associate with the SC-35 and 9G8 through its RS repeat region. Recombinant cyclin L2 protein can stimulate in vitro mRNA splicing. Overexpression of human cyclin L2 suppresses the growth of human hepatocellular carcinoma SMMC 7721 cells both in vitro and in vivo, inducing cellular apoptosis. This process involves up-regulation of p53 and Bax and decreased expression of Bcl-2. The data suggest that cyclin L2 represents a new member of the cyclin family, which might regulate the transcription and RNA processing of certain apoptosis-related factors, resulting in tumor cell growth inhibition and apoptosis.
文摘AIM: To investigate the effects of suberoylanilide hydroxamic acid(SAHA) on proliferation and apoptosis of a human hepatocellular carcinoma cell line(HepG2.2.15) and hepatitis B virus(HBV) replication.METHODS: HepG2.2.15 cells were treated with different concentrations of SAHA.Cell morphology was examined by confocal laser scanning microscopy,and cell proliferation was determined using a MTT colorimetric assay.Flow cytometry was used to detect apoptosis and determine cell cycle phase,while hepatitis B surface antigen and hepatitis B e antigen content were measured using chemiluminescence.Reverse transcription polymerase chain reaction was performed to measure HBV DNA in cell lysate.RESULTS: Cell proliferation rates were significantly reduced by the addition of SAHA.The inhibitory effect of SAHA on cell proliferation was both time-and dosedependent.After 24 h of treatment with SAHA,the early cell apoptotic rate increased from 3.25% to 21.02%(P = 0.041).The proportion of G0 /G1 phase cells increased from 50.3% to 65.3%(P = 0.039),while that of S phase cells decreased from 34.9% to 20.6%(P = 0.049).After 48 h of treatment,hepatitis B surface antigen and hepatitis B e antigen content increased from 12.33 ± 0.62 to 25.42 ± 2.67(P = 0.020) and 28.92 ± 1.24 to 50.48 ± 1.85(P = 0.026),respectively.Furthermore,HBV DNA content increased from 4.54 ± 0.46 to 8.34 ± 0.59(P = 0.029).CONCLUSION: SAHA inhibits HepG2.2.15 cell proliferation,promotes apoptosis,and stimulates HBV replication.In combination with anti-HBV drugs,SAHA may potentially be used cautiously for treatment of hepatocellular carcinoma.
基金Supported by the Basic Research Key Project of the Science Foundation of Shanghai Municipal Commission of Science and Technology, No. 02JC14001
文摘AIM: To study the in vitro and in vivo inhibitory effects of genistein on invasive potential of Bel 7402 hepatocellular carcinoma (HCC) cells and to explore the underlying mechanism.METHODS: Bel 7402 HCC cells were exposed to genistein. The invasive activity of tumor cells was assayed in transwell cell culture chamber. p125FAK expression and cell cycle were evaluated by a functional assay. Cell apoptosis analysis was performed with TUNEL method. In addition, bilateral subrenal capsule xenograft transplantation of HCC was performed in 10 nude mice.Genistein was injected and the invasion of HCC into the renal parenchyma was observed. Microvessels with immunohistochemical staining were detected.RESULTS: Genistein significantly inhibited the growth of Bel 7402 cells, the inhibitory rate of tumor cells was 26 -42%. The invasive potential of Bel 7402 cells in vitro was significantly inhibited, the inhibitory rate was 11-28%. Genistein caused G2/M cell cycle arrest, S phase decreased significantly. The occurrence of apoptosis in genistein group increased significantly. The expression of p125FAK in 5 μg/mL genistein group (15.26±0.16%)and 10 μg/mL genistein group (12.89±0.36%) was significantly lower than that in the control group (19.75± 1.12%,P<0.05). Tumor growth in genistein-treated nude mice was significantly retarded in comparison to control mice, the inhibitory rate of tumor growth was about 20%. Genistein also significantly inhibited the invasion of Bel 7402 cells into the renal parenchyma of nude mice with xenograft transplant. The positive unit value of microvessels in genistein-treated group (10.422±0.807)was significantly lower than that in control group (22.330 ± 5.696, P< 0.01).CONCLUSION: Genistein can effectively inhibit the invasive potential of Bel 7402 HCC cells by altering cell cycle, apoptosis and angiogenesis, inhibition of focal adhesion kinase may play a significant role in this process.
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 Iow 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-Iincreased gradually following tumor progression in LCID20 model, and correlated with tumor size and AFP level,Phasic expression of tissue intercellular adhesion molecule-I 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 antiangiogenesis, 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 vivo and in vitro study of HCC invasiveness, angiogenesis as well as intervention of HCC recurrence.
基金the National Science Foundation of China,No.39370198
文摘AIM: To study the viscoelastic properties of humanhepatocytss and helatocellulsr carcinoma (HCC) cellsunder cytoslelstal perturbation, and to further to study theviscoelastic properties and the adhesive properties of mousehepatorna cells (HTC) in different cell cycls.METHODS: Micropipette aspiration technique was adopted tomeasure viscoelastic coefficients and adhesion force tocollagen coated surface ofthe cells. Three kinds ofcytoskeleton perturbing agents, colchiclnes (Col),cytochalssin D (CD) and vinblastine (VBL), were used totreat HCC cells and hepatocytes and the effects of thesetreatent on cell viscoelastic coefficients were investigated.The experimental results were analyzed with a thres-elsmentstandard linear solid. Further, the viscoelastic properties ofHTC cells and the adhesion force of different cycle HTC cellswere also investigated. The synchronous G1 and S phasecells were achieved through thymine-2-desoryriboside andcolchicines sequential blockage method and thymine-2-desoryriboside blockage method respectively.RESULTS: The elastic coefficients, but not viscouscoefficient of HCC cells (k1 = 103.6± 12.6N.m-2, k2 =42.5±10.4N. m-2, μ = 4.5 ± 1.9Pa. s), were significantly higherthan the corresponding value for hepatocytes (K1 = 87.5 ±12.1N.m-2, k2 =33.3± 10.3N.m-2, μ=5.9±3.0Pa. s, P<0.01). Upon treatment with CD, the viscoelastic coefficients ofboth hepotocytes and HCC cells decreased consistently,with magnitudes for the decrease in elastic coefficients ofHCC cells (k1: 68.7 N.m-2 to 81.7N.m-2, 66.3 % to 78.9 %;k2: 34.5 N.m-2 to37.1N.m-2, 81.2% to 87.3 %, P<0.001)larger than those for normal hepatocytes (k1: 42.6N. m-2 to49.8N.nt-2, 48.7% to56.9 %; k2: 17.2N.m-2 to 20.4N.m-2,51.7 % to 61.3 %, P< 0.001). There was a little decrease inthe vlscous coefficient of HCC cells (2.0 to 3.4Pa. s, 44.4 to75.6 %, P<0.001) than that for hepatocytes (3.0 to 3.gPa.s, 50.8to 66.1% P<0.001). Upon trastment with Col andVBL, the elastic coefficients of hepatocytes generallyincreased or tended to increase while those of HCC cellsdecreased. HTC cells with 72.1% ofG1 phase and 98. 9 % ofS phase were achieved and high K1, k2 value and low μvalue were the general characlteristics of HTC cells. G1phase cells had higher K1 value and lower tμ value than Sphase cells had, and G1 phase HTC cells had strongeradhesive forces [(275.9±232.8) x 10-10N] than S phase cells[(161.2± 120.4) x l0-10N, P<0.001).CONCLUSION: The difference in both the pattern and themagnitude of the effect of cytoskeletal perturbing agent onthe viscoelastic properties between HCC cells andhepatocytes may reflect differencss in the state of thecytusieleton structure and function and in the sensitivity toperturbing agent treatment between trinse two types of cells.Change in the viscoelastic properties of cancer cells mayaffect significantly tumor cell invasion and metastasis as wellas interactions between tumor cells and their micro-mechanical environments.
文摘To investigate the modulating effects of survivn antisense oligonucletode (ASODN) on the cell cycle and apoptosis of human hepatocellular carcinoma (HCC) cell line SMMC-7721 and explore its mechanism.Methods Survivin ASODN was transfected into SMMC-7721 cells mediated by DOTAP liposomal reagent.Electron microscopy,flow cytometry and RT-PCR were used to detect the changes in cell ultrastructure,apoptosis,cell cycle and the expression of cyclinB1 mRNA,respectively.Results After transfection of survivin ASODN,the expression of cyclinB1 mRNA in the cells significantly increased and increase in G2-M arrest and apoptosis appeared.Meanwhile,the cell ultrastructure had apoptotic changes such as chromatin condensation and apoptotic body formation.Conclusion Survivin ASODN can induce the expression of cyclinB1 that may result in G2-M arrest.Consequently,apoptosis is triggered.Survivin ASODN transfection might be an improtant new treatment for HCC.14 refs,2 figs,1 tab.
基金Supported by Asahi Medical Foundation,No.00-2000-03
文摘AIM: To evaluate the effects of sulindac in inducing growth inhibition and apoptosis of human gastric cancer cells in comparison with human hepatocellular carcinoma (HCC)cells.METHODS: The human gastric cancer cell lines MKN45 and MKN28 and human hepatocellular carcinoma cell lines HepG2and SMMC7721 were used for the study. Anti-proliferative effect was measured by MTT assay, and apoptosis was determined by Hoechst-33258 staining, electronography and DNA fragmentation. The protein of cyclooxygenase-2 (COX(2) and Bcl-2 were detected by Westem dot blotting.RESULTS: Sulindac could initiate growth inhibition and apoptosis of MKN45, MKN28, HepG2 and SMMC7721 cells in a dose-and time-dependent manner. Growth inhibitory activity and apoptosis were more sensitive in HepG2 cells than in SMMC7721 cells, MKN45 and MKN28 cells. After 24hours incubation with sulindac at 2mmol. L-1 and 4mmol.L-1, the level of COX-2 and Bcl-2 protein were lowered in MKN45, SMMC7721 and HepG2 cells but not in MKN28 cells.CONCLUSION: Sulindac could inhibit the growth of gastric cancer cells and HCC cells effectively in vitro by apoptosis induction, which was associated with regression of COX-2and Bcl-2 expression. The growth inhibition and apoptosis of HCC cells were greater then that of human gastric cancer cells. The different effects of apoptosis in gastric cancer cells may be related to the differentiation of the cells.
文摘The prognosis of hepatocellular carcinoma (HCC) stillremains dismal, although many advances in its clinicalstudy have been made. It is important for tumor control toidentity the factors that predispose patients to death. Withnew discoveries in cancer biology, the pathological andbiological prognostic factors of HCC have been studied quiteextensively. Analyzing molecular markers (biomarkers) withprognostic significance is a complementary method. A largenumber of molecular factors have been shown to associatewith the invasiveness of HCC, and have potential prognosticsignificance. One important aspect is the analysis ofmolecular markers for the cellular malignancy phenotypeThese include alterations in DNA ploidy, cellularproliferation markers (PCNA, Ki-67, Mcm2, MIB1, MIA, andCSE1L/CAS protein), nuclear morphology, the p53 geneand its related molecule MDM2, other cell cycle regulators(cyclin A, cyclin D, cyclin E, cdc2, p27, p73), oncogenesand their receptors (such as ras, c-myc, c-fms, HGF, c-met, and erb-B receptor family members ), apoptosisrelated factors (Fas and FasL), as well as telomeraseactivity. Another important aspect is the analysis ofmolecular markers involved in the process of cancerinvasion and metastasis. Adhesion molecules (E-cadherin,catenins, serum intercellular adhesion molecule-1, CD44variants), proteinases involved in the clegradation ofextracellular matrix (MMP-2, MMP-9, uPA, uPAR, PAl), aswell as other molecules have been regarded as biomarkersfor the malignant phenotype of HCC, and are related toprognosis and therapeutic outcomes. Tumor angiogenesisis critical to both the growth and metastasis of cancersincluding HCC, and has drawn much attention in recentyears. Many angiogenesis-related markers, such as vascularendothelial growth factor (VEGF), basic fibroblast growthfactor (bFGF), platelet-derived endothelial cell growth factor( PD-ECGF ), thrombospondin ( TSP ), angiogenin,pleiotrophin, and endostatin (ES) levels, as well asinratumor microvessel density (MVD) have been evaluatedand found to be of prognostic significance. Body fluid(particularly blood and urinary) testing for biomarkers iseasily accessible and useful in clinical patients. Theprognostic significance of circulating DNA in plasma orserum, and its genetic alterations in HCC are otherimportant trends. More attention should be paid to thesetwo areas in future. As the progress of the human genomeproject advances, so does a clearer understanding of tumorbiology, and more and more new prognostic markers withhigh sensitivity and specificity will be found and used inclinical assays. However, the combination of some items, i.e., the pathological features and some biomarkersmentioned above, seems to be more practical for now.
基金Supported by The National Nature Science Foundation of China,No. 30070338
文摘AIM: To evaluate the receptor protein which can specifically bind to β2GPⅠon the membrane of hepatocellular carcinoma (HCC) cell line SMMC-7721, and to study the biological function of the receptor.METHODS: Through β2GPⅠ-affinity chromatography column, the peptid-polysome-mRNA complex, which can specially bind to β2GPⅠ, stayed with the column and was separated from the whole polysome of liver cells, and then eluted and collected. Using cDNA synthesis kit and cDNA PCR kit, the corresponding cDNA was obtained and sequenced. RT-PCR was used to amplify annexinⅡ, and flow cytometry was used to study the competitive binding of annexinⅡ with β2GPⅠto SMMC-7721.RESULTS: A total of 1.1 kb of the cDNA fragment of the specific binding protein of β2GPⅠon liver cell membrane was obtained. The sequence of cDNA shared high homology with human annexinⅡ (98%). AnnexinⅡ was expressed on the membrane of SMMC-7721, and could compete with β2GPⅠfor combining with SMMC-7721.CONCLUSION: The receptor for β2GPⅠon membrane of SMMC-7721 cells is annexinⅡ, which might bridge HBV to infect hepatocytes.
文摘AIM: Cyclooxygenase-2 (COX-2) has been suggested to beassociated with carcinogenesis. We sought to investigatethe effect of the selective COX-2 inhibitor, Nimesulide onproliferation and apoptosis of SMMC-7721 human hepatomacells.METHODS: This study was carried out on the culture ofhepatic carcinoma SMMC-7721 cell line. Variousconcentrations of Nimesulide (0、200 μmol/L、300 μmol/L、400μmol/L) were added and incubated. Cell proliferation wasdetected with MTT colorimetric assay, cell apoptosis byelectron microscopy, flow cytometry and TUNEL.RESULTS: Nimesulide could significantly inhibit SMMC-7721cells proliferation dose-dependent and in a dependentmanner compared with that of the control group. Theduration lowest inhibition rate produced by Nimesulide inSMMC-7721 cells was 19.06 %, the highest inhibition ratewas 58.49 %. After incubation with Nimesulide for 72 h, themost highest apoptosis rate and apoptosis index of SMMC-7721 cells comparing with those of the control were 21. 20 %+1.62% vs2.24% +0.26% and21.23+ 1.78 vs2.01+0.23(p<0.05).CONCLUSION: The selective COX-2 inhibitor, Nimesulide caninhibit the proliferation of SMMC-7721 cells and increaseapoptssis rate and apoptosis index of SMMC-7721 cells. Theapoptoois rate and the apoptosis index are dose-dependent.Under electron microscope SMMC-7721 cells incubated with 300μmol and 400 μmol Nimesulide show apoptotic characteristics.With the clarification of the mechanism of selective COX-2inhibitors, These COX-2 selective inhibitors can become thechoice of prevention and treatment of cancers.
文摘AIM To demonstrate the relationship betweenH-ras oncogene and hepatocellular carcinoma(HCC) metastasis.METHODS Activated H-ras oncogene wastransfected into SMMC 7721, a cell line derivedfrom human HCC, by calcium phosphatetransfection method. Some metastasis-relatedparameters were detected in vitro, includingadhesion assay, migration assay, expression ofcollagenase ⅣV (c ⅣV ase) and epidermal growthfactor receptor (EGFR).RESULTS The abilities of H-ras-transfected cellclones in adhesion to laminin (LN) or fibronectin(FN), migration, c Ⅳ ase secretion increasedmarkedly, and the expression of EGFR elevatedmoderately. More importantly, these alterationswere consistent positively with the expressionof p21, the protein product of H-ras oncogene.CONCLUSION H-ras oncogene could inducethe metastatic phenotype of HCC cell in vitro toraise its metastatic potential.
基金National Natural Science Foundation of China,No.39760077
文摘AIM: The goal of this study was to characterize the AlPreceptor, its possible signal transduction pathway and itsproliferative functions in human hepatoma cell line Bel 7402.METHODS: Cell proliferation enhanced by AFlP was detectedby MTT assay, 3H-thymidine incorporation and S-stsgepercentage of cell cycle analysis. With radioactive labeled 125 I-AFP for receptor binding assay; cAMP acctmuation, ProteinKinase A activity were detected by radioactive immunosorbentassay and the change of intracellular free calcium ([Ca2+ ], )was monitored by scanning fluorescence intensity under TCS-NT confocal microscope. The expression of oncxgenes N- ras,p53, and p21ras in the cultured cells in vitro were detected byNorthem blotting and Western blotting respectively.RESULTS: It was demonstrated that AFP enhanced theproliferation of human hepatoma Bel 7402 cell in a dosedependlent fashion asshown in MTT assay, 3H-thymidineincorporation and S-phase percentage up to 2-fold. Twosubtypes of AFP receptors were identified in the cells withKds of 1.3 x 10-9 mol. L-1 and 9.9 x 10-8 mol. L-1 respectively.Pretreatnent of cells with AFP resulted-in a significantincrease (625 %) in cAMP accumulation. The activity ofprotein kinase A activity were increased up to 37.5, 122.6,73.7 and 61.2 % at treatment time point 2, 6, 12 and 24hours. The level of intracellular calcium were elevated afterthe treatment of alpha-fetoprotsin and achieved to 204 % at 4min. The results also showed that AFP (20 mg. L-1 ) couldupregulate the expression of N-ras oncogenes and p53 andp21ras in Bel 7402 cells. In the later case, the alteration ware 81.1%(12 h) and 97.3 %(12 h) respectively compared with control.CONCLUSION: These results demonstrate that AFP is apotential growth factor to promote the proliferation of humanhepatoma Bel 7402 cells. Its growth-regulatory effects aremediated by its specific plasma membrane receptorscoupled with its transmembrane signaling transductionthrough the pathway of cAMP-PKA and intracellular calciumto regulate the expression of oncogenes.
基金Supported by Natural Science Foundation of China No.39970290
文摘AIM: To explore the role of SF/HGF-Met autocrine and parscrine in metastasis of hepatocellular carcinoma (HCC).METHODS: SF/HGF and c-met transcription and protein expression in HCC were examined by RT-PCR and Western Blot in 4 HCC cell lines, including HepG2, Hep3B,SMMC7721 and MHCC-1, the last cell line had a higher potential of metastasis. Sf/hgf cDNA was transfected by the method of Lipofectin into SMMC7721. SF/HGF and c-met antibody were used to stimulate and block SF/HGF-c-met signal transduction. Cell morphology, mobility, and proliferation were respectively compared by microscopic observation, wound healing assay and cell growth curve.RESULTS: HCC malignancy appeared to be relative to its met-SF/HGF expression. In MHCC-1, c-met expression was much stronger than that in other cell lines with lower potential of metastasis and only SF/HGF autocrine existed in MHCC-1. After sf/hgf cDNA transfection or conditioned medium of MHCC-1 stimulation, SMMC7721 changed into elongated morphology, and the abilities of proliferation ( P < 0.05) and mobility increased. Such bio-activity could he blocked by c-met antibody ( P< 0.05).CONCLUSION: The system of SF/HGF-c-met autocrine and paracrine played an important role in development and metastasis potential of HCC. Inhibition of SF/HGF-c-met signal transduction system may reduce the growth and metastasis of HCC.
基金Project supported partly by the National Natural Science Foundation of China, No. 39870344
文摘INTRODUCTIONHepatocellular carcinoma (HCC) is one of the mostcommon human malignancies worldwide[1,2], and isclosely associated with infection of HBV and HCVand contamination of aflatoxin B1[3-6]. Althoughthe molecular mechanisms of hepatocarcinogenesisremain poorly understood, an increasing number ofgenetic abnormalities have been recognized[7-10],for example, the p16 gene[11,12] the p53gene[13-18], the E-cadherin gene[19], and the c-mycgene[20].