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Prostate Apoptosis Response-4 Is Expressed in Normal Cholangiocytes, Is Down-Regulated in Human Cholangiocarcinoma, and Promotes Apoptosis of Neoplastic Cholangiocytes When Induced Pharmacologically

2010; Elsevier BV; Volume: 177; Issue: 4 Linguagem: Inglês

10.2353/ajpath.2010.091171

1525-2191

Antonio Franchitto, A. Torrice, R. Semeraro, Cristina Napoli, Gennaro Nuzzo, Felice Giuliante, Gianfranco Alpini, Guido Carpino, P.B. Berloco, Luciano Izzo, Antonio Bolognese, Paolo Onori, Anastasia Renzi, Alfredo Cantàfora, Eugenio Gaudio, Domenico Alvaro,

MicroRNA in disease regulation

Prostate apoptosis response-4 (Par-4) is a tumor suppressor protein that sensitizes cells to apoptosis; therefore, Par-4 modulation has therapeutic potential. No data currently exist on Par-4 expression in cholangiocarcinoma (CCA). We evaluated the expression of Par-4 in normal and neoplastic cholangiocytes and the effects of its pharmacological or genetic modulation. The study was performed in human and rat liver, CCA patient biopsies, and two CCA cell lines. PAR-4 was expressed in normal rat and human cholangiocytes, but its expression levels decreased in both human CCA and CCA cell lines. In both intrahepatic and extrahepatic CCA, Par-4 expression (as shown by immunohistochemistry) was inversely correlated with markers of proliferation (eg, proliferating cellular nuclear antigen) and directly correlated with apoptotic markers (eg, Bax and Bax/BCL2 ratio). Par-4 expression was decreased during CCA cell proliferation but was enhanced after apoptosis induction. Pharmacological induction of Par-4 expression in CCA cell lines by diindolymethane or withaferin A promoted activation of apoptosis and inhibition of proliferation. In contrast, specific Par-4 silencing by small-interfering RNA determined activation of CCA cell line proliferation. Par-4 is expressed in rat and human cholangiocytes and is down-regulated in both human CCA and CCA cell lines. Par-4 protein levels decrease during cell proliferation but increase during apoptosis. Pharmacological or genetic induction of Par-4 determines apoptosis of CCA cells, suggesting Par-4 targeting as a CCA treatment strategy. Prostate apoptosis response-4 (Par-4) is a tumor suppressor protein that sensitizes cells to apoptosis; therefore, Par-4 modulation has therapeutic potential. No data currently exist on Par-4 expression in cholangiocarcinoma (CCA). We evaluated the expression of Par-4 in normal and neoplastic cholangiocytes and the effects of its pharmacological or genetic modulation. The study was performed in human and rat liver, CCA patient biopsies, and two CCA cell lines. PAR-4 was expressed in normal rat and human cholangiocytes, but its expression levels decreased in both human CCA and CCA cell lines. In both intrahepatic and extrahepatic CCA, Par-4 expression (as shown by immunohistochemistry) was inversely correlated with markers of proliferation (eg, proliferating cellular nuclear antigen) and directly correlated with apoptotic markers (eg, Bax and Bax/BCL2 ratio). Par-4 expression was decreased during CCA cell proliferation but was enhanced after apoptosis induction. Pharmacological induction of Par-4 expression in CCA cell lines by diindolymethane or withaferin A promoted activation of apoptosis and inhibition of proliferation. In contrast, specific Par-4 silencing by small-interfering RNA determined activation of CCA cell line proliferation. Par-4 is expressed in rat and human cholangiocytes and is down-regulated in both human CCA and CCA cell lines. Par-4 protein levels decrease during cell proliferation but increase during apoptosis. Pharmacological or genetic induction of Par-4 determines apoptosis of CCA cells, suggesting Par-4 targeting as a CCA treatment strategy. Cholangiocarcinoma (CCA) is a devastating cancer with a bad prognosis and scarce response to chemotherapy.1Blechacz B Gores GJ Cholangiocarcinoma: advances in pathogenesis, diagnosis, and treatment.Hepatology. 2008; 48: 308-321Crossref PubMed Scopus (539) Google Scholar, 2Khan SA Thomas HC Davidson BR Taylor-Robinson SD Cholangiocarcinoma.Lancet. 2005; 366: 1303-1314Abstract Full Text Full Text PDF PubMed Scopus (1008) Google Scholar, 3Patel T Worldwide trends in mortality from biliary tract malignancies.BMC Cancer. 2002; 2: 10Crossref PubMed Scopus (400) Google Scholar CCA arises from the neoplastic transformation of cholangiocytes, the epithelial cells lining the biliary tree. Although important advances have been obtained in the last few years, the mechanisms leading to neoplastic transformation, growth, and spreading of CCA cells are undefined.4Fava G Marzioni M Benedetti A Glaser S DeMorrow S Francis H Alpini G Molecular pathology of biliary tract cancers.Cancer Lett. 2007; 250: 155-167Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 5Alvaro D Mancino MG New insights on the molecular and cell biology of human cholangiopathies.Mol Aspects Med. 2008; 29: 50-57Crossref PubMed Scopus (32) Google Scholar As in other cancers, dysregulation of multiple mechanisms modulating cell proliferation and apoptosis have been described in CCA.1Blechacz B Gores GJ Cholangiocarcinoma: advances in pathogenesis, diagnosis, and treatment.Hepatology. 2008; 48: 308-321Crossref PubMed Scopus (539) Google Scholar, 2Khan SA Thomas HC Davidson BR Taylor-Robinson SD Cholangiocarcinoma.Lancet. 2005; 366: 1303-1314Abstract Full Text Full Text PDF PubMed Scopus (1008) Google Scholar, 4Fava G Marzioni M Benedetti A Glaser S DeMorrow S Francis H Alpini G Molecular pathology of biliary tract cancers.Cancer Lett. 2007; 250: 155-167Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 5Alvaro D Mancino MG New insights on the molecular and cell biology of human cholangiopathies.Mol Aspects Med. 2008; 29: 50-57Crossref PubMed Scopus (32) Google Scholar Unfortunately, a progressive increase in incidence and mortality for CCA has been reported worldwide, which mainly affect the intrahepatic form of CCA.3Patel T Worldwide trends in mortality from biliary tract malignancies.BMC Cancer. 2002; 2: 10Crossref PubMed Scopus (400) Google Scholar, 6Alvaro D Crocetti E Ferretti S Bragazzi MC Capocaccia R the AISF “Cholangiocarcinoma” Committee Descriptive epidemiology of cholangiocarcinoma in Italy.Dig Liver Dis. 2009, Dec 18; ([Epub ahead of print])Google Scholar Half of CCA are not candidate for surgical resection at the time of diagnosis1Blechacz B Gores GJ Cholangiocarcinoma: advances in pathogenesis, diagnosis, and treatment.Hepatology. 2008; 48: 308-321Crossref PubMed Scopus (539) Google Scholar, 2Khan SA Thomas HC Davidson BR Taylor-Robinson SD Cholangiocarcinoma.Lancet. 2005; 366: 1303-1314Abstract Full Text Full Text PDF PubMed Scopus (1008) Google Scholar, 3Patel T Worldwide trends in mortality from biliary tract malignancies.BMC Cancer. 2002; 2: 10Crossref PubMed Scopus (400) Google Scholar and no efficacious treatment exists. Therefore, research aimed to find a new therapeutic target is currently an important challenge. Prostate apoptosis response-4 (Par-4), a tumor suppressor protein that sensitizes cells to apoptotic stimuli,6Alvaro D Crocetti E Ferretti S Bragazzi MC Capocaccia R the AISF “Cholangiocarcinoma” Committee Descriptive epidemiology of cholangiocarcinoma in Italy.Dig Liver Dis. 2009, Dec 18; ([Epub ahead of print])Google Scholar, 7Sells SF Wood DP Joshi-Barve SS Muthukumar S Jacob RJ Crist SA Humphreys S Rangnekar VM Commonality of the gene programs induced by effectors of apoptosis in androgen-dependent and -independent prostate cells.Cell Growth Differ. 1994; 5: 457-466PubMed Google Scholar, 8Goswami A Qiu S Dexheimer TS Ranganathan P Burikhanov R Pommier Y Rangnekar VM Par-4 binds to topoisomerase 1 and attenuates its DNA relaxation activity.Cancer Res. 2008; 68: 1-9Crossref Scopus (27) Google Scholar, 9Lee TJ Lee JT Kim SH Choi YH Song KS Park JW Kwon TK Overexpression of Par-4 enhances thapsigargin-induced apoptosis via down-regulation of XIAP and inactivation of Akt in human renal cancer cells.J Cell Biochem. 2008; 103: 358-368Crossref PubMed Scopus (20) Google Scholar, 10Diaz-Meco MT Abu-Baker S The Par-4/PTEN connection in tumor suppression.Cell Cycle. 2009; 8: 2518-2522Crossref PubMed Scopus (19) Google Scholar, 11Fernandez-Marcos PJ Abu-Baker S Joshi J Galvez A Castilla EA Cañamero M Collado M Saez C Moreno-Bueno G Palacios J Leitges M Serrano M Moscat J Diaz-Meco MT Simultaneous inactivation of Par-4 and PTEN in vivo leads to synergistic NF-kappaB activation and invasive prostate carcinoma.Proc Natl Acad Sci USA. 2009; 106: 12962-12967Crossref PubMed Scopus (29) Google Scholar, 12Kline CL Shanmugavelandy SS Kester M Irby RB Delivery of PAR-4 plasmid in vivo via nanoliposomes sensitizes colon tumor cells subcutaneously implanted into nude mice to 5-FU.Cancer Biol Ther. 2009; 22: 1831-1837Crossref Scopus (28) Google Scholar, 13Burikhanov R Zhao Y Goswami A Qiu S Schwarze SR Rangnekar VM The tumor suppressor Par-4 activates an extrinsic pathway for apoptosis.Cell. 2009; 138: 377-388Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 14Moscat J Diaz-Meco MT Wooten MW Of the atypical PKCs, Par-4 and p62: recent understandings of the biology and pathology of a PB1-dominated complex.Cell Death Differ. 2009; 16: 1426-1437Crossref PubMed Scopus (62) Google Scholar, 15Moreno-Bueno G Fernandez-Marcos PJ Collado M Tendero MJ Rodriguez-Pinilla SM Garcia-Cao I Hardisson D Diaz-Meco MT Moscat J Serrano M Palacios J Inactivation of the candidate tumor suppressor par-4 in endometrial cancer.Cancer Res. 2007; 67: 1927-1934Crossref PubMed Scopus (72) Google Scholar, 16García-Cao I Duran A Collado M Carrascosa MJ Martín-Caballero J Flores JM Diaz-Meco MT Moscat J Serrano M Tumour-suppression activity of the proapoptotic regulator Par4.EMBO Rep. 2005; 6: 577-583Crossref PubMed Scopus (78) Google Scholar was first identified in prostate cancer cells that were induced to undergo apoptosis. Par-4 is a leucine zipper domain protein widely expressed in diverse normal and cancerous cell types and tissues and resides in both the cytoplasm and the nucleus. Endogenous PAR-4 itself does not cause apoptosis, yet it is essential for apoptosis induced by a variety of exogenous insults.6Alvaro D Crocetti E Ferretti S Bragazzi MC Capocaccia R the AISF “Cholangiocarcinoma” Committee Descriptive epidemiology of cholangiocarcinoma in Italy.Dig Liver Dis. 2009, Dec 18; ([Epub ahead of print])Google Scholar, 7Sells SF Wood DP Joshi-Barve SS Muthukumar S Jacob RJ Crist SA Humphreys S Rangnekar VM Commonality of the gene programs induced by effectors of apoptosis in androgen-dependent and -independent prostate cells.Cell Growth Differ. 1994; 5: 457-466PubMed Google Scholar, 8Goswami A Qiu S Dexheimer TS Ranganathan P Burikhanov R Pommier Y Rangnekar VM Par-4 binds to topoisomerase 1 and attenuates its DNA relaxation activity.Cancer Res. 2008; 68: 1-9Crossref Scopus (27) Google Scholar, 9Lee TJ Lee JT Kim SH Choi YH Song KS Park JW Kwon TK Overexpression of Par-4 enhances thapsigargin-induced apoptosis via down-regulation of XIAP and inactivation of Akt in human renal cancer cells.J Cell Biochem. 2008; 103: 358-368Crossref PubMed Scopus (20) Google Scholar, 10Diaz-Meco MT Abu-Baker S The Par-4/PTEN connection in tumor suppression.Cell Cycle. 2009; 8: 2518-2522Crossref PubMed Scopus (19) Google Scholar, 11Fernandez-Marcos PJ Abu-Baker S Joshi J Galvez A Castilla EA Cañamero M Collado M Saez C Moreno-Bueno G Palacios J Leitges M Serrano M Moscat J Diaz-Meco MT Simultaneous inactivation of Par-4 and PTEN in vivo leads to synergistic NF-kappaB activation and invasive prostate carcinoma.Proc Natl Acad Sci USA. 2009; 106: 12962-12967Crossref PubMed Scopus (29) Google Scholar, 12Kline CL Shanmugavelandy SS Kester M Irby RB Delivery of PAR-4 plasmid in vivo via nanoliposomes sensitizes colon tumor cells subcutaneously implanted into nude mice to 5-FU.Cancer Biol Ther. 2009; 22: 1831-1837Crossref Scopus (28) Google Scholar, 13Burikhanov R Zhao Y Goswami A Qiu S Schwarze SR Rangnekar VM The tumor suppressor Par-4 activates an extrinsic pathway for apoptosis.Cell. 2009; 138: 377-388Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 14Moscat J Diaz-Meco MT Wooten MW Of the atypical PKCs, Par-4 and p62: recent understandings of the biology and pathology of a PB1-dominated complex.Cell Death Differ. 2009; 16: 1426-1437Crossref PubMed Scopus (62) Google Scholar, 15Moreno-Bueno G Fernandez-Marcos PJ Collado M Tendero MJ Rodriguez-Pinilla SM Garcia-Cao I Hardisson D Diaz-Meco MT Moscat J Serrano M Palacios J Inactivation of the candidate tumor suppressor par-4 in endometrial cancer.Cancer Res. 2007; 67: 1927-1934Crossref PubMed Scopus (72) Google Scholar, 16García-Cao I Duran A Collado M Carrascosa MJ Martín-Caballero J Flores JM Diaz-Meco MT Moscat J Serrano M Tumour-suppression activity of the proapoptotic regulator Par4.EMBO Rep. 2005; 6: 577-583Crossref PubMed Scopus (78) Google Scholar Recent studies suggest how Par-4 serves as an intracellular repressor of topoisomerase 1 catalytic activity, and regulates DNA topology to suppress cellular transformation.7Sells SF Wood DP Joshi-Barve SS Muthukumar S Jacob RJ Crist SA Humphreys S Rangnekar VM Commonality of the gene programs induced by effectors of apoptosis in androgen-dependent and -independent prostate cells.Cell Growth Differ. 1994; 5: 457-466PubMed Google Scholar, 8Goswami A Qiu S Dexheimer TS Ranganathan P Burikhanov R Pommier Y Rangnekar VM Par-4 binds to topoisomerase 1 and attenuates its DNA relaxation activity.Cancer Res. 2008; 68: 1-9Crossref Scopus (27) Google Scholar Consistent with its tumor suppressor function, Par-4 is silenced or mutated in different types of cancers,11Fernandez-Marcos PJ Abu-Baker S Joshi J Galvez A Castilla EA Cañamero M Collado M Saez C Moreno-Bueno G Palacios J Leitges M Serrano M Moscat J Diaz-Meco MT Simultaneous inactivation of Par-4 and PTEN in vivo leads to synergistic NF-kappaB activation and invasive prostate carcinoma.Proc Natl Acad Sci USA. 2009; 106: 12962-12967Crossref PubMed Scopus (29) Google Scholar, 12Kline CL Shanmugavelandy SS Kester M Irby RB Delivery of PAR-4 plasmid in vivo via nanoliposomes sensitizes colon tumor cells subcutaneously implanted into nude mice to 5-FU.Cancer Biol Ther. 2009; 22: 1831-1837Crossref Scopus (28) Google Scholar, 13Burikhanov R Zhao Y Goswami A Qiu S Schwarze SR Rangnekar VM The tumor suppressor Par-4 activates an extrinsic pathway for apoptosis.Cell. 2009; 138: 377-388Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar and experimental models of Par-4 knockout spontaneously develop tumors in various organs.17Barradas M Monjas A Diaz-Meco MT Serrano M Moscat J The downregulation of the pro-apoptotic protein Par-4 is critical for Ras-induced survival and tumor progression.EMBO J. 1999; 18: 6362-6369Crossref PubMed Scopus (98) Google Scholar In contrast, transgenic mice overexpressing Par-4 showed resistance to development of spontaneous and oncogene-induced, autochthonous tumors.18Zhao Y Burikhanov R Qiu S Lele SM Jennings CD Bondada S Spear B Rangnekar VM Cancer resistance in transgenic mice expressing the SAC module of Par-4.Cancer Res. 2007; 67: 9276-9285Crossref PubMed Scopus (50) Google Scholar Therefore, Par-4 modulation has tremendous therapeutic potential and, indeed, genetic or pharmacological strategies to induce Par-4 expression are currently under investigation for cancer prevention or treatment.9Lee TJ Lee JT Kim SH Choi YH Song KS Park JW Kwon TK Overexpression of Par-4 enhances thapsigargin-induced apoptosis via down-regulation of XIAP and inactivation of Akt in human renal cancer cells.J Cell Biochem. 2008; 103: 358-368Crossref PubMed Scopus (20) Google Scholar, 19Srinivasan S Ranga RS Burikhanov R Han SS Chendil D Par-4-dependent apoptosis by the dietary compound withaferin A in prostate cancer cells.Cancer Res. 2007; 67: 246-253Crossref PubMed Scopus (176) Google Scholar, 20Azmi AS Ahmad A Banerjee S Rangnekar VM Mohammad RM Sarkar FH Chemoprevention of pancreatic cancer: characterization of Par-4 and its modulation by 3,3′ diindolylmethane (DIM).Pharm Res. 2008; 25: 2117-2124Crossref PubMed Scopus (46) Google Scholar, 21Azmi AS Wang Z Burikhanov R Rangnekar VM Wang G Chen J Wang S Sarkar FH Mohammad RM Critical role of prostate apoptosis response-4 in determining the sensitivity of pancreatic cancer cells to small-molecule inhibitor-induced apoptosis.Mol Cancer Ther. 2008; 7: 2884-2893Crossref PubMed Scopus (37) Google Scholar To this regard, it is noteworthy that different natural compounds inducing Par-4 expression have been identified and proposed for cancer chemoprevention.19Srinivasan S Ranga RS Burikhanov R Han SS Chendil D Par-4-dependent apoptosis by the dietary compound withaferin A in prostate cancer cells.Cancer Res. 2007; 67: 246-253Crossref PubMed Scopus (176) Google Scholar, 20Azmi AS Ahmad A Banerjee S Rangnekar VM Mohammad RM Sarkar FH Chemoprevention of pancreatic cancer: characterization of Par-4 and its modulation by 3,3′ diindolylmethane (DIM).Pharm Res. 2008; 25: 2117-2124Crossref PubMed Scopus (46) Google Scholar, 21Azmi AS Wang Z Burikhanov R Rangnekar VM Wang G Chen J Wang S Sarkar FH Mohammad RM Critical role of prostate apoptosis response-4 in determining the sensitivity of pancreatic cancer cells to small-molecule inhibitor-induced apoptosis.Mol Cancer Ther. 2008; 7: 2884-2893Crossref PubMed Scopus (37) Google Scholar No data exist on the role of Par-4 in modulating cholangiocyte proliferation and apoptosis, and the expression of Par-4 in human CCA is unknown. The aim of our study was to evaluate the expression of Par-4 in normal and neoplastic cholangiocytes and the effects of its pharmacological or genetic modulation on cell proliferation and apoptosis. Our findings demonstrate how Par-4 is involved in modulating apoptosis in CCA, suggesting Par-4 as a new potential therapeutic target for this devastating cancer. Reagents were purchased from Sigma Chemical Co (St. Louis, MO) unless otherwise indicated. HuH-28 cell line derived from intrahepatic CCA was acquired from Cancer Cell Repository, Tohoku University (Sendai, Japan) and maintained in CRML 1066 medium containing 10% fetal bovine serum (FBS). TFK-1 cell line (derived from extra-hepatic CCA) was kindly provided by Dr. Yoshiyuki Ueno form Cancer Cell Repository, Tohoku University, and maintained in RPMI medium containing 10% FBS. The hepatocellular carcinoma HepG2 cell line was acquired from Sigma Chemical Co and maintained in minimal essential medium (BioConcept's AMIMED, Switzerland) containing 10% FBS, 1% NEAA, 1% Na-pyruvate, and 1% l-glutamine. 3,3′-diindolylmethane (B-DIM) was obtained from BioResponse (Boulder, CO). Withaferin A (WA), a major constituent of the medicinal plant Withania somnifera, was obtained from Chromadex (Santa Ana, CA). Media and additives for cell culture were obtained from Gibco (BRL, Invitrogen Corporation s.r.l., S. Giuliano Milanese, Italy) unless otherwise indicated. The use of human material has been approved by the local Institutional Review Board. Samples of CCA and/or liver were obtained from (1) 10 patients (five female patients, aged 64 to 73 years, and five male patients, aged 67 to 75 years) with intrahepatic CCA presenting as a single mass lesion within the liver and submitted to surgical resection; (2) seven patients affected by extra-hepatic CCA (Klatskin tumor), (three female patients, aged 69 to 77 years, and four male patients, aged 68 to 78 years) submitted to radical surgery; and (3) five biopsies from liver donors with a normal histology (three female patients and two male patients, aged 49 to 54 years). Liver fragments (0.5 cm) were fixed in 10% buffered formalin for 2 to 4 hours, embedded in low-temperature-fusion paraffin (55°C to 57°C): 3- to 4-μm sections were stained with hematoxylin-eosin and Masson's trichrome. For immunohistochemistry (IHC), 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 PBS (pH 7.4) before applying the primary antibody. Sections were incubated overnight at 4°C with antibodies for (1) Par-4 (Santa Cruz, Inc., Santa Cruz, CA; sc-1666, mouse monoclonal; 1:100); (2) tumor suppressor phosphatase and tensin homolog deleted in chromosome 10 (PTEN; Dako, M3627; mouse monoclonal; 1:20); (3) proliferating cellular nuclear antigen (PCNA; Dako, PC10; mouse monoclonal; 1:100); (4) bcl-2-associated X protein (BAX; Santa Cruz, Inc., sc-7480, mouse monoclonal; 1:200); (5) B cell lymphoma gene-2 (pBCL2; Santa Cruz, Inc., sc-7382, mouse monoclonal; 1:100); and (6) nuclear factor-κB (pNFκB; Santa Cruz, Inc., sc-33039-R, rabbit polyclonal; 1:50). Samples were then rinsed with PBS for 5 minutes, incubated for 20 minutes at room temperature with secondary biotinylated antibody (Dako LSAB Plus System, HRP, Milan, Italy), then with Dako ABC (Dako LSAB Plus System, HRP) and finally developed with 3–3′ diaminobenzidine. For all immunoreactions, negative controls (the primary antibody was replaced with pre-immune serum) were also included. Light microscopy and IHC observation were taken by Olympus BX-5 1 Light Microscopy (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 IHC observations were independently performed by three pathologists in a blind fashion. Briefly, six slides were analyzed per each specimen of normal liver or CCA. Neoplastic or normal cholangiocytes were counted in a random, blinded fashion in six nonoverlapping fields (magnification ×20) of each slide and the data expressed as percent positive cells. Fragments of intrahepatic bile ducts, averaging 20 μm in diameter, were isolated from human liver (five liver donors, 1 Gram pieces) as previously described for isolation and characterization of bile duct units from rat liver.22Mennone A Alvaro D Cho W Boyer JL Isolation of small polarized bile duct units.Proc Natl Acad Sci USA. 1995; 92: 6527-6531Crossref PubMed Scopus (101) Google Scholar HuH-28 or TFK-1 cells were platted on six-well plates and maintained in appropriate medium supplemented with 10% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, in humidified atmosphere of 5% CO2. HuH-28 or TFK-1 cells were exposed to 17β-estradiol, insulin-like growth factor 1 (IGF1), Withaferin A or B-DIM, and proliferation or apoptosis evaluated as follows. 17β-estradiol, Withaferin A, and B-DIM were prepared as stock solutions in dimethyl sulfoxide (DMSO) while IGF1 was dissolved in saline. 17β-estradiol stock solutions were then diluted (1:1,000,000), Withaferin A or B-DIM stock solutions were diluted (1:10,000), and IGF1 stock solutions were then diluted 1:10,000 into serum-free culture medium. Control cells were exposed to DMSO or saline only. Cell proliferation was assessed as described23Alvaro D Barbaro B Franchitto A Onori P Glaser SS Alpini G Francis H Marucci L Sterpetti P Ginanni-Corradini S Onetti Muda A Dostal DE De Santis A Attili AF Benedetti A Gaudio E Estrogens and insulin-like growth factor 1 modulate neoplastic cell growth in human cholangiocarcinoma.Am J Pathol. 2006; 169: 877-888Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 24Mancino A Mancino MG Glaser SS Alpini G Bolognese A Izzo L Francis H Onori P Franchitto A Ginanni-Corradini S Gaudio E Alvaro D Estrogens stimulate the proliferation of human cholangiocarcinoma by inducing the expression and secretion of vascular endothelial growth factor.Dig Liver Dis. 2009; 41: 156-163Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar by both 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) proliferation assay and PCNA protein expression (Western blot). For MTS proliferation assay, we used a commercially available colorimetric cell proliferation assay (Cell Titer 96 AQueous Non-Radioactive 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. Apoptosis was induced in cell lines by different maneuvers including serum depletion for 48 hours, exposure to human recombinant TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), or to beauvericin. TRAIL was dissolved in PBS (10 μg/ml stock solution), stored at −20°C in multiple aliquots, and then added (1:1000, 100 ng/ml) for 24 hours into HuH-28 cell cultured in the appropriate medium containing 10% FBS. Beauvericin was dissolved in methanol (3 mmol/L stock solution), stored at −20°C in multiple aliquots, and then added (1:10,000; 25 μmol/L) for 8 hours in HuH-28 cells cultured in the appropriate medium containing 10% FBS. Control cells were incubated in a medium containing an equivalent amount of methanol for 8 hours. Apoptosis was evaluated23Alvaro D Barbaro B Franchitto A Onori P Glaser SS Alpini G Francis H Marucci L Sterpetti P Ginanni-Corradini S Onetti Muda A Dostal DE De Santis A Attili AF Benedetti A Gaudio E Estrogens and insulin-like growth factor 1 modulate neoplastic cell growth in human cholangiocarcinoma.Am J Pathol. 2006; 169: 877-888Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 24Mancino A Mancino MG Glaser SS Alpini G Bolognese A Izzo L Francis H Onori P Franchitto A Ginanni-Corradini S Gaudio E Alvaro D Estrogens stimulate the proliferation of human cholangiocarcinoma by inducing the expression and secretion of vascular endothelial growth factor.Dig Liver Dis. 2009; 41: 156-163Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar by measuring caspase-3 and −8 activity. For caspase-3, we used a colorimetric assay kit (Sigma-Aldrich), based on the hydrolysis of the peptide substrate acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) by caspase-3, resulting in the release of the p-nitroanilide (p-NA). 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 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and 5 mmol/L DTT). The spectrophotometric absorbance of each sample, calculated at 405 nm, was normalized with its protein content and expressed as caspase-3 activity in relative absorbance intensity. For caspase-8, we used a colorimetric assay kit (Sigma-Aldrich), based on the hydrolysis of the peptide substrate acetyl-lle-Glu-Thr-Asp p-nitroaniline by caspase 8, resulting in the release of the p-NA. 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 DTT). The spectrophotometric absorbance of each sample, calculated at 405 nm, was normalized with its protein content and expressed as caspase-8 activity in relative absorbance intensity. Pieces of dissected tissues (about 30 mg) were immediately submerged in RNAlater stabilization solution (Ambion, Inc., Austin, TX). Each sample was left 10 minutes at room temperature and then stored at −20°C until being submitted to RNA extraction. The tissue fragment removed from RNAlater was transferred into a clean Eppendorf tube where the total RNA was extracted by using the TRI REAGENT (Sigma-Aldrich) and 1-bromo-3-chloropropane by following the manufacturer's instructions. The isolated RNA was dissolved in 55 μl of RNase-free water. RNA quality and quantity was evaluated by the RNA 6000 Nano LabChip kit with the Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany) as previously described.25Schroeder A Mueller O Stocker S Salowsky R Leiber M Gassmann M Lightfoot S Menzel W Granzow M Ragg T The RIN: an RNA integrity number for assigning integrity values to RNA measurements.BMC Mol Biol. 2006; 7: 3Crossref PubMed Scopus (1816) Google Scholar An amount of 2.5 μg of total RNA was used for the RT reaction primed by the random hexamer (Invitrogen). This was conducted in a 20-μl volume with a genetically engineered version of the M-MuLV reverse transcriptase (Expand Reverse Trancriptase, Roche Diagnostics GmbH, Mannheim, Germany) according the manufacturer's directions. The quality of cDNA produced, ie, the length of the transcript and the lack of DNA contamination, was tested by PCR with the RNA/cDNA Inspector kit (Sigma-Aldrich) and the DNA1000 LabChip kit with the Agilent 2100 BioAnalyzer as we previously described.26Cantafora A Blotta I Rivabene R Pisciotta L Bertolini S Evaluation of RNA messengers involved in lipid trafficking of human intestinal cells by reverse-transcription polymerase chain reaction with competimer technology and microchip electrophoresis.S Electrophoresis. 2003; 24: 3748-3754Crossref PubMed Scopus (13) Google Scholar Gene expression was determined by real-time PCR with a MX3000P instrument (Stratagene, La Jolla, CA) by using the averaged cycle threshold (Ct) automatically computed by the built-in software from three replicas of each sample. PCR amplifications were conducted into a volume of 25 μl, with 1.0 μl of cDNA template, 12.5 μl of 2× SYBR Green Brilliant QPCR Master Mix (Stratagene), 3 pmoles each of upstream and downstream primer for the gene analyzed, and 0.3 μl of diluted reference dye (6-Carboxyl-X-Rhodamine [ROX] at a final concentration 30 nmol/L). All real-time PCR amplifications were conducted with the following cycling program: 10 minutes at 95°C followed by 40 cycles (30 seconds at 95°C, 30 seconds at 60°C, and 30 seconds at 72°C). The fluorescence detection was performed during the extension step of each cycle. The expression of the gene of interest, ie, the PRKC Apoptosis WT1 Regulator (PAWR), was normalized by three endogenous reference genes, ie, hypoxanthine phosphoribosyl-transferase I (HPRT1), hydroxymethyl-bilane synthase (HMBS), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as suggested.6Alvaro D Crocetti E Ferretti S Bragazzi MC Capocaccia R the AISF “Cholangiocarcinoma” Committee Descriptive epidemiology of cholangiocarcinoma in Ital