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Interaction between heat shock protein 72 and α-fetoprotein in human hepatocellular carcinomas
Clinica Chimica Acta 379 (2007) 158 – 162 www.elsevier.com/locate/clinchim
Short communication
Interaction between heat shock protein 72 and α-fetoprotein in human hepatocellular carcinomas ☆ Xiaoping Wang a,d,e,g,⁎, Yongxue Zhou b,c,d,e , Xiaoping Ying b,c,d , Lansheng Guo c,e,f , Yanhong Zhao c,d,f , Yan Fang c,f Department of Pathology, Shaanxi University of Chinese Medicine, Xianyang 712046, China Received 16 September 2006; received in revised form 12 December 2006; accepted 13 December 2006 Available online 29 December 2006
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 α-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 α-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. © 2007 Elsevier B.V. All rights reserved. Keywords: Heat shock protein 72(HSP72); α-fetoprotein(AFP); Immunohistochemistry; Confocal; Immunoprecipitation; Hepatocellular carcinoma
1. Introduction α-fetoprotein(AFP) is a major serum protein synthesized by fetal liver cells, yolk sac cells, and in trace amounts by the fetal gastrointestinal tract [1]. AFP appears to function as an osmotic and carrier protein in the fetus and to regulate the immune ☆ Supported by the Key Project of Ministry of Education of China (No.2005002) and Research Program of Beijing Education Committee (No.200410025002). ⁎ Corresponding author. Tel.: +86 29 38185359. E-mail address: [email protected] (X.P. Wang). a Study design. b Data collection. c Statistical analysis. d Data interpretation. e Manuscript preparation. f Literature search. g Funds collection.
0009-8981/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2006.12.015
system by immunosuppressive functions, such as preventing immunological attacks to the embryo by the maternal immune system [1]. Reappearance of AFP in adult serum often signals pathological conditions, particularly the presence of hepatocellular carcinoma (HCC) and germ cell tumors containing yolk sac cell elements [2]. Now AFP has been successfully used as a diagnostic and prognostic tool for HCC. Although many investigations for the function of AFP had been carried out, the biological role of AFP is still a riddle so far. Heat shock protein 72 (HSP72) belongs to the heat shock protein 70 family which are molecular chaperones emerging as biochemical regulators of cell growth, apoptosis, protein homeostasis and cellular targets of peptides [3–5]. Up-regulated expression of HSP72 during the growth of cancer cells has a close relationship with the epithelial carcinoma proliferation [6–8]. Several studies have showed that AFP plays some roles during
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cell survival and cell proliferation of hepatocellular carcinoma cells [9–11]. So there may be a possible correlation between the expression of HSP72 and AFP during the growth and differentiation of hepatocellular carcinoma cells. In this study, by immunohistochemistry, confocal laser microscopy and immunoprecipitation, Western immunoblots, we observed that HSP72 was associated with AFP in hepatocellular carcinoma cell cytoplasm. The interaction between HSP72 and AFP in human hepatocellular carcinoma cells will provide a new route for studying the pathogenesis and immunotherapy of hepatocellular carcinoma. 2. Materials and methods 2.1. Reagents Rabbit anti-human HSP72 antibody, mouse anti-human AFP monoclonal antibody, mouse anti-human β-actin monoclonal antibody, TRITC labeled goat anti-rabbit antibody and FITC labeled goat anti-mouse antibody were from Santa Crus Company. EnVisionTM kits were from Dako Biological Technology Company. ProteinA/G-agarose beads were from Gene Company.
2.2. Specimens Surgical specimens of 40 patients with primary hepatocellular cancer undergoing liver resection were collected from the Affiliated Hospital, Shaanxi University of Chinese Medicine, Xianyang, China from 2001 to 2005. The patients consist of 23 males and 17 females, with a mean age of 51.5 y
Fig. 2. Expressions of HSP72 and AFP in hepatocellular carcinoma tissues by immunofluorescence and confocal microscopy, ×200. A: HSP72 showed red immunofluorescence in cytoplasm; B: AFP expressed green immunofluorescence in cytoplasm. C: HSP72 and AFP showed yellow immunofluorescence in cytoplasm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) (rang 37–65 y). Routine pathological diagnosis showed that all cases were primary hepatocellular carcinoma. Among them, 16 cases were well-differential type and 24 cases were poor differential. The specimens were fixed in 10% buffered formalin and embedded in paraffin. Serial sections, 5-μm-thick were cut and placed on MAS-coated glass slides.
2.3. Staining methods Fig. 1. Expressions of HSP72 and AFP in hepatocarcinoma tissues by immunohistochemistry, ×400. A: HSP72 positive expression in cell nuclei and cytoplasm; B: AFP immunostained in cytoplasm.
All sections were deparaffinnized and dehydrated with graded alcohol. Endogenous peroxidase was then blocked with 0.3% H2O2 diluted in methanol for 30 min at room temperature. Antigen retrieval was performed by
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treating the slides in citrate buffer in a microwave for 10 min. The slides were incubated in a moist chamber with HSP72 rabbit antibody (1:100) or AFP mouse monoclonal antibody (1:100) at 4 °C overnight respectively. After a complete wash in PBS, the slides were treated with HRP labeled goat antirabbit and goat anti-mouse antibody (1:100) for 45 min at 37 °C. After a complete wash in PBS, the slides were developed in 0.05% freshly prepared diaminobenzedine solution (DAB, Sigma Co., St. Louis, MO) for 8 min, and then counterstained with hematoxylin, dehydrated, air dried, and mounted. CEA was used to substitute for the primary antibody as a negative control. All sections were studied by light microscopy, at × 400 magnification, with a 1cm × 1-cm ocular micrometer (a square defined a field). Under the microscope, one field was 250 × 250 μm2. The immunoreactive cells in the hepatocellular carcinoma were studied by continuous cell counting in the restricted field.
2.4. Immunofluorescence and confocal laser microscopy All sections were deparaffinized and dehydrated with graded alcohol. The tissues were blocked in 10 ml/l bovine serum albumin (BSA) for 30 min at room temperature, and then incubated with HSP72 rabbit antibody (1:100) or AFP mouse monoclonal antibody (1:100) at 4 °C overnight respectively. After a complete wash in PBS, the cells were treated with TRITC labeled goat anti-rabbit antibody or FITC labeled goat anti-mouse antibody (1:20) for 40 min at room temperature. After extensively washed, the stained cells were observed under a laser scanner confocal microscope Bio-Rad MRC 1024ES equipped with a Nikon (Tokyo, Japan) Diaphot inverted microscope. Anti-CEA was used to substitute for the primary antibody as a negative control.
2.5. Immunoprecipitation and Western blot The hepatocellular carcinoma tissues and normal liver tissues were dispersed through 100-well copper mesh. Then cells were treated with 0.5 g/l trypsin and 0.2 g/l EDTA. After washing with cold PBS, cells were centrifuged and harvested. Cells were then lysed with 500 μl of lysis buffer (10 mmol/l Tris–HCL, pH 7.4, 150 mmol/l NaCl, 1 ml/l Triton X-100, 10 g/l Na-deoxycholate) containing 1 μg/ml pepstatin and 1 mmol/l phenyl– methylsulfonyl fluoride (PMSF). Also, 5 units of apyrase (Sigma) were added to the lysate to deplete endogenous ATP. The cell lysates were sonicated and clarified by centrifugation. The supernatants were preabsorbed with 20 μl protein A/G-agarose beads at 4 °C for 4 h. After centrifuged, the supernatants were incubated with 100 μl HSP72 rabbit antibody(1:100) or 100 μL AFP mouse monoclonal antibody (1:100) respectively at 4 °C for 60 min. Then 50 μl protein A/G-agarose beads was added and incubated for 60 min at 4 °C. The immunoprecipitates were collected by centrifugation and washed five times with PBS. The precipitated protein complexes were released from the immunopellet with SDS-sample buffer (62.5 mmol/l Tris–HCL, pH 6.8, 25 g/l SDS, 50 ml/l β-mercaptoethanol, 100 ml/l glycerol) at 100 °C
for 5 min. The immunoprecipitates were then analyzed by 90 g/l SDSpolyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to nitrocellulose (NC) membrane (BioRad) and detected by immunoblotting with anti-AFP monoclonal antibody or anti-HSP72 antibody (1:100) at 4 °C overnight respectively. After a complete wash in PBS, the membranes were treated with HRP labeled goat anti-rabbit and goat anti-mouse antibody (1:100) for 45 min at 37 °C. After a complete wash in PBS, the membranes were developed in 0.5 g/l freshly prepared diaminobenzedine solution (DAB, Sigma) for 8 min. Anti-β-actin mouse monoclonal antibody was used as an internal reference to detect β-actin in hepatocellular carcinoma cells.
2.6. Statistical analysis HSP72, AFP expression differences between hepatocellular carcinoma and control groups were analyzed statistically using χ2 test. P b 0.05 was considered statistically significant.
3. Results 3.1. Expression of HSP72 and AFP in hepatocellular carcinoma tissues Positive expression of HSP72 and AFP showed brown staining in the nuclei or cytoplasm, more than 500 cells calculated in different microscopic fields of each well, and percentage of positive cells were evaluated. Immunohistochemical staining showed that the positive rate of HSP72 was 95.0% (450/500), and AFP was 96.0% (480/500), while the positive rate of HSP72 and AFP in adjacent normal tissues were only 12.0% (60/500) and 4.0% (20/500). There was a significant difference between carcinoma tissues and adjacent normal tissues (P b 0.01). HSP72 were stained in cell nuclei and cytoplasm respectively, while AFP stained in cell plasma (Fig. 1). Immunofluorescence and confocal laser microscopy showed that hepatocellular carcinoma synchronously coexpressed higher level of HSP72 and AFP. Both of them were localized in cell cytoplasm (Fig. 2). 3.2. Interaction between HSP72 and AFP in hepatocellular carcinoma cells Immunoprecipitate and Western blot showed that there were 2 clear protein bands in the transferred NC membrane. Under precipitated with anti-HSP72 antibody, there appeared an M r 69,000 clear α-fetoprotein band (lane 1) and under precipitated with anti-AFP mAb, there existed an M r 72,000 clear heat shock protein 72 band (lane 2), demonstrating that AFP existed in the immunoprecipitate of anti-HSP72 antibody and HSP72 existed in the immunoprecipitate of anti-AFP mAb. The results indicated that HSP72 interacted with AFP in hepatocellular carcinoma tissues (Fig. 3). 4. Discussion
Fig. 3. Analysis of immunoprecipitate of anti-HSP72 antibody and anti-AFP mAb by SDS-PAGE and Western blot (lane 1: precipitated with anti-HSP72 antibody; lane 2: precipitated with anti-AFP mAb; lane 3: anti-β-actin mAb (internal reference detected in hepatocellular carcinoma); lane 4: normal liver cells lysates supernatants; lane 5: hepatocellular carcinoma cells lysates supernatants; M: molecular mass markers).
We examined the expressions of HSP72 and AFP in hepatocellular carcinoma tissues by immunohistochemistry, confocal microscopy and immunoprecipitate analysis. The results showed that almost all hepatocellular carcinoma cells detected expressed high level of HSP72 and AFP, which had a
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significant difference compared with adjacent normal tissues. HSP72 associated with AFP mainly localizing in cell cytoplasm, the results were similar to cultured human hepatocellular carcinoma cell line [12]. AFP always accompanies with the growth of liver cells, and it is confirmed that AFP may be related to the proliferation of tumor or fetal cells [9,10]. The mechanism for growth-promoting activity of AFP is still unclear. Escaping from the surveillance of immune system is the primary cause for malignant growth of hepatocellular carcinoma cells [13,14]. Several investigations have showed that AFP could be individually synergy with other growth factors to promote the growth of many tumor cells [15,16]. AFP receptors have been found anchoring on the membrane of various tumor cells [17–19]. The receptor may mediate intercellular signal transduction which influences the expression of genes related to proliferation [17,18]. AFP can stimulate the expression of some oncogenes which control cell cycle, and then enhance the proliferation of human hepatocellular carcinoma [20,21]. When BEL-7402 cell line was treated with AFP, oncogene protein, such as c-fos, c-jun, c-ras and mutant p53 and p21ras increased rapidly, which have an important function in modulating growth and differentiation of the cells [22]. Heat shock protein 72, belonging to the family of HSP70, is a type of highly conserved protein synthesized under various stress. In non-transformed cells at normal conditions Hsp72 is expressed at very low levels. It is, however, present at elevated levels in the major fraction of tumors and in many transformed cell lines [23,24]. It is commonly assumed that in tumor cells the expression of HSP72 at elevated levels is the consequence of oncogenic transformation and enhanced expression of HSP72 has a close relationship with the epithelial carcinoma cells growth [6–8]. It is also found that AFP plays some roles during tumor cell survival and proliferation [9–11]. Some studies have suggested that high level expression of HSPs in HCC is correlated with AFP and poor prognosis [25–27]. Upregulated expression of HSP70 family in tumor cells may be required to serve as molecular chaperones in regulating and stabilizing oncofetal protein and mutant oncogene products during tumor growth process [28,29]. Therefore, overexpressed HSP72 may assist to stabilize AFP in cell cytoplasm, transports it to cell membrane and releases its monomer to the serum, which indirectly prompts and protects AFP function. Conversely, AFP itself stimulates hepatocellular carcinoma cells proliferation through membrane receptor mediating cellular signal transduction pathways. This indicates that the proliferating HCC cells need much more HSP72 to maintain the formation of AFP activities. Our results showed that human hepatocellular carcinoma cells existed high level expression of HSP72 and AFP. It indicates that co-expression of HSP72 and AFP is likely to have some relationship with proliferation, development and poor prognosis of human hepatocellular carcinoma. The observed fact that HSP72 was associated with AFP may be useful to study the pathogenesis and immunity of human hepatocellular carcinoma. Recent studies on the immunodominant epitopes of AFP have provided a solution to the obstacle of HCC immunotherapy. AFP is produced at low serum levels after birth throughout
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life. The majority of human HCC overexpress the oncofetal antigen AFP, M r 64,000–72,000 glycoprotein. Despite being exposed to high plasma levels of this oncofetal protein during embryonic development, body has a low immunity to it [1,2]. Butterfield et al. [30] recently found that four peptides of human AFP processed and presented in the context of HLA-A0201, could be recognized by the human T cell repertoire, and could be used to generate AFP-specific CTL in human T cell cultures. It was also found that murine immune system could generate Tcell responses to this oncofetal antigen [31]. Therefore, it may be a better target for immunotherapy. But AFP immunization alone still results in lower levels of specific response and poorly reproducible protective immunity [31,32]. How to enhance host's active immunity to AFP may be an interesting strategy for HCC therapy. In our previous study, tumor rejection assay demonstrated that recombinant DNA vaccine AFP/HSP70 elicited strong specific antitumor immunity against AFP-producing SP2/0 cells than AFP DNA vaccine. Results indicated that AFP immunogenicity can be improved greatly by HSP70 molecular and vaccination with DNA encoding HSP70 could increase both humoral and T-cell proliferation responses to α-fetoprotein. We attributed the successful α-fetoprotein specific T-cell responses in mice to the HSP70 molecular by mediating APCs to efficient uptake and process of AFP [33]. Numerous investigations have been verified that HSP72 is a better molecular chaperone and adjuvant which can process and present weak tumor antigen to MHC-I of host APCs, eliciting specific T-cell response and CTL reaction [34,35]. HSP72-associated peptides can also anchor antigen on the cell membrane and directly present it to nature killer cells or γδ T cells as superantigen without being dependent on the stimulation of MHC-I molecules [36,37]. The observed fact that HSP72 associated with AFP in hepatocellular carcinoma, may be useful to study tumor pathogenesis or design effective vaccine against liver cancers. 5. Conclusions Human hepatocellular carcinomas existed high level expression of HSP72 and AFP. HSP72 was associated with AFP mainly localizing in cell cytoplasm. Co-expression of HSP72 and AFP is likely to have some relationship with proliferation, development and poor prognosis of human hepatocellular carcinoma. The observed fact that HSP72 was associated with AFP may be useful to study the pathogenesis and design effective vaccine against liver cancers. References [1] Deutsch HF. Chemistry and biology of a-fetoprotein. Adv Cancer Res 1991;56:253–311. [2] Goldenberg DM, Kim EE, Deland F, et al. Clinical studies on the radioimmunodetection of tumors containing alpha-fetoprotein. Cancer 1980;45:2500–5. [3] Schlesinger MJ. Heat shock proteins. J Biol Chem 1990;265:12111–4. [4] Little E, Ramakrishnan M, Roy B, Gazit G, Lee AS. The glucose-regulated proteins (GRP60 and GRP94): functions, gene regulation, and applications. Crit Rev Eukaryot Gene Expr 1994;4:1–18.
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[5] Morimoto RI. Cells in stress: transcriptional activation of heat shock genes. Science 1993;259:1409–10. [6] Bausero MA, Page DT, Osinaga E, Asea A. Surface expression of Hsp25 and Hsp72 differentially regulates tumor growth and metastasis. Tumour Biol 2004;25:243–51. [7] Gabai VL, Budagova KR, Sherman MY. Increased expression of the major heat shock protein Hsp72 in human prostate carcinoma cells is dispensable for their viability but confers resistance to a variety of anticancer agents. Oncogene 2005;24:3328–38. [8] Volloch VZ, Sherman MY. Oncogenic potential of Hsp72. Oncogene 1999;18:3648–51. [9] Dudich E, Semonkova L, Gorbatova E, et al. Growth-regulative activity of human alpha-fetoprotein for different types of tumor and normal cells. Tumour Biol 1998;19:30–40. [10] Wang XW, Xie H. Alpha-fetoprotein enhances the proliferation of human hepatoma cells in vitro. Life Sci 1999;64:17–23. [11] Wang XW, Xu B. Stimulation of tumor-cell growth by alpha-fetoprotein. Int J Cancer 1998;75:596–9. [12] 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. [13] Semeniuk DJ, Boismenu R, Tam J, Weissenhofer W, Murgita RA. Evidence that immunosuppression is an intrinsic property of the alphafetoprotein molecule. Adv Exp Med Biol 1995;383:255–69. [14] Gotsman I, Israeli D, Alper R, Rabbani E, Engelhardt D, Ilan Y. Induction of immune tolerance toward tumor-associated enables growth of human hepatoma in mice. Int J Cancer 2002;97:52–7. [15] Keel BA, Eddy KB, Cho S, May JV. Synergistic action of purified alphafetoprotein and growth factors on the proliferation of porcine granulosa cells in monolayer culture. Endocrinology 1991;129:217–25. [16] Peng SY, Chen WJ, Lai PL, Jeng YM, Sheu JC, Hsu HC. High alphafetoprotein level correlates with high stage, early recurrence and poor prognosis of hepatocellular carcinoma: significance of hepatitis virus infection, age, p53 and beta-catenin mutations. Int J Cancer 2004;112:44–50. [17] Esteban C, Geuskens M, Uriel J. Activation of an alpha-fetoprotein (AFP)/ receptor autocrine loop in HT-29 human colon carcinoma cells. Int J Cancer 1991;49:425–30. [18] Villacampa MJ, Moro R, Naval J, Failly-Crepin C, Lempreave F, Uriel J. Alpha-fetoprotein receptors in a human breast cancer cell line. Biochem Biophys Res Commun 1984;122:1322–7. [19] Naval J, Villacampa MJ, Goguel AF, Uriel J. Cell-type-specific receptor for alpha-fetoprotein in mouse T-lymphoma cell line. Proc Natl Acad Sci U S A 1985;82:3301–5. [20] Caruso ML, Valentini AM. Overexpression of p53 in a large series of patients with hepatocellular carcinoma: a clinicopathological correlation. Anticancer Res 1999;19:3853–6. [21] Zhang XW, Xu B. Differential regulation of P53, c-Myc, Bcl-2, Bax and AFP protein expression, and caspase activity during 10-hydroxycamptothecininduced apoptosis in Hep G2 cells. Anticancer Drugs 2000;11:747–56. [22] Li MS, Li PF, He SP, Du GG, Li G. The promoting molecular mechanism of alpha-fetoprotein on the growth of human hepatoma Bel-7402 cell line. World J Gastroenterol 2002;8:469–75.
[23] Lopez-Cotarelo C, Sellhaus B, Baba HA, et al. Expression of heat shock proteins 72/73 in human peritoneal mesothelial cells in vivo and in vitro. Nephron 2000;85:148–55. [24] Kato K, Yamanaka K, Nakano M, Hasegawa A, Okada S. 72-kDa stress protein (hsp72) induced by administration of dimethylarsinic acid to mice accumulates in alveolar flat cells of lung, a target organ for arsenic carcinogenesis. Biol Pharm Bull 2000;23:1212–5. [25] Takashima M, Kuramitsu Y, Yokoyama Y, et al. Proteomic profiling of heat shock protein 70 family members as biomarkers for hepatitis C virusrelated hepatocellular carcinoma. Proteomics 2003;3:2487–93. [26] Feng JT, Liu YK, Song HY, et al. Heat-shock protein 27: a potential biomarker for hepatocellular carcinoma identified by serum proteome analysis. Proteomics 2005;5:4581–8. [27] Luk JM, Lam CT, Siu AF, et al. Proteomic profiling of hepatocellular carcinoma in Chinese cohort reveals heat-shock proteins (Hsp27, Hsp70, GRP78) up-regulation and their associated prognostic values. Proteomics 2006;6:1049–57. [28] Hwang TS, Han HS, Choi HK, et al. Differential, stage-dependent expression of Hsp70, Hsp110 and Bcl-2 in colorectal cancer. J Gastroenterol Hepatol 2003;18:690–700. [29] Dorsey WC, Tchounwou PB. CYP1a1, HSP70, P53, and c-fos expression in human liver carcinoma cells (HepG2) exposed to pentachlorophenol. Biomed Sci Instrum 2003;39:389–96. [30] Butterfield LH, Koh A, Meng W, et al. Generation of human T-cell responses to an HLA-A2.1-restricted peptide epitope derived from alphafetoprotein. Cancer Res 1999;59:3134–42. [31] Saeki A, Nakao K, Nagayama Y, et al. Diverse efficacy of vaccination therapy using the alpha-fetoprotein gene against mouse hepatocellular carcinoma. Int J Mol Med 2004;13:111–6. [32] Hanke P, Serwe M, Dombrowski F, Sauerbruch T, Caselmann WH. DNA vaccination with AFP-encoding plasmid DNA prevents growth of subcutaneous AFP-expressing tumors and does not interfere with liver regeneration in mice. Cancer Gene Ther 2002;9:346–55. [33] Wang XP, Liu GZ, Song AL, Li HY, Liu Y. Antitumor immunity induced by DNA vaccine encoding alpha-fetoprotein/heat shock protein 70. World J Gastroenterol 2004;10:3197–200. [34] Wells AD, Rai SK, Salvato MS, Band H, Malkovsky M. Hsp72-mediated augmentation of MHC class I surface expression and endogenous antigen presentation. Int Immunol 1998;10:609–17. [35] Faure O, Graff-Dubois S, Bretaudeau L, et al. Inducible Hsp70 as target of anticancer immunotherapy: identification of HLA-A⁎0201-restricted epitopes. Int J Cancer 2004;108:863–70. [36] Multhoff G, Botzler C, Jennen L, Schmidt J, Ellwart J, Issels R. Heat shock protein 72 on tumor cells: a recognition structure for natural killer cells. J Immunol 1997;158:4341–50. [37] Zhang H, Hu H, Jiang X, He H, Cui L, He W. Membrane HSP70: the molecule triggering gammadelta T cells in the early stage of tumorigenesis. Immunol Invest 2005;34:453–68.
Short communication
Interaction between heat shock protein 72 and α-fetoprotein in human hepatocellular carcinomas ☆ Xiaoping Wang a,d,e,g,⁎, Yongxue Zhou b,c,d,e , Xiaoping Ying b,c,d , Lansheng Guo c,e,f , Yanhong Zhao c,d,f , Yan Fang c,f Department of Pathology, Shaanxi University of Chinese Medicine, Xianyang 712046, China Received 16 September 2006; received in revised form 12 December 2006; accepted 13 December 2006 Available online 29 December 2006
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 α-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 α-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. © 2007 Elsevier B.V. All rights reserved. Keywords: Heat shock protein 72(HSP72); α-fetoprotein(AFP); Immunohistochemistry; Confocal; Immunoprecipitation; Hepatocellular carcinoma
1. Introduction α-fetoprotein(AFP) is a major serum protein synthesized by fetal liver cells, yolk sac cells, and in trace amounts by the fetal gastrointestinal tract [1]. AFP appears to function as an osmotic and carrier protein in the fetus and to regulate the immune ☆ Supported by the Key Project of Ministry of Education of China (No.2005002) and Research Program of Beijing Education Committee (No.200410025002). ⁎ Corresponding author. Tel.: +86 29 38185359. E-mail address: [email protected] (X.P. Wang). a Study design. b Data collection. c Statistical analysis. d Data interpretation. e Manuscript preparation. f Literature search. g Funds collection.
0009-8981/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2006.12.015
system by immunosuppressive functions, such as preventing immunological attacks to the embryo by the maternal immune system [1]. Reappearance of AFP in adult serum often signals pathological conditions, particularly the presence of hepatocellular carcinoma (HCC) and germ cell tumors containing yolk sac cell elements [2]. Now AFP has been successfully used as a diagnostic and prognostic tool for HCC. Although many investigations for the function of AFP had been carried out, the biological role of AFP is still a riddle so far. Heat shock protein 72 (HSP72) belongs to the heat shock protein 70 family which are molecular chaperones emerging as biochemical regulators of cell growth, apoptosis, protein homeostasis and cellular targets of peptides [3–5]. Up-regulated expression of HSP72 during the growth of cancer cells has a close relationship with the epithelial carcinoma proliferation [6–8]. Several studies have showed that AFP plays some roles during
X.P. Wang et al. / Clinica Chimica Acta 379 (2007) 158–162
159
cell survival and cell proliferation of hepatocellular carcinoma cells [9–11]. So there may be a possible correlation between the expression of HSP72 and AFP during the growth and differentiation of hepatocellular carcinoma cells. In this study, by immunohistochemistry, confocal laser microscopy and immunoprecipitation, Western immunoblots, we observed that HSP72 was associated with AFP in hepatocellular carcinoma cell cytoplasm. The interaction between HSP72 and AFP in human hepatocellular carcinoma cells will provide a new route for studying the pathogenesis and immunotherapy of hepatocellular carcinoma. 2. Materials and methods 2.1. Reagents Rabbit anti-human HSP72 antibody, mouse anti-human AFP monoclonal antibody, mouse anti-human β-actin monoclonal antibody, TRITC labeled goat anti-rabbit antibody and FITC labeled goat anti-mouse antibody were from Santa Crus Company. EnVisionTM kits were from Dako Biological Technology Company. ProteinA/G-agarose beads were from Gene Company.
2.2. Specimens Surgical specimens of 40 patients with primary hepatocellular cancer undergoing liver resection were collected from the Affiliated Hospital, Shaanxi University of Chinese Medicine, Xianyang, China from 2001 to 2005. The patients consist of 23 males and 17 females, with a mean age of 51.5 y
Fig. 2. Expressions of HSP72 and AFP in hepatocellular carcinoma tissues by immunofluorescence and confocal microscopy, ×200. A: HSP72 showed red immunofluorescence in cytoplasm; B: AFP expressed green immunofluorescence in cytoplasm. C: HSP72 and AFP showed yellow immunofluorescence in cytoplasm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) (rang 37–65 y). Routine pathological diagnosis showed that all cases were primary hepatocellular carcinoma. Among them, 16 cases were well-differential type and 24 cases were poor differential. The specimens were fixed in 10% buffered formalin and embedded in paraffin. Serial sections, 5-μm-thick were cut and placed on MAS-coated glass slides.
2.3. Staining methods Fig. 1. Expressions of HSP72 and AFP in hepatocarcinoma tissues by immunohistochemistry, ×400. A: HSP72 positive expression in cell nuclei and cytoplasm; B: AFP immunostained in cytoplasm.
All sections were deparaffinnized and dehydrated with graded alcohol. Endogenous peroxidase was then blocked with 0.3% H2O2 diluted in methanol for 30 min at room temperature. Antigen retrieval was performed by
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treating the slides in citrate buffer in a microwave for 10 min. The slides were incubated in a moist chamber with HSP72 rabbit antibody (1:100) or AFP mouse monoclonal antibody (1:100) at 4 °C overnight respectively. After a complete wash in PBS, the slides were treated with HRP labeled goat antirabbit and goat anti-mouse antibody (1:100) for 45 min at 37 °C. After a complete wash in PBS, the slides were developed in 0.05% freshly prepared diaminobenzedine solution (DAB, Sigma Co., St. Louis, MO) for 8 min, and then counterstained with hematoxylin, dehydrated, air dried, and mounted. CEA was used to substitute for the primary antibody as a negative control. All sections were studied by light microscopy, at × 400 magnification, with a 1cm × 1-cm ocular micrometer (a square defined a field). Under the microscope, one field was 250 × 250 μm2. The immunoreactive cells in the hepatocellular carcinoma were studied by continuous cell counting in the restricted field.
2.4. Immunofluorescence and confocal laser microscopy All sections were deparaffinized and dehydrated with graded alcohol. The tissues were blocked in 10 ml/l bovine serum albumin (BSA) for 30 min at room temperature, and then incubated with HSP72 rabbit antibody (1:100) or AFP mouse monoclonal antibody (1:100) at 4 °C overnight respectively. After a complete wash in PBS, the cells were treated with TRITC labeled goat anti-rabbit antibody or FITC labeled goat anti-mouse antibody (1:20) for 40 min at room temperature. After extensively washed, the stained cells were observed under a laser scanner confocal microscope Bio-Rad MRC 1024ES equipped with a Nikon (Tokyo, Japan) Diaphot inverted microscope. Anti-CEA was used to substitute for the primary antibody as a negative control.
2.5. Immunoprecipitation and Western blot The hepatocellular carcinoma tissues and normal liver tissues were dispersed through 100-well copper mesh. Then cells were treated with 0.5 g/l trypsin and 0.2 g/l EDTA. After washing with cold PBS, cells were centrifuged and harvested. Cells were then lysed with 500 μl of lysis buffer (10 mmol/l Tris–HCL, pH 7.4, 150 mmol/l NaCl, 1 ml/l Triton X-100, 10 g/l Na-deoxycholate) containing 1 μg/ml pepstatin and 1 mmol/l phenyl– methylsulfonyl fluoride (PMSF). Also, 5 units of apyrase (Sigma) were added to the lysate to deplete endogenous ATP. The cell lysates were sonicated and clarified by centrifugation. The supernatants were preabsorbed with 20 μl protein A/G-agarose beads at 4 °C for 4 h. After centrifuged, the supernatants were incubated with 100 μl HSP72 rabbit antibody(1:100) or 100 μL AFP mouse monoclonal antibody (1:100) respectively at 4 °C for 60 min. Then 50 μl protein A/G-agarose beads was added and incubated for 60 min at 4 °C. The immunoprecipitates were collected by centrifugation and washed five times with PBS. The precipitated protein complexes were released from the immunopellet with SDS-sample buffer (62.5 mmol/l Tris–HCL, pH 6.8, 25 g/l SDS, 50 ml/l β-mercaptoethanol, 100 ml/l glycerol) at 100 °C
for 5 min. The immunoprecipitates were then analyzed by 90 g/l SDSpolyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to nitrocellulose (NC) membrane (BioRad) and detected by immunoblotting with anti-AFP monoclonal antibody or anti-HSP72 antibody (1:100) at 4 °C overnight respectively. After a complete wash in PBS, the membranes were treated with HRP labeled goat anti-rabbit and goat anti-mouse antibody (1:100) for 45 min at 37 °C. After a complete wash in PBS, the membranes were developed in 0.5 g/l freshly prepared diaminobenzedine solution (DAB, Sigma) for 8 min. Anti-β-actin mouse monoclonal antibody was used as an internal reference to detect β-actin in hepatocellular carcinoma cells.
2.6. Statistical analysis HSP72, AFP expression differences between hepatocellular carcinoma and control groups were analyzed statistically using χ2 test. P b 0.05 was considered statistically significant.
3. Results 3.1. Expression of HSP72 and AFP in hepatocellular carcinoma tissues Positive expression of HSP72 and AFP showed brown staining in the nuclei or cytoplasm, more than 500 cells calculated in different microscopic fields of each well, and percentage of positive cells were evaluated. Immunohistochemical staining showed that the positive rate of HSP72 was 95.0% (450/500), and AFP was 96.0% (480/500), while the positive rate of HSP72 and AFP in adjacent normal tissues were only 12.0% (60/500) and 4.0% (20/500). There was a significant difference between carcinoma tissues and adjacent normal tissues (P b 0.01). HSP72 were stained in cell nuclei and cytoplasm respectively, while AFP stained in cell plasma (Fig. 1). Immunofluorescence and confocal laser microscopy showed that hepatocellular carcinoma synchronously coexpressed higher level of HSP72 and AFP. Both of them were localized in cell cytoplasm (Fig. 2). 3.2. Interaction between HSP72 and AFP in hepatocellular carcinoma cells Immunoprecipitate and Western blot showed that there were 2 clear protein bands in the transferred NC membrane. Under precipitated with anti-HSP72 antibody, there appeared an M r 69,000 clear α-fetoprotein band (lane 1) and under precipitated with anti-AFP mAb, there existed an M r 72,000 clear heat shock protein 72 band (lane 2), demonstrating that AFP existed in the immunoprecipitate of anti-HSP72 antibody and HSP72 existed in the immunoprecipitate of anti-AFP mAb. The results indicated that HSP72 interacted with AFP in hepatocellular carcinoma tissues (Fig. 3). 4. Discussion
Fig. 3. Analysis of immunoprecipitate of anti-HSP72 antibody and anti-AFP mAb by SDS-PAGE and Western blot (lane 1: precipitated with anti-HSP72 antibody; lane 2: precipitated with anti-AFP mAb; lane 3: anti-β-actin mAb (internal reference detected in hepatocellular carcinoma); lane 4: normal liver cells lysates supernatants; lane 5: hepatocellular carcinoma cells lysates supernatants; M: molecular mass markers).
We examined the expressions of HSP72 and AFP in hepatocellular carcinoma tissues by immunohistochemistry, confocal microscopy and immunoprecipitate analysis. The results showed that almost all hepatocellular carcinoma cells detected expressed high level of HSP72 and AFP, which had a
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significant difference compared with adjacent normal tissues. HSP72 associated with AFP mainly localizing in cell cytoplasm, the results were similar to cultured human hepatocellular carcinoma cell line [12]. AFP always accompanies with the growth of liver cells, and it is confirmed that AFP may be related to the proliferation of tumor or fetal cells [9,10]. The mechanism for growth-promoting activity of AFP is still unclear. Escaping from the surveillance of immune system is the primary cause for malignant growth of hepatocellular carcinoma cells [13,14]. Several investigations have showed that AFP could be individually synergy with other growth factors to promote the growth of many tumor cells [15,16]. AFP receptors have been found anchoring on the membrane of various tumor cells [17–19]. The receptor may mediate intercellular signal transduction which influences the expression of genes related to proliferation [17,18]. AFP can stimulate the expression of some oncogenes which control cell cycle, and then enhance the proliferation of human hepatocellular carcinoma [20,21]. When BEL-7402 cell line was treated with AFP, oncogene protein, such as c-fos, c-jun, c-ras and mutant p53 and p21ras increased rapidly, which have an important function in modulating growth and differentiation of the cells [22]. Heat shock protein 72, belonging to the family of HSP70, is a type of highly conserved protein synthesized under various stress. In non-transformed cells at normal conditions Hsp72 is expressed at very low levels. It is, however, present at elevated levels in the major fraction of tumors and in many transformed cell lines [23,24]. It is commonly assumed that in tumor cells the expression of HSP72 at elevated levels is the consequence of oncogenic transformation and enhanced expression of HSP72 has a close relationship with the epithelial carcinoma cells growth [6–8]. It is also found that AFP plays some roles during tumor cell survival and proliferation [9–11]. Some studies have suggested that high level expression of HSPs in HCC is correlated with AFP and poor prognosis [25–27]. Upregulated expression of HSP70 family in tumor cells may be required to serve as molecular chaperones in regulating and stabilizing oncofetal protein and mutant oncogene products during tumor growth process [28,29]. Therefore, overexpressed HSP72 may assist to stabilize AFP in cell cytoplasm, transports it to cell membrane and releases its monomer to the serum, which indirectly prompts and protects AFP function. Conversely, AFP itself stimulates hepatocellular carcinoma cells proliferation through membrane receptor mediating cellular signal transduction pathways. This indicates that the proliferating HCC cells need much more HSP72 to maintain the formation of AFP activities. Our results showed that human hepatocellular carcinoma cells existed high level expression of HSP72 and AFP. It indicates that co-expression of HSP72 and AFP is likely to have some relationship with proliferation, development and poor prognosis of human hepatocellular carcinoma. The observed fact that HSP72 was associated with AFP may be useful to study the pathogenesis and immunity of human hepatocellular carcinoma. Recent studies on the immunodominant epitopes of AFP have provided a solution to the obstacle of HCC immunotherapy. AFP is produced at low serum levels after birth throughout
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life. The majority of human HCC overexpress the oncofetal antigen AFP, M r 64,000–72,000 glycoprotein. Despite being exposed to high plasma levels of this oncofetal protein during embryonic development, body has a low immunity to it [1,2]. Butterfield et al. [30] recently found that four peptides of human AFP processed and presented in the context of HLA-A0201, could be recognized by the human T cell repertoire, and could be used to generate AFP-specific CTL in human T cell cultures. It was also found that murine immune system could generate Tcell responses to this oncofetal antigen [31]. Therefore, it may be a better target for immunotherapy. But AFP immunization alone still results in lower levels of specific response and poorly reproducible protective immunity [31,32]. How to enhance host's active immunity to AFP may be an interesting strategy for HCC therapy. In our previous study, tumor rejection assay demonstrated that recombinant DNA vaccine AFP/HSP70 elicited strong specific antitumor immunity against AFP-producing SP2/0 cells than AFP DNA vaccine. Results indicated that AFP immunogenicity can be improved greatly by HSP70 molecular and vaccination with DNA encoding HSP70 could increase both humoral and T-cell proliferation responses to α-fetoprotein. We attributed the successful α-fetoprotein specific T-cell responses in mice to the HSP70 molecular by mediating APCs to efficient uptake and process of AFP [33]. Numerous investigations have been verified that HSP72 is a better molecular chaperone and adjuvant which can process and present weak tumor antigen to MHC-I of host APCs, eliciting specific T-cell response and CTL reaction [34,35]. HSP72-associated peptides can also anchor antigen on the cell membrane and directly present it to nature killer cells or γδ T cells as superantigen without being dependent on the stimulation of MHC-I molecules [36,37]. The observed fact that HSP72 associated with AFP in hepatocellular carcinoma, may be useful to study tumor pathogenesis or design effective vaccine against liver cancers. 5. Conclusions Human hepatocellular carcinomas existed high level expression of HSP72 and AFP. HSP72 was associated with AFP mainly localizing in cell cytoplasm. Co-expression of HSP72 and AFP is likely to have some relationship with proliferation, development and poor prognosis of human hepatocellular carcinoma. The observed fact that HSP72 was associated with AFP may be useful to study the pathogenesis and design effective vaccine against liver cancers. References [1] Deutsch HF. Chemistry and biology of a-fetoprotein. Adv Cancer Res 1991;56:253–311. [2] Goldenberg DM, Kim EE, Deland F, et al. Clinical studies on the radioimmunodetection of tumors containing alpha-fetoprotein. Cancer 1980;45:2500–5. [3] Schlesinger MJ. Heat shock proteins. J Biol Chem 1990;265:12111–4. [4] Little E, Ramakrishnan M, Roy B, Gazit G, Lee AS. The glucose-regulated proteins (GRP60 and GRP94): functions, gene regulation, and applications. Crit Rev Eukaryot Gene Expr 1994;4:1–18.
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