AIM:To investigate whether Melatonin has synergistic effects with Doxorubicin in the growth-inhibition and apoptosis-induction of human hepatoma cell lines HepG2 and Bel-7402.METHODS:The synergism of Melatonin and Dox...AIM:To investigate whether Melatonin has synergistic effects with Doxorubicin in the growth-inhibition and apoptosis-induction of human hepatoma cell lines HepG2 and Bel-7402.METHODS:The synergism of Melatonin and Doxorubicin inhibited the cell growth and induced cell apoptosis in human hepatoma cell lines HepG2 and Bel-7402.Cell viability was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide(MTT)assay.Cell apoptosis was evaluated using TUNEL method and flow cytometry.Apoptosis-related protein Bax,Bcl-2 and caspase-3 expressions were measured by immunohistochemical staining.RESULTS:Treatment with Melatonin(10 -8 -10 -5 mol/L) alone had a dose-related inhibitory effect on cell proliferation but no cytotoxic effect on hepatoma cell lines HepG2 and Bel-7402.Interestingly,when combined with Doxorubicin,Melatonin significantly increased the effects of cell growth inhibition and cell apoptosis.Furthermore,TUNEL staining and flow cytometry revealed that cooperative apoptosis induction was associated with decreased expression of Bcl-2 as well as increased expression of Bax and Caspase3.CONCLUSION:The synergism of Melatonin and Doxorubicin inhibits hepatoma cell growth and induces cell apoptosis.展开更多
Objective To determine whether transforming growth factor betal (TGF-β1)/Smad signaling pathway mediates p53-dependent apoptosis in hepatoma cell lines.Methods Three human hepatic carcinoma cell lines, HepG2, Huh-7, ...Objective To determine whether transforming growth factor betal (TGF-β1)/Smad signaling pathway mediates p53-dependent apoptosis in hepatoma cell lines.Methods Three human hepatic carcinoma cell lines, HepG2, Huh-7, and Hep3B, were used in this study.TGF-β1-induced apoptosis in hepatic carcinoma cell lines was analyzed using TUNEL assay.For identifying the mechanism of apoptosis induced by TGF-β1, cell lines were transfected with a TGF-β1-inducible luciferase reportor plasmid containing Smad4 binding elements.After transfection, cells were treated with TGF-β1, then assayed for luciferase activity.Results The apoptosis rate of HepG2 cell lines (48.51%± 8.21%) was significantly higher than control ( 12.72%±2.18%, P<0.05).But TGF-β1 was not able to induce apoptosis of Huh-7 and Hep3B cell lines.The relative luciferase activity of TGF-β1-treated HepG2 cell lines (4.38) was significantly higher than control (1.00, P< 0.05).But the relative luciferase activity of TGF-β1-treated Huh-7 and Hep3B cell lines less increased compared with control.Conclusions HepG2 cells seem to be highly susceptible to TGF-β1-induced apoptosis compared with Hep3B and Huh-7 cell lines.Smad4 is a central mediator of TGF-β1 signaling transdution pathway.TGF-β1/Smad signaling pathway might mediate p53-dependent apoptosis in hepatoma cell lines.展开更多
To gain a better understanding of the anticancer effects of hydroxyapatite (HAP) nanoparticles in vivo and in vitro, the effects of the interaction of HAP nanoparticles with hepatoma cells were explored. HAP nanopar...To gain a better understanding of the anticancer effects of hydroxyapatite (HAP) nanoparticles in vivo and in vitro, the effects of the interaction of HAP nanoparticles with hepatoma cells were explored. HAP nanoparticles were prepared by homogeneous precipitation and characterized by laser particle analysis and transmission electron microscopy (TEM). HAP nanoparticles were observed to be uniformly distributed, with rod-like shapes and diameters in the range of 42.1-87.1 nm. Overnight attached, suspended, and proliferating Bel-7402 cells were incubated with HAP nanoparticles. Inverted microscopy observation revealed that HAP nanoparticles with a cell membrane showed good adsorption. TEM demonstrated that HAP nanoparticles were present on the surface of cells, continuously taken up by cells through endocytosis, and transported in vesicles close to the nucleus. Fluorescence microscopy showed that the concentrations of intracellular Ca2+ labeled with Fluo-3 calcium fluorescent probe were significantly enhanced. In addition, inverted microscopy observation revealed that suspended cells treated with HAP nanoparticles did not adhere to the culture bottle, resulting in cell death. After the overnight attached cells were treated with HAP nanoparticles for 96 h with increasing doses of HAP nanoparticles, inverted microscopy observation revealed that cell proliferation was slowed and ceU-ceU adhesion was weakened. Feulgen staining and image analysis indicated that the nuclear DNA content of the cells was markedly reduced, and argyrophilic nucleolar organizer region (AgNOR) staining and image analysis indicated that the number of AgNORs was significantly decreased. Therefore, hepatoma cells brought about the adsorption, uptake, transport and degradation of HAP nanoparticles. In addition, HAP nanoparticles affected hepatoma cells with regard to cell-cell adhesion, cell and extracellular matrix adhesion, and DNA and protein synthesis; thus inhibiting cell proliferation. This understanding of the effects of interaction between HAP nanoparticles and hepatoma cells is useful for further study of the anticancer mechanisms of HAP nanoparticles.展开更多
In order to .study the effect of tanshinone ⅡA on growth and apoptosis in human hepatoma cell line BEL-7402 in vitro, the human hepatoma cell line BEL-7402 was treated with tanshinone ⅡA at various concentrations fo...In order to .study the effect of tanshinone ⅡA on growth and apoptosis in human hepatoma cell line BEL-7402 in vitro, the human hepatoma cell line BEL-7402 was treated with tanshinone ⅡA at various concentrations for 72 h. Growth suppression was evaluated by MTT assay; apoptosis-relat-ed alterations in morphology and biochemistry were ascertained under cytochemical staining (Hoechst 33258), transmission electron microscopy (TEM), and DNA agarose gel electrophoresis. Apoptotic rate was quantified by flow cytometry (FCM). The results showed that Tanshinone ⅡA could inhibit the growth of hepatoma cells in a dose-dependent manner, with IC50 value being 6. 28μg/ml. After treatment with 1-10μg/ml tanshinone ⅡA for 72 h, BEL-7402 cells apoptosis with nuclear chro-matin condensation and fragmentation as well as cell shrinkage and the formation of apoptotic bodies were observed. DNA ladder could be demonstrated on DNA electrophoresis. FCM analysis showed hypodiploid peaks on histogram, and the apoptotic rates at μg/ml concentration for 12 h> 24 h, 36 h, 48 h and 72 h were (2. 32±0. 16)%, (3. 01±0. 35) %, (3. 87±0. 43)%, (6. 73±0. 58)% and (20. 85 ± 1. 74) % respectively, which were all significantly higher than those in the control group (1. 07±0. 13) %. It is concluded that Tanshinone ⅡA could induce human hepatoma cell line BEL-7402 apoptosis, which may be related to the mechanism of growth inhibition.展开更多
In this paper, the relationship between radiosensitivity, cell cycle alteration and the change of apoptosis in different human hepatoma cell lines irradiated by heavy ions were studied with the aim of building up the ...In this paper, the relationship between radiosensitivity, cell cycle alteration and the change of apoptosis in different human hepatoma cell lines irradiated by heavy ions were studied with the aim of building up the base data for clinical therapy. Exponentially growing hepatoma cell lines were irradiated by 80.55 MeV/u12C6+ ions at a dose of 0 Gy, 0.5 Gy, 1 Gy, 2 Gy, 4 Gy and 8 Gy. The radiosensitivity was assessed by means of the colony-forming assay. The DNA content, the percentage of each cell-cycle phase and the apoptosis rate were obtained with flow cytometry methods. After the irradiation, the SF2 (survival fraction at 2 gray) of SMMC-7721 cells were evidently lower than that of HepG2 cells. The S phase arrest, G2/M phase arrest delay and the apoptosis in the two hepatoma cell lines varied with the increase of the dose and repair time. The heavy ions could obviously kill the human hepatoma cell lines. Compared to HepG2 cells, SMMC-7721 cells were more radiosensitive to 12C^6+ ions.展开更多
Many particles are found in the cytoplasm area after the mixture of hydroxyapatite (HAP) nanoparticles and cultured cancer cells. The purpose of this study was to confirm whether these particles in cytoplasm are HAP...Many particles are found in the cytoplasm area after the mixture of hydroxyapatite (HAP) nanoparticles and cultured cancer cells. The purpose of this study was to confirm whether these particles in cytoplasm are HAP nanoparticles exactly. BEL7402 cells were incubated in HAP sol for 8 hours. Then, the cells were collected for specimen preparation. Transmission electron microscope (TEM), energy dispersing spectrum (EDS) and electronic diffraction (ED) attached to TEM were used to detect the properties of the particles. It is found that many particles similar to HAP in shape are in the cytoplasm under TEM. By EDS analysis, they are the particles containing calcium (Ca) and phosphorus (P). The classic rings of HAP crystal appear in the ED pictures of these particles. So the particles are confirmed as HAP nanoparticles. Thus, it is concluded that HAP nanoparticles as the crystal particles can be absorbed by hepatoma cells.展开更多
It is reported thut nanoporticles can be applied as carriers and anti-cancer medicines. But the interaction of nanoparticles and cells is unclear. The purpose of this study was to discuss whether inorganic crystal nan...It is reported thut nanoporticles can be applied as carriers and anti-cancer medicines. But the interaction of nanoparticles and cells is unclear. The purpose of this study was to discuss whether inorganic crystal nanoparticles can get through cells with intact crystal. BEL7402 heputoma cells and titanium dioxide ( TiO2 ) nanoporticles were selected and incubated together in vitro. All specimens were prepared and observed under a traasmission electron mieroscope (TEM). TiO2 nanopartieles were found not in the nuclear area but in the cytoplasma. TiO2 nanoponieles maintained the plate-like shape during absorbing. The result shows that hepatoma cells can endocytose the intact TiO2 crystal nanoparticles. It implies that novel nano-effect plays an important role in the biomedicinal application of inorganic crystal nanopartieles.展开更多
It's reported that hepatic stimulator substance (HSS) was extracte from the fetal liver of 4 - 6 months of fetus, and that the effect of HSS on the proliferation of human Alexender hepatoma cells was studied in th...It's reported that hepatic stimulator substance (HSS) was extracte from the fetal liver of 4 - 6 months of fetus, and that the effect of HSS on the proliferation of human Alexender hepatoma cells was studied in this paper. The results showed that proliferation of Alexender cells varied with the amount of HSS in the culture medium, and the former was positively correlated with the latter significantly (P<0. 01). The study indicated that HSS from the fetal liver can stimulate the proliferation of human Alexender hepatoma cells.展开更多
Having been passed for 160 generations, a cell linedesignated as H22-F25/L was established from a murine tumorlymphatic metastatlc model H22-F25 which had been set up in our college. The cell line was in suspension cu...Having been passed for 160 generations, a cell linedesignated as H22-F25/L was established from a murine tumorlymphatic metastatlc model H22-F25 which had been set up in our college. The cell line was in suspension culture with a rapid proliferation and stable growth. The peak tune of cell division and proliferation was 48 and 96 hours after culture. In a week, the cell number was Increased by 25 tunes. H22-F25/L still keeps the features of a poorly differentiated cancer. Its tumor inducing rate (in vivo)was 100% in 615 mice. Lymph node metastasis rate was 50% and pulmonary metastasis rate 10%. H22- F25/ L Is a population of heterogenetlc tumor cells Including 2 stem cell lines (the model number of chromosomes being 43 in 40% tumor cells and 86 in 32%) and some side lines. The common marker chromosomes M1, M2, M3 and M4 were present in all stem and side lines.展开更多
AIM:To study the effects of hypoxia-inducible factor1α(HIF-1α) silencing on the proliferation of hypoxic CBRH-7919 rat hepatoma cells.METHODS:The CBRH-7919 rat hepatoma cell line was used in this study and the hypox...AIM:To study the effects of hypoxia-inducible factor1α(HIF-1α) silencing on the proliferation of hypoxic CBRH-7919 rat hepatoma cells.METHODS:The CBRH-7919 rat hepatoma cell line was used in this study and the hypoxic model was constructed using CoCl2.The HIF-1α-specific RNAi sequences were designed according to the gene coding sequence of rat HIF-1α obtained from GeneBank.The secondary structure of the HIF-1α gene sequence was analyzed using RNA draw software.The small interfering RNA(siRNA) transfection mixture was produced by mixing the siRNA and Lipofectamine2000TM,and transfected into the hypoxic hepatoma cells.Real time reverse transcription-polymerase chain reaction(RTPCR) and Western blotting assay were used to detect the expression levels of mRNA and protein.HIF-1α and vascular endothelial growth factor(VEGF) mRNA was determined using real time RT-PCR;the protein expression levels of AKT,p-AKT,p21 and cyclinD1 were determined using Western blotting.The proliferation of hepatoma cells was observed using the methyl thiazolyl tetrazolium(MTT) assay and the bromodeoxyuridine(BrdU) incorporation cell proliferation assay.RESULTS:Under induced hypoxia,the viability of the hepatoma cells reached a minimum at 800 μmol/L CoCl2;the viability of the cells was relatively high at CoCl2 concentrations between 100 μmol/L and 200 μmol/L.Under hypoxia,the mRNA and protein expression levels of HIF-1α and VEGF were significantly higher than that of hepatoma cells that were cultured in normaxia.HIF-1α-specific RNAi sequences were successfully transfected into hepatoma cells.The transfection of specific siRNAs significantly inhibited the mRNA and protein expression levels of HIF-1α and VEGF,along with the protein expression levels of p-AKT and cyclinD1;the protein expression of p21 was significantly increased,and there was no significant difference in the expression of AKT.The MTT assay showed that the amount of hepatoma cells in S phase in the siRNA transfection group was obviously smaller than that in the control group;in the siRNA transfection group,the amount of hepatoma cells in G1 phase was more than that in the control group.The BrdU incorporation assay showed that the number of BrdU positive hepatoma cells in the siRNA transfection group was less than that in the control group.The data of the MTT assay and BrdU incorporation assay suggested that HIF-1α silencing using siRNAs significantly inhibited the proliferation of hepatoma cells.CONCLUSION:Hypoxia increases the expression of HIF-1α,and HIF-1α silencing significantly inhibits the proliferation of hypoxic CBRH-7919 rat hepatoma cells.展开更多
AIM: To investigate the possibility of recombinant highdensity lipoprotein (rHDL) being a carrier for delivering antitumoral drug to hepatoma cells.METHODS: Recombinant complex of HDL and aclacinomycin(rHDL-ACM) was p...AIM: To investigate the possibility of recombinant highdensity lipoprotein (rHDL) being a carrier for delivering antitumoral drug to hepatoma cells.METHODS: Recombinant complex of HDL and aclacinomycin(rHDL-ACM) was prepared by cosonication of apoproteins from HDL (Apo HDL) and ACM as well as phosphatidylcholine.Characteristics of the rHDL-ACM were elucidated by electrophoretic mobility, including the size of particles,morphology and entrapment efficiency. Binding activity of rHDL-ACM to human hepatoma cells was determined by competition assay in the presence of excess native HDL. The cytotoxicity of rHDL-ACM was assessed by MTT method.RESULTS: The density range of rHDL-ACM was 1.063-1.210g/mL, and the same as that of native HDL. The purity of all rHDL-ACM preparations was more than 92%.Encapsulated efficiencies of rHDL-ACM were more than90%. rHDL-ACM particles were typical sphere model of lipoproteins and heterogeneous in particle size. The average diameter was 31.26±5.62 nm by measure of 110rHDL-ACM particles in the range of diameter of lipoproteins.rHDL-ACM could bind on SMMC-7721 cells, and such binding could be competed against in the presence of excess native HDL. rHDL-ACM had same binding capacity as native HDL. The cellular uptake of rHDL-ACM by SMMC-7721 hepatoma cells was significantly higher than that of free ACM at the concentration range of 0.5-10 μg/mL(P<0.01). Cytotoxicity of rHDL-ACM to SMMC-7721 cells was significantly higher than that of free ACM at concentration range of less than 5 μg/mL (P<0.01) and IC50 of rHDL-ACM was lower than IC50 of free ACM(1.68 nmol/L vs3 nmol/L). Compared to L02 hepatocytes,a normal liver cell line, the cellular uptake of rHDL-ACM by SMMC-7721 cells was significantly higher (P<0.01) and in a dose-dependent manner at the concentration range of 0.5-10 μg/mL. Cytotoxicity of the rHDL-ACM to SMMC-7721 cells was significantly higher than that to L02 cells at concentration range of 1-7.5 μg/mL (P<0.01). IC50 for SMMC-7721 cells (1.68 nmol/L) was lower than that for L02 cells (5.68 nmol/L), showing a preferential cytotoxicity of rHDL-ACM for SMMC-7721 cells.CONCLUSION: rHDL-ACM complex keeps the basic physical and biological binding properties of native HDL and shows a preferential cytotoxicity for SMMC-7721hepatoma to normal L02 hepatocytes. HDL is a potential carrier for delivering lipophilic antitumoral drug to hepatoma cells.展开更多
A non-radioisotopic, quantitative TRAP-based telomerase activity assay was established mainly by using SYBR Green-I staining instead of radioisotope. Comparing with conventional radioisotope based method, it was bette...A non-radioisotopic, quantitative TRAP-based telomerase activity assay was established mainly by using SYBR Green-I staining instead of radioisotope. Comparing with conventional radioisotope based method, it was better in reproducibility and accuracy. Using this method, we found telomerase activities were absent in normal human liver cells, while detected in ail of four human hepatoma cell lines (BEL-7404, SMMC-7721, QGY-7903 and HCCM) without significant differences.展开更多
AIM: To investigate the apoptotic effect of photoexcited titanium dioxide (TiO2) nanoparticles in the presence of visible light on human hepatoma cell line (Bel 7402) and to study the underlying mechanism. METHODS: Ce...AIM: To investigate the apoptotic effect of photoexcited titanium dioxide (TiO2) nanoparticles in the presence of visible light on human hepatoma cell line (Bel 7402) and to study the underlying mechanism. METHODS: Cerium-element-doped titanium dioxide nanoparticles were prepared by impregnation method. Bel 7402 human hepatoma cells were cultured in RPMI 1640 medium in a humidified incubator with 50 mL/L CO2 at 37℃. A 15 W fluorescent lamp with continuous wavelength light was used as light source in the photocatalytic test. Fluorescence morphology and agarose gel eletrophoresis pattern were performed to analyze apoptotic cells. RESULTS: The Ce (Ⅳ)-doped TiO2 nanoparticles displayed their superiority. The adsorption edge shifted to the 400-450 nm region. With visible light illuminated for 10 min, 10 μg/cm3 Ce (Ⅳ)-doped TiO2 induced micronuclei and significant apoptosis in 4 and 24 h, respectively. Hochest 33 258 staining of the fixed cells revealed typical apoptotic structures (apoptotic bodies), agarose gel electrophoresis showed typical DNA ladder pattern in treated cells but not in untreated ones. CONCLUSION: Ce (Ⅳ) doped TiO2 nanoparticles can induce apoptosis of Bel 7402 human hepatoma cells in the presence of visible light.展开更多
AIM: To investigate the adhesive mechanical properties of different cell cycle human hepatoma cells (SMMC-7721)to human umbilical vein endothelial cells (ECV-304),expression of adhesive molecule integrinβ1 in SMMC-77...AIM: To investigate the adhesive mechanical properties of different cell cycle human hepatoma cells (SMMC-7721)to human umbilical vein endothelial cells (ECV-304),expression of adhesive molecule integrinβ1 in SMMC-7721cells and its contribution to this adhesive course.METHODS: Adhesive force of SMMC-7721 cells to endothelialcells was measured using micropipette aspiration technique.Synchronous G1 and S phase SMMC-7721 cells wereachieved by thymine-2-deoxyriboside and colchicinessequential blockage method and double thymine-2-deoxyriboside blockage method, respectively. Synchronousrates of SMMC-7721 cells and expression of integrinβ1 inSMMC-7721 cells were detected by flow cytometer.RESULTS: The percentage of cell cycle phases of generalSMMC-7721 cells was 11.01% in G2/M phases, 53.51% inG0/G1 phase, and 35.48% in S phase. The synchronous ratesof G1 and S phase SMMC-7721 cells amounted to 74.09%and 98.29%, respectively. The adhesive force of SMMC-7721cells to endothelial cells changed with the variations ofadhesive time and presented behavior characteristics ofadhesion and de-adhesion. S phase SMMC-7721 cells had higheradhesive forces than G1 phase cells [(307.65±92.10)× 10-10Nvs (195.42±60.72)×10-10N, P<0.01]. The expressivefluorescent intensity of integrinβ1 in G1 phase SMMC-7721cells was depressed more significantly than the values ofS phase and general SMMC-7721cells. The contribution ofadhesive integrinβ1 was about 53% in this adhesive course.CONCLUSION: SMMC-7721 cells can be synchronizedpreferably in G1 and S phases with thymine-2-deoxyribosideand colchicines. The adhesive molecule integrinβ1 expressesa high level in SMMC-7721 cells and shows differences invarious cell cycles, suggesting integrin β1 plays an importantrole in adhesion to endothelial cells. The change of adhesiveforces in different cell cycle SMMC-7721 cells indicatesthat S phase cells play predominant roles possibly whilethey interact with endothelial cells.展开更多
AIM:To investigate the effect of hepatoma cells on up-regulation of programmed cell death-1 (PD-1),and the function of PD-1 on T cells. METHODS:HepG2 or HepG2.2.1.5 cells were co-cultured with a lymphoma cell line-Jur...AIM:To investigate the effect of hepatoma cells on up-regulation of programmed cell death-1 (PD-1),and the function of PD-1 on T cells. METHODS:HepG2 or HepG2.2.1.5 cells were co-cultured with a lymphoma cell line-Jurkat cells. PD-1 expression was detected by flow cytometry. IL-2,INF-γ and IL-10 in culture supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Cytotoxic action of T cells was determined by MTT reduction assay-direct mononuclear cell cytotoxicity assay. RESULTS:The PD-1 expression on Jurkat cells increased by 16.17% ± 2.5% and 17.43% ± 2.2% after HepG2 or HepG2.2.1.5 cells were co-cultured for 48 h. The levels of IL-2,INF-γ and IL-10 in the culture supernatant were 202.9 ± 53.0 pg/mL,88.6 ± 4.6 pg/mL and 63.7 ± 13.4 pg/mL respectively,which were signif icantly higher than those (102.9 ± 53 pg/mL,39.3 ± 4.2 pg/mL,and 34.6 ±13.7 pg/mL) in the control group (P < 0.05). The OD value for MTT assay in the blocking group (0.29 ± 0.06) was significantly higher than that (0.19 ± 0.09) in the control group (P < 0.05). CONCLUSION:PD-1 expression on Jurkat cells is up-regulated by hepatoma cells,cytokines and cytotoxic action are elevated after PD-1/PD-L1 is blocked.展开更多
The inhibitory effect of parvovirus H-1 on the colonyforming ability in vitro of QGY-7703, a cultured human hepatoma cell line, and on the formation and growth of its tumors in nude mice was studied. With higher multi...The inhibitory effect of parvovirus H-1 on the colonyforming ability in vitro of QGY-7703, a cultured human hepatoma cell line, and on the formation and growth of its tumors in nude mice was studied. With higher multiplicity of infection (MOI) of H-1 given, survival of the QGY-7703 cells was found to be decreased. H-1 DNA amplification level at 30 h postinfection(p.i.) was detected to be 7.4 times higher than that at 2 h by dispersed cells assay, while the cells were delayed to enter into S phase.Plaques were formed in the indicator cells (new-born human kidney cell line, NBK) by progeny H-1 virus particles released from the infected QGY-7703 cells by infectious cell center assay. The formation of tumors in nude mice by QGY-7703 cells which were injected s c at 2 h postinfection was observed to be prevented in 2 groups with given MOI 25 and 50. The tumor growth of MOI 10 group occurred at a lower exponential rate than that of control,after a 20 d latent period. It was evident that parvovirus H-1 exhibited a direct inhibitory effect on the formation and growth of human hepatoma cells in vivo as well as in vitro.展开更多
Effects of antisense epidermal growth factor receptor (EGFR) sequence on apoptotic cell death were examined in a human hepatoma cell line BEL-7404 cells. In the cells of JX-1, a sub clone of BEL-7404 stably transfecte...Effects of antisense epidermal growth factor receptor (EGFR) sequence on apoptotic cell death were examined in a human hepatoma cell line BEL-7404 cells. In the cells of JX-1, a sub clone of BEL-7404 stably transfected with antisense EGFR vector (Cell Research, 3:75, 1993), an enhanced rate (9.5%) of spontaneous apoptosis was detected by flow cytometry, whereas the rates of spontaneous apoptosis in JX-0 cells, a sub-clone of BEL-7404 transfected by control vector, and the parellt BEL-7404 cells were almost equal and about 1.7%. Serum-starvation for 72 h increased the rate of apoptosis of JX-1cells up to 33.7%, while JX-0 and BEL-7404 cells, under the same condition, produced less than 5% of apoptotic cells. Observation with electron microscope demonstrated that condensation and fragmentation of chromatin and formation of apoptotic bodies of ten occurred in JX-1 cells, especially during serumstarvation. These results, combined with the data of DNA fragmentation Elisa test, suggested that antisense EGFR sequence enhances apoptosis in the human hepatoma cells.Comparison of intracellular Ca2+ level and the responsiveness of JX-1 cells to the induced action of EGF and tharpsigargin (TG) treatment with that of control JX-0cells indicated that antisense EGFR might interrupt the EGF/EGFR signaling pathway resulting in the decreass of intracellular Ca2+ pool content as well as the responsiveness of these cells to the extracellular signals. These findings suggest that antisense EGFR either directly or indirectly reglllates Ca2+ storage in endoplasmic reticulum,thereby enhances apoptosis in the hnman hepatoma cells.展开更多
Objective Cucurbitacins are the highly oxygenated tetracyclic triterpenes,which are predominantly found in the Cucurbitaceae family but are also present in several other families of the plant kingdom.A number of compo...Objective Cucurbitacins are the highly oxygenated tetracyclic triterpenes,which are predominantly found in the Cucurbitaceae family but are also present in several other families of the plant kingdom.A number of compounds of this group have been investigated for their cytotoxic,hepatoprotective,anti-inflammatory,cardiovascular and anti-diabetic activities.In China,the cucurbitacin preparation,which contains mostly cucurbitacin B and cucurbitacin E,has been clinically used for the treatment of the primary liver carcinoma.It has been previously reported that cucurbitacin E could produce cytotoxicity against a variety of cancer cells,and various mechanisms were implicated in its cytotoxic effect.The present study is to investigate the effect of cucurbitacin E on hepatoma cells in vitro and in vivo and to study their potential mechanisms of action.Methods The MTT assay was used to assess the viability of human HepG2 and BEL7402 hepatoma cells in vitro after treatment with different concentrations of cucurbitacin E.The cell cycle distribution was determined by flowcytometric analysis after propidium iodide(PI)staining.The cell cycle-related proteins were detected using western blotting analysis.Implanted mouse hepatoma H22 model was built to evaluate the growth inhibitory effect of cucurbitacin E in vivo in mice.Results Our studies found that cucurbitacin E(10-300 nM)produced anti-proliferative effect on human HepG2 and BEL7402 hepatoma cells in vitro without cytotoxicity.According to flowcytometric analysis,cucurbitacin E arrested the cell cycle at G2/M phase in both HepG2 and BEL7402 hepatoma cells after 24 h treatment.Cucurbitacin E induced the decrease in the level of CDK1 protein and the increase in the level of p21 protein,but had no effect on the levels of cyclin A,cyclin B1 and Cdc25C protein.In in vivo anti-tumor experiment,cucurbitacin E had significant inhibitory effects on the growth of mouse H22 hepatoma cells.Conclusions Cucurbitacin E inhibited the proliferation of hepatoma cells in vitro and in vivo,at least in part,through induction of cell cycle arrest at G2/M phase,which was mediated by concomitant upregulation of p21 and downregulation of CDK1.We consider that cucurbitacin E may be useful in the treatment of liver cancer.展开更多
文摘AIM:To investigate whether Melatonin has synergistic effects with Doxorubicin in the growth-inhibition and apoptosis-induction of human hepatoma cell lines HepG2 and Bel-7402.METHODS:The synergism of Melatonin and Doxorubicin inhibited the cell growth and induced cell apoptosis in human hepatoma cell lines HepG2 and Bel-7402.Cell viability was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide(MTT)assay.Cell apoptosis was evaluated using TUNEL method and flow cytometry.Apoptosis-related protein Bax,Bcl-2 and caspase-3 expressions were measured by immunohistochemical staining.RESULTS:Treatment with Melatonin(10 -8 -10 -5 mol/L) alone had a dose-related inhibitory effect on cell proliferation but no cytotoxic effect on hepatoma cell lines HepG2 and Bel-7402.Interestingly,when combined with Doxorubicin,Melatonin significantly increased the effects of cell growth inhibition and cell apoptosis.Furthermore,TUNEL staining and flow cytometry revealed that cooperative apoptosis induction was associated with decreased expression of Bcl-2 as well as increased expression of Bax and Caspase3.CONCLUSION:The synergism of Melatonin and Doxorubicin inhibits hepatoma cell growth and induces cell apoptosis.
文摘Objective To determine whether transforming growth factor betal (TGF-β1)/Smad signaling pathway mediates p53-dependent apoptosis in hepatoma cell lines.Methods Three human hepatic carcinoma cell lines, HepG2, Huh-7, and Hep3B, were used in this study.TGF-β1-induced apoptosis in hepatic carcinoma cell lines was analyzed using TUNEL assay.For identifying the mechanism of apoptosis induced by TGF-β1, cell lines were transfected with a TGF-β1-inducible luciferase reportor plasmid containing Smad4 binding elements.After transfection, cells were treated with TGF-β1, then assayed for luciferase activity.Results The apoptosis rate of HepG2 cell lines (48.51%± 8.21%) was significantly higher than control ( 12.72%±2.18%, P<0.05).But TGF-β1 was not able to induce apoptosis of Huh-7 and Hep3B cell lines.The relative luciferase activity of TGF-β1-treated HepG2 cell lines (4.38) was significantly higher than control (1.00, P< 0.05).But the relative luciferase activity of TGF-β1-treated Huh-7 and Hep3B cell lines less increased compared with control.Conclusions HepG2 cells seem to be highly susceptible to TGF-β1-induced apoptosis compared with Hep3B and Huh-7 cell lines.Smad4 is a central mediator of TGF-β1 signaling transdution pathway.TGF-β1/Smad signaling pathway might mediate p53-dependent apoptosis in hepatoma cell lines.
基金Funded by the National Natural Science Foundation of China(Nos.81190133,51172171 and 51002109)the National Natural Science Foundation of Hubei Province((No.2013CFB354)the Excellent Youth Innovative Research Team Foundation and Talents Project of Hubei Polytechnic University(No.13xtz01)
文摘To gain a better understanding of the anticancer effects of hydroxyapatite (HAP) nanoparticles in vivo and in vitro, the effects of the interaction of HAP nanoparticles with hepatoma cells were explored. HAP nanoparticles were prepared by homogeneous precipitation and characterized by laser particle analysis and transmission electron microscopy (TEM). HAP nanoparticles were observed to be uniformly distributed, with rod-like shapes and diameters in the range of 42.1-87.1 nm. Overnight attached, suspended, and proliferating Bel-7402 cells were incubated with HAP nanoparticles. Inverted microscopy observation revealed that HAP nanoparticles with a cell membrane showed good adsorption. TEM demonstrated that HAP nanoparticles were present on the surface of cells, continuously taken up by cells through endocytosis, and transported in vesicles close to the nucleus. Fluorescence microscopy showed that the concentrations of intracellular Ca2+ labeled with Fluo-3 calcium fluorescent probe were significantly enhanced. In addition, inverted microscopy observation revealed that suspended cells treated with HAP nanoparticles did not adhere to the culture bottle, resulting in cell death. After the overnight attached cells were treated with HAP nanoparticles for 96 h with increasing doses of HAP nanoparticles, inverted microscopy observation revealed that cell proliferation was slowed and ceU-ceU adhesion was weakened. Feulgen staining and image analysis indicated that the nuclear DNA content of the cells was markedly reduced, and argyrophilic nucleolar organizer region (AgNOR) staining and image analysis indicated that the number of AgNORs was significantly decreased. Therefore, hepatoma cells brought about the adsorption, uptake, transport and degradation of HAP nanoparticles. In addition, HAP nanoparticles affected hepatoma cells with regard to cell-cell adhesion, cell and extracellular matrix adhesion, and DNA and protein synthesis; thus inhibiting cell proliferation. This understanding of the effects of interaction between HAP nanoparticles and hepatoma cells is useful for further study of the anticancer mechanisms of HAP nanoparticles.
基金This project was supported by a grant from Natural Sciences Foundation of Hubei Province(No.2000J064).
文摘In order to .study the effect of tanshinone ⅡA on growth and apoptosis in human hepatoma cell line BEL-7402 in vitro, the human hepatoma cell line BEL-7402 was treated with tanshinone ⅡA at various concentrations for 72 h. Growth suppression was evaluated by MTT assay; apoptosis-relat-ed alterations in morphology and biochemistry were ascertained under cytochemical staining (Hoechst 33258), transmission electron microscopy (TEM), and DNA agarose gel electrophoresis. Apoptotic rate was quantified by flow cytometry (FCM). The results showed that Tanshinone ⅡA could inhibit the growth of hepatoma cells in a dose-dependent manner, with IC50 value being 6. 28μg/ml. After treatment with 1-10μg/ml tanshinone ⅡA for 72 h, BEL-7402 cells apoptosis with nuclear chro-matin condensation and fragmentation as well as cell shrinkage and the formation of apoptotic bodies were observed. DNA ladder could be demonstrated on DNA electrophoresis. FCM analysis showed hypodiploid peaks on histogram, and the apoptotic rates at μg/ml concentration for 12 h> 24 h, 36 h, 48 h and 72 h were (2. 32±0. 16)%, (3. 01±0. 35) %, (3. 87±0. 43)%, (6. 73±0. 58)% and (20. 85 ± 1. 74) % respectively, which were all significantly higher than those in the control group (1. 07±0. 13) %. It is concluded that Tanshinone ⅡA could induce human hepatoma cell line BEL-7402 apoptosis, which may be related to the mechanism of growth inhibition.
基金the Ministry of Science and Technology,China(No.2003CCB00200)National Natural Science Foundation of China(No.10335050)
文摘In this paper, the relationship between radiosensitivity, cell cycle alteration and the change of apoptosis in different human hepatoma cell lines irradiated by heavy ions were studied with the aim of building up the base data for clinical therapy. Exponentially growing hepatoma cell lines were irradiated by 80.55 MeV/u12C6+ ions at a dose of 0 Gy, 0.5 Gy, 1 Gy, 2 Gy, 4 Gy and 8 Gy. The radiosensitivity was assessed by means of the colony-forming assay. The DNA content, the percentage of each cell-cycle phase and the apoptosis rate were obtained with flow cytometry methods. After the irradiation, the SF2 (survival fraction at 2 gray) of SMMC-7721 cells were evidently lower than that of HepG2 cells. The S phase arrest, G2/M phase arrest delay and the apoptosis in the two hepatoma cell lines varied with the increase of the dose and repair time. The heavy ions could obviously kill the human hepatoma cell lines. Compared to HepG2 cells, SMMC-7721 cells were more radiosensitive to 12C^6+ ions.
基金the National Natural Science Foundation of China (No.50472040)
文摘Many particles are found in the cytoplasm area after the mixture of hydroxyapatite (HAP) nanoparticles and cultured cancer cells. The purpose of this study was to confirm whether these particles in cytoplasm are HAP nanoparticles exactly. BEL7402 cells were incubated in HAP sol for 8 hours. Then, the cells were collected for specimen preparation. Transmission electron microscope (TEM), energy dispersing spectrum (EDS) and electronic diffraction (ED) attached to TEM were used to detect the properties of the particles. It is found that many particles similar to HAP in shape are in the cytoplasm under TEM. By EDS analysis, they are the particles containing calcium (Ca) and phosphorus (P). The classic rings of HAP crystal appear in the ED pictures of these particles. So the particles are confirmed as HAP nanoparticles. Thus, it is concluded that HAP nanoparticles as the crystal particles can be absorbed by hepatoma cells.
文摘It is reported thut nanoporticles can be applied as carriers and anti-cancer medicines. But the interaction of nanoparticles and cells is unclear. The purpose of this study was to discuss whether inorganic crystal nanoparticles can get through cells with intact crystal. BEL7402 heputoma cells and titanium dioxide ( TiO2 ) nanoporticles were selected and incubated together in vitro. All specimens were prepared and observed under a traasmission electron mieroscope (TEM). TiO2 nanopartieles were found not in the nuclear area but in the cytoplasma. TiO2 nanoponieles maintained the plate-like shape during absorbing. The result shows that hepatoma cells can endocytose the intact TiO2 crystal nanoparticles. It implies that novel nano-effect plays an important role in the biomedicinal application of inorganic crystal nanopartieles.
文摘It's reported that hepatic stimulator substance (HSS) was extracte from the fetal liver of 4 - 6 months of fetus, and that the effect of HSS on the proliferation of human Alexender hepatoma cells was studied in this paper. The results showed that proliferation of Alexender cells varied with the amount of HSS in the culture medium, and the former was positively correlated with the latter significantly (P<0. 01). The study indicated that HSS from the fetal liver can stimulate the proliferation of human Alexender hepatoma cells.
文摘Having been passed for 160 generations, a cell linedesignated as H22-F25/L was established from a murine tumorlymphatic metastatlc model H22-F25 which had been set up in our college. The cell line was in suspension culture with a rapid proliferation and stable growth. The peak tune of cell division and proliferation was 48 and 96 hours after culture. In a week, the cell number was Increased by 25 tunes. H22-F25/L still keeps the features of a poorly differentiated cancer. Its tumor inducing rate (in vivo)was 100% in 615 mice. Lymph node metastasis rate was 50% and pulmonary metastasis rate 10%. H22- F25/ L Is a population of heterogenetlc tumor cells Including 2 stem cell lines (the model number of chromosomes being 43 in 40% tumor cells and 86 in 32%) and some side lines. The common marker chromosomes M1, M2, M3 and M4 were present in all stem and side lines.
基金Supported by Natural Science Foundation of Guangdong Province People’s Republic of China,No. 10151008901000182
文摘AIM:To study the effects of hypoxia-inducible factor1α(HIF-1α) silencing on the proliferation of hypoxic CBRH-7919 rat hepatoma cells.METHODS:The CBRH-7919 rat hepatoma cell line was used in this study and the hypoxic model was constructed using CoCl2.The HIF-1α-specific RNAi sequences were designed according to the gene coding sequence of rat HIF-1α obtained from GeneBank.The secondary structure of the HIF-1α gene sequence was analyzed using RNA draw software.The small interfering RNA(siRNA) transfection mixture was produced by mixing the siRNA and Lipofectamine2000TM,and transfected into the hypoxic hepatoma cells.Real time reverse transcription-polymerase chain reaction(RTPCR) and Western blotting assay were used to detect the expression levels of mRNA and protein.HIF-1α and vascular endothelial growth factor(VEGF) mRNA was determined using real time RT-PCR;the protein expression levels of AKT,p-AKT,p21 and cyclinD1 were determined using Western blotting.The proliferation of hepatoma cells was observed using the methyl thiazolyl tetrazolium(MTT) assay and the bromodeoxyuridine(BrdU) incorporation cell proliferation assay.RESULTS:Under induced hypoxia,the viability of the hepatoma cells reached a minimum at 800 μmol/L CoCl2;the viability of the cells was relatively high at CoCl2 concentrations between 100 μmol/L and 200 μmol/L.Under hypoxia,the mRNA and protein expression levels of HIF-1α and VEGF were significantly higher than that of hepatoma cells that were cultured in normaxia.HIF-1α-specific RNAi sequences were successfully transfected into hepatoma cells.The transfection of specific siRNAs significantly inhibited the mRNA and protein expression levels of HIF-1α and VEGF,along with the protein expression levels of p-AKT and cyclinD1;the protein expression of p21 was significantly increased,and there was no significant difference in the expression of AKT.The MTT assay showed that the amount of hepatoma cells in S phase in the siRNA transfection group was obviously smaller than that in the control group;in the siRNA transfection group,the amount of hepatoma cells in G1 phase was more than that in the control group.The BrdU incorporation assay showed that the number of BrdU positive hepatoma cells in the siRNA transfection group was less than that in the control group.The data of the MTT assay and BrdU incorporation assay suggested that HIF-1α silencing using siRNAs significantly inhibited the proliferation of hepatoma cells.CONCLUSION:Hypoxia increases the expression of HIF-1α,and HIF-1α silencing significantly inhibits the proliferation of hypoxic CBRH-7919 rat hepatoma cells.
基金Supported by the National Natural Science Foundation of China,No. 39770164
文摘AIM: To investigate the possibility of recombinant highdensity lipoprotein (rHDL) being a carrier for delivering antitumoral drug to hepatoma cells.METHODS: Recombinant complex of HDL and aclacinomycin(rHDL-ACM) was prepared by cosonication of apoproteins from HDL (Apo HDL) and ACM as well as phosphatidylcholine.Characteristics of the rHDL-ACM were elucidated by electrophoretic mobility, including the size of particles,morphology and entrapment efficiency. Binding activity of rHDL-ACM to human hepatoma cells was determined by competition assay in the presence of excess native HDL. The cytotoxicity of rHDL-ACM was assessed by MTT method.RESULTS: The density range of rHDL-ACM was 1.063-1.210g/mL, and the same as that of native HDL. The purity of all rHDL-ACM preparations was more than 92%.Encapsulated efficiencies of rHDL-ACM were more than90%. rHDL-ACM particles were typical sphere model of lipoproteins and heterogeneous in particle size. The average diameter was 31.26±5.62 nm by measure of 110rHDL-ACM particles in the range of diameter of lipoproteins.rHDL-ACM could bind on SMMC-7721 cells, and such binding could be competed against in the presence of excess native HDL. rHDL-ACM had same binding capacity as native HDL. The cellular uptake of rHDL-ACM by SMMC-7721 hepatoma cells was significantly higher than that of free ACM at the concentration range of 0.5-10 μg/mL(P<0.01). Cytotoxicity of rHDL-ACM to SMMC-7721 cells was significantly higher than that of free ACM at concentration range of less than 5 μg/mL (P<0.01) and IC50 of rHDL-ACM was lower than IC50 of free ACM(1.68 nmol/L vs3 nmol/L). Compared to L02 hepatocytes,a normal liver cell line, the cellular uptake of rHDL-ACM by SMMC-7721 cells was significantly higher (P<0.01) and in a dose-dependent manner at the concentration range of 0.5-10 μg/mL. Cytotoxicity of the rHDL-ACM to SMMC-7721 cells was significantly higher than that to L02 cells at concentration range of 1-7.5 μg/mL (P<0.01). IC50 for SMMC-7721 cells (1.68 nmol/L) was lower than that for L02 cells (5.68 nmol/L), showing a preferential cytotoxicity of rHDL-ACM for SMMC-7721 cells.CONCLUSION: rHDL-ACM complex keeps the basic physical and biological binding properties of native HDL and shows a preferential cytotoxicity for SMMC-7721hepatoma to normal L02 hepatocytes. HDL is a potential carrier for delivering lipophilic antitumoral drug to hepatoma cells.
文摘A non-radioisotopic, quantitative TRAP-based telomerase activity assay was established mainly by using SYBR Green-I staining instead of radioisotope. Comparing with conventional radioisotope based method, it was better in reproducibility and accuracy. Using this method, we found telomerase activities were absent in normal human liver cells, while detected in ail of four human hepatoma cell lines (BEL-7404, SMMC-7721, QGY-7903 and HCCM) without significant differences.
文摘AIM: To investigate the apoptotic effect of photoexcited titanium dioxide (TiO2) nanoparticles in the presence of visible light on human hepatoma cell line (Bel 7402) and to study the underlying mechanism. METHODS: Cerium-element-doped titanium dioxide nanoparticles were prepared by impregnation method. Bel 7402 human hepatoma cells were cultured in RPMI 1640 medium in a humidified incubator with 50 mL/L CO2 at 37℃. A 15 W fluorescent lamp with continuous wavelength light was used as light source in the photocatalytic test. Fluorescence morphology and agarose gel eletrophoresis pattern were performed to analyze apoptotic cells. RESULTS: The Ce (Ⅳ)-doped TiO2 nanoparticles displayed their superiority. The adsorption edge shifted to the 400-450 nm region. With visible light illuminated for 10 min, 10 μg/cm3 Ce (Ⅳ)-doped TiO2 induced micronuclei and significant apoptosis in 4 and 24 h, respectively. Hochest 33 258 staining of the fixed cells revealed typical apoptotic structures (apoptotic bodies), agarose gel electrophoresis showed typical DNA ladder pattern in treated cells but not in untreated ones. CONCLUSION: Ce (Ⅳ) doped TiO2 nanoparticles can induce apoptosis of Bel 7402 human hepatoma cells in the presence of visible light.
基金Supported by the National Natural Science Foundation of China, No.19972077 and No.10372121
文摘AIM: To investigate the adhesive mechanical properties of different cell cycle human hepatoma cells (SMMC-7721)to human umbilical vein endothelial cells (ECV-304),expression of adhesive molecule integrinβ1 in SMMC-7721cells and its contribution to this adhesive course.METHODS: Adhesive force of SMMC-7721 cells to endothelialcells was measured using micropipette aspiration technique.Synchronous G1 and S phase SMMC-7721 cells wereachieved by thymine-2-deoxyriboside and colchicinessequential blockage method and double thymine-2-deoxyriboside blockage method, respectively. Synchronousrates of SMMC-7721 cells and expression of integrinβ1 inSMMC-7721 cells were detected by flow cytometer.RESULTS: The percentage of cell cycle phases of generalSMMC-7721 cells was 11.01% in G2/M phases, 53.51% inG0/G1 phase, and 35.48% in S phase. The synchronous ratesof G1 and S phase SMMC-7721 cells amounted to 74.09%and 98.29%, respectively. The adhesive force of SMMC-7721cells to endothelial cells changed with the variations ofadhesive time and presented behavior characteristics ofadhesion and de-adhesion. S phase SMMC-7721 cells had higheradhesive forces than G1 phase cells [(307.65±92.10)× 10-10Nvs (195.42±60.72)×10-10N, P<0.01]. The expressivefluorescent intensity of integrinβ1 in G1 phase SMMC-7721cells was depressed more significantly than the values ofS phase and general SMMC-7721cells. The contribution ofadhesive integrinβ1 was about 53% in this adhesive course.CONCLUSION: SMMC-7721 cells can be synchronizedpreferably in G1 and S phases with thymine-2-deoxyribosideand colchicines. The adhesive molecule integrinβ1 expressesa high level in SMMC-7721 cells and shows differences invarious cell cycles, suggesting integrin β1 plays an importantrole in adhesion to endothelial cells. The change of adhesiveforces in different cell cycle SMMC-7721 cells indicatesthat S phase cells play predominant roles possibly whilethey interact with endothelial cells.
基金Supported by National Natural Science Foundation of China, No. 30771905
文摘AIM:To investigate the effect of hepatoma cells on up-regulation of programmed cell death-1 (PD-1),and the function of PD-1 on T cells. METHODS:HepG2 or HepG2.2.1.5 cells were co-cultured with a lymphoma cell line-Jurkat cells. PD-1 expression was detected by flow cytometry. IL-2,INF-γ and IL-10 in culture supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Cytotoxic action of T cells was determined by MTT reduction assay-direct mononuclear cell cytotoxicity assay. RESULTS:The PD-1 expression on Jurkat cells increased by 16.17% ± 2.5% and 17.43% ± 2.2% after HepG2 or HepG2.2.1.5 cells were co-cultured for 48 h. The levels of IL-2,INF-γ and IL-10 in the culture supernatant were 202.9 ± 53.0 pg/mL,88.6 ± 4.6 pg/mL and 63.7 ± 13.4 pg/mL respectively,which were signif icantly higher than those (102.9 ± 53 pg/mL,39.3 ± 4.2 pg/mL,and 34.6 ±13.7 pg/mL) in the control group (P < 0.05). The OD value for MTT assay in the blocking group (0.29 ± 0.06) was significantly higher than that (0.19 ± 0.09) in the control group (P < 0.05). CONCLUSION:PD-1 expression on Jurkat cells is up-regulated by hepatoma cells,cytokines and cytotoxic action are elevated after PD-1/PD-L1 is blocked.
文摘The inhibitory effect of parvovirus H-1 on the colonyforming ability in vitro of QGY-7703, a cultured human hepatoma cell line, and on the formation and growth of its tumors in nude mice was studied. With higher multiplicity of infection (MOI) of H-1 given, survival of the QGY-7703 cells was found to be decreased. H-1 DNA amplification level at 30 h postinfection(p.i.) was detected to be 7.4 times higher than that at 2 h by dispersed cells assay, while the cells were delayed to enter into S phase.Plaques were formed in the indicator cells (new-born human kidney cell line, NBK) by progeny H-1 virus particles released from the infected QGY-7703 cells by infectious cell center assay. The formation of tumors in nude mice by QGY-7703 cells which were injected s c at 2 h postinfection was observed to be prevented in 2 groups with given MOI 25 and 50. The tumor growth of MOI 10 group occurred at a lower exponential rate than that of control,after a 20 d latent period. It was evident that parvovirus H-1 exhibited a direct inhibitory effect on the formation and growth of human hepatoma cells in vivo as well as in vitro.
文摘Effects of antisense epidermal growth factor receptor (EGFR) sequence on apoptotic cell death were examined in a human hepatoma cell line BEL-7404 cells. In the cells of JX-1, a sub clone of BEL-7404 stably transfected with antisense EGFR vector (Cell Research, 3:75, 1993), an enhanced rate (9.5%) of spontaneous apoptosis was detected by flow cytometry, whereas the rates of spontaneous apoptosis in JX-0 cells, a sub-clone of BEL-7404 transfected by control vector, and the parellt BEL-7404 cells were almost equal and about 1.7%. Serum-starvation for 72 h increased the rate of apoptosis of JX-1cells up to 33.7%, while JX-0 and BEL-7404 cells, under the same condition, produced less than 5% of apoptotic cells. Observation with electron microscope demonstrated that condensation and fragmentation of chromatin and formation of apoptotic bodies of ten occurred in JX-1 cells, especially during serumstarvation. These results, combined with the data of DNA fragmentation Elisa test, suggested that antisense EGFR sequence enhances apoptosis in the human hepatoma cells.Comparison of intracellular Ca2+ level and the responsiveness of JX-1 cells to the induced action of EGF and tharpsigargin (TG) treatment with that of control JX-0cells indicated that antisense EGFR might interrupt the EGF/EGFR signaling pathway resulting in the decreass of intracellular Ca2+ pool content as well as the responsiveness of these cells to the extracellular signals. These findings suggest that antisense EGFR either directly or indirectly reglllates Ca2+ storage in endoplasmic reticulum,thereby enhances apoptosis in the hnman hepatoma cells.
文摘Objective Cucurbitacins are the highly oxygenated tetracyclic triterpenes,which are predominantly found in the Cucurbitaceae family but are also present in several other families of the plant kingdom.A number of compounds of this group have been investigated for their cytotoxic,hepatoprotective,anti-inflammatory,cardiovascular and anti-diabetic activities.In China,the cucurbitacin preparation,which contains mostly cucurbitacin B and cucurbitacin E,has been clinically used for the treatment of the primary liver carcinoma.It has been previously reported that cucurbitacin E could produce cytotoxicity against a variety of cancer cells,and various mechanisms were implicated in its cytotoxic effect.The present study is to investigate the effect of cucurbitacin E on hepatoma cells in vitro and in vivo and to study their potential mechanisms of action.Methods The MTT assay was used to assess the viability of human HepG2 and BEL7402 hepatoma cells in vitro after treatment with different concentrations of cucurbitacin E.The cell cycle distribution was determined by flowcytometric analysis after propidium iodide(PI)staining.The cell cycle-related proteins were detected using western blotting analysis.Implanted mouse hepatoma H22 model was built to evaluate the growth inhibitory effect of cucurbitacin E in vivo in mice.Results Our studies found that cucurbitacin E(10-300 nM)produced anti-proliferative effect on human HepG2 and BEL7402 hepatoma cells in vitro without cytotoxicity.According to flowcytometric analysis,cucurbitacin E arrested the cell cycle at G2/M phase in both HepG2 and BEL7402 hepatoma cells after 24 h treatment.Cucurbitacin E induced the decrease in the level of CDK1 protein and the increase in the level of p21 protein,but had no effect on the levels of cyclin A,cyclin B1 and Cdc25C protein.In in vivo anti-tumor experiment,cucurbitacin E had significant inhibitory effects on the growth of mouse H22 hepatoma cells.Conclusions Cucurbitacin E inhibited the proliferation of hepatoma cells in vitro and in vivo,at least in part,through induction of cell cycle arrest at G2/M phase,which was mediated by concomitant upregulation of p21 and downregulation of CDK1.We consider that cucurbitacin E may be useful in the treatment of liver cancer.