端粒酶反义寡聚脱氧核苷酸对人肝癌细胞株端粒酶活性作用的实验研究

观察端粒酶反义寡聚脱氧核苷酸对肝癌细胞株的作用。用端粒酶反义寡聚脱氧核苷酸与肝癌细胞株共同孵育一定时间后 ,观察细胞形态变化、细胞DNA含量的分布 ,测定端粒酶活性。端粒酶反义寡聚脱氧核苷酸能诱导肝癌细胞凋亡 ,抑制端粒酶活性 ,在形态学上表现为细胞膜起泡、染色质固缩、核碎裂、凋亡小体形成 ;电泳呈凋亡特征性Ladder带 ;流式细胞仪分析显示 ,在G1期前出现亚 2倍体凋亡峰 ;端粒酶活性抑制。端粒酶反义寡聚脱氧核苷酸能诱导肝癌细胞凋亡 ,抑制端粒酶活性 ,抑制端粒合成 ,从而抑制肝癌细胞的生长。

Apoptosis of Human Hepatoma Cells Induced by Gynostemma Pentaphyllum Makino

Objective： To identify the proapoptotic effects of Gynostemma pentaphyllum Makino （GpM） on human hepatoma cells. Methods： The effects of GpM on the cell apoptosis of human hepatoma cell line Huh-7 was assessed by flow cytomety. The expression of Bcl-2, Bcl-XL, Bax and Bad molecules in hepatoma cells treated with GpM was detected by Western blot. Results： After treatment with 20 mg/mL GpM for 24 h, 56% of Huh-7 cells were undergoing apoptosis, while cell death was only observed in 12% of humaa fibroblast cells treated with GpM. Western blot demonstrated that, Bcl-2 was markedly decreased in Huh-7 cells treated with GpM. Whereas, Bax was significantly up-regulated in Huh-7 cells treated with GpM. Conclusion： Treatment of human hepatoma cells with GpM induced apoptosis through the down-regulation of Bcl-2, and up-regulation of Bax.

Regulation of Bcl-2 Family in TIP30-Induced Apoptosis in Hepatoblastoma Cells

Objective: To investigate the role of TIP30 in the apoptotic signal pathwayin HepG2, and Hep3B and Hu-7 hepatoblastoma cell lines. Methods: In order to confirm whether TIP30conducted Bcl-2 family was involved in apoptosis signal pathway, MTT assay, in situ 3' end labellingof DNA assay and Western blot were carried out to detect the diverse apoptotic function of TIP30and the regulation of Bcl-2 family. Results: TIP30 induced apoptosis as evidenced by morphologicalchanges in hepatoblastoma cells, which was accompanied by up-regulating Bax and Bad proteins andstimulating them from cytoplasm to mitochondria, and down-regulating Bcl-xl, while it had no effecton the level of Bak protein. Conclusion: TIP30 induced apoptosis partly by modulating the proteinlevels of members of Bcl-2 family in hepatoblastoma cells. Elucidating the mechanism by which TIP30induces cell death might establish it as an anticancer factor.

Experimental Study on Double Blocking of T Lymphocytes Apoptosis Induced by Fas/FasL

Objective： To block the apoptosis of T lymphocytes induced by Fas/FasL in order to establish a method of the large-scale preparation of large amounts of tumor-specific cytoxic T-lymphocytes （CTL）. Methods： Liver cancer cells and tumor infiltrating lymphocytes （TIL） were isolated from FasL positive fresh specimens, and co-cultured. Specific CTL were activated and prepared in the presence of the co-stimulation of monoclonal antibody CD28. Then the blocking and activation of apoptosis of T lymphocytes was activated by soluble Fas receptor, which was detected by cytometry and DNA ladder test simultaneously. The apoptosis-blocking effect was compared with the control group. Furthermore, the changes of T cell proliferation and killing activity were detected by the method of ^3H thymidine incorporation and ^51Cr release test. Results： There was a significant increase in apoptosis rate in unblocking group compared with blocking group and quiescent group, with the unblocking group of 47.82%±0.13%, quiescent group of 3.76%±0.25%, and the blocking group of 8.22%±0.26% respectively （P〈0.01）. T cell-ladder appeared in unblocking group by DNA ladder test. Both the killing ability and proliferation rate of T cells were significantly increased after blocking. There was significant difference among blocking group, unblocking group and quiescent group （P〈0.01）. Conclusion： With this method we obtained large amounts of tumor-specific T lymphocytes, which was able to kill liver cancer cells effectively.

Hot water-extracted Lycium barbarum and Rehmannia glutinosa inhibit proliferation and induce apoptosis of hepatocellular carcinoma cells

AIM： To investigate the effect of hot water-extracted Lydurn barbarum （LBE） and Rehrnannia glutinosa （RGE） on cell proliferation and apoptosis in rat and/or human hepatocellular carcinoma （HCC） cells. METHODS： Rat （H-4-Ⅱ-E） and human HCC （HA22T/ VGH） cell lines were incubated with various concentrations （0-10 g/L） of hot water-extracted LBE and RGE. After 6-24 h incubation, cell proliferation （n = 6） was measured by a colorimetric method. The apoptotic cells （n = 6） were detected by flow cytometry. The expression of p53 protein （n = 3） was determined by SDS-PAGE and Western blotting. RESULTS： Crude LBE （2-5 g/L） and RGE （2-10 g/L） dose-dependently inhibited proliferation of H-4-Ⅱ-E cells by 11% （P 〈 0.05） to 85% （P 〈 0.01） after 6-24 h treatment. Crude LBE at a dose of 5 g/L suppressed cell proliferation of H-4-Ⅱ-E cells more effectively than crude RGE after 6-24 h incubation （P 〈 0.01）. Crude LBE （2-10 g/L） and RGE （2-5 g/L） also dose-dependently inhibited proliferation of HA22T/VGH cells by 14%-43% （P 〈 0.01） after 24 h. Crude LBE at a dose of 10 g/L inhibited the proliferation of HA22T/VGH cells more effectively than crude RGE （56.8% ＋ 1.6% vs 70.3% ＋ 3.1% of control, P = 0.0003 〈 0.01）. The apoptotic cells significantly increased in H-4-Ⅱ-E cells after 24 h treatment with higher doses of crude LBE （2-5 g/L） and RGE （5-10 g/L） （P 〈 0.01）. The expression of p53 protein in H-4-Ⅱ-E cells was 119% and 143% of the control group compared with the LBE-treated （2, 5 g/L） groups, and 110% and 132% of the control group compared with the RGE -treated （5, 10 g/L） groups after 24 h. CONCLUSION： Hot water-extracted crude LBE （2-5 g/L） and RGE （5-10 g/L） inhibit proliferation and stimulate p53-mediated apoptosis in HCC cells.

Knockdown of survivin gene expression by RNAi induces apoptosis in human hepatocellular carcinoma cell line SMMC-7721

AIM: To investigate the survivin gene expression in human hepatocellular carcinoma cell line SMMC-7721 and the effects of survivin gene RNA interference (RNAi) on cell apoptosis and biological behaviors of SMMC-7721 cells. METHODS: Eukaryotic expression vector of survivin gene RNAi and recombinant plasmid pSuppressorNeo-survivin (pSuNeo-SW), were constructed by ligating into the vector, pSupperssorNeo (pSuNeo) digested with restriction enzymes Xba I and Sail and the designed double-chain RNAi primers. A cell model of SMMC-7721 after treatment with RNAi was prepared by transfecting SMMC-7721 cells with the lipofectin transfection method. Strept-avidin-biotin-complex (SABC) immunohistochemical staining and RT-PCR were used to detect survivin gene expressions in SMMC-7721 cells. Flow cytometry was used for the cell cycle analysis. Transmission electron microscopy was performed to determine whether RNAi induced cell apoptosis, and the method of measuring the cell growth curve was utilized to study the growth of SMMC-7721 cells before and after treatment with RNAi. RESULTS: The eukaryotic expression vector of survivin gene RNAi and pSuNeo-SW, were constructed successfully. The expression level of survivin gene in SMMC-7721 cells was observed. After the treatment of RNAi, the expression of survivin gene in SMMC-7721 cells was almost absent, apoptosis index was increased by 15.6%, and the number of cells was decreased in G2/M phase and the cell growth was inhibited. CONCLUSION: RNAi can exert a knockdown of survivin gene expression in SMMC-7721 cells, and induce apoptosis and inhibit the growth of carcinoma cells.

Pro-apoptotic effects of tectorigenin on human hepatocellular carcinoma HepG2 cells

AIM:To investigate the effects of tectorigenin on human hepatocellular carcinoma(HCC)HepG2 cells.METHODS:Tectorigenin,one of the main components of rhizome of Iris tectorum,was prepared by simple methods,such as extraction,filtration,concentration,precipitation and recrystallization.HepG2 cells were incubated with tectorigenin at different concentrations,and their viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.Apoptosis was detected by morphological observation of nuclear change,agarose gel electrophoresis of DNA ladder,and flow cytometry with Hoechst 33342,Annexin V-EGFP and propidium iodide staining.Generation of reactive oxygen species was quantified using DCFH-DA.Intracellular Ca2+was monitored by Fura 2-AM.Mitochondrial membrane potential was monitored using Rhodamine 123.Release of cytochrome c from mitochondria to cytosol was detected by Western blotting.Activities of caspase-3,-8 and-9 were investigated by Caspase Activity Assay Kit.RESULTS:The viability of HepG2 cells treated by tectorigenin decreased in a concentration-and timedependent manner.The concentration that reduced the number of viable HepG2 cells by 50%(IC50)after 12,24 and 48 h of incubation was 35.72 mg/L,21.19 mg/L and 11.06 mg/L,respectively.However,treatment with tectorigenin at 20 mg/L resulted in a very slight cytotoxicity to L02 cells after incubation for 12,24 or 48 h.Tectorigenin at a concentration of 20 mg/L greatly inhibited the viability of HepG2 cells and induced the condensation of chromatin and fragmentation of nuclei.Tectorigenin induced apoptosis of HepG2 cells in a time-and dose-dependent manner.Compared with the viability rate,induction of apoptosis was the main mechanism of the anti-proliferation effect of tectorigenin in HepG2 cells.Furthermore,tectorigenininduced apoptosis of HepG2 cells was associated with the generation of reactive oxygen species,increased intracellular[Ca2+]i,loss of mitochondrial membrane potential,translocation of cytochrome c,and activation of caspase-9 and-3.CONCLUSION:Tectorigenin induces apoptosis of HepG2 cells mainly via mitochondrial-mediated pathway,and produces a slight cytotoxicity to L02 cells.

Octreotide induces caspase activation and apoptosis in human hepatoma HepG2 cells

AIM: To investigate the role of octreotide on cellular proliferation and apoptosis of human hepatoma (HepG2) cells. METHODS: We studied cellular proliferation, apoptosis and the possible internal caspase-mediated apoptosis pathway involved, after treatment of HepG2 carcinoma cells with octreotide in comparison with the apoptosis caused by tumor necrosis factor-α (TNF-α). Activities of caspase-3, caspase-9, caspase-8 and caspase-2 were studied, while apoptosis was investigated through detection of DNA fragmentation and through identification of apoptotic cells with the annexin-V/propidium iodide flow cytometric method. RESULTS: After an initial increase in HepG2 cellular proliferation, a significant inhibition was observed with 10-8 mol/L octreotide, while TNF-α dose-dependently decreased proliferation. Early and late apoptosis was significantly increased with both substances. Octreotide significantly increased caspase-3, caspase-8 and caspase-2 activity. TNF-α signifi cantly increased only caspase-2. Cellular proliferation was decreased after treatment with octreotide or TNF-α alone but, in contrast to TNF-α, octreotide decreased proliferation only at concentrations of 10-8 mol/L, while lower concentrations increased proliferation. CONCLUSION: Our findings are suggestive of caspasemediated signaling pathways of octreotide antitumor activity in HepG2 cells, and indicate that measurements of serum octreotide levels may be important, at least in clinical trials, to verify optimal therapeutic drug concentrations.

WWOX induces apoptosis and inhibits proliferation of human hepatoma cell line SMMC-7721

AIM: To investigate the effects of the WWOX gene on the human hepatic carcinoma cell line SMMC-7721. METHODS: Full-length WWOX cDNA was amplified from normal human liver tissues. Full-length cDNA was subcloned into pEGFP-N1, a eukaryotic expression vector. After introduction of the WWOX gene into cancer cells using liposomes, the WWOX protein level in the cells was detected through Western blotting. Cell growth rates were assessed by methyl thiazolyl tetrazolium (MTT) and colony formation assays. Cell cycle progression and cell apoptosis were measured by flow cytometry. The phosphorylated protein kinase B (AKT) and activated fragments of caspase-9 and caspase-3 were examined by Western blotting analysis. RESULTS: WWOX significantly inhibited cell proliferation, as evaluated by the MTT and colony formation assays. Cells transfected with WWOX showed significantly higher apoptosis ratios when compared with cells transfected with a mock plasmid, and overexpression of WWOX delayed cell cycle progression from G1 to S phase, as measured by flow cytometry. An increase in apoptosis was also indicated by a remarkable activation of caspase-9 and caspase-3 and a dephosphorylation of AKT (Thr308 and Ser473) measured with Western blotting analysis. CONCLUSION: Overexpression of WWOX induces apoptosis and inhibits proliferation of the human hepatic carcinoma cell line SMMC-7721.

Effect of Bcl-2 and Bax on survival of side population cells from hepatocellular carcinoma cells

AIM： To understand the role and significance of side population （SP） cells from hepatocellular carcinoma （HCC） in hepatocarcinogenesis, development, relapse and metastasis, we simulated the denutrition conditions that cancer cells experience in clinical therapy, observed the different anti-apoptosis ability of SP cells and non-SP cells under such conditions, and established the possible effects of P53, Bcl-2 and Bax on survival of SP cells. METHODS： We used flow cytometry to analyze and sort the SP and non-SP cells in established HCC lines MHCC97 and hHCC. We evaluated cell proliferation by methyl thiazolyl tetrazolium （MTT） assay and investigated the expression of p53, bcl-2 and bax genes during denutrition, by RT-PCR and immunofluorescence staining. RESULTS： The percentage of SP cells in the two established HCC lines was 0.25% and 0.5%, respectively. SP cells had greater anti-apoptosis and proliferation ability than non-SP cells. Expression of Bcl-2 and Bax in SP and non-SP cells differed during denutrition. The former was up-regulated in SP cells, and the latter was up-regulated in non-SP cells. CONCLUSION： It may be that different upstream molecules acted and led to different expression levels of Bcl-2 and Bax in these two cell lines. There was a direct relationship between up-regulation of Bcl-2 and down-regulation of Bax and higher anti-apoptosis ability in SP cells. It may be that the existence and activity of SP cells are partly responsible for some of the clinical phenomena which are seen in HCC, such as relapse or metastasis. Further research on SP cells may have potential applications in the field of anticancer therapy.

Lentivirus mediated shRNA interference targeting MAT2B induces growth-inhibition and apoptosis in hepatocelluar carcinoma

AIM: To investigate the effects of lentivirus vector mediated short hairpin RNA interference targeting methionine adenosyltransferase 2β gene (LV-shMAT2B) on hepatocelluar carcinoma (HCC) cells. METHODS: We constructed four plasmids of RNA interference targeting the MAT2B gene. After LV-shMAT2B was transfected with L-02 cells and two kinds of HCC cells, cell viability and proliferation were measured with MTT and [3H]thymidine assays respectively. Flow cytometry was used to assess cell apoptosis. The level of S-adenosyl methionine (SAMe) in HepG2 cells was evaluated. The expressions of cyclin D1, cyclin D2, bcl-xL and bcl-xS were detected with western blot. RESULTS: We constructed LV-shMAT2B successfully. LV-shMAT2B was safe for human normal liver cells. LV-shMAT2B caused dramatic reduction in proliferation compared with controls in HCC cells Bel-7402 (P = 0.054) and HepG2 (P = 0.031). Flow cytometry analysis showed that cell apoptosis caused by LV-shMAT2B was greater in HCC cells Bel-7402 and HepG2 than in control induced by scrambled siRNA (P = 0.047), but apoptosis rates in L-02 induced by LV-shMAT2B and scrambled siRNA respectively had no significant difference. Moreover, LV-shMAT2B significantly suppressed expression of MAT2B leading to growth-inhibition effect on HCC cells by down-regulating cyclin D1. Apoptosis induced by LV-shMAT2B was involved indown-regulating bcl-xL and up-regulating bcl-xS. CONCLUSION: LV-shMAT2B can induce cell apoptosis and growth-inhibition in HCC cells. MAT2B may be a therapy target in HCC in the future.

Synergistic effect of all-trans-ret inoic acid and arsenic trioxide on growth inhibition and apoptosis in human hepatoma, breast cancer, and lung cancer cells in vitro

AIM： To investigate the effect of all-trans-retinoic acid （ATRA） on arsenic trioxide （As2O3）-induced apoptosis of human hepatoma, breast cancer, and lung cancer cells in an attempt to find a better combination therapy for solid tumors. METHODS： Human hepatoma cell lines HepG2, Hep3B, human breast cancer cell line MCF-7, and human lung adenocarcinoma cell line AGZY-83-a were treated with As203 together with ATRA. Cell survival fraction was determined by MTT assay, cell viability and apoptosis were measured by annexin V-fluorescein isothiocyanate （FITC） and PI staining, and intracellular glutathione （GSH） and glutathione-S-transferase （GST） activities were determined using commercial kits. RESULTS： Cytotoxicity of ATRA was low. ATRA （0.1, 1, and 10 μmol/L） could synergistically potentiate As2O3 to exert a dose-dependent inhibition of growth and to induce apoptosis in each of the cell lines. HepG2 and Hep3B with low intracellular GSH or GST activities were remarkably sensitive to As2O3 or As2O3＋ATRA, while AGZY-83-a with higher GSH or GST activities was less sensitive to As2O3 or As2O3＋ATRA. Treatment with 2 μmol/L As2O3 for 72 h significantly decreased intracellular GSH and GST levels in each of the cell lines, and 1 μmol/L ATRA alone reduced minimal intracellular GSH and GST levels. ATRA potentiated the effect of As2O3 on intracellular GSH levels, but intracellular GST levels were not significantly affected by the combination of As2O3 and ATRA for 72 h as compared to As2O3 alone.CONCLUSION： ATRA can strongly potentiate As2O3- induced growth-inhibition and apoptosis in each of the cell lines, and two drugs can produce a significant synergic effect. The sensitivity to As2O3 or As2O3＋ATRA is inversely proportional to intracellular GSH or GST levels in each of the cell lines. The GSH redox system may be the possible mechanism by which ATRA synergistically potentiates As203 to exert a dose-dependent inhibition of growth and to induce apoptosis. 2005 The WJG Press and Elsevier Inc. All rights reserved.

Antiproliferation and apoptosis induction of paeonol in HepG_2 cells

AIM： To investigate the antiproliferative effect of paeonol （Pae） used alone or in combination with chemotherapeutic agents [cisplatin （CDDP）, doxorubicin （DOX） and 5-fluorouracil （5-FU）] on human hepatoma cell line HepG2 and the possible mechanisms. METHODS： The cytotoxic effect of drugs on HepG2 cells was measured by 3-（4, 5-dimethylthiazol-2- yl）-2, 5-diphenyltetra-zolium bromide （MTT） assay. Morphologic changes were observed by acridine orange （AO） fuorescence staining. Cell cycle and apoptosis rate were detected by flow cytometry （FCM）. Drug-drug interactions were analyzed by the coefficient of drug RESULTS： Pae （7.81-250 mg/L） had an inhibitory effect on the proliferation of HepG2 cells in a dose-dependent manner, with the IC50 value of （104.77±7.28） mg/L. AO fluorescence staining and FCM assays showed that Pae induced apoptosis and arrested cell cycle at S phase in HepG2 cells. Further, different extent synergisms were observed when Pae （15.63, 31.25, 62.5 rag/L） was combined with CDDP （0.31-2.5 mg/L）, DOX （0.16-1.25 mg/L）, or 5-FU （12.5-100 mg/L） at appropriate concentrations. The IC50 value of the three drugs decreased dramatically when combined with Pae （P 〈 0.01）. Of the three different combinations, the sensitivity of cells to drugs was considerably different.CONCLUSION： Pae had a significant growth-inhibitory effect on the human hepatoma cell line HepG2, which may be related to apoptosis induction and cell cycle arrest. It also can enhance the cytotoxicity of chemotherapeutic agents on HepG2 cells, and the S phase arrest induced by Pae may be one of the mechanisms of these interactions.

Dynamic expression of apoptosis-related genes during development of laboratory hepatocellular carcinoma and its relation to apoptosis

AIM： To explore the expression of p53, bcl-2, bax, survivin and the cell apoptosis during the development of tree shrew hepatocellular carcinoma （HCC）, the relationship between expression of these genes, its impact on HCC development, and its relation to cell apoptosis. METHODS： Tree shrew HCC was induced with aflatoxin B1 （AFB1）, and regular biopsy of liver tissues was carried out and the biopsy tissues were collected during cancer inducement. Liver biopsy tissue and HCC tissue were collected from 35 pre-cancerous experimental animals at wk 30 and 60 and at the 30^th, 60^th, and 90^th -wk. Liver biopsy tissues were collected from 13 blank control animals at wk 30, 60, and 90. Expression of p53, bcl-2, bax, and survivin at each stage was examined by immunohistochemistry method. Apoptotic cells were detected in situ by the terminal deoxynucleotidyl transferase-mediated nick end labeling （TUNEL） technique. RESULTS： The apoptosis rate of normal hepatic cells was extremely low, whereas it increased during the formation of HCC. Expression of the apoptosis-related genes p53, bcl-2, bax, and survivin during the formation of HCC presented an increasing tendency. Expression of p53 did not noticeably relate to that of bcl-2, bax, and survivin, whereas expression of bcl-2 and bax was closely related. In HCC, p53 did not present a distinct relation to cell apoptosis, whereas its high level expression was probably related to liver cell proliferation. Survivin negatively correlated apoptosis index, and its overexpression could inhibit cell apoptosis. CONCLUSION： Apoptosis-related genes p53, bcl-2, bax, and survivin are all related to the occurrence of HCC. The anti-apoptosis effect of bcl-2 is influenced by bax, and ratio bcl/bax reflects more correctly the extent of cell apoptosis.

Tea pigments induce cell-cycle arrest and apoptosis in HepG2 cells

AIM： To investigate the molecular mechanisms by which tea pigments exert preventive effects on liver carcinogenesis. METHODS： HepG2 cells were seeded at a density of 5×10^5/well in six-well culture dishes and incubated overnight. The cells then were treated with various concentrations of tea pigments over 3 d, harvested by trypsinization, and counted using a hemocytometer. Flow cytometric analysis was performed by a flow cytometer after propidium iodide labeling. Bd-2 and p21^WAF1 proteins were determined by Western blotting. In addition, DNA laddering assay was performed on treated and untreated cultured HepG2 cells. RESULTS： Tea pigments inhibited the growth of HepG2 cells in a dose-dependent manner. Flow-cytometric analysis showed that tea pigments arrested cell cycle progression at G1 phase. DNA laddering was used to investigate apoptotic cell death, and the result showed that 100 mg/L of tea pigments caused typical DNA laddering. Our study also showed that tea pigments induced upregulation of p21^WAF1 protein and downregulation of Bcl-2 protein. CONCLUSION： Tea pigments induce cell-cycle arrest and apoptosis. Tea pigments may be used as an ideal chemopreventive agent.

NT4(Si)-p53(N15)-antennapedia induces cell death in a human hepatocellular carcinoma cell line

AIM： To construct the recombinant lentivirus expression plasmid, pLenti6/V5-NT4 p53（N 15）-antennapedia （Ant）, and study its effect on HepG2 cells. METHODS： Plasmid pLenti6/V5-NT4 p53（N15）-Ant was constructed incorporating the following functional regions, including signal peptide sequence and proregion of neurotrophin 4, N-terminal residues 12-26 of p53 and 17 amino acid drosophila carrier protein, Ant. Hepatocellular carcinoma （HepG2） cells were used for transfection. 3-[4,5-climethyl-thiazol-2yl]-2,5 diphenyl tetrazolium bromide （MI-I） assay, lactate dehydrogenase （LDH） release assay, transmission electron microscopy （TEM） and flow cytometric analysis （FCM） were employed to investigate the effects of LV-NT4（Si）- p53（N15）-Ant in vitro on HepG2 cells. In vivo experiment was also performed to investigate the inhibitory effect of LV-NT4（Si）-p53（N15）-Ant on tumor growth in nude mice.RESULTS： LV-NT4（Si）-p53（N15）-Ant significantly suppressed the growth of HepG2 cells. MTT assay showed that the growth of HepG2 cells was mucj more significantly inhibited by LV-NT4（Si）-p53（N15）-Ant than by LV-EGFP. The inhibition rate for HepG2 cell growth in the two groups was 46.9% and 94.5%, respectively, 48 h after infection with LV-NT4（Si）-p53（N15）-Ant, and was 33.9% and 95.8%, respectively, 72 h after infection with LV-NT4（Si）-p53（N15）-Ant （P 〈 0.01）. Light microscopy and TEM showed morphological changes in HepG2 cells infected with LV-NT4（Si）-p53（N15）-Ant, but no significant changes in HepG2 cells infected with LV-EGFR Changes were observed in ultra-structure of HepG2 cells infected with LV-NT4（Si）-p53（N15）-Ant, with degraded membranes, resulting in necrosis. LDH release from HepG2 cells was analyzed at 24, 48, 72 and 96 h after infection with LV-NT4（Si）-p53（N15）-Ant and LV-EGFP, which showed that LDH release was significantly higher in LV-NT4（Si）-p53（N15）-Ant treatment group （682 IU/L） than in control group （45 IU/L, P 〈 0.01）. The longer the time was after infection, the bigger the difference was in LDH release. FCM analysis showed that LV-NT4（Si）- p53（N15）-Ant could induce two different kinds of cell death： necrosis and apoptosis, with apoptosis being the minor type and necrosis being the main type, suggesting that LV-NT4（Si）-p53（N15）-Ant exerts its anticancer effect on HepG2 cells by inducing necrosis. The in vivo study showed that LV-NT4（Si）-p53（N15）-Ant significantly inhibited tumor growth with an inhibition rate of 66.14% in terms of tumor size and weight. CONCLUSION： LV-NT4（Si）-p53（N15）-Ant is a novel recombinant lentivirus expression plasmid and can be used in gene therapy for cancer.

Nano-cerium-element-doped titanium dioxide induces apoptosis of Bel 7402 human hepatoma cells in the presence of visible light

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 COL 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/cm^3 Ce （lV）-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.

Effect of ethanol on pro-apoptotic mechanisms in polarizedhepatic cells

Chronic ethanol consumption is associated with serious and potentially fatal alcohol-related liver injuries such as hepatomegaly, alcoholic hepatitis and cirrhosis. Moreover, it has been documented that the clinical progression of alcohol-induced liver damage may be associated with an increase in hepatocellular death that involves apoptotic mechanisms. Although much information has been learned about the clinical manifestations associated with alcohol-related diseases, the search continues for a better understanding of the molecular and/or cellular mechanisms by which ethanol exerts its deleterious effects such as the induction of pro-apoptotic mechanisms and related cell damaging events. As part of the effort to enhance our understanding of those particular cellular pathways and mechanisms associated with ethanol toxicity, researchers over the years have utilized a variety of model systems. Recently, work has come forth demonstrating the utility of a hybrid cell line (WIF-B) as a cell culture model system for the study of alcohol-associated alterations in hepatocellular mechanisms. Success with such emerging model systems could aid in the development of potential therapeutic treatments for the prevention of alcohol- induced apoptotic cell death that may ultimately serve as a significant target in delaying the onset and/or progression of clinical symptoms of alcohol-mediated liver disease. This review article summarizes the current understanding of ethanol-mediated modifications in cell survival and thus the promotion of pro-apoptotic events with emphasis on analyses made in various experimental model systems, particularly the more recently characterized WIF-B cell system.

Secretory Transactivating Transcription-apoptin fusion protein induces apoptosis in hepatocellular carcinoma HepG2 cells

AIM:To determine whether SP-TAT-apoptin induces apoptosis and also maintains its tumor cell specificity. METHODS:In this study,we designed a secretory protein by adding a secretory signal peptide(SP) to the N terminus of Transactivating Transcription(TAT)-apoptin(SP-TAT-apoptin),to test the hypothesis that it gains an additive bystander effect as an anti-cancer therapy. We used an artificial human secretory SP whose amino acid sequence and corresponding cDNA sequence were generated by the SP hidden Markov model. RESULTS:In human liver carcinoma HepG2 cells,SP-TAT-apoptin expression showed a diffuse pattern in the early phase after transfection. After 48 h,however,it translocated into the nuclear compartment and caused massive apoptotic cell death,as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay and annexin-V binding assay. SP-TAT-apoptin did not,however,cause any cell death in non-malignant human umbilical vein endothelial cells(HUVECs). Most importantly,the conditioned medium from Chinese hamster ovary(CHO) cells transfected with SP-TAT-apoptin also induced significant cell deathin HepG2 cells,but not in HUVECs. CONCLUSION:The data demonstrated that SP-TAT-apoptin induces apoptosis only in malignant cells,and its secretory property might greatly increase its potency once it is delivered in vivo for cancer therapy.

Casticin-induced apoptosis involves death receptor 5 upregulation in hepatocellular carcinoma cells

AIM:To investigate the apoptotic activities of casticin in hepatocellular carcinoma(HCC) cells and its molecular mechanisms.METHODS:PLC/PRF/5 and Hep G2 cell lines were cultured in vitro and the inhibitory effect of casticin on the growth of cells was detected by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolim bromide(MTT) assay.The apoptotic cell death was examined using the cell apoptosis enzyme linked immunosorbent assay(ELISA) detection kit,flow cytometry(FCM) after propidium iodide(PI) staining and DNA agarose gel electrophoresis.The caspase activities were measured using ELISA.Reactive oxygen species(ROS) production was evaluated by FCM after dichlorodihydrofluorescein diacetate(DCFH-DA) probe labeling.Intracellular glutathione(GSH) content was measured using a glutathione assay kit.The expression of death receptor(DR)4 and DR5 proteins was analyzed by Western blotting and FCM.RESULTS:Casticin significantly inhibited the growth of human HCC(PLC/PRF/5 and Hep G2) cells in a dosedependent manner(P < 0.05).Casticin increased the percentage of the sub-G1 population in HCC cells in a concentration-dependent manner.The potency of casticin to PLC/PRF/5 cells was higher than that of 5-flurouracil(26.8% ± 4.8% vs 17.4% ± 5.1%) at 10 μmol/L for 24 h.Casticin increased the levels of Histone/DNA fragmentation and the levels of active caspase-3,-8 and-9 in a concentration-dependent manner(P < 0.05).Treatment with 30 μmol/L casticin for 24 h resulted in the formation of a DNA ladder.Casticin reduced the GSH content(P < 0.05),but did not affect the level of intracellular ROS in PLC/PRF/5 and Hep G2 cells.The thiol antioxidants,acetylcysteine(NAC) and GSH restored GSH content and attenuated casticin-induced apoptosis.In contrast,the nonthiol antioxidants,butylated hydroxyanisole and mannitol failed to do so.In the HCC cells treated with casticin for 24 h,DR5 protein level was increased.The expression of DR5 protein induced by casticin was inhibited by NAC.Pretreatment with DR5/Fc chimera protein,a blocking antibody,effectively attenuated the induction of apoptosis by casticin.CONCLUSION:Casticin-induced apoptosis of HCC cells is involved in GSH depletion and DR5 upregulation.