Telomerase Inhibition Decreases Alpha-Fetoprotein Expression and Secretion by Hepatocellular Carcinoma Cell Lines: In Vitro and In Vivo Study

Alpha-fetoprotein (AFP) is a diagnostic marker for hepatocellular carcinoma (HCC). A direct relationship between poor prognosis and the concentration of serum AFP has been observed. Telomerase, an enzyme that stabilizes the telomere length, is expressed by 90% of HCC. The aim of this study was to investigate the effect of telomerase inhibition on AFP secretion and the involvement of the PI3K/Akt/mTOR signaling pathway. Proliferation and viability tests were performed using tetrazolium salt. Apoptosis was determined through the Annexin V assay using flow cytometry. The concentrations of AFP were measured using ELISA kits. The AFP mRNA expression was evaluated using RT-PCR, and cell migration was evaluated using a Boyden chamber assay. The in vivo effect of costunolide on AFP production was tested in NSG mice. Telomerase inhibition by costunolide and BIBR 1532 at 5 and 10 μM decreased AFP mRNA expression and protein secretion by HepG2/C3A cells. The same pattern was obtained with cells treated with hTERT siRNA. This treatment exhibited no apoptotic effect. The AFP mRNA expression and protein secretion by PLC/PRF/5 was decreased after treatment with BIBR1532 at 10 μM. In contrast, no effect was obtained for PLC/PRF/5 cells treated with costunolide at 5 or 10 μM. Inhibition of the PI3K/Akt/mTOR signaling pathway decreased the AFP concentration. In contrast, the MAPK/ERK pathway appeared to not be involved in HepG2/C3A cells, whereas ERK inhibition decreased the AFP concentration in PLC/PRF/5 cells. Modulation of the AFP concentration was also obtained after the inhibition or activation of PKC. Costunolide (30 mg/kg) significantly decreased the AFP serum concentration of NSG mice bearing HepG2/C3A cells. Both the inhibition of telomerase and the inhibition of the PI3K/Akt/mTOR signaling pathway decreased the AFP production of HepG2/C3A and PLC/PRF/5 cells, suggesting a relationship between telomerase and AFP expression through the PI3K/Akt/mTOR pathway     

Affinity chromatography of human alpha-fetoprotein on immobilized estrogens

Human alpha-fetoprotein was isolated from abortive material with the help of affinity chromatography on immobilized estrogens. After butanol extraction from the abortive material human AFP obtained the ability for affinity binding with immobilized estrogens. The addition of estrogens to the extract of isolated AFP preparation and incubation with them did not lower AFP binding with immobilized estrogens during the experiments, using affinity chromatography. A 10% buffered aqueous butanol solution was most optimal for elution. The data obtained can suggest that AFP in biological fluids is bound to estrogens, and butanol extraction deestrogenizes human AFP. The mechanism of AFP binding to estrogens in vivo is, evidently, carried out with the help of specific unknown carrier, as AFP does not bind free estrogens.     

Rat alpha-fetoprotein: isolation, radioimmunoassay and fetal-maternal distribution during pregnancy.

A method is described for the isolation of mg quantities of two forms of rat alpha-fetoprotein (AFP) from amniotic fluid by preparative disc-gel column electrophoresis using a continuous elution system. AFP isolated by this method is suitable for use as an antigen, can be labelled for use in a radioimmunoassay and serves as a reference standard. The characteristics of a new antiserum to AFP are also described. The protocol for a radioimmunoassay is outlined which permits the measurement of AFP in several fetal-maternal physiological compartments throughout gestation. Levels of AFP in fetal liver and fetal plasma suggest that secretion of AFP from liver occurs soon after synthesis with minimal hepatic storage. The pattern for AFP in maternal serum parallels that observed in amniotic fluid and fluctuations in maternal serum levels of AFP appear to be buffered by AFP accumulation in amniotic fluid. Fetal clearance of AFP under normal conditions may be relatively constant from Days 11-20 of gestation since an amniotic fluid: maternal serum AFP ratio of 30:1 is maintained during this period.     

Monoclonal antibodies against human alpha-fetoprotein more reactive to cell-surface alpha-fetoprotein than to free alpha-fetoprotein.

This paper describes the finding of monoclonal antibody (MoAb) more reactive to cell-surface alpha-fetoprotein (AFP) than to free AFP by using a simple in vitro system. Twelve mouse MoAbs, ten IgG1, one IgG2a and one IgG2b, against human AFP from hepatocellular carcinoma were obtained by the cell fusion technique. Each hybridoma supernatant was assayed by enzyme-linked immunosorbent assay (ELISA) to solid-phase AFP. The assay results showed that two MoAbs, 67D and 80G, were most reactive to AFP. 80G had a higher affinity constant than 67D, while the both reactions were similarly difficult to inhibit by free AFP in ELISA. 67D and 80G reacted with AFP on the surface of ethanol-fixed cells from the human hepatoma cell line HuH-7 and this reaction was also difficult to inhibit by free AFP in Cell ELISA. Furthermore, Western blot analysis showed that 67D and 80G were more reactive to membrane-bound AFP than other antibodies. These findings first suggest that there could be anti-AFP MoAbs preferably binding to cell-surface AFP rather than to serum AFP.     

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.     

Secretion and glycosylation of alpha-foetoprotein by the mouse yolk sac.

Secretion and glycosylation of alpha-foetoprotein (AFP) by mouse yolk sac were studied by using yolk-sac explants cultured in vitro. Yolk-sac explants rapidly incorporated [35S]methionine into AFP, whereas radioactively labelled AFP was not found in the medium until 30 min after incubation was initiated. Electrophoretic analysis revealed that microheterogeneity of AFP synthesized in explants increased in parallel with the gestational age of the yolk sacs. The change in microheterogeneity was noted by the formation of increasingly acidic forms. Only the most acidic forms of AFP were found to be present in the medium on each gestational day studied. Tunicamycin reduced the incorporation of glucosamine into AFP with a concomitant decrease in molecular weight and microheterogeneity. However, the relative amount of AFP released into the medium was not altered by the presence of tunicamycin. The presence of under-glycosylated AFP in the medium indicates that glycosylation of AFP is not essential for its secretion from the yolk sac. In light of these and previous findings, it is suggested that the glycosylation of AFP may be important for the turnover of this glycoprotein in serum.     

甲胎蛋白携带药物靶向治疗肿瘤

甲胎蛋白(alpha fetoprotein,AFP)是一种在胎儿发育时期高表达的蛋白质,它又是一种穿梭蛋白质,能够将营养物质输送给胚胎细胞.相似的是,在肝癌等恶性肿瘤发展时期,肿瘤细胞也高表达AFP及其受体,它们通过AFP受体摄取AFP及其运载的物质.因此,可以将AFP与抗癌药物结合,选择性攻击肿瘤细胞.AFP与药物...     

Development of an inexpensive and rapid two site microenzyme linked immunosorbent assay kit for human alpha fetoprotein

Laboratory scale development of a two site micro enzyme linked immuno assay kit is described. The kit comprises rabbit anti human alphafetoprotein (AFP), anti human AFP IgG peroxidase conjugate and standard AFP. All the above reagents were prepared in the laboratory. The kit is eminently suitable for early screening of blood sample of pregnant women for neural tube defects of their fetuses and for the quantitation of AFP as a tumor marker. The assay kit was used to determine AFP in 76 sera from women at different stages of pregnancy. During 1st trimester AFP level was 18 to 119 ng/ml, during 2nd trimester the concentration varied from 85 to 302 ng/ml and during 3rd from 103 to 580 ng/ml. No evidence for maternal antibody to AFP was found. The above data agree with AFP level in pregnant women reported by earlier workers, using RIA or ELISA. The present ELISA kit would hopefully be much cheaper than internationally available ELISA kits for human AFP.     

The antifungal protein AFP from Aspergillus giganteus inhibits chitin synthesis in sensitive fungi

The antifungal protein AFP from Aspergillus giganteus is highly effective in restricting the growth of major human- and plant-pathogenic filamentous fungi. However, a fundamental prerequisite for the use of AFP as an antifungal drug is a complete understanding of its mode of action. In this study, we performed several analyses focusing on the assumption that the chitin biosynthesis of sensitive fungi is targeted by AFP. Here we show that the N-terminal domain of AFP (amino acids 1 to 33) is sufficient for efficient binding of AFP to chitin but is not adequate for inhibition of the growth of sensitive fungi. AFP susceptibility tests and SYTOX Green uptake experiments with class III and class V chitin synthase mutants of Fusarium oxysporum and Aspergillus oryzae showed that deletions made the fungi less sensitive to AFP and its membrane permeabilization effect. In situ chitin synthase activity assays revealed that chitin synthesis is specifically inhibited by AFP in sensitive fungi, indicating that AFP causes cell wall stress and disturbs cell integrity. Further evidence that there was AFP-induced cell wall stress was obtained by using an Aspergillus niger reporter strain in which the cell wall integrity pathway was strongly induced by AFP.     

Hormonal regulation during secretion of alpha-fetoprotein in hepatoma cells grown in synthetic medium

The variant cell line of H4-II-E-C3 cells derived from the Reuber H-35 hepatoma cells has been established using protein- and lipid-free synthetic medium. This H4-II-E-C3-V line can synthesize and secrete considerable amounts of alpha-fetoprotein (AFP) and albumin. The addition of 5 X 10(-7) M dexamethasone to the medium stimulated the excretion of AFP without increasing total AFP synthesis, whereas 8.7 X 10(-8) M insulin inhibited the excretion of AFP without a significant inhibition of intracellular AFP synthesis. However, neither dexamethasone nor insulin altered either the cellular or secreted levels of albumin. Cells were pulse labeled with [35S]methionine and then chased after addition of excess unlabeled methionine. AFP appeared in the medium after 10 min, and 50% of the protein was secreted after 110 min. The rate of secretion of AFP was much slower than that of albumin, 50% of which was secreted after 25 min. Dexamethasone, 5 X 10(-7) M, caused a marked enhancement in the rate of AFP secretion, with 50% released after 75 min. Insulin, 8.7 X 10(-8) M, by contrast, caused a marked delay in AFP secretion with only 20% released after 180 min and then a plateau was approached. Since the intracellular AFP was excreted 55% after 180 min the remaining 25% of newly made AFP was suggested to be degraded during secretion. The kinetics of movement of AFP during secretion and endoglycosidase H treatment of intracellular and secreted AFP suggested that insulin impeded the transport of AFP from the rough endoplasmic reticulum to the Golgi apparatus.     

A silent antifungal protein (AFP)-like gene lacking two introns in the mould Trichoderma viride

In cells of the mould Trichoderma viride, the existence of an antifungal protein (AFP)-like gene consisting of 285 bp was confirmed by Southern analysis that genomic DNA of T. viride could hybridize with the cDNA of mature AFP of Aspergillus giganteus MDH 18894. Except for the absence of two introns, the nucleotide sequence of the AFP-like gene was identical to that of the AFP gene of A. giganteus in positions 336-479, 568-649, and 706-765. The AFP-like gene could not be transcribed into its mRNA in T. viride cells as examined by RT-PCR using total RNAs of T. viride as template. Furthermore, AFP could not be detected either directly from the culture medium of T. viride or by Western analysis. However, the AFP-like gene could be actively expressed like the cDNA of AFP in Escherichia coli cell. Recombinant AFP exhibited similar antifungal activity as native AFP.     

生理性及病理性人体甲种胎儿蛋白分子异质性研究

为阐明人体组织处于生理及病理状态时合成及分泌的各种甲种胎儿蛋白(AFP)分子中糖基组成上的差异,本文采用刀豆球蛋白A(ConA)及扁豆凝集素(LCA)亲和双向放射免疫电泳(Aff-RIEP)分析人体羊水、孕妇、急性肝炎及原发性肝癌患者血清中各种不同来源的AFP。实验表明孕妇及肝炎血清中AFP在ConA及LCA电泳中呈现相似的电泳图谱,但与肝癌AFP有显著差异。在ConA电泳中,肝癌血清显示二种分子变异体,其中以ConA结合型为主,兼有小部分非结合型AFP,而肝炎及孕妇AFP仅为一种ConA结合型分子。然而,在LCA电泳中,肝炎及孕妇血清AFP却以LCA非结合型为主,兼有小部分结合型,但肝癌AFP却与之相反,以结合型为主,非结合型为次。上述结果提示癌性与非癌性AFP分子之间存在糖基组成及结构上的差异。     

Affinity chromatography of mouse and rat alpha-fetoproteins on immobilized diethylstilbestrol]

Alpha-fetoproteins (AFP) from amniotic fluid of mouse and rat demonstrate high affinity and specificity during their binding with immobilized diethylstilbestrol, which allows to isolate these two proteins by one step using the method of affinity chromatography on Sepharose with immobilized diethylstilbestrol. Meanwhile the yield of mouse AFP was 42%, and rat AFP--75%. The preliminary incubation of the amniotic fluid of rat and mouse with free estradiol results in abrupt fall of AFP outcome, which may testify to the binding of estradiol and diethylstilbestrol by the same receptor sites on AFP molecule.     

A leucine-rich repeat protein of carrot that exhibits antifreeze activity

A gene encoding an antifreeze protein (AFP) was isolated from carrot (Daucus carota) using sequence information derived from the purified protein. The carrot AFP is highly similar to the polygalacturonase inhibitor protein (PGIP) family of apoplastic plant leucine-rich repeat (LRR) proteins. Expression of the AFP gene is rapidly induced by low temperatures. Furthermore, expression of the AFP gene in transgenic Arabidopsis thaliana plants leads to an accumulation of antifreeze activity. Our findings suggest that a new type of plant antifreeze protein has recently evolved from PGIPs.     

Role of alpha-fetoprotein regulatory elements in transcriptional activation in transient heterokaryons.

The requirements for activation of the mouse alpha-fetoprotein (AFP) gene in transient heterokaryons were investigated. For this purpose, the 7-kilobases of DNA flanking the 5' end of the AFP gene were linked to a mouse major histocompatibility complex (MHC) class I structural gene. The fusion gene was stably integrated at different sites into mouse L-cells, which do not transcribe the AFP gene. Transient heterokaryon fusions demonstrated that the silent AFP-MHC gene and the endogenous AFP gene were activated by factors present in HepG2 cells, a liver-derived cell line, but not by those in HeLa cells. Activation was detected at the protein level in single heterokaryons by using monoclonal antibodies against the cell surface protein and at the mRNA level in populations of cells. The AFP promoter alone was sufficient for activation could be used for DNA transfer strategies to identify genes which can activate AFP promoter elements in trans.     

Alpha-fetoprotein expression in intra- and extraembryonic fluids of developing chick embryo

The ontogeny of alpha-fetoprotein (AFP) has been studied in the chicken (from 7 days of incubation until 2 days after hatching) using (1) the two-dimensional immunoelectrophoresis technique, (2) the polyacrylamide gel electrophoresis, and (3) the high resolution two-dimensional polyacrylamide gel electrophoresis. The molecular weight of AFP was estimated at 71,000. AFP was seen as a heterogeneous population composed of four isoforms which slightly differ by their isoelectric points. Up to the 18th day of development, qualitative changes in AFP heterogeneity do not occur. Only traces of the two alkaline isoforms were observed in plasma of 2 days post-hatching chickens. AFP has been identified in allantoic and cerebrospinal fluids but is not present in amniotic fluid. At 7 days of embryonic age, all the plasma AFP species are present in cerebrospinal fluid.