Growth inhibitory effects of pegylated IFN alpha-2b on human liver cancer cells in vitro and in vivo.

PURPOSE: We investigated the effects of pegylated IFN-alpha2b (PEG-IFN-alpha2b) on the growth of human liver cancer cells. METHODS: The effect of PEG-IFN-alpha2b on the proliferation of 13 liver cancer cell lines was investigated in vitro. Chronological changes in growth and IFN-alpha receptor-2 (IFNAR-2) expression were monitored in hepatocellular carcinoma (HCC) cells (HAK-1B) cultured with PEG-IFN-alpha2b. After HAK-1B cells were transplanted into nude mice, various doses of PEG-IFN-alpha2b or IFN-alpha2b were administered, and tumor volume, weight, histology, and IFNAR-2 expression were examined. RESULTS: PEG-IFN-alpha2b inhibited the growth of nine cell lines with apoptosis in a dose- and time-dependent manner. Continuous contact with PEG-IFN-alpha2b induced time-dependent growth inhibition and down-regulation of IFNAR-2 expression. PEG-IFN-alpha2b induced a dose-dependent decrease in tumor volume and weight, a significant increase of apoptotic cells, and a decrease in IFNAR-2 expression in the tumor. The clinical dose for chronic hepatitis C was also effective. The antitumor effect of PEG-IFN-alpha2b was significantly stronger than that of non-PEG-IFN-alpha2b in vivo. CONCLUSIONS: Continuous contact with PEG-IFN-alpha2b induces strong antitumor effects and the down-regulation of IFNAR-2 in HCC cells. The data suggest potential clinical application of PEG-IFN-alpha2b for the prevention and treatment of HCC.     

Expression pattern of mda-7/IL-24 receptors in liver cancer cell lines

BACKGROUND:The mda-7/IL-24 receptor belongs to the typeⅡcytokine receptor family,and its two heterodimeric receptors are IL-22R1/IL-20R2 and IL-20R1/IL-20R2. Mda-7/IL-24 receptor expression in liver cancer cell lines has not yet been described.This information may be helpful for further clinical gene therapy. METHODS:With normal skin total RNA as template,the cDNA sequences of IL-20R1,IL-20R2 and IL-22R were amplified by RT-PCR.Total RNA was extracted from cultured liver cancer cell lines and a normal liver...     

Combined intra-arterial 5-fluorouracil and subcutaneous interferon-alpha therapy for highly advanced hepatocellular carcinoma

Because of the difficulties of low sensitivity for anticancer agents and giving sufficient dose because of poor liver function, chemotherapy may not play a central role for treatment of hepatocellular carcinoma (HCC) patients, especially those with liver cirrhosis. However, chemotherapy must be one of the important possibilities of multimodal treatment for advanced HCC, for which hepatic resection, percutaneous ablation, transcatheter arterial embolization and other general therapies would not be effective or even possible. Also, intra-arterial perfusion chemotherapy is a common therapy for HCC and it is not difficult to maintain; but the effective rate is not sufficient. Recently, the combination therapy of s.c. interferon (IFN)-α and intra-arterial 5-fluorouracil (5-FU) showed an outstandingly effective rate for intractable HCC (with portal vein thrombosis). In addition,recent preclinical and clinical studies have revealed that the mechanism of combination therapy may concern direct antitumor effects (through cell-cycle arrest and induction of apoptosis) and indirect actions (through immunocompetent cells and anti-angiogenic effect). For the further advance of HCC treatment and prognosis, this therapy might be a promising treatment modality and is expected to develop. In this review, we summarize recent clinical and preclinical data regarding IFN-α and 5-FU combination therapy and discuss the further prospects of this therapy.     

Growth inhibitory effects of flavonoids in human thyroid cancer cell lines.

Previous studies have indicated that flavonoids exhibit antiproliferative properties on some hormone-dependent cancer cell lines, such as breast and prostate cancer. In the present study, the effects of some selected flavonoids, genistein, apigenin, luteolin, chrysin, kaempferol, and biochanin A on human thyroid carcinoma cell lines, UCLA NPA-87-1 (NPA) (papillary carcinoma), UCLA RO-82W-1 (WRO) (follicular carcinoma), and UCLA RO-81A-1 (ARO) (anaplastic carcinoma) have been examined. Among the flavonoids tested, apigenin and luteolin are the most potent inhibitors of these cell lines with IC50 (concentration at which cell proliferation was inhibited by 50%) values ranging from 21.7 microM to 32.1 microM. The cells were viable at these concentrations. Using NPA cells known to be estrogen receptor positive (ER+), it was shown that no significant [3H]-E2 displacement occurred with these flavonoids at the IC50 concentration. In WRO cells that are known to have an antiestrogen binding site (AEBS), biochanin A caused a stronger inhibitory growth effect (IC50 = 64.1 microM) than in NPA and ARO cells. In addition, it was observed that biochanin A has an appreciable binding affinity for the AEBS as indicated by the displacement of [3H]-tamoxifen from the WRO cells. In summary, flavonoids have potent antiproliferative activity in vitro against various human thyroid cancer cell lines. The inhibitory activity of certain flavonoid compounds may be mediated via the AEBS and/or type II EBS. The observation that ARO cells that lack both the AEBS and the ER are effectively inhibited by apigenin and luteolin suggest that other mechanisms of action are operative as well. The present study suggests that flavonoids may represent a new class of therapeutic agents in the management of thyroid cancer.     

Expression of interferon-alpha subtypes in peripheral mononuclear cells from patients with chronic hepatitis C: a role for interferon-alpha5

Interferon (IFN)-α is a family of antiviral proteins encoded by different genes. The biological significance of the existence of various IFN-α subtypes is not clear. We have investigated the interferon system in chronic hepatitis C virus (HCV) infection, a disease that responds to interferon-α2 therapy in only a limited proportion of cases. We analysed the expression of interferon regulatory factor (IRF)-1, IRF-2, and IFN-α subtypes in nonstimulated and Sendai virus-stimulated peripheral blood mononuclear cells (PBMC) from HCV infected patients and healthy controls. We observed that the IRF-1 mRNA and IRF-1/IRF-2 ratios were increased in PBMC from hepatitis C patients with respect to normal subjects. Sendai virus stimulation of PBMC led to a significant increase in the levels of IRF-1, IRF-2 and IFN-α mRNAs and in the production of IFN-α protein with respect to basal values in healthy controls as well as in patients with HCV infection. In addition, we found that while natural HCV infection induced increased IFN-α5 expression in PBMC, in vitro infection of these cells with Sendai virus caused a raise in the expression of IFN-α8 in both patients and normal controls. In summary, our results indicate that virus-induced activation of the IFN system in human PBMC is associated with selective expression of individual IFN-α subtypes, IFN-α5 being the specific subtype induced in PBMC from patients with chronic HCV infection.     

Reduced interferon-alpha production by Epstein-Barr virus transformed B-lymphoblastoid cell lines and lectin-stimulated lymphocytes in congenital dyserythropoietic anaemia type I

The concentrations of interferon-α (IFN-α) in supernatants from cultures of Epstein-Barr virus (EBV) transformed B-lymphoblastoid cell lines derived from seven patients with congenital dyserythropoietic anaemia (CDA) type I were below the 95% confidence limits for those derived from six healthy subjects. In contrast, the concentrations of IFN-α in supernatants from cultures of EBV-transformed lymphoblastoid cell lines derived from four patients with other types of CDA and four patients with hereditary sideroblastic anaemia were normal. Supernatants from cultures of peripheral blood lymphocytes stimulated with phytohaemagglutinin or pokeweed mitogen contained less IFN-α when the cells were derived from patients with CDA type I than when derived from healthy subjects. Since patients with CDA type I show a substantial haematological response to treatment with IFN-α, the data suggest that impaired IFN-α production may be an important pathogenetic mechanism in CDA type I.     

Treatment of chronic hepatitis C virus infection in patients with cirrhosis

Chronic hepatitis C virus (HCV) infection eventually leads to cirrhosis in 20–30% of patients and to hepatocellular carcinoma (HCC) in 1–5% of patients. Rates of sustained virological response with standard interferon-α (IFN-α) are low in patients without cirrhosis (generally < 20%) and are even lower in those with cirrhosis. Combination therapy with IFN and ribavirin improves response rates in patients with chronic hepatitis C without cirrhosis, and the results from subgroups of HCV-infected patients with advanced fibrosis or cirrhosis are encouraging. Importantly, treatment with IFN slows progression of liver fibrosis, regardless of HCV genotype or early response to therapy, and reduces the risk of HCC by two- to fivefold. The risk of development of HCC is also lower in patients who show at least a partial response to IFN therapy compared with those who show no response. There is a clear need for more definitive studies of treatment in patients with chronic hepatitis C and cirrhosis, ideally using therapies with greater efficacy. Nonetheless, based on the potential to slow the progression of liver fibrosis (regardless of treatment response) and to reduce the risk of HCC, a greater number of HCV-infected patients with cirrhosis should be considered as candidates for IFN treatment. Preliminary data indicate that pegylated IFNs have improved virological response rates and may have additional clinical benefits in the prevention or reduction of fibrosis and retardation of progression of cirrhosis and HCC in these patients.     

Growth inhibitory effects of Phyllanthus niruri extracts in combination with cisplatin on cancer cell lines

AIM:To investigate the cytotoxic effects of spray-dried extracts of Phyllanthus niruri in combination with cisplatin on two cancer cell lines.METHODS:Colorectal carcinoma(HT29) and human hepatocellular carcinoma(HepG2) cells were treated with spray-dried extracts of Phyllanthus niruri(SDEPN) either alone or in combination with cisplatin at different concentrations(0.5 mg/mL and 1 mg/mL) for 4 h and 24 h.To verify and quantify cancer cells treated with these products as well as identify the cell cycle stage and cell viability,we stained the cells with propidium iodide and assessed them by flow cytometry.The percentage of cells in different cell cycle phases was quantified and data were expressed as histograms.Significant differences between groups were determined using analysis of variance and Bonferroni's test,as indicated.A value of P 0.05 was considered to be statistically significant.RESULTS:SDEPN had significantly different cytotoxic effects on HT29(2.81 ± 0.11 vs 3.51 ± 1.13,P 0.05) and HepG2(5.07 ± 0.3 vs 15.9 ± 1.04,P 0.001) cells when compared to control cells for 4 h.SDEPN also had significantly different cytotoxic effects on HT29(1.91 ± 0.57 vs 4.53 ± 1.22,P 0.05) and HepG2(14.56 ± 1.6 vs 35.67 ± 3.94,P 0.001) cells when compared to control cells for 24 h.Both cell lines were killed by cisplatin in a dose-dependent manner compared to control cells(HepG2 cells for 4 h:10.78 ± 1.58 vs 53.89 ± 1.53,P 0.001;24 h:8.9 ± 1.43 vs 62.78 ± 1.87,P 0.001 and HT29 cells for 4 h:9.52 ± 0.913 vs 49.86 ± 2.89,P 0.001;24 h:11.78 ± 1.05 vs 53.34 ± 2.65,P 0.001).In HT29 cells,pretreatment with SDEPN and subsequent treatment with cis-platin resulted in a greater number of cells being killed(12.78 ± 1.01 vs 93.76 ± 1.6,P 0.001).HepG2 cells showed significant cell killing with treatment with SDEPN when combined with cisplatin(12.87 ± 2.78 vs 78.8 ± 3.02,P 0.001).CONCLUSION:SDEPN is selectively toxic against two cancer cell lines.Moreover,SDEPN in combination with cisplatin induces a synergistic increase in the cell death of both HT29 and HepG2 cells.     

生长抑素类似物诱导人肝癌细胞凋亡的研究

目的研究生长抑素类似物对肝癌细胞(H epG2)生长及凋亡的影响。方法用奥曲肽(0.25μg/m l)分别作用人H epG20、6、12和24 h,以0 h作为样本对照组,采用DNA琼脂糖凝胶电泳、流式细胞仪检测细胞凋亡。结果奥曲肽作用H epG2细胞6 h DNA电泳无明显异常,作用12和24 h后DNA琼脂糖凝胶电泳出现典型的梯状条带图谱(DNA ladder)。奥曲肽作用H epG2细胞6、12和24 h后,A nnex in V-F ITC标记的凋亡细胞分别为(25.5±5.5)%,(42.6±2.3)%和(52.1±1.6)%。结论生长抑素类似物奥曲肽能诱导H epG2发生凋亡,抑制H epG2生长。     

热休克蛋白27高表达与肝细胞癌耐药相关

目的应用蛋白质组学技术从蛋白质水平寻找肝癌耐药相关的蛋白质分子。方法以长春新碱（VCR）诱导HepG2细胞建立的耐药肝癌细胞HepG2／VCR为研究对象,用二维电泳技术分离两种细胞的总蛋白,对差异表达的蛋白质点用基质辅助激光解吸电离飞行时间质谱及电喷雾串联质谱进行分析鉴定。再用反义核酸抑制该差异蛋白质表达以研究其与耐药的关系。结果鉴定出一个在肝癌耐药细胞株HepG2／VCR中高丰度表达的蛋白质为热休克蛋白27（HSP27）,反义核酸抑制HSP27的表达后增强了HepG2／VCR对VCR的化疗敏感性（P〈0．05）。结论HSP27可能是与肝癌细胞株HepG2／VCR耐药密切相关的蛋白质。     

原花青素对人肝癌细胞SMMC-7721增殖及凋亡的作用

目的探讨葡萄籽提取物原花青素（PA）对人肝癌细胞SMMC-7721增殖和凋亡的影响。方法取对数生长期人肝癌细胞SMMC-7721,分别加人20、40、60mg／L的PA培养24h后,采用MTI＂法检测细胞增殖抑制率、流式细胞仪分析细胞凋亡率,并测定细胞内超氧化物歧化酶（SOD）活力、丙二醛（MDA）和活性氧簇（ROS）等指标。结果20、40、60mg／L的PA对人肝癌细胞SMMC-7721的增殖抑制率分别为（22．7±1．8）％、（38．6±1。9）％、（47．6±2．5）％,细胞凋亡率分别为（19．7±5．1）％、（29．7±2．9）％、（48．5±4．5）％,两者均随PA浓度的升高而增高（P均〈0．01）;与对照组比较,各剂量组人肝癌细胞SMMC-7721中MDA、ROS水平逐渐下降（P〈0．05）;SOD活力逐渐上升（P〈0．05）。结论PA在体外可呈浓度依赖性抑制人肝癌细胞SMMC-7721的增殖及凋亡,其作用机制可能与清除ROS、提高SOD的活性、降低脂质过氧化反应等有关。     

Antiproliferative effects of 5-fluorouracil and interferon-alpha in combination on a hepatocellular carcinoma cell line in vitro and in vivo

Background and Aim: We investigated the antiproliferative effects of interferon‐alpha (IFN‐α) and 5‐fluorouracil (5‐FU) in combination on a hepatocellular carcinoma (HCC) cell line. Method: In the in vitro study, IFN‐α and/or 5‐FU was added to the culture of the poorly differentiated‐type HCC cell line, HAK‐1B, and their antiproliferative effects and additional or synergic effects in combination treatment were examined. In the in vivo study, HAK‐1B cells were transplanted into nude mice and the changes in tumor volume and weight, apoptosis, BrdU and cyclin A positive cells, and artery‐like blood vessels were investigated. Expressions of angiogenesis factors and IFN‐α receptor (IFNAR‐2) were examined in the developed tumors. Results: In vitro growth of HAK‐1B cells was suppressed dose‐dependently to 5‐FU, but the addition of IFN‐α did not induce additional or synergic effects. In vivo growth in terms of tumor diameter and weight was suppressed at most in the IFN‐α + 5‐FU (combination) group, that is, the tumor volume became 29.3% and the tumor weight became 54.7% of the control. In the combination group, numbers of BrdU‐positive S‐phase cells and cyclin A positive cells increased together with the increase in apoptotic cells, but there was no significant relation between the tumor shrinkage effects and angiogenesis factors or artery‐like blood vessels. In the combination group, INFAR‐2 decreased significantly in comparison to the other groups. Conclusion: The synergic growth‐suppression effects in the current in vivo study using the combination treatment are attributable to the enhanced induction of S‐phase arrest and of apoptosis.     

Expression of hepatitis B surface antigen subtypes in liver of patients with hepatocellular carcinoma; comparison of subtypes in serum and liver

ABSTRACT— To clarify the discrepancy in hepatitis B surface antigen (HBsAg) subtypes present in the serum and liver, as well as among hepatocytes, liver specimens which were resected from 37 HBsAg-positive patients with hepatocellular carcinoma (HCC) were examined. We evaluated HBsAg and the subtypic determinants of HBsAg and hepatitis B core antigen (HBcAg) using the peroxidase-antiperoxidase (PAP) staining method. Hepatitis B antigens were more frequently detected in small tumors (HBsAg in 67%, HBcAg in 40%) than in large ones (HBsAg in 36%, HBcAg in 14%). The prevalence of each subtypic determinant in the HBsAg positive non-tumorous vs. tumorous areas was 100% vs. 67% in a, 100% vs. 57% in d, 100% vs. not tested in y, 100% vs. 53% in r and 25% vs. 0% in w (a, d, y, r and w represent subtypic determinants). There was virtually no difference in a set of subtypic determinants between the serum and liver. However, there were some variations in a set of subtypic determinants among the hepatocytes. On the other hand, liver tissue of compound subtype adyr in serum contained both cells with a,d,r and with a,y,r as well as a few cells with a,d,y,r. These findings suggest that HBV genomes in hepatocytes of type B chronic liver disease may differ genetically among cells even in the same liver tissue.     

Low-molecular-weight protein (LMP)2/LMP7 abnormality underlies the downregulation of human leukocyte antigen class I antigen in a hepatocellular carcinoma cell line

BACKGROUND: Tumor cells may alter the expression of numerous components involved in antigen-processing machinery to decrease human leukocyte antigen (HLA) class I expression, allowing the tumor cells to escape immune surveillance. The purpose of the present study was to investigate the involvement of these components in the downregulation of HLA class I expression in human hepatocellular carcinoma cell line BEL7,404. METHODS: Expression of HLA-I and antigen presentation-related genes were analyzed by flow cytometry and polymerase chain reaction. The HLA class I-deficient BEL7,404 cell was transfected with the low-molecular-weight protein (LMP) 2 and LMP7 gene and were analyzed by flow cytometry for restoration of surface HLA class I expression. RESULTS: The BEL7,404 cells downregulated the expression of HLA class I antigen and lacked expression of LMP2 and LMP7. Interferon (IFN)-gamma treatment increased the expression of LMP2 but not LMP7. The LMP2-transfected BEL7,404 cells or LMP2 and LMP7-cotransfected cells restored surface HLA class I expression while LMP7-transfected cells did not. However, in IFN-gamma-treated BEL7,404 cells, transfection with the LMP7 gene induced more HLA class I expression than mock transfection. CONCLUSIONS: The LMP2 gene was required for the expression of HLA class I molecules in BEL7,404. The LMP7 was not the major reason for loss of HLA class I in BEL7,404 cells, although the supply of exogenous LMP7 could increase surface expression of HLA class I antigen.     

Induction of apoptosis and cell cycle arrest by imexon in human pancreatic cancer cell lines

Imexon is an aziridine-containing small molecule currently in Phase I clinical trials. This agent has been shown to bind to thiols and increase intracellular oxidants, inducing apoptosis in hematologic cancer cells. Pancreatic cancers are known to be sensitive to oxidation, suggesting this disease may be an appropriate target for this agent. The current report examines the activity of imexon in pancreatic cells. Imexon induced concentration-dependent and time-dependent apoptosis in a panel of six human pancreatic carcinoma cell (PCC) lines. The mean IC₅₀ (SD) for growth inhibition by the SRB assay was 200 (101) μM for a 48 h exposure with a range of 64–358 μM. Cell killing was schedule-dependent, favoring exposure times ≥48 h. Imexon-treated MiaPaCa-2 cells underwent non-lethal growth arrest following exposure to concentrations ≤200 μM for 48 h. When concentrations were increased to 300 μM for ≥48 h, the MiaPaCa-2 cells arrested in G₂ phase and activated caspases 3, 8, and 9 were detected. After a 72 h exposure to the IC₈₀ concentration of imexon, cells exhibited a loss of mitochondrial membrane potential detected by CMXRos staining. However, there was no loss of reduced cellular thiols unless very high concentrations of ≥400 μM were used. In contrast, reactive oxygen species (ROS) were elevated in a dose-dependent fashion, starting at very low imexon concentrations. Imexon also significantly inhibited MiaPaCa-2 tumor growth in SCID mice at 100 mg/kg/d for 9 d. The tumor growth inhibition (% T/C) was 27% of control, and the tumor growth delay was 21 d, indicating an active agent by NCI standards. The levels of imexon that are cytotoxic in human PCC’s are achievable based on the preliminary results of the ongoing Phase I trial. Imexon appears to be active against PCCs in vitro and has an entirely novel mechanism of action involving G₂ arrest, accumulation of ROS, and the induction of apoptosis.     

Silencing thioredoxin induces liver cancer cell senescence under hypoxia

Aim: Although thioredoxin 1 (TXN) has pleiotropic cellular functions as a redox-sensitive protein, very little is known about its role in tumor survival and growth under hypoxia. MHCC97H hepatocellular carcinoma cells have a high metastatic potential and high thioredoxin expression levels compared with their parent cell line, MHCC97. Thus, we used this cell line to explore the functional connections between TXN and hypoxia. Methods: MHCC97H cells were cultured under normoxia and hypoxia for specific periods after nucleofection with TXN siRNA or control siRNA. We assessed the β-phenylethyl isothiocyanate (PEITC) sensitivity of the cells, cell proliferation, cell cycle and senescence, and DNA damage response by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, colony formation assays, flow cytometry, β-galactosidase staining, western blotting, and immunohistochemistry. Results: β-phenylethyl isothiocyanate treatment shifted reduced TXN to oxidized TXN in MHCC97H cells. Although silencing of TXN via siRNA had no effect on the PEITC sensitivity of the cells, it suppressed cell proliferation and colony formation under both normoxia and hypoxia. Under hypoxia, silencing TXN did not induce apoptosis but induced DNA damage response and cellular senescence. Conclusions: High TXN levels in MHCC97H cells protect them from DNA damage and cellular senescence under hypoxia. Targeting TXN might enhance the chemotherapeutic effects of some DNA-damaging agents against hepatocellular carcinoma.