Effect of alpha-fetoprotein on the growth of human hepatoma cells in vitro]

The stimulatory activity of human alpha-fetoprotein (AFP) on the growth of mouse hepatoma-22 cells had been reported in our previous paper. The present work aimed at further investigation of the effect of AFP on human hepatoma cell growth by MTT colorimetric assay. The results showed that AFP could stimulate the growth of SMMC-7721 human hepatoma cells in vitro. The present results also showed that the stimulatory effect of AFP on the growth of SMMC-7721 cells was decreased by the anti-serum of AFP. The anti-AFP antibody alone could suppress the growth of SMMC-7721 cells. On the other hand, AFP and anti-AFP antibody had no effect on the growth of HL-60 human leukemia cells, indicating that the tumor cell growth stimulating effect of AFP was not simply due to non-specific addition of exogenous protein and this effect of AFP showed strict tumor cell specificity. In addition, MCF-7 human breast cancer cell growth was also promoted by AFP and inhibited by anti-AFP antibody. Because AFP cell-surface receptors have been detected in MCF-7 breast cancer cells, and AFP could also be produced and secreted by MCF-7 cells, the possibility may be considered: AFP may bind with its receptors on tumor cell membrane for the purposes of growth stimulation.     

甲胎蛋白在体外对人肝癌细胞生长的影响

本文用MTT比色法观察了甲胎蛋白(AFP)在体外对人肝癌细胞生长的影响。结果表明,AFP能促进SMMC-7721人肝癌细胞的生长。当AFP与AFP抗体合用时,AFP抗体能减弱AFP对SMMC-7721细胞生长的促进作用;AFP抗体单用对此种细胞的生长亦有抑制作用。另一方面,在相同的实验条件下,AFP和AFP抗体对HL-60人白血病细胞的生长无明显影响;提示AFP的促生长作用具有一定的肿瘤细胞特异性,并非一种蛋白质对培养细胞的非特异性营养作用。此外,AFP亦能促进MCF-7人乳腺癌细胞的生长,AFP抗体对此种细胞的生长有抑制作用。由于MCF-7细胞存在功能性AFP受体,也能合成和分泌AFP。这就提示,人肝癌细胞中的AFP很可能与其受体特异性结合,产生促生长效应。确切机制尚待进一步阐明。     

BEL-7404人肝癌细胞甲胎蛋白基因表达的原位杂交与免疫细胞化学研究

本文用地高辛标记的甲胎蛋白（ＡＦＰ）ｃＤＮＡ探针原位杂交和免疫细胞化学方法观察到,几乎所有的ＢＥＬ－７４０４人肝癌细胞均能表达ＡＦＰｍＲＮＡ和相应的蛋白。     

外源NF-IL6(C/EBPβ)对肝癌细胞系BEL-7404的肿瘤抑制效应

目的:探讨核转录因子NFIL6对肝癌细胞系BEL7404恶性度的影响。方法:用磷酸钙介导转染技术,将NFIL6表达载体(pCN)和空载体质粒(pCN空)分别导入肝癌细胞系BEL7404,并借助细胞生长曲线,软琼脂集落形成试验,裸鼠成瘤试验对转染细胞的恶性度进行了检测。结果:与原细胞系BEL7404和空载体转染的该细胞系相比较,转染了NFIL6基因的BEL7404的各细胞克隆生长速度减慢,在软琼脂中集落形成率恶性度下降,裸鼠成瘤试验显示成瘤性明显降低。结论:表明外源转染的NFIL6对肝癌细胞系BEL7404具有明显的肿瘤抑制作用 。     

An insight into the mechanism of cytotoxicity of ricin to hepatoma cell: roles of Bcl-2 family proteins, caspases, Ca(2+)-dependent proteases and protein kinase C

The ability of ricin, a type II ribosome-inactivating protein, to induce hepatoma cell (BEL7404) to apoptosis in vitro was examined by fluorescence microscopy, flow cytometry, and DNA fragmentation assay. As a Bcl-2 lacking model, BEL7404 bore unique advantage to study the effect of over-expressing Bcl-2 on the apoptosis induced by the inhibitor of protein synthesis. By establishing a Bcl-2 over-expressing cell line (BEL7404/ Bcl-2), we found that Bcl-2 could promote the survival of the hepatoma cell against ricin insult. The ricin-induced apoptosis of BEL7404 was accompanied by increased expression of Bak and decreased levels of Bcl-xl and Bax. Caspases and PARP cleavage activity were found to be implicated in the death process. Through the inhibitor tests, our results excluded the participation of calcium-dependent proteases or protein kinase C in the apoptotic process induced by ricin, though an elevation of intracellular calcium did occur as an immediate response to ricin treatment. Cycloheximide, another protein synthesis inhibitor, did synergistically enhance rather than inhibit the cytotoxicity of ricin to hepatoma cell BEL7404. Actually, cycloheximide alone was able to induce hepatoma cell BEL7404 to death that could also be inhibited by over-expressing Bcl-2. The elevation of apoptotic protein Bak was discussed to challenge the notion that ricin exerted its cytotoxicity through nonspecific inhibition of all the de novo protein synthesis.     

Hepatocyte growth factor down-regulates the alpha-fetoprotein gene expression in PLC/PRF/5 human hepatoma cells.

Hepatocyte growth factor (HGF) is a potent mitogen for hepatocytes; however, in certain human hepatoma cell lines, the growth is inhibited by HGF. In the present study, the effect of HGF on the alpha-fetoprotein (AFP) gene expression was analyzed in PLC/PRF/5 human hepatoma cells. HGF did not inhibit cell proliferation, but dose-dependently suppressed AFP secretion at the concentrations of 10 ng/ml or less. By Northern blot analysis, the levels of AFP mRNA were suppressed by HGF, whereas the levels of beta-actin mRNA used as a control did not show any significant changes. In the transient chloramphenicol acetyltransferase plasmid transfection assays, the AFP promoter activity was repressed by HGF, in contrast, the AFP enhancer activity was not affected by HGF. These results suggest that the AFP gene expression is down-regulated by HGF through the suppression of its promoter activity in human hepatoma cells.     

Monitoring of cell growth and assessment of cytotoxicity using electrochemical impedance spectroscopy

Electrochemical impedance spectroscopy (EIS) was used to monitor the growth of mammalian cancer cells and evaluate the cytotoxicity of chemicals using Fe(CN)6(3-/4-) as a redox probe. Cancer cells, the human hepatocarcinoma cell line (BEL7404), were grown on optically transparent indium tin oxide (ITO) semiconductor slides, which were used as the working electrodes in electrochemical experiments. Attachment and proliferation of cancer cells on ITO surfaces resulted in increase of electron-transfer resistance (R(et)) between the redox probe of Fe(CN)6(3-/4-) in electrolyte solution and ITO electrode surface. For cytotoxicity assessment, cells grown on ITO substrates were further cultured in the presence of different cytotoxicants and electrochemical impedance measurements were carried out at different time intervals. Gemcitabine, a promising antineoplastic drug showing activity against a wide spectrum of human solid tumors, was selected as a model for long-term cytotoxicity effect study, whereas mercury chloride represented a model for acute toxicants. The inhibitions of gemcitabine and mercury chloride on the viability and proliferation of BEL7404 cells were observed from the electrochemical impedance experiments, and the different action modes were discriminated. Additionally, microscope images were also used to observe the effects of these two chemicals on the morphology of the cells. General consistency has been found between the electrochemical impedance response and the morphological observation. Such an impedance method provides a simple and inexpensive way for in vitro assessment of chemical cytotoxicity.     

mad—overexpression down regulates the malignant growth and p53 mediated apoptosis in human hepatocellular carcinoma BEL—7404 cells

Mad protein has been shown as an antagonist of cMyc protein in some cell lines.The effect of Mad protein to the malignant phenotype of human hepatoma BEL-7404 cell line was investigated experimentally.An eukarryotic vector pCDNA Ⅲ containing full ORF fragment of mad cDNA was transfected into targeted cells.Under G418 selection,stable Mad-overexpressed cells were cloned.Studies on the effect of Mad over-expression in cell proliferation and cell cycle revealed that cell morphology of the Mad-overexpressed BEL-7404-M1 cells was significantly different from the parent and control vector transfected cells.DNA synthesis,cell proliferation and anchorage-independent growth in soft-agar of the madtransfected cells were partially inhibited in comparison to control cells.Flos cytometry analysis indicated that mad over-expression might block more transfectant cells at G0/G1 phase,resulting in the retardation of cell proliferation.RT-PCR detected a marked inhibition of the expression of cdc25A,an important regulator gene of G0/G1 to S phase in cell cycle.It was also found that Mad protein overexpression could greatly suppress p53-mediated apoptosis in BEL-74040M1 cells in the absence of serume.Thus,Mad proteins may function as a negative regulator antagonizing c-Myc activity in the control of cell growth and apoptosis in human hepatocellular carcinoma BEL-7404 cells.     

Synergistic antitumor effect of TRAIL and IL-24 with complete eradication of hepatoma in the CTGVT-DG strategy

The ZD55-tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and ZD55-interleukin (IL)-24 were constructed by inserting TRAIL or IL-24 gene separately into the oncolytic adenovirus named ZD55 (with adenovirus E1B-55kD deletion). The resulting ZD55-TRAIL and ZD55-IL-24 were used in combination to treat xenograft tumors in nude mice model. The results showed that it can not only completely eliminate BEL7404 hepatoma xenograft but also have excellent antitumor effect against gaster, lung, prostate, and breast carcinomas. It was also found that ZD55-TRAIL could not only suppress the tumor growth promoting effect by ZD55-IL-24 at lower dosage, but also substantially reduce the cancer cell viability in their combined use. This is because ZD55-IL-24 and ZD55-TRAIL could mutually enhance each other's antitumor effect greatly. All these findings conspicuously showed the synergistic antitumor effect of TRAIL and IL-24, which is also the reason for the antitumor effect by the combined use of TRAIL and IL-24 in vitro and also in vivo.     

Elevated serum α-fetoprotein level of nude mice bearing hepatoma cells by treatment with monoclonal antibodies to α-fetoprotein

This study was carried out to clarify the reason for elevation of serum α-fetoprotein (AFP) level of nude mice bearing hepatoma cells after treatment with monoclonal antibodies (MoAbs) to AFP. MoAbs to AFP showed no effect on the cumulative amounts of AFP secreted from human hepatoma cell line, HuH-7, in vitro. However, the treatment of nude mice bearing HuH-7N cells (HuH-7 xenograft) with MoAbs to AFP led to elevation of the serum AFP level in spite of the fact that the growth curve of HuH-7N cells was similar to that for PBS treatment. This apparent elevation of the serum AFP level is thought to be due to the slow elimination of AFP-MoAb immune complexes with little lattice structure from circulation, but not the enhancement of AFP secretion of HuH-7N cells. Thus, when using a MoAb alone or MoAb-drug conjugate, the serum AFP level should only be cautiously used as a tumor marker for evaluating the targeting immunotherapy.     

EGFR expression and EGF stimulation of proliferation in human liver carcinoma cells

Epidermal growth factor receptor (EGFR) gene expression and growth stimulation of EGF on human hepatoma cells of cell lines BEL-7404 and SMMC-7721 were studied. 125I-EGF binding assay was used to measure the binding characteristics and the amounts of EGFR on these cells. The binding time course and the binding competition assay showed that the binding of 125I-EGF to 7404 cells was saturable and specific. Scatchard analysis of EGF binding curve indicated that 7404 and 7721 cells expressed approximately 1.1 x 10(5) and 0.7 x 10(5) EGFRs per cell with binding affinity (Kd) 2.1 nM and 1.8 nM respectively. Northern hybridization and immunoblotting analysis showed the EGFR gene expression products in 7404 and 7721 cells were 5.6 Kb mRNA and 170 Kilo-dalton glycoprotein. Anchorage-dependent growth of 7404 and 7721 cells was stimulated in the presence of nanogram quantities of EGF in medium containing 10% calf serum or 0.5% calf serum. The factors in serum appeared to act synergitically in stimulating of cell proliferation. EGF also stimulated the anchorage-independent growth of 7404 and 7721 cells in soft agar. The results suggest that EGFR is actively expressed in human hepatoma 7404 and 7721 cells and EGF may be one of the mitogens needed for the growth of hepatoma cells.     

Human mammary tumor cell proliferation: primary role of platelet-derived growth factor and possible synergism with human alpha-fetoprotein.

Human mammary medullary carcinoma cells (passages 16 to 21) were cultured for 2 days to allow for attachment, followed by 6 days of culture in either fetal calf serum, human cord blood, human amniotic fluid, or growth factors in the presence or absence of purified human alpha-fetoprotein (AFP). When growth factors were tested alone, only platelet-derived growth factor produced a significant increase in cell proliferation. Although up to 40% amniotic fluid had no effect on cell proliferation, human cord blood was two-fold more potent than fetal calf serum at similar concentrations. The addition of 10 ng/ml of platelet-derived growth factor increased the proliferative activity of human cord blood 1.5- to 2.5-fold. Ablation of endogenous AFP by affinity chromatography reduced the proliferative activity of cord blood by 75%. Similarly, the mitogenic activity of cord blood plus platelet-derived growth factor was reduced by 56% when AFP was removed. Purified AFP dose-dependently enhanced the proliferative activity of platelet-derived growth factor. This synergistic effect was specific for platelet-derived growth factor. We conclude that platelet-derived growth factor is a major growth factor controlling the proliferation of these tumor cells and that AFP may enhance growth factor proliferative activity and human mammary tumor growth.     

紫芝酸性三萜类化合物体外抑癌和抑菌作用的研究

采用噻唑蓝比色(MTT)法研究紫芝酸性三萜对几种癌细胞体外增殖的影响,并用管碟法检测了紫芝胞内酸性三萜对几种细菌和霉菌的体外抑菌作用。结果表明,紫芝胞内酸性三萜和胞外酸性三萜在250μg/mL时,对人肝癌细胞BEL7402和人乳腺癌细胞MCF-7均有显著抑制作用(P<0.05),但对人胃癌细胞SGC-7901没有显著抑制作用(P>0.05)。BEL7402细胞的生长曲线试验表明,胞内酸性三萜组的细胞受到显著抑制,未出现指数增长期,且BEL7402细胞培养3d后,对照组细胞数目多、均匀,而胞内酸性三萜组的细胞数目明显减少,且细胞变小。抑菌试验结果表明,胞内酸性三萜在40mg/mL时,对大肠杆菌Escherichia coli和金黄色葡萄球菌Staphylococcus aureus的生长均具有显著抑制作用(P<0.01),对枯草芽孢杆菌Bacillus subtitis和青霉的Penicillium chrysogenum抑制作用较弱;而在此浓度下对黑曲霉Aspergillus niger没有抑制作用。该样品对大肠杆菌、金黄色葡萄球菌、枯草芽孢杆菌、黑曲霉和青霉的MIC分别为20mg/mL、20mg/mL、40mg/mL、80mg/mL和40mg/mL。此外,该酸性三萜的抑菌成分在60℃下(处理2h)较稳定,但在80℃以上,热稳定性较差,活性降低。     

Interaction of bovine epithelial lens (BEL) cells with extracellular matrix (ECM) and eye-derived growth factor (EDGF): I. Effects on short-term adhesiveness and on long-term organization of the culture

A growth factor (EDGF) derived from the retina controls the proliferation and shape of adult bovine epithelial lens (BEL) cells in vitro as well as extracellular matrix (ECM) assembly. In order to analyse this mechanism and the specificity of the interactions between BEL cells and the extracellular matrix we have investigated the adhesion and growth of BEL cells on various substrata (fibronectin, laminin, ECM). BEL cells treated with EDGF adhered more slowly to plastic Petri dishes than untreated cells, in part due to EDGF inhibition of fibronectin deposition. The untreated BEL cells spread less well on ECM or laminin than on fibronectin-coated plastic. The preferential adhesiveness of BEL cells on fibronectin vs laminin was confirmed by attachment experiments performed on replicas of SDS-PAGE of these proteins. However, in long-term cultures, 8 days after seeding, BEL cells were very differently arranged on plastic or on ECM. ECM by itself did not increase the proliferation rate but helped to restore an organized cell monolayer. BEL cells stimulated to grow on ECM by treatment with EDGF exhibited at least transiently contact inhibition producing a perfectly organized epithelium similar to the one observed in vivo. These results suggest specific interactions between ECM or ECM components with BEL cell that restrain excessive cell spreading and restore an original polarized phenotype of the cells seen in vivo.     

Role of transforming growth factor-alpha (TGF-alpha) in basal and hormone-stimulated growth by estradiol, prolactin and progesterone in human and rat mammary tumor cells: studies using TGF-alpha and EGF receptor antibodies

The biological role of transforming growth factor-alpha (TGF-alpha) in basal and hormone-stimulated proliferation of primary human and rat mammary tumor cells was studied using antibodies against TGF-alpha and its receptor. A monoclonal antibody, MAb-425 against human EGF receptor was added to in vitro soft agar, clonogenic cultures of human breast carcinoma cells under basal and estradiol(E2)-stimulated conditions. The antibody had an antagonist effect on colony growth in 4 of 10 tumors and an agonist effect in 4 (72 and 153% of control). E2-stimulated colony growth in 5 tumors (167% of control) and the antibody blocked E2-stimulation in 3 of the 5. Inhibition of E2-stimulated growth in 3 and basal growth in 4 other tumors by the EGF receptor antibody suggest that endogenously secreted TGF-alpha has a role as an autocrine/paracrine growth factor in constitutive and E2-stimulated tumor cell proliferation in a majority of human tumors. A polyclonal antibody against TGF-alpha was used to study the role of TGF-alpha in E2-, prolactin(Prl)- and progesterone(Prog)-stimulated proliferation of NMU(nitrosomethylurea)-induced rat mammary tumor cells under similar culture conditions. TGF-alpha, E2, Prl and Prog stimulated colony growth equally to 176, 187, 168 and 181% of control. The antibody produced significant and similar inhibition of TGF-alpha and E2-stimulated growth (95 and 83%). In contrast, inhibition of Prl- and Prog-stimulated growth by the antibody was only 24 and 37%. The TGF-alpha ligand antibody did not have an agonist or antagonist effect when added alone. Thus, TGF-alpha seems to be a major stimulatory growth factor mediating E2-induced tumor cell proliferation in rat mammary tumors. It is less important in Prl- and Prog-induced tumor growth and not essential for basal growth in these tumors. We conclude that TGF-alpha is a biologically important autocrine/paracrine growth factor in primary human breast cancer cell proliferation and in E2-induced rat mammary tumor growth.     

蒽贝素(Embelin)通过阻断NF-κB信号通路抑制肝癌细胞Bel-7404生长的研究

蒽贝素(Embelin)是一种X连锁凋亡抑制蛋白(X-linked inhibitor of apoptosis protein, XIAP)的小分子抑制剂,可以抑制XIAP的生成和活性,从而解除XIAP的抗凋亡作用,使凋亡顺利进行．研究了Embelin抑制肝癌细胞Bel-7404增殖的机制．采用不同浓度梯度,通过荧光显微镜、Hochest33342染色、MTT检测、AnnexinⅤ/PI流式细胞术和Western blot分别检测Embelin对Bel-7404细胞的形态学变化、凋亡小体形成、细胞存活率、细胞凋亡水平、凋亡信号转导相关蛋白表达的影响．结果显示,Embelin能明显抑制Bel-7404细胞增殖,并通过激活caspase通路以及阻断NF-κB信号通路诱导Bel-7404细胞凋亡,为进一步利用Embelin进行肝癌治疗的研究打下一定的基础．     

体内表达的休眠蛋白抑制裸鼠肿瘤的生长

休眠蛋白 (restin)是内皮抑素 (endostatin)的同源蛋白 ,它们之间相同的氨基酸占整个蛋白质的 6 2 %。休眠蛋白位于胶原XV羧端的非胶原结构域。在大肠杆菌中重组表达的休眠蛋白经复性后可以专一地抑制牛主动脉内皮细胞的生长 ,并引起凋亡。将鼠源的休眠蛋白基因与信号肽连接后克隆入真核表达载体 pCDNA3,取名为pCDNAXV并转染Bel74 0 4细胞。利用RT PCR和Western印迹证实休眠蛋白在稳定转染株中表达。将这一稳定转染株细胞接入裸鼠皮下观察肿瘤生长 ,发现转染有休眠蛋白基因的肿瘤生长显著慢于对照组 ;由于休眠蛋白基因对肿瘤细胞的增殖没有影响 ,这种抑制作用很可能是通过抑制血管生长来实现的。同时 ,这种方法为研究抑制血管生长蛋白提供了新的手段     

Targeting of hepatoma cell and suppression of tumor growth by a novel 12mer peptide fused to superantigen TSST-1

Hepatocellular carcinoma (HCC), one of the most common and malignant tumors worldwide, is unresponsive to any of the available therapies. Using intact HCC cells as therapeutic targets, we isolated a novel peptide, denoted HCC79 (KSLSRHDHIHHH), from a phage display peptide library. HCC79 can bind to hepatoma cell membranes with high affinity and specificity. Remarkably, competitive binding assays demonstrated that HCC79 competed with HAb25, a specific antibody for HCC, in binding to hepatoma cells. The corresponding synthetic peptide did not inhibit tumor proliferation directly, but repressed tumor invasion significantly in a cell migration assay. Moreover, we explored the potential of the selected peptide to deliver a superantigen (SAg) to cancer cells, to attain a significant cell-targeting effect. When the peptide is fused to the TSST-1 SAg, the resulting fusion protein could bind to hepatoma cells with high affinity in vitro and improved the tumor inhibition effect by activating T lymphocyte cells in vitro and in vivo, compared with TSST-1 alone. Taken together, our results indicate that this peptide and its future derivatives may have the potential to be developed into highly specific therapeutic agents against cancer.     

甲胎蛋白对HeLa细胞N-ras、p53和p21~(ras)表达的促进作用

大量研究已证明甲胎蛋白 (alpha fetoprotein ,AFP)对肿瘤细胞的增殖具有调节作用 .为探讨AFP对细胞生长促进作用的分子机理 ,采用从人脐带血中提取的AFP作用于体外培养的HeLa细胞 ,用Northern印迹分析法分析不同作用时间时细胞N rasmRNA的表达以及用Western印迹分析法分析p5 3、p2 1ras的表达 .结果发现 ,在AFP(2 0mg L)作用后 ,HeLa细胞的N rasmRNA、p5 3蛋白质和p2 1ras蛋白质的表达量与对照组比较在 12h和 2 4h时都有明显增加 .AFP的作用均可被抗AFP单克隆抗体所拮抗 .实验结果提示 ,AFP对细胞生长的调节作用可能通过促进这些原癌基因的表达来实现 .