Estrogens and Insulin-Like Growth Factor 1 Modulate Neoplastic Cell Growth in Human Cholangiocarcinoma
2006; Elsevier BV; Volume: 169; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2006.050464
ISSN1525-2191
AutoresDomenico Alvaro, Barbara Barbaro, Antonio Franchitto, Paolo Onori, Shannon Glaser, Gianfranco Alpini, Heather Francis, Luca Marucci, Paola Sterpetti, Stefano Ginanni Corradini, Andrea Onetti Muda, David E. Dostal, A. De Santis, A.F. Attili, A. Benedetti, Eugenio Gaudio,
Tópico(s)Cholangiocarcinoma and Gallbladder Cancer Studies
ResumoWe investigated the expression of estrogen receptors (ERs), insulin-like growth factor 1 (IGF-1), and IGF-1R (receptor) in human cholangiocarcinoma and cholangiocarcinoma cell lines (HuH-28, TFK-1, Mz-ChA-1), evaluating the role of estrogens and IGF-1 in the modulation of neoplastic cell growth. ER-α, ER-β, IGF-1, and IGF-1R were expressed (immunohistochemistry) in all biopsies (18 of 18) of intrahepatic cholangiocarcinoma. ER-α was expressed (Western blot) only by the HuH-28 cell line (intrahepatic cholangiocarcinoma), whereas ER-β, IGF-1, and IGF-1R were expressed in the three cell lines examined. In serum-deprived HuH-28 cells, serum readmission induced stimulation of cell proliferation that was inhibited by ER and IGF-1R antagonists. 17β-Estradiol and IGF-1 stimulated proliferation of HuH-28 cells to a similar extent to that of MCF7 (breast cancer) but greater than that of TFK-1 and Mz-ChA-1, inhibiting apoptosis and exerting additive effects. These effects of 17β-estradiol and IGF-1 were associated with enhanced protein expression of ER-α, phosphorylated (p)-ERK1/2 and pAKT but with decreased expression of ER-β. Finally, transfection of IGF-1R anti-sense oligonucleotides in HuH-28 cells markedly decreased cell proliferation. In conclusion, human intrahepatic cholangiocarcinomas express receptors for estrogens and IGF-1, which cooperate in the modulation of cell growth and apoptosis. Modulation of ER and IGF-1R could represent a strategy for the management of cholangiocarcinoma. We investigated the expression of estrogen receptors (ERs), insulin-like growth factor 1 (IGF-1), and IGF-1R (receptor) in human cholangiocarcinoma and cholangiocarcinoma cell lines (HuH-28, TFK-1, Mz-ChA-1), evaluating the role of estrogens and IGF-1 in the modulation of neoplastic cell growth. ER-α, ER-β, IGF-1, and IGF-1R were expressed (immunohistochemistry) in all biopsies (18 of 18) of intrahepatic cholangiocarcinoma. ER-α was expressed (Western blot) only by the HuH-28 cell line (intrahepatic cholangiocarcinoma), whereas ER-β, IGF-1, and IGF-1R were expressed in the three cell lines examined. In serum-deprived HuH-28 cells, serum readmission induced stimulation of cell proliferation that was inhibited by ER and IGF-1R antagonists. 17β-Estradiol and IGF-1 stimulated proliferation of HuH-28 cells to a similar extent to that of MCF7 (breast cancer) but greater than that of TFK-1 and Mz-ChA-1, inhibiting apoptosis and exerting additive effects. These effects of 17β-estradiol and IGF-1 were associated with enhanced protein expression of ER-α, phosphorylated (p)-ERK1/2 and pAKT but with decreased expression of ER-β. Finally, transfection of IGF-1R anti-sense oligonucleotides in HuH-28 cells markedly decreased cell proliferation. In conclusion, human intrahepatic cholangiocarcinomas express receptors for estrogens and IGF-1, which cooperate in the modulation of cell growth and apoptosis. Modulation of ER and IGF-1R could represent a strategy for the management of cholangiocarcinoma. Cholangiocarcinoma is a malignant tumor arising from the epithelial cells (cholangiocytes) lining the biliary tree and characterized by a poor prognosis and scarce response to current therapies.1Gores GJ A spotlight on cholangiocarcinoma.Gastroenterology. 2003; 125: 1536-1538Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 2Gores GJ Cholangiocarcinoma: current concepts and insights.Hepatology. 2003; 37: 961-969Crossref PubMed Scopus (255) Google Scholar The incidence and mortality for cholangiocarcinoma are increasing worldwide.3Blendis L Halpern Z An increasing incidence of cholangio-carcinoma: why?.Gastroenterology. 2004; 127: 1008-1009Abstract Full Text Full Text PDF PubMed Google Scholar Estrogens are positive growth modulators for normal and neoplastic cells expressing estrogen receptors (ERs).4Migliaccio A Castoria G Di Domenico M de Falco A Bilancio A Lombardi M Bottero D Varricchio L Nanayakkara M Rotondi A Auricchio F Sex steroid hormones act as growth factors.J Steroid Biochem Mol Biol. 2002; 83: 31-35Crossref PubMed Scopus (90) Google Scholar, 5Koike S Sakai M Muramatsu M Molecular cloning and characterization of rat estrogen receptor.Nucleic Acids Res. 1987; 15: 2499-2513Crossref PubMed Scopus (469) Google Scholar, 6Kuiper GG Enmark E Pelto-Huikko M Nilsson S Gustafsson JA Cloning of a novel receptor expressed in rat prostate and ovary.Proc Natl Acad Sci USA. 1996; 93: 5925-5930Crossref PubMed Scopus (4270) Google Scholar, 7Mosselman S Polman J Dijkema R ERβ: identification and characterization of a novel human estrogen receptor.FEBS Lett. 1996; 392: 49-53Abstract Full Text PDF PubMed Scopus (2073) Google Scholar They bind ER-α and/or ER-β subtypes and modulate cell growth by both direct genomic and nongenomic pathways, in which different intracellular transduction signals are involved but with a major role played by MAP kinases.4Migliaccio A Castoria G Di Domenico M de Falco A Bilancio A Lombardi M Bottero D Varricchio L Nanayakkara M Rotondi A Auricchio F Sex steroid hormones act as growth factors.J Steroid Biochem Mol Biol. 2002; 83: 31-35Crossref PubMed Scopus (90) Google Scholar, 5Koike S Sakai M Muramatsu M Molecular cloning and characterization of rat estrogen receptor.Nucleic Acids Res. 1987; 15: 2499-2513Crossref PubMed Scopus (469) Google Scholar, 6Kuiper GG Enmark E Pelto-Huikko M Nilsson S Gustafsson JA Cloning of a novel receptor expressed in rat prostate and ovary.Proc Natl Acad Sci USA. 1996; 93: 5925-5930Crossref PubMed Scopus (4270) Google Scholar, 7Mosselman S Polman J Dijkema R ERβ: identification and characterization of a novel human estrogen receptor.FEBS Lett. 1996; 392: 49-53Abstract Full Text PDF PubMed Scopus (2073) Google Scholar The role played by estrogens and their receptors in the growth of ER-positive neoplasms represents the basis for the pharmacological treatment and/or prevention of different cancers (mainly breast cancer) with ER antagonists.8Platet N Cathiard AM Gleizes M Garcia M Estrogens and their receptors in breast cancer progression: a dual role in cancer proliferation and invasion.Crit Rev Oncol Hematol. 2004; 51: 55-67Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 9Thomas T Gallo MA Thomas TJ Estrogen receptors as targets for drug development for breast cancer, osteoporosis and cardiovascular diseases.Curr Cancer Drug Targets. 2004; 4: 483-499Crossref PubMed Scopus (41) Google Scholar We have recently shown that 1) human and rat cholangiocytes express both ER-α and/or ER-β subtypes, 2) estrogens positively modulate cholangiocyte proliferation, and 3) ERs are overexpressed during cholangiocyte proliferation associated with human cholangiopathies.10Alvaro D Invernizzi P Onori P Franchitto A De Santis A Crosignani A Sferra R Ginanni-Corradini S Mancino MG Maggioni M Attili AF Podda M Gaudio E Estrogen receptors in cholangiocytes and the progression of primary biliary cirrhosis.J Hepatol. 2004; 41: 905-912Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 11Alvaro D Alpini G Onori P Perego L Svegliati Baroni G Franchitto A Baiocchi L Glaser SS Le Sage G Folli F Gaudio E Estrogens stimulate proliferation of intrahepatic biliary epithelium in rats.Gastroenterology. 2000; 119: 1681-1691Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 12Alvaro D Alpini G Onori P Franchitto A Glaser SS Le Sage G Gigliozzi A Attili AF Gaudio E Effect of ovariectomy on the proliferative capacity of intrahepatic biliary epithelium.Gastroenterology. 2002; 123: 336-344Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 13Alvaro D Onori P Drudi Metalli V Svegliati-Baroni G Folli F Franchitto A Alpini G Mancino MG Attili AF Gaudio E Intracellular pathways mediating estrogen-induced cholangiocyte proliferation in the rat.Hepatology. 2002; 36: 297-304Crossref PubMed Scopus (83) Google Scholar Furthermore, studies in rat cholangiocytes indicate that estrogens interact with and potentiate the effect of growth factors on cholangiocyte proliferation.14Gigliozzi A Alpini G Baroni GS Marucci L Metalli VD Glaser SS Francis H Mancino MG Ueno Y Barbaro B Benedetti A Attili AF Alvaro D Nerve growth factor modulates the proliferative capacity of the intrahepatic biliary epithelium in experimental cholestasis.Gastroenterology. 2004; 127: 1198-1209Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 1515. Alvaro D, Metalli VD, Alpini G, Onori P, Franchitto A, Barbaro B, Glaser SS, Francis H, Cantafora A, Blotta I, Attili AF, Gaudio E: The intrahepatic biliary epithelium is a target of the growth hormone/insulin-like growth factor 1 axis. J Hepatol 43:875–883Google Scholar Specifically, by interacting at both receptor and postreceptor levels, 17β-estradiol markedly potentiates the proliferating effect of insulin-like growth factor 1 (IGF-1) on isolated rat cholangiocytes.1515. Alvaro D, Metalli VD, Alpini G, Onori P, Franchitto A, Barbaro B, Glaser SS, Francis H, Cantafora A, Blotta I, Attili AF, Gaudio E: The intrahepatic biliary epithelium is a target of the growth hormone/insulin-like growth factor 1 axis. J Hepatol 43:875–883Google Scholar Similar interactions between IGF-1 and estrogens modulate neoplastic cell growth of tumors expressing ERs, which may include breast, ovary, and endometrial cancers.16Helle SI The insulin-like growth factor system in advanced breast cancer.Best Pract Res Clin Endocrinol Metab. 2004; 18: 67-79Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 17Wimalasena J Meehan D Dostal R Foster JS Cameron M Smith M Growth factors interact with estradiol and gonadotropins in the regulation of ovarian cancer cell growth and growth factor receptors.Oncol Res. 1993; 5: 325-337PubMed Google Scholar, 18Hata H Hamano M Watanabe J Kuramoto H Role of estrogen and estrogen-related growth factor in the mechanism of hormone dependency of endometrial carcinoma cells.Oncology. 1998; 55: 35-44Crossref PubMed Scopus (17) Google Scholar Little information exists on the role of estrogens and IGF-1 in the modulation of growth and progression of cholangiocarcinoma. In the present study, we investigated the expression of ER and IGF-1R in human cholangiocarcinoma and human cholangiocarcinoma cell lines and evaluated the role of estrogens and IGF-1 in the modulation of neoplastic cell growth. Reagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise indicated. Media and serum for cell culture were obtained from Life Technologies, Inc. (Gaithersburg, MD). The IGF-1R blocking antibody αIR3 was obtained from Oncogene-DBA (DBA Italia, srl, Segrate, Milan, Italy). We investigated 18 patients (nine females, age 60 to 75 years, and nine males, age 63 to 73 years) with intrahepatic cholangiocarcinoma presenting as a single mass lesion within the liver. In 10 of 18 patients, US-guided liver biopsies were investigated, whereas in 8 of 18 patients (four female, four male) specimens were obtained after surgical resection (four patients) or liver transplantation (four patients). As normal controls, we investigated 10 liver biopsies with a normal histology from patients (five females, age 58 to 69 years, and five males, age 60 to 72 years) submitted to laparotomy. Liver fragments (0.5 cm) were fixed in 10% buffered formalin for 2 to 4 hours and embedded in low-temperature fusion paraffin (55 to 57°C), and 3- to 4-μm sections were stained with hematoxylin and eosin and Masson's trichrome. For immunohistochemistry, sections were mounted on glass slides coated with 0.1% poly-l-lysine. After deparaffination, endogenous peroxidase activity was blocked by a 30-minute incubation in methanolic hydrogen peroxide (2.5%). The endogen biotin was then blocked by Biotin Blocking System (DAKO, code X0590; DAKO, Copenhagen, Denmark) according to the instructions supplied by the vendor. Sections were hydrated in graded alcohol and rinsed in phosphate-buffered saline (PBS, pH 7.4) before applying the primary antibody. Sections were incubated overnight at 4°C with antibodies for cytokeratin 19 (monoclonal antibody CK-19; DAKO), proliferating cellular nuclear antigen (PCNA) (PC10; DAKO), ER-α [a cocktail of three monoclonal antibodies: SC-314, D12, F10 (33% of each); Santa Cruz Biotechnology. Inc., Santa Cruz, CA], ER-β (monoclonal antibody; GenTex, San Antonio, TX), IGF-1 (Santa Cruz), or IGF-1R (Santa Cruz). Samples were then rinsed with PBS for 5 minutes, incubated for 10 minutes at room temperature with secondary biotinylated antibody (DAKO LSAB Plus System; HRP, Milan, Italy), incubated with DAKO ABC (DAKO LSAB Plus System; HRP, Milan, Italy), and finally developed with 3-3′ diaminobenzidine. For all immunoreactions, negative controls were also included. Light microscopy and immunohistochemistry observation were taken by BX-5 1 light microscopy (Olympus, Tokyo, Japan) with a videocam (Spot Insight; Diagnostic Instrument, Inc., Sterling Heights, MI) and processed with an Image Analysis System (IAS; Delta Sistemi, Rome, Italy). Light microscopy and immunohistochemical observations were independently performed by three pathologists in a blind manner. ER-α and ER-β immunohistochemical expression was evaluated as previously described.11Alvaro D Alpini G Onori P Perego L Svegliati Baroni G Franchitto A Baiocchi L Glaser SS Le Sage G Folli F Gaudio E Estrogens stimulate proliferation of intrahepatic biliary epithelium in rats.Gastroenterology. 2000; 119: 1681-1691Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 12Alvaro D Alpini G Onori P Franchitto A Glaser SS Le Sage G Gigliozzi A Attili AF Gaudio E Effect of ovariectomy on the proliferative capacity of intrahepatic biliary epithelium.Gastroenterology. 2002; 123: 336-344Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 13Alvaro D Onori P Drudi Metalli V Svegliati-Baroni G Folli F Franchitto A Alpini G Mancino MG Attili AF Gaudio E Intracellular pathways mediating estrogen-induced cholangiocyte proliferation in the rat.Hepatology. 2002; 36: 297-304Crossref PubMed Scopus (83) Google Scholar Briefly, six slides were analyzed per each specimen of normal liver or cholangiocarcinoma. Neoplastic or normal cholangiocytes were counted in a random, blinded manner in six nonoverlapping fields (magnification ×20) of each slide and the data expressed as percentage of positive cells. The use of human material has been approved by the local institutional review board. Mz-ChA-1 cells (gallbladder origin)19Knuth A Gabbert H Dippold W Klein O Sachsse W Bitter-Suermann D Prellwitz W Meyer zum Buschenfelde KH Biliary adenocarcinoma. Characterization of three new human tumor cell lines.J Hepatol. 1985; 1: 579-596Abstract Full Text PDF PubMed Scopus (171) Google Scholar were a gift from Dr. J.G. Fitz (University of Texas, Southwest Medical Center, Dallas, TX). HuH-28 (intrahepatic bile duct)20Kusaka Y Tokiwa T Sato J Establishment and characterization of a cell line from a human cholangiocellular carcinoma.Res Exp Med (Berl). 1988; 188: 367-375Crossref PubMed Scopus (45) Google Scholar and TFK-1 (extrahepatic bile duct)21Saijyo S Kudo T Suzuki M Katayose Y Shinoda M Muto T Fukuhara K Suzuki T Matsuno S Establishment of a new extrahepatic bile duct carcinoma cell line, TFK-1.Tohoku J Exp Med. 1995; 177: 61-71Crossref PubMed Scopus (131) Google Scholar cholangiocarcinoma cell lines were acquired from Cancer Cell Repository, Tohoku University, Tohoku, Japan. Mz-ChA-1, TFK-1, and HuH-28 cells were maintained in CRML 1066 medium containing 10% fetal bovine serum. The human HCC cell line Alex (PRF/PLC/5) was a gift from Prof. R. Mazzanti (University of Florence, Florence, Italy) and was maintained in Eagle's minimum essential medium containing 10% fetal bovine serum. The human colon carcinoma SW 480 cell line (a gift from Dr. E. Porfiri, Polytechnic University of Ancona, Ancona, Italy) was cultured in Leibovitiz's L15 medium containing 10% fetal bovine serum. Cell lines, cultured in the appropriate medium containing 10% fetal bovine serum, were deprived of serum for 48 hours. Then, cells were maintained in serum-deprived conditions for an additional 48 hours (controls = C) or exposed to serum, 17β-estradiol, IGF-1, and/or receptor antagonists for an additional 48 hours. Specifically, cell medium was replaced with fresh serum-free or serum-containing medium to which the tested agent was added. 17β-Estradiol and ICI 182,780 were dissolved in dimethyl sulfoxide whereas IGF-1 and αIR3 were dissolved in saline as a stock solution that was then added (dilution, 1:100,000) to serum-free culture medium. In these experimental conditions, proliferation, apoptosis, and immunoblots were evaluated as described below. Cell proliferation was assessed by a commercially available colorimetric cell proliferation assay (CellTiter 96 aqueous nonradioactive cell proliferation assay, MTS kit; Promega, Madison, WI), by following the manufacturer's instructions. Proliferation index was calculated as the ratio (multiplied × 100) between cell numbers in unstimulated and stimulated cultures as described.22Park J Tadlock L Gores GJ Patel T Inhibition of interleukin 6-mediated mitogen-activated protein kinase activation attenuates growth of a cholangiocarcinoma cell line.Hepatology. 1999; 30: 1128-1133Crossref PubMed Scopus (175) Google Scholar In selected experiments, proliferation was also evaluated by PCNA protein expression (Western blot) or by [3H]thymidine incorporation as previously described.23Kanno N LeSage G Phinizy JL Glaser SS Francis H Alpini G Stimulation of α2-adrenergic receptor inhibits cholangiocarcinoma growth through modulation of Raf-1 and B-Raf activities.Hepatology. 2002; 35: 1329-1340Crossref PubMed Scopus (50) Google Scholar In these latter experiments, [3H]thymidine was added into the culture medium (1 μCi/ml) for the last 2 hours of each treatment. Apoptosis was evaluated by a caspase 3 colorimetric assay kit (Sigma Chemical Co.), based on the hydrolysis of the peptide substrate acetyl-Asp-Glu-Val-Asp p-nitro-anilide (Ac-DEVD-pNA) by caspase 3, resulting in the release of the p-nitroaniline (pNA). For this assay, cells were lysed in the appropriate lysis buffer provided by the vendor (50 mmol/L HEPES, pH 7.4, 5 mmol/L CHAPS, and 5 mmol/L dithiothreitol). The concentration of the pNA released from the substrate was calculated from the absorbance values at 405 nm. Caspase 3 activity resulting from the measured concentration of pNA [controls (48 hours plus 48 hours serum-free) = 1.66 ± 0.11 μmol pNA/minute/ml] was expressed as percent changes with respect to controls. For Western blot analysis, cells were solubilized in lysis buffer containing 15 mmol/L Tris-HCl (pH 7.4), 5 mmol/L ethylenediaminetetraacetic acid, 100 mmol/L NaCl, 1% Igepal, 2 mmol/L phenylmethyl sulfonyl fluoride, 2 mmol/L benzamidine, and 1% aprotinin on ice for 30 minutes. After centrifugation at 10,000 × g for 20 seconds at 4°C, the supernatant was recovered, and protein concentration was determined with the protein assay-dye reagent (Bio-Rad Laboratories GmbH, Segrate, Milan, Italy). Cell extracts (10 μg) were diluted in 6× LSB (Laemmli sample buffer) containing 0.3 mol/L 2-mercaptoethanol and resolved by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Western blotting was performed as described12Alvaro D Alpini G Onori P Franchitto A Glaser SS Le Sage G Gigliozzi A Attili AF Gaudio E Effect of ovariectomy on the proliferative capacity of intrahepatic biliary epithelium.Gastroenterology. 2002; 123: 336-344Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 13Alvaro D Onori P Drudi Metalli V Svegliati-Baroni G Folli F Franchitto A Alpini G Mancino MG Attili AF Gaudio E Intracellular pathways mediating estrogen-induced cholangiocyte proliferation in the rat.Hepatology. 2002; 36: 297-304Crossref PubMed Scopus (83) Google Scholar, 14Gigliozzi A Alpini G Baroni GS Marucci L Metalli VD Glaser SS Francis H Mancino MG Ueno Y Barbaro B Benedetti A Attili AF Alvaro D Nerve growth factor modulates the proliferative capacity of the intrahepatic biliary epithelium in experimental cholestasis.Gastroenterology. 2004; 127: 1198-1209Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 1515. Alvaro D, Metalli VD, Alpini G, Onori P, Franchitto A, Barbaro B, Glaser SS, Francis H, Cantafora A, Blotta I, Attili AF, Gaudio E: The intrahepatic biliary epithelium is a target of the growth hormone/insulin-like growth factor 1 axis. J Hepatol 43:875–883Google Scholar by using the following primary antibodies from Santa Cruz: anti-PCNA, specific mouse monoclonal antibody (1:300 dilution); anti-IGF-1, goat polyclonal antibody (1:100 dilution); anti-IGF-1Rb, rabbit polyclonal antibody (1:400 dilution); anti-ER-α rabbit polyclonal antibody (1:200 dilution); anti-ER-β rabbit polyclonal antibody (1:200 dilution); anti-tERK, rabbit polyclonal antibody (1:1000 dilution); anti-pERK, mouse monoclonal antibody (1:800 dilution); anti-AKT, mouse monoclonal antibody (1:200 dilution); and anti-pAKT, rabbit polyclonal antibody (1:300 dilution). As secondary antibodies, anti-mouse IgG peroxidase-conjugated (1:2000; Sigma), anti-rabbit IgG peroxidase-conjugated (1:10,000; Sigma), or anti-goat IgG peroxidase-conjugated (1:10,000; Sigma) antibody was used. The intensity of the bands was determined by scanning video densitometry (Ultra Violet Products, Cambridge, UK) and expressed as arbitrary densitometric units normalized to β-actin expression (ie, tested protein/β-actin × 100). Total cellular RNA was extracted by the Micro-Fast Track II kit (Invitrogen, San Diego, CA) according to the instructions of the vendor. Total RNA (1 μg) was used for first strand cDNA synthesis by AMV reverse transcriptase (Roche Diagnostics, Mannheim, Germany). PCR primers for ER-α, 5′-AAGGAGACTCGCTACTGT-3′ (sense) and 5′-TCAAAGATCTCCACCATGCC-3′ (anti-sense), were based on the published sequence.24Inoue S Hoshino S Miyoshi H Akishita M Hosoi T Orimo H Ouchi Y Identification of a novel isoform of estrogen receptor, a potential inhibitor of estrogen action, in vascular smooth muscle cells.Biochem Biophys Res Com. 1996; 219: 766-772Crossref PubMed Scopus (53) Google Scholar GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as housekeeping gene.25Alpini G Glaser S Robertson W Phinizy JL Rodgers R Caligiuri A LeSage G Bile acids stimulate proliferative and secretory events in large but not small cholangiocytes.Am J Physiol. 1997; 273: G518-G529PubMed Google Scholar Primers were synthesized by Invitrogen. PCR conditions used were as follows: 30 cycles of 1 minute at 94°C, 1 minute at 57°C, and 2 minutes at 72°C. An 18-mer anti-sense phosphorothioate oligonucleotide (S-ODN) targeted against codons 2 to 7 of the prepropeptide IGF-I receptor sequence,26Muller M Dietel M Turzynski A Wiechen K Antisense phosphorothioate oligodeoxynucleotide down-regulation of the insulin-like growth factor 1 receptor in ovarian cancer cells.Int J Cancer. 1998; 77: 567-571Crossref PubMed Scopus (38) Google Scholar, 27Ullrich A Gray A Tam AW Yang-Feng T Tsubokawa M Collins C Henzel W Le Bon T Kathuria S Chen E Insulin-like growth factor I receptor primary structure: comparison with insulin receptor suggests structural determinants that define functional specificity.EMBO J. 1986; 5: 2503-2512Crossref PubMed Scopus (1642) Google Scholar sense, and mismatch control oligonucleotides were obtained from Invitrogen (Invitrogen S.R.L San Giuliano Milanese, Milan, Italy). The sequences were 5′-TCCTCCGGAGCCAGACTT-3′ (anti-sense), 5′-AAGTCTGGCTCCGGAGGA-3′ (sense), 5′-TGAGCCCTCCTCCGTAGA-3′ (mismatch primer 1) and 5′-CTCTGAGCCAGACGTCTC-3′ (mis-match primer 2). HuH-28 cells were kept in CMRL 1066 medium with penicillin-streptomycin-glutamine 1% + 10% fetal bovine serum (Invitrogen). Per protocol instructions, 1 day before transfection, HuH-28 cells were plated in growth medium (10% fetal bovine serum + 0.5% antibiotics) to obtain 50% confluency at the time of transfection. Phosphorothioate oligonucleotides were transfected into cells using oligofectamine reagent (Invitrogen). Per protocol instructions, the cells were washed two times with serum-free medium and then incubated with S-ODN-oligofectamine solution at 37°C in a CO2 incubator for 4 hours (serum-free medium). Then, a medium containing 30% serum and 1% antibiotics was added to the cells without removing the transfection mixture, and after 2 days (30% serum) the protein expression of IGF-1R and PCNA (proliferation marker) was analyzed. Data are presented as arithmetic mean ± SEs. Statistical analysis was conducted by using one-way analysis of the variance with pair-wise comparison by the Fisher's protected least-significant difference test. In all cases, P < 0.05 was considered significant. Cholangiocytes of intrahepatic bile ducts of normal human liver (n = 10 biopsies) were all negative at the immunohistochemical analysis for ER-α, ER-β, IGF-1, and IGF-1R (Figure 1A and Figure 2A). In contrast, all 18 human intrahepatic cholangiocarcinomas showed a marked positivity for both ER-α and ER-β, which involves 81.0 ± 3.5% and 82.5 ± 3.7% cells, respectively, with a staining located at both the cytoplasmic and nuclear level (Figure 1B). The 18 biopsies of human cholangiocarcinoma showed an intense positivity for both IGF-1 and IGF-1R, which involved 60.8 ± 2.8% and 64.4 ± 3.2% cells, respectively, with staining predominantly located at the cytoplasmic level (Figure 2B).Figure 2Immunohistochemistry for IGF-1 and IGF-1R in human normal liver and cholangiocarcinoma. A: Intrahepatic bile ducts of the normal liver were negative at immunohistochemical analysis for IGF-1 (left) and IGF-1R (right). B: Biopsies of human cholangiocarcinoma showing an intense positivity for both IGF-1 and IGF-1R at cytoplasmatic level (arrows). Light microscopy. Original magnifications, ×20.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Western blot analysis shows (Figure 3A) that ER-α was expressed by the HuH-28 (human intrahepatic) cell line but not by the TFK-1 (human extrahepatic) or Mz-ChA-1 (human gallbladder) cell lines (five independent experiments). In contrast, by qualitative RT-PCR the message for ER-α was also detected in TFK-1 and Mz-ChA-1 cells (Figure 3B). Therefore, Western blot data should be indicative for a very low level of ER-α protein expression in these two cell lines. In comparison with the MCF7 breast cancer cell line used as control, the expression (Western blot) of ER-α was significantly lower (P < 0.05, five independent experiments; Figure 3A) in HuH-28 cells. ER-β was similarly expressed in HuH-28 (intrahepatic) and in MCF7 breast cancer (positive control) cell lines but highly expressed in both TFK-1 (human extrahepatic) and Mz-ChA-1 (human gallbladder) cell line (P < 0.05 versus MCF7 or HuH-28, five independent experiments; Figure 3A). The protein mass of IGF-1 and IGF-1R (five independent experiments; Figure 3A) was similar in HuH-28 (intrahepatic) and TFK-1 (human extrahepatic) cholangiocarcinoma cell lines without significant differences with respect to cell lines derived from human hepatocellular carcinoma (Alex) or human colon carcinoma (SW480) used as positive controls. In contrast, the Mz-ChA-1 (gallbladder) cell line showed a protein mass of IGF-1 and IGF-1R significantly lower (P < 0.01, five independent experiments; Figure 3A) than all of the other cell lines investigated. Because the HuH-28 (intrahepatic) cell line, similar to human intrahepatic cholangiocarcinoma, expresses the protein for both ER-α and ER-β, we mainly focused on this cell line to evaluate the role and mechanism by which estrogens and IGF-1 modulate cell proliferation and apoptosis. For this purpose, HuH-28 cells were deprived of serum for 48 hours, a maneuver that elicited a 65.6 ± 8% decrease of proliferation index (10 independent experiments) and a 45 ± 5% (10 independent experiments) increase of apoptosis. Serum-deprived HuH-28 cells were left without serum (controls) or exposed to serum, 17β-estradiol, IGF-1, and/or receptor antagonists for an additional 48 hours. In controls, 48 + 48 hours serum-free starvation elicited a 75.4 ± 6% (10 independent experiments) decrease of proliferation index and a 54 ± 4% (10 independent experiments) increase of apoptosis with respect to HuH-28 cells cultured in serum-containing media. [3H]Thymidine incorporation decreased from 7560 ± 586 dpm/mg protein to 3050 ± 209 dpm/mg protein after 48 hours (P < 0.01) and to 2035 ± 197 dpm/mg protein after 48 + 48 hours (P < 0.05 versus 48 hours) serum-free starvation (10 independent experiments for each protocol). The significant [3H]thymidine incorporation demonstrates that a certain rate of proliferation still persists in HuH-28 cells starved without serum for 48 or 48 + 48 hours. We also performed flow cytometry analysis of propidium iodide-stained HuH-28 cells (not shown), indicating that after 48 hours of serum-free starvation HuH-28 cells do not completely arrest in G1 phase, but at least 24% of cells are still under proliferation (S phase). When serum-deprived HuH-28 cells were exposed for 48 hours to the ER antagonists tamoxifen (1 μmol/L, 10 independent experiments) and ICI 182,780 (1 μmol/L, 10 independent experiments) or to the IGF-1R blocking antibody αIR3 (1 μg/ml, 10 independent experiments), no significant changes in proliferation index were seen (Figure 4A). Readmission of serum for 48 hours induced a marked increase of proliferation (+67 ± 7%, 10 independent experiments) which was inhibited (P < 0.01) for ∼80% (10 independent experiments) by the two ER antagonists, tamoxifen and ICI 182,780, and to a higher extent (93% inhib
Referência(s)