18α-甘草酸二铵对丝裂霉素C诱导的HepG2细胞凋亡的影响及机制

目的探讨18α-甘草酸二铵对丝裂霉素(MMC)诱导的HepG2细胞凋亡的影响及机制。方法 18α-甘草酸二铵不同浓度与低剂量MMC(50μg/ml)MMC联合作用,采用MTT比色法观察MMC对HepG2细胞活性率的影响;流式细胞术分析凋亡细胞的百分率、线粒体膜电位的改变及Caspase-3的活性变化;Western Blot检测细胞内细胞色素C的表达及凋亡相关蛋白Bcl-2/Bax的表达情况。结果单独MMC(50μg/ml)处理的HepG2细胞活性率明显降低,凋亡率显著增加,线粒体膜电位下降,Caspase-3活性增加,细胞内细胞色素C表达增加,抗凋亡蛋白Bcl-2表达减少,而促凋亡蛋白Bax表达增加。不同浓度的18α-甘草酸二铵与MMC合用组,这些指标的变化均受到抑制。结论 18α-甘草酸二铵可抑制MMC诱导的HepG2细胞凋亡,其机制可能通过调控凋亡相关蛋白的表达而实现。

Survivin antisense compound inhibits proliferation and promotes apoptosis in liver cancer cells

AIM: To evaluate the effects of survivin on cell proliferation and apoptosis in liver cancer. METHODS: MTT assay was used to generate and optimize phosphorothioate antisense oligonucleotides (ODNs)-LipofectamineTM2000 (LiP) compound by varying ODNs (μg): LiP (μL) ratios from 1:0.5 to 1:5. Then, liver cancer cells (HepG2) were transfected with the compound. By using RT-PCR and Western blot, the expression levels of survivin mRNA and proteins were detected in HepG2 cells treated with antisense compounds (ODNs:LiP=1:4), and compared with those treated with sense compounds (1:4) as control. MTT assay was applied to the determination of cell proliferation in HepG2 cells. Active caspase-3 was evaluated by flow cytometric analysis. The morphological changes were assessed by electron microscopy. Laser scanning confocal microscopy was performed to detect the subcellular localization of survivin proteins in treated and untreated cells. RESULTS: Antisense compounds (1:4) down-regulated survivin expression (mRNA and protein) in a dose-dependent manner with an IC50 of 250 nmol/L. Its maximum effect was achieved at a concentration of 500 nmol/L, at which mRNA and protein levels were down-regulated by 80%. The similar results were found in MTT assay. Antisense compound (l:4)-treated cells revealed increased caspase-3-like protease activity compared with untreated cells. Untreated cells as control were primarily negative for the presence of active-caspase-3. As shown by transmission electron microscopy, treated cells with antisense compounds (1:4) resulted in morphological changes such as blebbing and loss of microvilli, vacuolization in the cytoplasm, condensation of the cytoplasm and nuclei, and fragmented chromatin. Immunofluorescence analysis confirmed the presence of survivin protein pool inside the cytoplasm in untreated cells. Labeled-FITC immunofluorescence staining of survivin clearly showed that survivin was distributed mainly in a spotted form inside the cytoplasm. Whereas cells treated with antisense compounds were rare and weak inside the cytoplasm. CONCLUSION: Down-regulation of survivin expression induced by the antisense compounds reduces tumor growth potential, promotes apoptosis and affects the localization of survivin proteins in HepG2 cells. Furthermore, survivin protein is a key molecule associated with proliferation and apoptosis, and antisense oligonucleotides targeting survivin have a bright prospect in the therapy of liver cancer.

Alpha-fetoprotein triggers hepatoma cells escaping from immune surveillance through altering the expression of Fas/FasL and tumor necrosis factor related apoptosis-inducing ligand and its receptor of lymphocytes and liver cancer cells

AIM: To investigate the mechanism of α-fetoprotein (AFP)in escaping from the host immune surveillance of hepatocellular carcinoma.METHODS: AFP purified from human umbilical blood was administrated into the cultured human lymphoma Jurkat T cell line or hepatoma cell line, Bel7402 in vitro. The expression of tumor necrosis factor related apoptosisinducing ligand (TRAIL) and its receptor (TRAILR) mRNA were analyzed by Northern blot and Western blot wasused to detect the expression of Fas and Fas ligand (FasL)protein.RESULTS: AFP (20 mg/L) could promote the expression of FasL and TRAIL, and inhibit the expression of Fas and TRAILR of Bel7402 cells. For Jurkat cell line, AFP could suppress the expression of FasL and TRAIL, and stimulate the expression of Fas and TRAILR. AFP also could synergize with Bel7402 cells to inhibit the expression of FasL protein and TRAIL mRNA in Jurkat cells. The monoclonal antibody against AFP (anti-AFP) could abolish these functions of AFP.CONCLUSION: AFP is able to promote the expression of FasL and TRAIL in hepatoma cells and enhance the expression of Fas and TRAILR in lymphocytes. These could elicit the escape of hepatocellular carcinoma cells from the host's lymphocytes immune surveillance.

Fiber-modified adenoviral vector expressing the tumor necrosis factor-related apoptosis-inducing ligand gene from the human telomerase reverse transcriptase promoter induces apoptosis in human hepatocellular carcinoma cells

AIM: Because of a major resistance to chemotherapy, prognosis of hepatocellular carcinoma (HCC) is still poor. New treatments are required and gene therapy may be an option. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in multiple malignant tumors, and using adenoviral vectors has shown a targeted tumor-specific therapy. However, repeated administration of adenoviral vectors can lead to cell resistance, which may be caused by the initial coxsackie-adenovirus receptor (CAR). One technique to overcome resistance is the use of modified adenoviral vectors containing an Arg-Gly-Asp (RGD) sequence. In this study we constructed an adenoviral vector (designated Ad/TRAIL-F/RGD) with RGD-modified fibers, expressing the TRAIL gene from the human telomerase reverse transcriptase (hTERT) promoter, and evaluated its antitumor activity in HCC cell lines.METHODS: To investigate the effects of Ad/TRAIL-F/RGD in human HCC cell lines Hep G2 and Hep 3b, cells were infected with Ad/CMV-GFP (vector control), Ad/gTRAIL (positive control), and Ad/TRAIL-F/RGD. Phosphatebuffered saline (PBS) was used as control. Cell viability was determined by proliferation assay (XTT), and apoptosis induction by fluorescence activated cell sorting (FACS).RESULTS: Cells treated with Ad/TRAIL-F/RGD and Ad/ gTRAIL showed a significantly reduced cell viability in comparison to PBS and Ad/CMV-GFP treatment in both cell lines. Whereas, treatment with PBS and Ad/CMVGFP had no cell-killing effect. The reduced cell viability was caused by induction of apoptosis as shown by FACS analysis. The amount of apoptotic cells was similar after incubation with Ad/gTRAIL and Ad/TRAIL-F/RGD. CONCLUSION: The new RGD modified vector Ad/TRAILF/RGD could become a potent therapeutic agent for the treatment of HCC, adenovirus resistant tumors, and CAR low or negative cancer cells.

Hepatic progenitor cells in human liver tumor development

In recent years, the results of several studies suggest that human liver tumors can be derived from hepatic progenitor cells rather than from mature cell types. The available data indeed strongly suggest that most combined hepatocellular-cholangiocarcinomas arise from hepatic progenitor cells that retained their potential to differentiate into the hepatocytic and biliary lineages. Hepatic progenitor cells could also be the basis for some hepatocellular carcinomas and hepatocellular adenomas, although it is very difficult to determine the origin of an individual hepatocellular carcinoma. There is currently not enough data to make statements regarding a hepatic progenitor cell origin of cholangiocarcinoma. The presence of hepatic progenitor cell markers and the presence and extent of the cholangiocellular component are factors that are related to the prognosis of hepatocellular carcinomas and combined hepatocellular- cholangiocarcinomas, respectively.

Tumor vaccine against recurrence of hepatocellular carcinoma

AIM: To investigate the effects of autologous tumor vaccine on recurrence of hepatocellular carcinoma (HCC). METHODS: Sixty patients with HCC who had undergone curative resection, were randomly divided into HCC vaccine group and control group. Three vaccinations at 2-wk intervals were performed after curative hepatic resection. Delayed-type- hypersensitivity (DTH) test was performed before and after vaccination. Primary endpoints were the time of recurrence. RESULTS: Four patients in control group and 6 patients in HCC vaccine group were withdrawn from the study. The vaccine containing human autologous HCC fragments showed no essential adverse effect in a phase II clinical trial and 17 of 24 patients developed a DTH response against the fragments. Three of 17 DTH-positive response patients and 5 of 7 DTH- negative response patients had recurrences after curative resection. After the operation, 1-, 2- and 3-year recurrence rates of HCC vaccine group were 16.7%, 29.2% and 33.3%, respectively. But, 1-, 2- and 3-year recurrence rates of the control group were 30.8%, 53.8% and 61.5%, respectively. The time before the first recurrence in the vaccinated patients was significantly longer than that in the control patients (P<0.05). CONCLUSION: Autologous tumor vaccine is of promise in decreasing recurrence of human HCC.

Significance and relationship between infiltrating inflammatory cell and tumor angiogenesis in hepatocellular carcinoma tissues

AIM: To investigate the relationship between infiltrating inflammatory cell and tumor angiogenesis in hepatocellular carcinoma (HCC) tissues and their clinicopathological features.METHODS: The paraffin-embedded specimens from 70 cases with HCC were stained using EliVision immunohistochemistry with mAbs against CD68, tryptase,and CD34. The counts of tumor-associated macrophage (TAM), mast cell (MC) and tumor microvessel (MV) were performed in the tissue sections.RESULTS: The mean counts of TAM, MC, and MV in HCC tissues were significantly higher than those in pericarcinomatous liver tissues (TAM: 69.31± 11.58 vs 40.23±10.36; MC: 16.74±5.67 vs 7.59±4.18; MV:70.11±12.45 vs 38.52± 11.16, P＜0.01). The MV count in the patients with metastasis was markedly higher than that with non-metastasis (P＜0.01). In addition, the MC count in the patients with poorly differentiated HCC was obviously higher than that with well differentiated HCC (P＜ 0.01). The correlation analysis showed that the TAM count was significantly correlated with the count of MV(r=0.712, P＜0.01), and the MC count was obviously correlated with the MV count (r= 0.336, P＜ 0.05).CONCLUSION: TAM and MC might be closely related to the enhancement of tumor angiogenesis. The MV count might be associated with tumor invasion and metastasis.Moreover, the MC count might be associated with tumor differentiation and prognosis of HCC.

Inhibition of hepatic tumor cell proliferation in vitro and tumor growth in vivo by taltobulin,a synthetic analogue of the tripeptide hemiasterlin

AIM： To investigate the inhibitory effects of taltobulin （HTI-286）, a synthetic analogue of natural hemiasterlin derived from marine sponges, on hepatic tumor growth in vitro and in vivo. METHODS： The potential anti-proliferative effects of HTI-286 on different hepatic tumor cell lines in vitro and in vivo were examined. RESULTS： HTI-286 significantly inhibited proliferation of all three hepatic tumor cell lines （mean IC50 = 2 nmol/L ±1 nmol/L） in vitro. Interestingly, no decrease in viable primary human hepatocytes （PHH） was detected under HTI-286 exposure. Moreover, intravenous administration of HTI-286 significantly inhibited tumor growth in vivo （rat allograft model）. CONCLUSION： HFI-286 might be considered a potent promising drug in treatment of liver malignancies. HTI-286 is currently undergoing clinical evaluation in cancer patients.

Tissue microarray for high-throughput analysis of gene expression profiles in hepatocellular carcinoma

AIM: To study the expression profiles of HBsAg, HBcAg,p21WAF1/CIP1 (p21), Rb genes in hepatocellular carcinoma (HCC) and to investigate their roles in the hepatocarcinogenesis.METHODS: HCC tissue microarray containing 120-min tissues of 40 HCC cases was constructed. HBsAg, HBc Ag,p21 and Rb proteins were immunohistochemically stained by streptavidin-peroxidase conjugated method (S-P). The expression loss of these genes in cancerous, paracancerous tissues and adjacent normal liver tissues of 40HCCs were comparatively examined.RESULTS: The positive rate of HBsAg expression in cancerous tissues of 40 HCCs was 7.5%, which was lower than that in para-cancerous and adjacent normal liver tissues (x2 =12.774, P＜0.01; x2= 18.442, P＜0.01). The positive rate of HBcAg expression in cancerous tissues of 40 HCCs was 20.0%, which was also lower than that in para-cancerous and adjacent normal liver tissues(x2= 9.482, P＜0.01; x2= 14.645, P＜0.01). p21 protein deletion rate in cancerous tissues of 40 HCCs was 27.5%,which was higher than that in para-cancerous and adjacent normal liver tissues (x2 = 7.439, P＜0.01; x2 = 11.174,P＜0.01). p21 protein deletion correlated remarkably with the pathological grade of HCC (x2 = 0.072, P＜0.05). Rb protein deletion rate in cancerous tissues of 40 HCCs was42.5%, which was also higher than that in para-cancerous and adjacent normal liver tissues (x2 = 10.551, P＜0.01;x2= 18.353, P＜0.01). Rb protein deletion rate did not correlate remarkably with tumor size or pathological grade of HCC (x2 = 0.014, P＞0.05; x2 = 0.017, P＞0.05).CONCLUSION: Expression deletion of HBsAg, HBcAg, p21and Rb proteins in HCCs may play important roles in the carcinogenesis of HCC. Tissue microarray is an effective high-throughput technique platform for cancer research.

Augmentation of tumor necrosis factor family-induced apoptosis by E3330 in human hepatocellular carcinoma cell lines via inhibition of NFκB

AIM- To investigate the reduction of cell viability in human hepatocellular carcinoma （HCC） cell lines induced by inhibition of nuclear factor kB （NFkB）. METIIOI）S： HLE, SKHep1, and HepG2 were incubated and E3330 was used to compare the stimulation of some chemotherapeutic drugs with that of TNF family, Fas ligand, TNF（x and TNF-related apoptosis-inducing ligand （TRAIL） at the point of the reduction of cell viability by inhibiting NFkB. RESULTS： E3330 decreased NFKB levels in HLE cells stimulated by TNF and TRAIL. The cytotoxicity of the combination of TRAIL, TNFa, Fas ligand, and E3330 increased synergistically in a dose-dependent manner compared to either E3330 alone in all HCC cell lines by MTT assay. However, the combination of some chemotherapeutic drugs and E3330 did not decrease the cell viability. CONCLUSION： Inhibition of NFd3 sensitizes human HCC cell lines to TNF-mediated apoptosis including TRAIL, and TRAIL-based tumor therapy might be a powerful potential therapeutic tool in the treatment of human HCC.

5-Fluorouracil concentration in blood, liver and tumor tissues and apoptosis of tumor cells after preoperative oral 5'-deoxy-5-fluorouridine in patients with hepatocellular carcinoma

AIM: To study the levels of 5-fluorouracail (5-FU) in plasma, liver and tumor in patients with hepatoceilular carcinoma after oral administration of 5'-deoxy-5-fluorouridine (5'-DFUR). METHODS: Thirty-nine patients with hepatoceilular carcinoma were treated with oral 5'-DFUR for more than 4 d before operation. The contents of 5-FU in plasma, liver and tumor were measured by high performance liquid chromatography (HPLC) and apoptosis of tumor cells was evaluated by in-situ TUNEL after resection of tumor. RESULTS: The concentrations of 5-FU were 1.1 μg/mL, 5.6, 5.9, and 10.5 μg/g in plasma, the liver tissue, the center of tumor and the periphery of tumor, respectively. 5-FU concentration was significantly higher in the periphery of tumor than that in the liver tissue and the center of tumor (10.5±1.6 μg/g vs 5.6±0.8 μg/g, t = 21.38, P<0.05; 10.5±1.6 μg/g vs 5.9±0.9 μg/g, t = 20.07, P<0.05). 5-FU level was significantly lower in plasma than that in the liver and the tumor (1.1±0.3 μg/mL vs 5.6±0.8 μg/g, t = 19.63, P<0.05; 1.1±0.3 μg/mL vs 10.5±1.6 μg/g, t= 41.01, P<0.05). Apoptosis of tumor cells was significantly increased after oral 5'-DFUR compared to the control group without 5-DFUR treatment. CONCLUSION: There is a higher concentration of 5-FU distributed in the tumor compared with liver tissue and apoptosis of tumor cells is increased following oral 5'-DFUR compared with the control group. The results indicate that 5'-DFUR is hopeful as neo-adjuvant chemotherapy to prevent recurrence after resection of hepatoceilular carcinoma.

Clinical use of hepatic carcinoma associated membrane protein antigen (HAg18-1) as reagent of enzyme linked immunosorbent assay for detection of primary hepatocellular carcinoma

AIMS To evaluate the sensitivity,specificity and clinical value of hepatic carcinoma associated mem- brane protein antigen (HAg18-1) used as a reagent of serum quick enzyme linked immunosorbent assay (ELISA) for the detection of primary hepatocellular car- cinoma (PHCC). METHODS Serum quick enzyme linked immunosor- bent assay (ELISA) with HAg18-1 as reagent,which was prepared by monoclonal antibody against human hepatic carcinoma,was performed on 100 cases of pri- mary hepatocellular carcinoma (PHCC),5 cases of hepatic biliary carcinoma (HBC),10 cases of metastatic hepatic carcinoma (MHC),20 cases of hep- atitis B (HB),20 cases of liver cirrhosis (LC),20 cas- es of gastrointestinal malignant tumours and 20 cases of gastronintestinal inflammatory diseases (including ulcers). Alpha-fetoprotein (AFP) detection concurrent- ly for each case. Twenty samples of bank blood were tested as controls. RESULTS Positive rate of HAg18-1 ELISA and AFP detection were 81% and 68% in PHCC,20% and 40% in HBC,19% and 20% in MHC,10% and 20% in BH, 10% and 20% in LC,10% and 15% in gastrointestinal malignant tumors,and 5% and 10% in gastrointestinal inflammatory diseases. No positive result of HAg18-1 ELISA and AFP detection occurred in any sample of bank blood. CONCLUSIONS HAg18-1 ELISA has high sensitivity and specificity in the detection of PHCC. HAg18-1 ELISA and AFP detection,if used together,may be complementary to each other in the diagnosis of PHCC.