Melatonin and Doxorubicin synergistically induce cell apoptosis in human hepatoma cell lines

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.

TGF-β1/SMAD SIGNALING PATHWAY MEDIATES p53-DEPENDENT APOPTOSIS IN HEPATOMA CELL LINES

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.

Inhibitory effect of parvovirus H-1 on the formation of colonies of human hepatoma cell line in vitro and its tumors in nude mice

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.

Growth Inhibition and Apoptosis Induction in Human Hepatoma Cells by Tanshinone Ⅱ_A

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.

Effects of Terminalia arjuna bark extract on apoptosis of human hepatoma cell line HepG2

AIM：To investigate the effects of Terminalia arjuna （T. arjuna） extract on human hepatoma cell line （HepG2） and its possible role in induction of apoptosis.METHODS： Human hepatoma cells were treated with different concentrations of ethanolic extract of T. arjuna and its cytotoxicity effect was measured by trypan blue exclusion method and lactate dehydrogenase leakage assay. Apoptosis was analyzed by light and fluorescence microscopic methods, and DNA fragmentation. The mechanism of apoptosis was studied with expression of p53 and caspase-3 proteins. Glutathione （GSH） content was also measured in HepG2 cells after T. arjuna treatment.RESULTS： T. arjuna inhibited the proliferation of HepG2 cells in a concentration-dependent manner. Apoptotic morphology was observed in HepG2 cells treated with T. arjuna at the concentrations of 60 and 100 mg/L. DNA fragmentation, accumulation of p53 and cleavage of procaspase-3 protein were observed in HepG2 cells after the treatment with T. arjuna. The depletion of GSH was observed in HepG2 cells treated with T. arjuna.CONCLUSION： T. arjuna induced cytotoxicity in HepG2 cells in vitro. Apoptosis of HepG2 cells may be due to the DNA damage and expression of apoptotic proteins. Depletion of GSH may be involved in the induction of apoptosis of HepG2 cells.

Effect of 80.55 MeV//u^(12)C^(6+) Ions on Radiosensitivity and Cell Cycle of Human Hepatoma Cell Lines

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.

EFFECT OF HEPATIC STIMULATOR SUBSTANCE (HSS) EXTRACTED FROM FETAL LIVER ON THE PROLIFERATION OF HUMAN ALEXENDER HEPATOMA CELLS

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.

ESTABLISHMENT OF A MURINE ASCITES HEPATOMA CELL LINE H_(22)-F_(25)/L AND ITS BIOLOGICAL CHARACTERISTICS

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.

Effect of Calmodulin and Voltage-dependent Ca^(2+) Channel on the Proliferation of Heptoma Cells Induced by Epidermal Growth Factor

The effect of thyrosine kinase, calmodulin and voltage-dependent Ca 2+ channel on the proliferation of hepatoma cells induced by EGF was studied. Hepatoma cell line SMMC7721 was cultured in RPMI1640 serum-free medium. DNA synthesis rate of hepatoma cells was measured by 3H-TdR incorporation. 10 -9 mol/L EGF could significantly stimulate the proliferation of hepatoma cells (P<0.05), and this effect might be significantly inhibited by tyrosine kinase inhibitor (P<0.001). Calmodulin inhibitor W-7 had no effect on the basic phase of cultured hepatoma cells (P> 0.05), but it had very significantly inhibitory effect on the proliferation of hepatoma cells induced by EGF (P<0.001). Voltage-dependent Ca 2+ channel inhibitor Varapamil had no inhibition on the proliferation of hepatoma cells induced by EGF (P>0.05). It had no effect on the basic phase of cultured hepatoma cells (P>0.05). It is suggested that tyrosine kinase and Ca 2+-calmodulin-dependent pathway may play a critical role on the proliferation of heptoma cells induced by EGF, and voltage-dependent Ca 2+ channel is independent of the effect of EGF.

In vitro study on the morphology of human blood dendritic cells and LPAK cells inducing apoptosis of the hepatoma cell line

Objective To observe in vitro effects and morphological changes of human peripheral blood dendritic cells (DCs) on the ability of lymphokine and phytohaemagglutininum (PHA) activated killer (LPAK) cells to induce apoptosis of the human hepatoma cell line (BEL-7402， B).Methods Experimental groups were divided into LD group (DCs+L+B), L group (L+B)， D group (DCs+B) and B group. The methods of neutral red uptake, ordinary light microscopy, electron microscopy, TDT mediated X-dUTP nick end labeling (TUNEL) were used. Results The difference between the D group and the B group was not distinct (P>0.05). The difference between the LD group and the L group was distinct， with DCs+LPAK >LPAK (P<0.01) in cytotoxity. Apoptotic cells were TUNEL positive in light microscopy, and apoptotic nuclei were stained yellow brown and dark brown, with size and shape varying from cell to cell. Ultrastructural change in apoptotic tumor cells comprised of compaction and condensation of nuclear chromatin， and condensation of cytoplasm and apoptotic bodies. At the same time, LPAK cells manifested the characteristics of autophagic apoptosis, and there were some autophagic bodies in it. Conclusions The combination of human blood DCs and LPAK cells could induce apoptosis of BEL-7402 cells effectively, with some LPAK cells manifesting the characteristics of autophagic apoptosis.

Connective tissue growth factor is overexpressed in human hepatocellular carcinoma and promotes cell invasion and growth

AIM:To determine the expression characteristics of connective tissue growth factor(CTGF/CCN2) in human hepatocellular carcinoma(HCC) in histology and to elucidate the roles of CCN2 on hepatoma cell cycle progression and metastasis in vitro.METHODS:Liver samples from 36 patients(who underwent hepatic resection for the first HCC between 2006 and 2011) and 6 normal individuals were examined for transforming growth factor β1(TGF-β1) or CCN2 mRNA by in situ hybridization.Computer image analysis was performed to measure integrated optimal density of CCN2 mRNA-positive cells in carcinoma foci and the surrounding stroma.Fibroblast-specific protein-1(FSP-1) and E-cadherin were examined to evaluate the process of epithelial to mesenchymal transition,α-smooth muscle actin and FSP-1 were detected to identify hepatic stellate cells,and CD34 was measured to evaluate the extent of vascularization in liver tissues by immunohistochemical staining.CCN2 was assessed for its stimulation of HepG2 cell migration and invasion using commercial kits while flow cytometry was used to determine CCN2 effects on HepG2 cell-cycle.RESULTS:In situ hybridization analysis showed that TGF-β1 mRNA was mainly detected in connective tissues and vasculature around carcinoma foci.In comparison to normal controls,CCN2 mRNA was enhanced 1.9-fold in carcinoma foci(12.36 ± 6.08 vs 6.42 ± 2.35) or 9.4-fold in the surrounding stroma(60.27 ± 28.71 vs 6.42 ± 2.35),with concomitant expression of CCN2 and TGF-β1 mRNA in those areas.Epithelial-mesenchymal transition phenotype related with CCN2 was detected in 12/36(33.3%) of HCC liver samples at the edges between carcinoma foci and vasculature.Incubation of HepG2 cells with CCN2(100 ng/mL) resulted in more of the cells transitioning into S phase(23.85 ± 2.35 vs 10.94 ± 0.23),and induced a significant migratory(4.0-fold) and invasive(5.7-fold) effect.TGF-β1-induced cell invasion was abrogated by a neutralizing CCN2 antibody showing that CCN2 is a downstream mediator of TGF-β1-induced hepatoma cell invasion.CONCLUSION:These data support a role for CCN2 in the growth and metastasis of HCC and highlight CCN2 as a potential novel therapeutic target.