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Zhang M, Liu K, Zhang Q, Xu J, Liu J, Lin H, Lin B, Zhu M, Li M. Alpha fetoprotein promotes polarization of macrophages towards M2-like phenotype and inhibits macrophages to phagocytize hepatoma cells. Front Immunol 2023; 14:1081572. [PMID: 36911723 PMCID: PMC9995430 DOI: 10.3389/fimmu.2023.1081572] [Citation(s) in RCA: 13] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2022] [Accepted: 02/13/2023] [Indexed: 02/25/2023] Open
Abstract
Alpha-fetoprotein(AFP) is a cancer biomarker for the diagnosis of hepatocellular carcinoma(HCC); however, its role in macrophage polarization and phagocytosis remains unclear. In the present study, we explored the correlation between AFP regulation of macrophage function and the possible regulatory mechanisms. Human mononuclear leukemia cells (THP-1) and monocytes from healthy donors were used to analyze the effect of AFP on the macrophages' phenotype and phagocytosis. THP-1 cells and healthy human donor-derived monocytes were polarized into M0 macrophages induced by phorbol ester (PMA), and M0 macrophages were polarized into M1 macrophages induced by lipopolysaccharide(LPS) and interferon-γ(IFN-γ). Interleukin-4(IL-4) and interleukin-13(IL-13) were used to induce M0 macrophage polarization into M2 macrophages. Tumor-derived AFP(tAFP) stimulated M0 macrophage polarization into M2 macrophages and inhibited M1 macrophages to phagocytize HCC cells. The role of AFP in promoting macrophage polarization into M2 macrophages and inhibiting the M1 macrophages to phagocytize HCC cells may be involved in activating the PI3K/Akt signaling pathway. AFP could also enhanced the migration ability of macrophages and inhibited the apoptosis of HCC cells when co-cultured with M1-like macrophages. AFP is a pivotal cytokine that inhibits macrophages to phagocytize HCC cells.
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Affiliation(s)
- Minni Zhang
- Hainan Provincial Key Laboratory of Carcinogenesis and Intervention, Hainan Medical College, Hiakou, Hainan, China
| | - Kun Liu
- Hainan Provincial Key Laboratory of Carcinogenesis and Intervention, Hainan Medical College, Hiakou, Hainan, China
| | - Qiuyue Zhang
- Hainan Provincial Key Laboratory of Carcinogenesis and Intervention, Hainan Medical College, Hiakou, Hainan, China
| | - Junnv Xu
- Department of Medical Oncology, Second Affiliated Hospital, Hainan Medical College, Haikou, Hainan, China
| | - Jinchen Liu
- Hainan Provincial Key Laboratory of Carcinogenesis and Intervention, Hainan Medical College, Hiakou, Hainan, China
| | - Haifeng Lin
- Department of Medical Oncology, Second Affiliated Hospital, Hainan Medical College, Haikou, Hainan, China
| | - Bo Lin
- Hainan Provincial Key Laboratory of Carcinogenesis and Intervention, Hainan Medical College, Hiakou, Hainan, China
| | - Mingyue Zhu
- Hainan Provincial Key Laboratory of Carcinogenesis and Intervention, Hainan Medical College, Hiakou, Hainan, China
| | - Mengsen Li
- Hainan Provincial Key Laboratory of Carcinogenesis and Intervention, Hainan Medical College, Hiakou, Hainan, China
- Department of Medical Oncology, Second Affiliated Hospital, Hainan Medical College, Haikou, Hainan, China
- Institution of Tumor, Hainan Medical College, Hiakou, Hainan, China
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Li W, Liu K, Chen Y, Zhu M, Li M. Role of Alpha-Fetoprotein in Hepatocellular Carcinoma Drug Resistance. Curr Med Chem 2021; 28:1126-1142. [PMID: 32729413 DOI: 10.2174/0929867327999200729151247] [Citation(s) in RCA: 25] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2020] [Revised: 07/04/2020] [Accepted: 07/11/2020] [Indexed: 01/30/2023]
Abstract
Hepatocellular carcinoma (HCC) is a major type of primary liver cancer and a major cause of cancer-related deaths worldwide because of its high recurrence rate and poor prognosis. Surgical resection is currently the major treatment measure for patients in the early and middle stages of the disease. Because due to late diagnosis, most patients already miss the opportunity for surgery upon disease confirmation, conservative chemotherapy (drug treatment) remains an important method of comprehensive treatment for patients with middle- and late-stage liver cancer. However, multidrug resistance (MDR) in patients with HCC severely reduces the treatment effect and is an important obstacle to chemotherapeutic success. Alpha-fetoprotein (AFP) is an important biomarker for the diagnosis of HCC. The serum expression levels of AFP in many patients with HCC are increased, and a persistently increased AFP level is a risk factor for HCC progression. Many studies have indicated that AFP functions as an immune suppressor, and AFP can promote malignant transformation during HCC development and might be involved in the process of MDR in patients with liver cancer. This review describes drug resistance mechanisms during HCC drug treatment and reviews the relationship between the mechanism of AFP in HCC development and progression and HCC drug resistance.
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Affiliation(s)
- Wei Li
- Hainan Provincial Key Laboratory of Carcinogenesis and Intervention, Hainan Medical College, Haikou 571199, Hainan Province, China
| | - Kun Liu
- Hainan Provincial Key Laboratory of Carcinogenesis and Intervention, Hainan Medical College, Haikou 571199, Hainan Province, China
| | - Yi Chen
- Hainan Provincial Key Laboratory of Carcinogenesis and Intervention, Hainan Medical College, Haikou 571199, Hainan Province, China
| | - Mingyue Zhu
- Hainan Provincial Key Laboratory of Carcinogenesis and Intervention, Hainan Medical College, Haikou 571199, Hainan Province, China
| | - Mengsen Li
- Hainan Provincial Key Laboratory of Carcinogenesis and Intervention, Hainan Medical College, Haikou 571199, Hainan Province, China
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Linson EA, Hanauer SB. More Than a Tumor Marker…A Potential Role for Alpha-Feto Protein in Inflammatory Bowel Disease. Inflamm Bowel Dis 2019; 25:1271-1276. [PMID: 30624658 DOI: 10.1093/ibd/izy394] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/24/2018] [Revised: 11/05/2018] [Indexed: 02/07/2023]
Abstract
BACKGROUND Human alpha-fetoprotein (hAFP) is a glycoprotein derived from the gut entoderm and expressed sequentially by cells of the yolk sac, fetal liver, and gastrointestinal tract. By adulthood, serum levels of alpha-fetoprotein (AFP) are undetectable in healthy, nonpregnant adults. Despite the clinical utilities of AFP monitoring in pregnancy and malignancy, much remains to be determined regarding its potential physiological functions. METHODS We focused on literature related to AFP's immunoregulatory role and its ability to modulate disease activity both in animal models of autoimmune disorders and in human clinical studies. RESULTS Evidence suggests that AFP plays an important role in immunoregulation by inducing T-cell suppressor activity, downregulating dendritic-like cell antigen expression, and impairing the function of macrophages. Studies evaluating AFP and its effects in rodent models of autoimmune diseases have shown that AFP is associated with downregulation of inflammation. Observations in studies of pregnant patients with immune-mediated inflammatory diseases have also described potential correlations between AFP expression and disease activity during different stages of pregnancy and postpartum. CONCLUSIONS We propose further prospective evaluations of AFP expression during pregnancy in inflammatory bowel disease patients to further correlate with disease activity and consider the potential of AFP as a novel therapeutic agent.
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Affiliation(s)
| | - Stephen B Hanauer
- Northwestern University, Feinberg School of Medicine, Chicago Illinois
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4
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Recombinant human alpha fetoprotein synergistically potentiates the anti-cancer effects of 1'-S-1'-acetoxychavicol acetate when used as a complex against human tumours harbouring AFP-receptors. Oncotarget 2016; 6:16151-67. [PMID: 26158863 PMCID: PMC4599262 DOI: 10.18632/oncotarget.3951] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2014] [Accepted: 04/08/2015] [Indexed: 01/01/2023] Open
Abstract
Purpose Previous in vitro and in vivo studies have reported that 1′-S-1′-acetoxychavicol acetate (ACA) isolated from rhizomes of the Malaysian ethno-medicinal plant Alpinia conchigera Griff (Zingiberaceae) induces apoptosis-mediated cell death in tumour cells via dysregulation of the NF-κB pathway. However there were some clinical development drawbacks such as poor in vivo solubility, depreciation of biological activity upon exposure to an aqueous environment and non-specific targeting of tumour cells. In the present study, all the problems above were addressed using the novel drug complex formulation involving recombinant human alpha fetoprotein (rhAFP) and ACA. Experimental Design To study the synergistic effect of both agents on human cancer xenografts, athymic nude (Nu/Nu) mice were used and treated with various combination regimes intraperitoneally. Serum levels of tumour markers for carcinoembryonic antigen (CEA) and prostate specific antigen (PSA) were assessed using sandwich ELISA. IHC and Western blotting were also conducted on in vivo tumour biopsies to investigate the involvement of NF-κB regulated genes and inflammatory biomarkers. Quantification and correlation between drug efficacies and AFP-receptors were done using IF-IC and Pearson's correlation analysis. Results Mice exposed to combined treatments displayed higher reductions in tumour volume compared to stand alone agents, consistent with in vitro cytotoxicity assays. Milder signs of systemic toxicity, such as loss in body weight and inflammation of vital organs were also demonstrated compared to stand alone treatments. Tumour marker levels were consistent within all rhAFP/ACA treatment groups where levels of CEA and PSA were initially elevated upon commencement of treatment, and consecutively reduced corresponding to a decrease in tumour bulk volume. Both IHC and Western blotting results indicated that the combined action of rhAFP/ACA was not only able to down-regulate NF-κB activation, but also reduce the expression of NF-κB regulated genes and inflammatory biomarkers. The efficacy of rhAFP/ACA complex was also found to be weakly negatively correlated to the level of surface AFP-receptors between tumour types. Conclusions This drug complex formulation shows great therapeutic potential against AFP-receptor positive tumours, and serves as a basis to overcome insoluble and non-specific anti-neoplastic molecules.
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5
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Zhang C, Li G. Role of alpha-fetoprotein in hepatitis B virus-induced hepatocellular carcinoma: Prospect in clinical application. Shijie Huaren Xiaohua Zazhi 2015; 23:3171-3181. [DOI: 10.11569/wcjd.v23.i20.3171] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Mammalian alpha-fetoprotein (AFP) as a fetal specific alpha-globulin that has been used as a serum fetal defect/tumor marker for diagnosis and prediction of liver disease. Over the past decade, research indicates that AFP as an intracellular signal molecule is not only a biomarker but also interacts with hepatitis B virus (HBV) and hepatitis B virus protein x and plays multifarious roles in the development of hepatocellular carcinoma, especially in HBV-induced liver cancer.
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6
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Pardee AD, Shi J, Butterfield LH. Tumor-derived α-fetoprotein impairs the differentiation and T cell stimulatory activity of human dendritic cells. THE JOURNAL OF IMMUNOLOGY 2014; 193:5723-32. [PMID: 25355916 DOI: 10.4049/jimmunol.1400725] [Citation(s) in RCA: 71] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
Several tumor-derived factors have been implicated in dendritic cell (DC) dysfunction in cancer patients. α-fetoprotein (AFP) is an oncofetal Ag that is highly expressed in abnormalities of prenatal development and several epithelial cancers, including hepatocellular carcinoma (HCC). In HCC patients exhibiting high levels of serum AFP, we observed a lower ratio of myeloid/plasmacytoid circulating DCs compared with patients with low serum AFP levels and healthy donors. To test the effect of AFP on DC differentiation in vitro, peripheral blood monocytes from healthy donors were cultured in the presence of cord blood-derived normal AFP (nAFP) or HCC tumor-derived AFP (tAFP), and DC phenotype and function were assessed. Although the nAFP and tAFP isoforms only differ at one carbohydrate group, low (physiological) levels of tAFP, but not nAFP, significantly inhibited DC differentiation. tAFP-conditioned DCs expressed diminished levels of DC maturation markers, retained a monocyte-like morphology, exhibited limited production of inflammatory mediators, and failed to induce robust T cell proliferative responses. Mechanistic studies revealed that the suppressive activity of tAFP is dependent on the presence of low molecular mass (LMM) species that copurify with tAFP and function equivalently to the LMM fractions of both tumor and nontumor cell lysates. These data reveal the unique ability of tAFP to serve as a chaperone protein for LMM molecules, both endogenous and ubiquitous in nature, which function cooperatively to impair DC differentiation and function. Therefore, novel therapeutic approaches that antagonize the regulatory properties of tAFP will be critical to enhance immunity and improve clinical outcomes.
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Affiliation(s)
- Angela D Pardee
- Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213
| | - Jian Shi
- Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213
| | - Lisa H Butterfield
- Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213; Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213; Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213; and University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213
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7
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Development of an Active Site Peptide Analog of α-Fetoprotein That Prevents Breast Cancer. Cancer Prev Res (Phila) 2014; 7:565-73. [DOI: 10.1158/1940-6207.capr-13-0405] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
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Abstract
Alpha-fetoprotein (AFP) is a major mammalian embryo-specific and tumor-associated protein that is also present in small quantities in adults at normal conditions. Discovery of the phenomenon of AFP biosynthesis in carcinogenesis by G. Abelev and Yu. Tatarinov 50 years ago, in 1963, provoked intensive studies of this protein. AFPs of some mammalian species were isolated, purified and physico-chemically and immunochemically characterized. Despite the significant success in study of AFP, its three-dimensional structure, mechanisms of receptor binding along with a structure of the receptor itself and, what is the most important, its biological role in embryo- and carcinogenesis remain still obscure. Due to difficulties linked with methodological limitations, research of AFP was to some extent extinguished by the 1990 s. However, over the last decade a growing number of investigations of AFP and its usage as a tumor-specific biomarker have been observed. This was caused by the use of new technologies, primarily, computer-based and genetic engineering approaches in studying of this very important oncodevelopmental protein. Our review summarizes efforts of different scientific groups throughout the world in studying AFP for 50 years with emphasis on detailed description of recent achievements in this field.
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9
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Wang XP, Wang QX, Lin HP, Wang YL, Yang Y. Glycoprotein 96 and α-fetoprotein cross-linking complexes elicited specific antitumor immunity. Cancer Biother Radiopharm 2013; 28:406-14. [PMID: 23484810 DOI: 10.1089/cbr.2012.1404] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
Hepatocellular carcinoma (HCC) is one of the most common malignant gastroenterological cancers over the world. α-fetoprotein (AFP) is an oncofetal protein produced during HCC development that could generate weaker and less reproducible antitumor protection, and it may serve as a target for immunotherapy. Therefore, it is imperative to enhance its immunogenicity and develop therapeutic vaccines to eliminate AFP-expressing tumors. In this study, by way of glutaraldehyde cross-linking, we constructed a potential therapeutic protein vaccine, glycoprotein 96 (gp96)/AFP. Our results demonstrated that AFP and gp96 synergistically exhibited significant increase in AFP-specific CD8⁺ T-cell responses and impressive cytotoxic antitumor effect against AFP-expressing tumors. Priming mice with the reconstructed vaccine, we elicited robust strong protective immunity. Our study suggests that tumor vaccine by cross-linking tumor antigen and gp96 is a promising approach to cancer therapy.
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Affiliation(s)
- Xiao-Ping Wang
- Laboratory of Molecular Biology & Pathology, Shaanxi University of Chinese Medicine, Xianyang, China.
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10
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Zubkova ES, Semenkova LN, Dudich IV, Dudich EI, Khromykh LM, Makarevich PI, Parfenova EV, Men'shikov MI. [Recombinant human alpha-fetoprotein as a regulator of adipose tissue stromal cell activity]. RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY 2013; 38:524-34. [PMID: 23342486 DOI: 10.1134/s1068162012050147] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Abstract
Recombinant human alpha-fetoprotein (rhAFP) expressed in yeast system as a glycoprotein, was isolated and purified to 98% by multistep method. The testing of the rhAFP in the culture of adipose tissue stromal cells (hASC) has revealed its ability to enhance hASC proliferation and migration as well as vascular endothelial growth factor production, with no significant influence on cell invasion and matrix metalloproteinase-2 and -9 secretion. It has been also estimated that rhAFP is internalized in hASC via clathrin-dependent mechanism. A study in the murine experimental model of hindlimb ischemia has shown the capability of rhAFP to enhance blood flow recovery. These data suggest that rhAFP is a promising agent for enhancement of the hASC regenerative ability.
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11
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Wang X, Wang Q, Lin H, Li S, Sun L, Yang Y. HSP72 and gp96 in gastroenterological cancers. Clin Chim Acta 2012; 417:73-9. [PMID: 23266770 DOI: 10.1016/j.cca.2012.12.017] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2012] [Revised: 12/14/2012] [Accepted: 12/15/2012] [Indexed: 11/30/2022]
Abstract
Heat shock protein 72 (HSP72) and glycoprotein 96 (gp96) are highly expressed in cancer tissues. Recent studies indicate the possible roles of HSP72 and gp96 in the development and progression of gastrointestinal carcinomas but detailed mechanisms are still ambiguous. Human esophageal cancer, gastric cancer, colon cancer and liver cancer are common gastrointestinal malignant carcinomas in the world. The studies indicated that there existed a significant correlation between the expression of HSP72, gp96 and the development and progression of digestive carcinomas. HSP72 and gp96 expression were significantly associated with the presence of tumor infiltration, lymph node and remote metastasis. Interestingly, studies have found that HSP72 chaperoned alpha-fetoprotein (AFP), HBx in hepatocellular carcinoma, and CD44 in colonic carcinomas. The further researches demonstrated that HSP72-AFP or gp96-AFP recombined vaccine could elicit specific anti-tumor immunity. The high-level expression of HSP72 and gp96 may be not only used as diagnostic or prognostic markers for gastrointestinal carcinomas but also as better immunotherapeutic vaccines in the cancers.
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Affiliation(s)
- Xiaoping Wang
- Key Laboratory of Molecular Biology and Pathology, Shaanxi University of Chinese Medicine, Xianyang, Shaanxi 712046, PR China.
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12
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Dudich E, Dudich I, Semenkova L, Benevolensky S, Morozkina E, Marchenko A, Zatcepin S, Dudich D, Soboleva G, Khromikh L, Roslovtceva O, Tatulov E. Engineering of the Saccharomyces cerevisiae yeast strain with multiple chromosome-integrated genes of human alpha-fetoprotein and its high-yield secretory production, purification, structural and functional characterization. Protein Expr Purif 2012; 84:94-107. [PMID: 22561245 DOI: 10.1016/j.pep.2012.04.008] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2011] [Revised: 04/11/2012] [Accepted: 04/14/2012] [Indexed: 11/20/2022]
Abstract
Alpha-fetoprotein (AFP) is a biological drug candidate of high medicinal potential in the treatment of autoimmune diseases, cancer, and regenerative medicine. Large-scale production of recombinant human alpha-fetoprotein (rhAFP) is desirable for structural and functional studies and applied research. In this study we cloned and expressed in the secreted form wild-type glycosylated human rhAFP and non-glycosylated mutant rhAFP(0) (N233S) in the yeast strain Saccharomyces cerevisiae with multiple chromosome-integrated synthetic human AFP genes. RhAFP and rhAFP(0) were successfully produced and purified from the culture liquids active naturally folded proteins. Elimination of the glycosylation by mutation reduced rhAFP(0) secretion about threefold as compared to the wild-type protein showing critical role of the N-linked glycan for heterologous protein folding and secretion. Structural similarity of rhAFP and rhAFP(0) with natural embryonic eAFP was confirmed by circular dichroism technique. Functional tests demonstrated similar type of tumor suppressive and immunosuppressive activity for both recombinant species rhAFP and rhAFP(0) as compared to natural eAFP. It was documented that both types of biological activities attributed to rhAFP and rhAFP(0) are due to the fast induction of apoptosis in tumor cells and mitogen-activated lymphocytes. Despite the fact that rhAFP and rhAFP(0) demonstrated slightly less effective tumor suppressive activity as compared to eAFP but rhAFP(0) had produced statistically notable increase in its ability to induce inhibition of in vitro lymphocyte proliferation as compared to the glycosylated rhAFP and eAFP. We conclude that N-linked glycosylation of rhAFP is required for efficient folding and secretion. However the presence of N-linked sugar moiety was shown to be unimportant for tumor suppressive activity but was critically important for its immunoregulative activity which demonstrates that different molecular mechanisms are involved in these two types of biological functional activities attributed to AFP.
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Affiliation(s)
- Elena Dudich
- Institute of Immunological Engineering, Lyubuchany, Moscow Region, Chekhov District 142380, Russia.
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Wang XP, Lin HP, Wang QX, Gu Y. Specific Antitumor Immunity Induced by Cross-linking Complex Heat Shock Protein 72 and Alpha-fetoprotein. Cancer Biother Radiopharm 2012; 27:189-97. [DOI: 10.1089/cbr.2011.1135] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023] Open
Affiliation(s)
- Xiao-Ping Wang
- Laboratory of Molecular Pathology, Shaanxi University of Chinese Medicine, Xianyang, China
- Department of Pharmaceutical Sciences, North Dakota State University, Fargo, North Dakota
| | - Huan-Ping Lin
- Laboratory of Molecular Pathology, Shaanxi University of Chinese Medicine, Xianyang, China
| | - Qiao-Xia Wang
- Laboratory of Molecular Pathology, Shaanxi University of Chinese Medicine, Xianyang, China
| | - Yan Gu
- Department of Pharmaceutical Sciences, North Dakota State University, Fargo, North Dakota
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14
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Peterson ML, Ma C, Spear BT. Zhx2 and Zbtb20: novel regulators of postnatal alpha-fetoprotein repression and their potential role in gene reactivation during liver cancer. Semin Cancer Biol 2011; 21:21-7. [PMID: 21216289 PMCID: PMC3313486 DOI: 10.1016/j.semcancer.2011.01.001] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2010] [Revised: 12/22/2010] [Accepted: 01/04/2011] [Indexed: 12/21/2022]
Abstract
The mouse alpha-fetoprotein (AFP) gene is abundantly expressed in the fetal liver, normally silent in the adult liver but is frequently reactivated in hepatocellular carcinoma. The basis for AFP expression in the fetal liver has been studied extensively. However, the basis for AFP reactivation during hepatocarcinogenesis is not well understood. Two novel factors that control postnatal AFP repression, Zhx2 and Zbtb20, were recently identified. Here, we review the transcription factors that regulate AFP in the fetal liver, as well as Zhx2 and Zbtb20, and raise the possibility that the loss of these postnatal repressors may be involved in AFP reactivation in liver cancer.
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Affiliation(s)
- Martha L Peterson
- Department of Microbiology, Immunology & Molecular Genetics and Markey Cancer Center, University of Kentucky, Lexington, KY 40536, USA
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15
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Belyaev NN, Bogdanov AY, Savvulidi PG, Krasnoshtanov VK, Tleulieva RT, Alipov GK, Sekine I, Bae JS, Lee JB, Min YK, Yang HM. The Influence of Alpha-fetoprotein on Natural Suppressor Cell Activity and Ehrlich Carcinoma Growth. THE KOREAN JOURNAL OF PHYSIOLOGY & PHARMACOLOGY : OFFICIAL JOURNAL OF THE KOREAN PHYSIOLOGICAL SOCIETY AND THE KOREAN SOCIETY OF PHARMACOLOGY 2008; 12:193-7. [PMID: 19967055 DOI: 10.4196/kjpp.2008.12.4.193] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
The influence of alpha-fetoprotein (AFP) on the bone marrow (BM) natural suppressor (NS) cells of intact Ehrlich carcinoma -bearing CBA mice was studied. Bone marrow NS cells were fractionated into three fractions by isopycnic centrifugation on percoll gradients: NS1 (rho=1.080 g/ml), NS2 (rho=1.090 g/ml) and NS3 (1.100>rho>1.090 g/ml). These fractions were highly different in their sensitivity to known NS cell inductors (interleukin (IL)-2, IL-3 or histamine). None of the NS fractions isolated from the intact mice spontaneously produced antiproliferative activity, however, they showed a high level of NS (antiproliferative and natural killer cell inhibitory) activity under the influence of AFP. A single injection of AFP to intact mice led to an increase of spontaneous NS activity and the inhibition of natural killer cell activity. NS activity, especially NS2, was increased in when tumor cells were subcutaneously inoculated three days after AFP injection. In the AFP-treated mice, the tumor mass at 14 days was 60% larger than that in the untreated mice. Our data confirmed that AFP is a tumor marker that can inhibit cancer immunity and plays a role in cancer pathogenesis.
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16
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Evdokimova VN, Butterfield LH. Alpha-fetoprotein and other tumour-associated antigens for immunotherapy of hepatocellular cancer. Expert Opin Biol Ther 2008; 8:325-36. [PMID: 18294103 DOI: 10.1517/14712598.8.3.325] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
BACKGROUND Hepatocellular carcinoma (HCC) is a leading cause of cancer death, with few treatment options for advanced disease. OBJECTIVES Here, we review the aetiology of HCC and focus on recent data on tumour-associated antigens (TAA) for HCC, their functions and potential use as immunological targets for immune-based therapy for HCC. In addition, we examine some aspects of antigen presentation within the liver. RESULTS/CONCLUSIONS alpha-Fetoprotein (AFP) has been investigated for many years as a TAA, and has been tested in recent clinical trials. More recently, additional TAA have been identified and new therapeutic approaches have been investigated which may be testable clinically in this difficult disease setting.
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Affiliation(s)
- Viktoria N Evdokimova
- University of Pittsburgh, Hillman Cancer Center, Department of Medicine, Hematology/Oncology, Research Pavilion, Room 1.32, 5117 Centre Avenue, Pittsburgh, PA 15213, USA
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17
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Bösze Z, Baranyi M, Whitelaw CBA. Producing recombinant human milk proteins in the milk of livestock species. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2008; 606:357-93. [PMID: 18183938 DOI: 10.1007/978-0-387-74087-4_15] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/19/2023]
Abstract
Recombinant human proteins produced by the mammary glands of genetically modified transgenic livestock mammals represent a special aspect of milk bioactive components. For therapeutic applications, the often complex posttranslational modifications of human proteins should be recapitulated in the recombinant products. Compared to alternative production methods, mammary gland production is a viable option, underlined by a number of transgenic livestock animal models producing abundant biologically active foreign proteins in their milk. Recombinant proteins isolated from milk have reached different phases of clinical trials, with the first marketing approval for human therapeutic applications from the EMEA achieved in 2006.
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18
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Evdokimova VN, Liu Y, Potter DM, Butterfield LH. AFP-specific CD4+ helper T-cell responses in healthy donors and HCC patients. J Immunother 2007; 30:425-37. [PMID: 17457217 PMCID: PMC3612834 DOI: 10.1097/cji.0b013e31802fd8e2] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023]
Abstract
Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and is often diagnosed at an advanced stage. We have investigated alpha-fetoprotein (AFP) as a tumor-associated antigen for HCC. We identified major histocompatibility complex class I-restricted peptide epitopes derived from AFP and studied CD8 T-cell responses in vivo and in vitro in ongoing immunotherapy studies. Helper T cells are of critical importance in shaping the immune response; therefore, we investigated the frequency and function of AFP-specific CD4 T cells in the general population and among HCC patients. CD4 T-cell responses were assessed by direct ex vivo multicytokine enzyme-linked immunospot assay and by measurement of cytokine levels using a multicytokine assay. Our analysis indicates that healthy donors have very low frequencies of AFP-specific CD4 T-cell responses, which are of TH1 type, detectable ex vivo. In contrast, these T cells were either reduced or eliminated in HCC patients at advanced stages of disease. To better activate these cells, we compared the stimulatory capacity of both AFP protein-fed and AdVhAFP-engineered dendritic cells (DC). Healthy donors have CD4 T-cell responses, which were activated in response to AFP protein-fed DC whereas HCC patients do not demonstrate significant responses to AFP protein. AdVhAFP-transduced DC were capable of activating higher frequency TH1 CD4 responses to AFP in both healthy donors and AFP-positive HCC patients. Importantly, CD4 T-cell cytokine expression profiles were skewed towards interleukin-2 and interferon-gamma production when activated by adenovirally engineered DC, which has therapeutic implications for vaccination efforts.
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Affiliation(s)
- Viktoria N. Evdokimova
- Department of Medicine, Division of Hematology/Oncology, University of Pittsburgh Cancer Institute, Pittsburgh, PA
| | - Yang Liu
- Department of Medicine, Division of Hematology/Oncology, University of Pittsburgh Cancer Institute, Pittsburgh, PA
| | - Douglas M. Potter
- Biostatistics Department, Graduate School of Public Health, University of Pittsburgh Cancer Institute, Pittsburgh, PA
- Biostatistics Facility, University of Pittsburgh Cancer Institute, Pittsburgh, PA
| | - Lisa H. Butterfield
- Department of Medicine, Division of Hematology/Oncology, University of Pittsburgh Cancer Institute, Pittsburgh, PA
- Department of Surgery and Immunology, University of Pittsburgh Cancer Institute, Pittsburgh, PA
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Wang X, Zhou Y, Ying X, Guo L, Zhao Y, Fang Y. Interaction between heat shock protein 72 and alpha-fetoprotein in human hepatocellular carcinomas. Clin Chim Acta 2006; 379:158-62. [PMID: 17250819 DOI: 10.1016/j.cca.2006.12.015] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2006] [Revised: 12/12/2006] [Accepted: 12/13/2006] [Indexed: 10/23/2022]
Abstract
BACKGROUND AFP in adult serum often signals pathological conditions, particularly the presence of hepatocellular carcinoma (HCC) and germ cell tumors containing yolk sac cell elements. Heat shock protein 72 (HSP72) as a molecular chaperone has been confirmed to overexpress in epithelial carcinoma cells. There may be a possible correlation between the expression of HSP72 and AFP during the growth and differentiation of hepatocellular carcinoma cells. We investigated the interaction between heat shock protein 72 (HSP72) and alpha-fetoprotein (AFP) in human hepatocellular carcinomas. METHODS The expression and localization of HSP72 and AFP in human hepatocellular carcinomas were determined by immunohistochemistry and confocal laser microscopy. The interaction between HSP72 and AFP in hepatocellular carcinoma cells was analyzed by immunoprecipitation and Western immunoblots. RESULTS Hepatocellular carcinoma synchronously co-expressed higher level of HSP72 and AFP than in adjacent normal liver tissues. HSP72 were stained in cell nuclei and cytoplasm respectively, while AFP stained in cell plasma. Based on Western blotting methods, AFP was detected in the immunoprecipitate of anti-HSP72 monoclonal antibody (mAb), while HSP72 existed in the immunoprecipitate of anti-AFP mAb. CONCLUSIONS HSP72 and AFP expression are higher in hepatocellular carcinoma tissues. HSP72 was associated with alpha-fetoprotein in human hepatocellular carcinoma cells. The interaction between HSP72 and AFP in human hepatocellular carcinoma cells can be a new route for studying the pathogenesis and immunotherapy of hepatocellular carcinoma.
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Affiliation(s)
- Xiaoping Wang
- Department of Pathology, Shaanxi University of Chinese Medicine, Xianyang 712046, China.
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20
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Tsuboi S, Taketa K, Nouso K, Fujikawa T, Manabe K, Ohmori H, Higashi T, Shiratori Y. High level of expression of alpha-fetoprotein receptor in gastric cancers. Tumour Biol 2006; 27:283-8. [PMID: 17028464 DOI: 10.1159/000096071] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2005] [Accepted: 12/27/2005] [Indexed: 11/19/2022] Open
Abstract
The expression of the receptor for alpha-fetoprotein (AFP-R) was examined immunohistochemically in 47 cancer and 14 benign human gastric tissues. Rabbit polyclonal antibody against human AFP-R was used for immunohistochemical staining. Thirty-four of the 47 cancer tissues expressed AFP-R showing granular or reticular staining on the cancer cell surface, while only 2 of 61 control cases (14 benign gastric tissues and 47 nonmalignant tissues adjacent to cancer) showed faint and homogeneous staining in the cytoplasm of noncancerous cells. There was a significant difference in staining intensity between the cancerous and noncancerous groups. However, no statistically significant difference in staining intensity was found among the groups of well-differentiated, moderately differentiated and poorly differentiated adenocarcinomas. On the other hand, the staining intensity of signet ring cell carcinoma was significantly weaker than that of the three adenocarcinoma groups. The high level of AFP-R expression in gastric cancers may allow the use of AFP-R as a new clinically useful marker of gastric cancer in the tissue level.
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Affiliation(s)
- So Tsuboi
- Department of Hepatology, Shigei Medical Research Hospital, 2117 Yamada, Okayama, Japan.
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Levy O, Coughlin M, Cronstein BN, Roy RM, Desai A, Wessels MR. The adenosine system selectively inhibits TLR-mediated TNF-alpha production in the human newborn. THE JOURNAL OF IMMUNOLOGY 2006; 177:1956-66. [PMID: 16849509 PMCID: PMC2881468 DOI: 10.4049/jimmunol.177.3.1956] [Citation(s) in RCA: 188] [Impact Index Per Article: 9.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
Abstract
Human newborns are susceptible to microbial infection and mount poor vaccine responses, yet the mechanisms underlying their susceptibility are incompletely defined. We have previously reported that despite normal basal expression of TLRs and associated signaling intermediates, human neonatal cord blood monocytes demonstrate severe impairment in TNF-alpha production in response to triacylated (TLR 2/1) and diacylated (TLR 2/6) bacterial lipopeptides (BLPs). We now demonstrate that in marked contrast, BLP-induced synthesis of IL-6, a cytokine with anti-inflammatory and Th2-polarizing properties, is actually greater in neonates than adults. Remarkably, newborn blood plasma confers substantially reduced BLP-induced monocyte synthesis of TNF-alpha, while preserving IL-6 synthesis, reflecting the presence in neonatal blood plasma of a soluble, low molecular mass inhibitory factor (<10 kDa) that we identify as adenosine, an endogenous purine metabolite with immunomodulatory properties. The neonatal adenosine system also inhibits TNF-alpha production in response to whole microbial particles known to express TLR2 agonist activity, including Listeria monocytogenes, Escherichia coli (that express BLPs), and zymosan particles. Selective inhibition of neonatal TNF-alpha production is due to the distinct neonatal adenosine system, including relatively high adenosine concentrations in neonatal blood plasma and heightened sensitivity of neonatal mononuclear cells to adenosine A3 receptor-mediated accumulation of cAMP, a second messenger that inhibits TLR-mediated TNF-alpha synthesis but preserves IL-6 production. We conclude that the distinct adenosine system of newborns polarizes TLR-mediated cytokine production during the perinatal period and may thereby modulate their innate and adaptive immune responses.
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Affiliation(s)
- Ofer Levy
- Infectious Diseases, Children's Hospital, Boston, MA 02115, USA.
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Abstract
The effect of glycosylation on AFP foldability was investigated by parallel quantitative and qualitative analyses of the refolding of glycosylated and nonglycosylated AFP variants. Both variants were successfully refolded by dialysis from the denatured-reduced state, attaining comparable "refolded peak" profiles and refolding yields as determined by reversed-phase HPLC analysis. Both refolded variants also showed comparable spectroscopic fingerprints to each other and to their native counterparts, as determined by circular dichroism spectroscopy. Inclusion body-derived AFP was also readily refolded via dilution under the same redox conditions as dialysis refolding, showing comparable circular dichroism fingerprints as native nonglycosylated AFP. Quantitative analyses of inclusion body-derived AFP showed sensitivity of AFP aggregation to proteinaceous and nonproteinaceous inclusion body contaminants, where refolding yields increased with increasing AFP purity. All of the refolded AFP variants showed positive responses in ELISA that corresponded with the attainment of a bioactive conformation. Contrary to previous reports that the denaturation of cord serum AFP is an irreversible process, these results clearly show the reversibility of AFP denaturation when refolded under a redox-controlled environment, which promotes correct oxidative disulfide shuffling. The successful refolding of inclusion body-derived AFP suggests that fatty acid binding may not be required for the attainment of a rigid AFP tertiary structure, contrary to earlier studies. The overall results from this work demonstrate that foldability of the AFP molecule from its denatured-reduced state is independent of its starting source, the presence or absence of glycosylation and fatty acids, and the refolding method used (dialysis or dilution).
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Affiliation(s)
- Susanna S J Leong
- Centre for Biomolecular Engineering, The University of Queensland, St. Lucia, Australia.
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Liang Z, Luo RC, You CX, Zheng H. Infection of rAAV-AFP enhances immunostimulatory effect of human peripheral blood monocyte-derived dendritic cells. Shijie Huaren Xiaohua Zazhi 2006; 14:1362-1366. [DOI: 10.11569/wcjd.v14.i14.1362] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To study the immunostimulatory effect of rAAV-AFP (recombinant adeno-associated virus expressing α-fetoprotein antigen) transfection on dendritic cells (DCs) derived from human peripheral blood monocytes.
METHODS: Newly isolated dendritic cells were infected with rAAV-AFP. The percentage of viable DCs was observed by trypan blue exclusion every 24 h. After transfection, the alterations of surface markers on mature DCs, including CD80, CD86, CD83, CD40, CD1a, HLA-DR and α-fetoprotein (AFP), were detected by flow cytometry. Meanwhile, 3H-thymidine incorporation method was used to measure the capacity of T-cell proliferation before and after transfection. The specific killing activity of T cells was evaluated by MTT assay.
RESULTS: About 77.7% mature DCs expressed AFP protein. The viable DCs percentages and surface marker expression showed no significant changes after transfection (P > 0.05). Transfected DCs still had strong potential of stimulating the proliferation of T lymphatic cells, and there was no significant difference between transfected and non-transfected group (P > 0.05). Transfected DCs were capable of inducing specific killing effect on the target cells, and the activity was significantly higher than those in the non-transfected cells when the DCs and T cells were mixed at the ratios of 80:1, 40:1, and 20:1 (35.5 ± 5.5 vs 20.6 ± 4.7; 28.7 ± 3.6 vs 15.3 ± 2.5; 16.2 ± 2.8 vs 9.6 ± 1.8; all P < 0.01).
CONCLUSION: AFP gene, which is carried by recombinant adeno-associated virus, can be transferred into DCs with high efficiency. The function of mature DCs is not affected significantly by AFP transfection.
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24
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Terentiev AA, Moldogazieva NT. Structural and functional mapping of alpha-fetoprotein. BIOCHEMISTRY (MOSCOW) 2006; 71:120-32. [PMID: 16489915 DOI: 10.1134/s0006297906020027] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Alpha-fetoprotein (AFP) is a major mammalian oncofetal protein, which is also present in small quantities in adults. It is a member of the albuminoid gene superfamily, which consists of AFP, serum albumin, vitamin D binding protein, and alpha-albumin (afamin). Although physicochemical and immunological properties of AFP have been well-studied, its biological role in embryo- and carcinogenesis and in adult organisms as well as mechanisms underlying its functioning remain unclear. During the recent decades, the biological role of AFP has been evaluated by identification of its functionally important sites. Comparison of primary structure of AFP and some physiologically active proteins revealed similarity of some polypeptide regions. This has been used for prediction of AFP functions (i.e., its multifunctionality). Localization of functionally important sites followed by determination of their amino acid composition and type of biological activity has provided valuable information for structural-functional mapping of AFP. Some peptide fragments of AFP have been synthesized and tested for biological activity. This review summarizes data on structural-functional interrelationships. We also describe functionally important AFP sites found by various groups during the last decade of structural-functional mapping of AFP with experimentally confirmed and putative biologically active sites.
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Affiliation(s)
- A A Terentiev
- Russian State Medical University, 117997 Moscow, Russia.
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25
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Wang XP, Wang QX, Li HY, Chen RF. Heat shock protein 70 chaperoned alpha-fetoprotein in human hepatocellular carcinoma cell line BEL-7402. World J Gastroenterol 2005; 11:5561-4. [PMID: 16222756 PMCID: PMC4320373 DOI: 10.3748/wjg.v11.i35.5561] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the interaction between heat shock protein 70 (HSP70) and α-fetoprotein (AFP) in human hepatocellular carcinoma (HCC) cell line BEL-7402.
METHODS: The expression and localization of HSP70 and AFP in human HCC cell line BEL-7402 were determined by immunocytochemistry and indirect immunofluorescence cytochemical staining. The interaction between HSP70 and AFP in HCC cells was analyzed by immunoprecipitation and Western blot.
RESULTS: Immunocytochemical staining detection showed that HCC cell BEL-7402 expressed a high level of HSP70 and AFP synchronously. Both were stained in cell plasma. AFP existed in the immunoprecipitate of anti-HSP70 mAb, while there was HSP70 in the immunoprecipitate of anti-AFP mAb.
CONCLUSION: HSP70 chaperones AFP in human HCC cell BEL-7402. The interaction between HSP70 and AFP in human HCC cell can be a new route to study the pathogenesis and immunotherapy of HCC.
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Affiliation(s)
- Xiao-Ping Wang
- Department of Pathology, Capital University of Medical Sciences, Beijing 100054, China.
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26
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Li MS, Ma QL, Chen Q, Liu XH, Li PF, Du GG, Li G. 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. World J Gastroenterol 2005; 11:2564-2569. [PMID: 15849812 PMCID: PMC4305744 DOI: 10.3748/wjg.v11.i17.2564] [Citation(s) in RCA: 38] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/07/2004] [Revised: 06/08/2004] [Accepted: 06/17/2004] [Indexed: 02/06/2023] Open
Abstract
AIM To investigate the mechanism of alpha-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 apoptosis-inducing ligand (TRAIL) and its receptor (TRAILR) mRNA were analyzed by Northern blot and Western blot was used 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.
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Affiliation(s)
- Meng-Sen Li
- Department of Biochemistry, Hainan Medical College, Haikou, China.
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27
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Parker MH, Birck-Wilson E, Allard G, Masiello N, Day M, Murphy KP, Paragas V, Silver S, Moody MD. Purification and characterization of a recombinant version of human α-fetoprotein expressed in the milk of transgenic goats. Protein Expr Purif 2004; 38:177-83. [PMID: 15555933 DOI: 10.1016/j.pep.2004.07.007] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2004] [Revised: 07/07/2004] [Indexed: 10/26/2022]
Abstract
Alpha-Fetoprotein (AFP) is a 68 kDa glycoprotein expressed at high levels by the fetal liver and yolk with transcription repressed to very low levels after birth. Transfer of fetal AFP through the placenta into the circulation of the mother is correlated with remission of rheumatoid arthritis, multiple sclerosis, and other autoimmune disorders. AFP is therefore under development as a biopharmaceutical for the treatment of autoimmune diseases. The clinical evaluation of AFP requires the production of hundreds of grams of highly purified and biologically active protein. We have produced goats that express a form of the human AFP transgene under the control of the beta-casein promoter. In this form of rhAFP, the single N-linked glycosylation site was removed by mutagenesis (N233Q). Here, we describe a purification protocol for this recombinant human (rh)AFP from the milk of these transgenic goats. A three-column procedure was developed to produce gram quantities of highly purified rhAFP. Near- and far-UV circular dichroism spectra of human umbilical cord blood AFP and rhAFP were essentially identical, suggesting that the structure is not affected by removal of the glycosylation site. Furthermore, the cell binding and pharmacokinetics of purified rhAFP were similar to human AFP isolated from cord blood. Our results demonstrate that an active form of rhAFP can be produced on industrial scale by expression in transgenic goat milk.
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Affiliation(s)
- Matthew H Parker
- Merrimack Pharmaceuticals, Inc., 101 Binney St., Cambridge, MA 02142, USA
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28
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Um SH, Mulhall C, Alisa A, Ives AR, Karani J, Williams R, Bertoletti A, Behboudi S. Alpha-fetoprotein impairs APC function and induces their apoptosis. THE JOURNAL OF IMMUNOLOGY 2004; 173:1772-8. [PMID: 15265907 DOI: 10.4049/jimmunol.173.3.1772] [Citation(s) in RCA: 92] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
alpha-Fetoprotein (AFP) is a tumor-associated Ag, and its serum level is elevated in patients with hepatocellular carcinoma (HCC). In vitro, AFP induces functional impairment of dendritic cells (DCs). This was demonstrated by the down-regulation of CD40 and CD86 molecules and the impairment of allostimulatory function. Also, AFP was found to induce significant apoptosis of DCs, and AFP-treated DCs produced low levels of IL-12 and TNF-alpha, a cytokine pattern that could hamper an efficient antitumor immune response. Ex vivo, APCs of patients with HCC and high levels of AFP produced lower levels of TNF-alpha than that of healthy individuals. In conclusion, these results illustrate that AFP induces dysfunction and apoptosis of APCs, thereby offering a mechanism by which HCC escapes immunological control.
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Affiliation(s)
- Soon Ho Um
- Institute of Hepatology, University College London, United Kingdom
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29
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Li MS, Li PF, Chen Q, Du GG, Li G. Alpha-fetoprotein stimulated the expression of some oncogenes in human hepatocellular carcinoma Bel 7402 cells. World J Gastroenterol 2004; 10:819-824. [PMID: 15040024 PMCID: PMC4726995 DOI: 10.3748/wjg.v10.i6.819] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/18/2003] [Revised: 09/23/2003] [Accepted: 11/15/2003] [Indexed: 12/15/2022] Open
Abstract
AIM To investigate the molecular mechanism of alpha-fetoprotein (AFP) on regulating the proliferation of human hepatocellular carcinoma cells. METHODS Alpha-fetoprotein purified from human umbilical blood was added to cultured human hepatocellular carcinoma Bel 7402 cells in vitro for various treatment periods. The expression of c-fos, c-jun, and N-ras mRNA involved in proliferation and differentiation of cells was analyzed by Northern blot, and the expression of mutative p53 and p21(ras) proteins was determined by Western blot. RESULTS The results showed that AFP (20 mg/L) stimulated mRNA expression of these oncogenes in Bel 7402 cells. The expression of c-fos mRNA increased by 51.1%, 60.9%, 96.0%, and 25.5% at 2, 6, 12, and 24 h, respectively. The expression of c-jun and N-ras mRNA reached to the maximum which increased by 81.3% and 59.9% as compared with the control after 6 h and 24 h incubation with AFP, respectively. Western blot assay also demonstrated that AFP promoted the expression of mutative p53 and p21(ras) proteins, and the increased rate of those proteins was 13.0%, 39.9%, and 70.9%, as well as 35.2%, 102.6%, and 46.8% at 6, 12, and 24 h, respectively, as compared with the control. Both human serum albumin (the same dosage as AFP) and monoclonal anti-AFP antibody failed to stimulate the expression of these oncogenes, but anti-AFP antibody could block the functions of AFP. CONCLUSION The data indicate that AFP can stimulate the expression of some oncogenes to enhance the proliferation of human hepatocellular carcinoma Bel 7402 cells.
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Affiliation(s)
- Meng-Sen Li
- Department of Biochemistry, Hainan Medical College, Haikou 571101, China.
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30
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Umar A, Ooms MP, Luider TM, Grootegoed JA, Brinkmann AO. Proteomic profiling of epididymis and vas deferens: identification of proteins regulated during rat genital tract development. Endocrinology 2003; 144:4637-47. [PMID: 12960072 DOI: 10.1210/en.2003-0404] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Epididymis and vas deferens form part of the male internal genital tract and are dependent on androgens for their growth and development. To better understand the molecular action of androgens during male genital tract development, protein expression profiles were generated using two-dimensional gels, for rat epididymides and vasa deferentia isolated on embryonic days (E) 17-21. Proteins that were differentially expressed between E17 and E21 were cut from the gels, digested into tryptic peptides and analyzed on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer. Using this approach, 20 proteins could be identified that were regulated in time and were categorized into cytoskeletal proteins, nuclear proteins, transport proteins, chaperones, and enzymes (mainly glycolytic). Furthermore, epididymides and vasa deferentia isolated on E19 were cultured in vitro in the absence or presence of 10 nm of the synthetic androgen R1881, for 9, 24, and 48 h. Under these conditions, regulation and posttranslational modification were observed for glyceraldehyde 3-phosphate dehydrogenase, triosephosphate isomerase, heterogeneous nuclear ribonucleoprotein A2/B1 and heterogeneous nuclear ribonucleoprotein A3, similar to the observed changes in vivo. In addition, posttranslational modification of RhoGDI1 (also named RhoGDIalpha) was found in response to androgen. Androgen-induced posttranslational modification of RhoGDI1 and glycolytic enzymes may be an important functional link between signaling pathways and cytoskeletal rearrangements in control of growth and development of the male internal genital tract.
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Affiliation(s)
- Arzu Umar
- Department of Reproduction and Development, Erasmus MC, P.O. Box 1738, Rotterdam 3000 DR, The Netherlands.
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31
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Dan Q, Sanchez R, Delgado C, Wepsic HT, Morgan K, Chen Y, Jeffes EW, Lowell CA, Morgan TR, Jadus MR. Non-immunogenic murine hepatocellular carcinoma Hepa1-6 cells expressing the membrane form of macrophage colony stimulating factor are rejected in vivo and lead to CD8+ T-cell immunity against the parental tumor. Mol Ther 2001; 4:427-37. [PMID: 11708879 DOI: 10.1006/mthe.2001.0477] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Hepatocellular carcinoma is a lethal disease and methods that develop effective cellular-based immunotherapy are needed. We retrovirally transduced non-immunogenic mouse Hepa1-6 hepatoma cells with the gene encoding the membrane form of macrophage colony stimulating factor (mM-CSF). Excess recombinant M-CSF and phagocytosis-inhibiting chemicals blocked macrophage-mediated killing of cloned mM-CSF transfected Hepa1-6 hepatoma cells. Macrophages derived from Hck(-/-)Fgr(-/-) and Lyn(-/-) triple knockout mice, which are incapable of performing phagocytosis, failed to kill the mM-CSF transduced cells. The mM-CSF transfected tumor clones failed to grow when injected into C57BL/6 or C57L/J mice. Splenocytes from these vaccinated mice displayed cytotoxicity against parental Hepa1-6 cells, but not against B16 and CT-26 tumor cells in vitro. Mice that rejected the mM-CSF transfected Hepa1-6 tumor subsequently rejected parental Hepa1-6 cells but not the B16 melanoma cells when rechallenged. Elimination of the CD8+ effector cells by an anti-CD8 antibody and complement treatment prevented the adoptive transfer of anti-Hepa1-6-specific immunity into naive animals. Thus, mM-CSF provides a method of generating effective anti-tumor immune responses by macrophages and cytotoxic T cells against the parental Hepa1-6 cells. Our work suggests that mM-CSF transduced hepatoma cells could be used as a tumor vaccine to stimulate immune responses against hepatocellular carcinoma.
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Affiliation(s)
- Q Dan
- Diagnostic and Molecular Medicine Health Care Group, Veterans Affairs Medical Center, University of California, 5901 E. 7th Street, Long Beach, CA 90822, USA
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Dudich E, Semenkova L, Dudich I, Gorbatova E, Tochtamisheva N, Tatulov E, Nikolaeva M, Sukhikh G. alpha-fetoprotein causes apoptosis in tumor cells via a pathway independent of CD95, TNFR1 and TNFR2 through activation of caspase-3-like proteases. EUROPEAN JOURNAL OF BIOCHEMISTRY 1999; 266:750-61. [PMID: 10583368 DOI: 10.1046/j.1432-1327.1999.00868.x] [Citation(s) in RCA: 47] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
alpha-Fetoprotein (AFP) is an oncoembryonal protein with multiple cell growth regulating, differentiating and immunosuppressive activities. Previous studies have shown that treatment of tumor cells in vitro with 1-10 microM AFP produces significant suppression of tumor cell growth by inducing dose-dependent cytotoxicity, but the molecular mechanisms underlying these AFP functions are obscure. Here, we show that AFP cytotoxicity is closely related to apoptosis, as shown by cell morphology, nuclear DNA fragmentation and caspase-3-like activity resulting in cleavage of poly(ADP-ribose) polymerase. Apoptosis was significantly inhibited by a CPP32 family protease inhibitor whereas a general caspase inhibitor had no inhibitory effect, showing some enhancement of AFP-mediated cell death. Using fluorogenic caspase substrates, we found that caspase-3-like proteases were activated as early as 4 h after treatment of Raji cells with 15 microM AFP, whereas caspase-1, caspase-8, and caspase-9-like activity was not detected during the time interval 0.5-17 h. AFP treatment of Raji cells increased Bcl-2 protein, showing that AFP-induced apoptosis is not explained by downregulation of the Bcl-2 gene. This also suggests that AFP operates downstream of the Bcl-2-sensitive step. AFP notably decreased basal levels of soluble and membrane-bound Fas ligand. Incubation of AFP-sensitive tumor cells (HepG2, Raji) with neutralizing anti-Fas, anti-tumor necrosis factor receptor (TNFR)1 or anti-TNFR2 mAb did not prevent AFP-induced apoptosis, demonstrating its independence of Fas-dependent and TNFR-dependent signaling. In addition, it was found that cells resistant to TNF-induced (Raji) or Fas-induced (MCF-7) apoptosis are, nevertheless, sensitive to AFP-mediated cell death. In contrast, cells sensitive to Fas-mediated cell death (Jurkat) are completely resistant to AFP. Taken as a whole, our data demonstrate that: (a) AFP induces apoptosis in tumor cells independently of Fas/Fas ligand or TNFR/TNF signaling pathways, and (b) AFP-mediated cell death involves activation of the effector caspase-3-like proteases, but is independent of upstream activation of the initiator caspase-1, caspase-8, and caspase-9-like proteases.
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Affiliation(s)
- E Dudich
- Institute of Engineering Immunology, Lyubuchany, Moscow Region, Russia.
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Bennett JA, Semeniuk DJ, Jacobson HI, Murgita RA. Similarity between natural and recombinant human alpha-fetoprotein as inhibitors of estrogen-dependent breast cancer growth. Breast Cancer Res Treat 1997; 45:169-79. [PMID: 9342442 DOI: 10.1023/a:1005841032371] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
Alpha-fetoprotein (AFP) isolated from rodent amniotic fluid or human cord sera, upon incubation with a molar excess of estradiol, is converted to a form which inhibits estrogen-stimulated tissue growth. The purpose of this study was to determine whether recombinant human AFP produced in an E. coli expression system retained this function. The recombinant protein was similar to the natural protein isolated from pooled human cord sera in all functional aspects evaluated. It was detected by monoclonal and polyclonal antibodies to the natural protein. Following exposure to estradiol, it was converted to an inhibitor of estrogen-stimulated growth of immature mouse uterus yielding a dose/response curve similar to that of the natural protein. It inhibited the growth of estrogen-dependent (MCF-7) but not estrogen-independent (MDA-MB-231) breast cancer xenografts with the same schedule dependency and resultant histological changes as the natural protein. Availability of large quantities of homogeneous, biologically active recombinant human AFP will facilitate further studies of structure/function, mechanism, and therapeutic potential of this agent as a regular of breast cancer growth.
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