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Longati P, Jia X, Eimer J, Wagman A, Witt MR, Rehnmark S, Verbeke C, Toftgård R, Löhr M, Heuchel RL. 3D pancreatic carcinoma spheroids induce a matrix-rich, chemoresistant phenotype offering a better model for drug testing. BMC Cancer 2013; 13:95. [PMID: 23446043 PMCID: PMC3617005 DOI: 10.1186/1471-2407-13-95] [Citation(s) in RCA: 270] [Impact Index Per Article: 22.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2012] [Accepted: 02/24/2013] [Indexed: 12/22/2022] Open
Abstract
Background Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of cancer related death. It is lethal in nearly all patients, due to an almost complete chemoresistance. Most if not all drugs that pass preclinical tests successfully, fail miserably in the patient. This raises the question whether traditional 2D cell culture is the correct tool for drug screening. The objective of this study is to develop a simple, high-throughput 3D model of human PDAC cell lines, and to explore mechanisms underlying the transition from 2D to 3D that might be responsible for chemoresistance. Methods Several established human PDAC and a KPC mouse cell lines were tested, whereby Panc-1 was studied in more detail. 3D spheroid formation was facilitated with methylcellulose. Spheroids were studied morphologically, electron microscopically and by qRT-PCR for selected matrix genes, related factors and miRNA. Metabolic studies were performed, and a panel of novel drugs was tested against gemcitabine. Results Comparing 3D to 2D cell culture, matrix proteins were significantly increased as were lumican, SNED1, DARP32, and miR-146a. Cell metabolism in 3D was shifted towards glycolysis. All drugs tested were less effective in 3D, except for allicin, MT100 and AX, which demonstrated effect. Conclusions We developed a high-throughput 3D cell culture drug screening system for pancreatic cancer, which displays a strongly increased chemoresistance. Features associated to the 3D cell model are increased expression of matrix proteins and miRNA as well as stromal markers such as PPP1R1B and SNED1. This is supporting the concept of cell adhesion mediated drug resistance.
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Affiliation(s)
- Paola Longati
- CLINTEC, Karolinska Institutet, Stockholm 14186, Sweden
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Koutsounas I, Giaginis C, Patsouris E, Theocharis S. Current evidence for histone deacetylase inhibitors in pancreatic cancer. World J Gastroenterol 2013; 19:813-28. [PMID: 23430136 PMCID: PMC3574878 DOI: 10.3748/wjg.v19.i6.813] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/09/2011] [Revised: 10/18/2011] [Accepted: 01/05/2013] [Indexed: 02/06/2023] Open
Abstract
Pancreatic cancer is one of the most aggressive human cancers, with more than 200 000 deaths worldwide every year. Despite recent efforts, conventional treatment approaches, such as surgery and classic chemotherapy, have only slightly improved patient outcomes. More effective and well-tolerated therapies are required to reverse the current poor prognosis of this type of neoplasm. Among new agents, histone deacetylase inhibitors (HDACIs) are now being tested. HDACIs have multiple biological effects related to acetylation of histones and many non-histone proteins that are involved in regulation of gene expression, apoptosis, cell cycle progression and angiogenesis. HDACIs induce cell cycle arrest and can activate the extrinsic and intrinsic pathways of apoptosis in different cancer cell lines. In the present review, the main mechanisms by which HDACIs act in pancreatic cancer cells in vitro, as well as their antiproliferative effects in animal models are presented. HDACIs constitute a promising treatment for pancreatic cancer with encouraging anti-tumor effects, at well-tolerated doses.
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Haraguchi N, Ohkuma M, Sakashita H, Matsuzaki S, Tanaka F, Mimori K, Kamohara Y, Inoue H, Mori M. CD133+CD44+ population efficiently enriches colon cancer initiating cells. Ann Surg Oncol 2008; 15:2927-33. [PMID: 18663533 DOI: 10.1245/s10434-008-0074-0] [Citation(s) in RCA: 162] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2008] [Revised: 06/25/2008] [Accepted: 06/25/2008] [Indexed: 12/13/2022]
Abstract
BACKGROUND Previous reports have demonstrated that CD133(+) cells or CD44(+) cells might be cancer initiating cells (CIC) of colon cancer. However, the association between the two cell types is unclear. In this study, we evaluated the tumorigenicity of each population of human colon cancer divided by CD133 and CD44 using non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. METHODS Using the colon cancer cell lines HT29 and Caco2 we evaluated the change of expression status of CD133 or CD44 by a treatment with sodium butyrate (NaBT) that can induce cellular differentiation. Next, we prepared ten clinical samples of colon cancer and analyzed the expression and tumorigenicity of CD133 and CD44. RESULTS With NaBT treatment, CD44 expression was greatly downregulated in both HT29 and Caco2 (HT29: nontreatment versus treatment; 77.8% versus 0.6%, Caco2: 14.0% versus 0.4%, respectively), more than CD133 expression (HT29: nontreatment versus treatment; 90.1% versus 67.7%, Caco2: 98.9% versus 76.3%, respectively). In clinical samples, the percentages of CD133(+) cells and CD44(+) cells varied from 0.3% to 82.0% (mean 35.5%), and from 11.5% to 58.4% (mean 30.0%), respectively. Subcutaneous injection of CD133(+) or CD44(+) cells made a tumor in all mice (3/3 and 4/4, respectively). The combined analysis of CD133 and CD44 revealed that only the CD133(+)CD44(+) population had the ability to produce a tumor (3/3). CONCLUSION The findings demonstrate that, at present, the CD133(+)CD44(+ ) population may be the best to identify tumor initiating cells of human colon cancer.
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Affiliation(s)
- Naotsugu Haraguchi
- Department of Gastroenterological Surgery, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, 565-0871, Japan
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Ceni E, Mello T, Tarocchi M, Crabb DW, Caldini A, Invernizzi P, Surrenti C, Milani S, Galli A. Antidiabetic thiazolidinediones induce ductal differentiation but not apoptosis in pancreatic cancer cells. World J Gastroenterol 2005; 11:1122-30. [PMID: 15754392 PMCID: PMC4250701 DOI: 10.3748/wjg.v11.i8.1122] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: Thiazolidinediones (TZD) are a new class of oral antidiabetic drugs that have been shown to inhibit growth of same epithelial cancer cells. Although TZD were found to be ligands for peroxisome proliferator-activated receptor γ (PPARγ), the mechanism by which TZD exert their anticancer effect is presently unclear. In this study, we analyzed the mechanism by which TZD inhibit growth of human pancreatic carcinoma cell lines in order to evaluate the potential therapeutic use of these drugs in pancreatic adenocarcinoma.
METHODS: The effects of TZD in pancreatic cancer cells were assessed in anchorage-independent growth assay. Expression of PPARγ was measured by reverse-transcription polymerase chain reaction and confirmed by Western blot analysis. PPARγ activity was evaluated by transient reporter gene assay. Flow cytometry and DNA fragmentation assay were used to determine the effect of TZD on cell cycle progression and apoptosis respectively. The effect of TZD on ductal differentiation markers was performed by Western blot.
RESULTS: Exposure to TZD inhibited colony formation in a PPARγ-dependent manner. Growth inhibition was linked to G1 phase cell cycle arrest through induction of the ductal differentiation program without any increase of the apoptotic rate.
CONCLUSION: TZD treatment in pancreatic cancer cells has potent inhibitory effects on growth by a PPAR-dependent induction of pacreatic ductal differentiation.
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Affiliation(s)
- Elisabetta Ceni
- Gastroenterology Unit, Department of Clinical Pathophysiology, University of Florence, Viale Morgani 85, 50134 Firenze, Italy
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Pettersson F, Colston KW, Dalgleish AG. Differential and antagonistic effects of 9-cis-retinoic acid and vitamin D analogues on pancreatic cancer cells in vitro. Br J Cancer 2000; 83:239-45. [PMID: 10901377 PMCID: PMC2363480 DOI: 10.1054/bjoc.2000.1281] [Citation(s) in RCA: 45] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022] Open
Abstract
Retinoids and vitamin D are known to exert important anti-tumour effects in a variety of cell types. In this study the effects of 9-cis-retinoic acid (9cRA) the vitamin D analogues EB1089 and CB1093 on three pancreatic adenocarcinoma cell lines were investigated. All compounds caused inhibition of in vitro growth but the vitamin D analogues were generally the more potent growth inhibitors. They were also more effective on their own than in combination with 9cRA. Growth arrest correlated with an increased proportion of cells in the G0/G1 phase. Apoptosis was induced in the three cell lines by 9cRA, whereas neither EB1089 nor CB1093 had this effect. Furthermore, addition of EB1089 or CB1093 together with 9cRA resulted in significantly reduced apoptosis. Our results show that retinoic acids as well as vitamin D analogues have inhibitory effects on pancreatic tumour cells but different and antagonistic mechanisms seem to be employed.
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Affiliation(s)
- F Pettersson
- Department of Oncology, Gastroenterology, Endocrinology and Metabolism, St George's Hospital Medical School, London, UK
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Reynolds S, Rajagopal S, Chakrabarty S. Differentiation-inducing effect of retinoic acid, difluoromethylornithine, sodium butyrate and sodium suramin in human colon cancer cells. Cancer Lett 1998; 134:53-60. [PMID: 10381130 DOI: 10.1016/s0304-3835(98)00242-0] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
Abstract
We investigated the relative effectiveness of four differentiation-inducing chemicals to induce a more normal or benign phenotype in the human colon cancer cell lines Moser and HT29. The differentiation-inducing capability of all-trans retinoic acid (ATRA), difluoromethylornithine (DFMO), sodium butyrate (NaB) and sodium suramin (NaS) was evaluated in terms of the efficacy of these chemicals in inhibiting cellular proliferation, growth in soft agarose, invasion of matrigel and induction of morphological alteration. The relative ability of these chemicals to induce production of the differentiation-related molecules fibronectin and carcinoembryonic antigen was also determined. Overall, ATRA was found to be the most effective chemical in inducing differentiation as measured by these parameters. The Moser cells were more susceptible to differentiation induction by comparison with the HT29 cells. Both similarities and differences in the cellular responses to DFMO, NaB and NaS were also observed for the Moser and HT29 cells. The differences in cellular responses to these chemicals may be due to different phenotypic properties of these two cell lines and different mechanisms of action of these chemicals.
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Affiliation(s)
- S Reynolds
- Division of Laboratory Medicine, The University of Texas, M.D. Anderson Cancer Center, Houston 77030, USA
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Rosewicz S, Stier U, Brembeck F, Kaiser A, Papadimitriou CA, Berdel WE, Wiedenmann B, Riecken EO. Retinoids: effects on growth, differentiation, and nuclear receptor expression in human pancreatic carcinoma cell lines. Gastroenterology 1995; 109:1646-60. [PMID: 7557150 DOI: 10.1016/0016-5085(95)90655-x] [Citation(s) in RCA: 49] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
BACKGROUND & AIMS Advanced pancreatic carcinoma has a dismal prognosis despite extensive chemotherapeutic trials. The aim of this study was to evaluate the role of retinoids as an experimental therapeutic approach for pancreatic cancer. METHODS Four ductal and one acinar pancreatic tumor cell lines were investigated. Growth was determined by cell number and a human tumor clonogenic assay. In vivo growth was assessed by xenografts transplanted into nude mice. Differentiation was characterized by immunofluorescence microscopy and carbonic anhydrase II gene expression. Retinoid receptors were characterized by Northern blotting and reverse-transcriptase polymerase chain reaction. RESULTS Retinoid treatment results in a time- and dose-dependent growth inhibition in vitro and in vivo of ductal but not acinar pancreatic tumor cells. Retinoid treatment induces a more differentiated phenotype in ductal tumor cells as shown by morphological criteria and increased expression of carbonic anhydrase II. All pancreatic tumor cell lines expressed a broad panel of cellular retinoid binding proteins and nuclear retinoid receptors. Retinoic acid receptor gamma and cellular retinoic acid binding protein II were found in all retinoid-sensitive ductal tumor cell lines but not in the retinoid-resistant acinar cell lines. CONCLUSIONS Detailed knowledge of nuclear retinoid receptor expression may provide rational strategies for retinoid treatment of pancreatic cancer.
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Affiliation(s)
- S Rosewicz
- Department of Gastroenterology, Klinikum Benjamin Franklin, Berlin, Germany
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Miszczuk-Jamska B, Merten M, Renaud W, Guy-Crotte O, Figarella C. Trypsinogen expression by two human pancreatic cell lines CFPAC-1 and CAPAN-1. Modulation during spontaneous and induced cell growth. INTERNATIONAL JOURNAL OF PANCREATOLOGY : OFFICIAL JOURNAL OF THE INTERNATIONAL ASSOCIATION OF PANCREATOLOGY 1994; 16:61-9. [PMID: 7806913 DOI: 10.1007/bf02925611] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
We previously demonstrated that two human pancreatic adenocarcinoma cell lines, CFPAC-1 (established from a patient with cystic fibrosis) and CAPAN-1, were able to secrete trypsinogens 1 and 2 specifically. In order to analyze the relation of trypsin secretion to differentiation and cell growth, we undertook a comparative study of immunoreactive trypsin 1 (IRT) secretion by the two cell lines during cell growth in the presence and in the absence of various differentiating agents: sodium butyrate (NaBut), dimethylsulfoxide (DMSO), and dexamethasone (DX). In the presence of NaBut, IRT levels in the supernatants of both cell lines were slightly increased, whereas the cellular growth of both cell lines decreased significantly. In the presence of DX, IRT levels in cell culture conditioned media immediately and dramatically decreased, but the cell growth of neither cell line was affected by DX. An important increase in IRT levels was observed when CFPAC-1 cells and CAPAN-1 cells were grown in the presence of DMSO, but for both cell lines the cellular growth decreased in the presence of DMSO. Our data show that neither the IRT secretion level nor the differentiation state of these cell lines correlates with cellular growth, and suggests that the expression of pancreatic proteases by these two tumor cell lines could be either related to a common stem cell with this potential or to a possible acinar origin of pancreatic cancer, as recently proposed by others.
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Affiliation(s)
- B Miszczuk-Jamska
- Groupe de Recherche sur les Glandes Exocrines, Faculté de Médecine, Marseille, France
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Goodwin TJ, Jessup JM, Wolf DA. Morphologic differentiation of colon carcinoma cell lines HT-29 and HT-29KM in rotating-wall vessels. IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY : JOURNAL OF THE TISSUE CULTURE ASSOCIATION 1992; 28A:47-60. [PMID: 1730571 DOI: 10.1007/bf02631079] [Citation(s) in RCA: 77] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
A new low shear stress microcarrier culture system has been developed at NASA's Johnson Space Center that permits three-dimensional tissue culture. Two established human colon adenocarcinoma cell lines, HT-29, an undifferentiated, and HT-29KM, a stable, moderately differentiated subline of HT-29, were grown in new tissue culture bioreactors called Rotating-Wall Vessels (RWVs). RWVs are used in conjunction with multicellular cocultivation to develop a unique in vitro tissue modeling system. Cells were cultivated on Cytodex-3 microcarrier beads, with and without mixed normal human colonic fibroblasts, which served as the mesenchymal layer. Culture of the tumor lines in the absence of fibroblasts produced spheroidlike growth and minimal differentiation. In contrast, when tumor lines were co-cultivated with normal colonic fibroblasts, initial growth was confined to the fibroblast population until the microcarriers were covered. The tumor cells then commenced proliferation at an accelerated rate, organizing themselves into three-dimensional tissue masses that achieved 1.0- to 1.5-cm diameters. The masses displayed glandular structures, apical and internal glandular microvilli, tight intercellular junctions, desmosomes, cellular polarity, sinusoid development, internalized mucin, and structural organization akin to normal colon crypt development. Differentiated samples were subjected to transmission and scanning electron microscopy and histologic analysis, revealing embryoniclike mesenchymal cells lining the areas around the growth matrices. Necrosis was minimal throughout the tissue masses. These data suggest that the RWV affords a new model for investigation and isolation of growth, regulatory, and structural processes within neoplastic and normal tissue.
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Affiliation(s)
- T J Goodwin
- Biomedical Operations and Research Branch, NASA Johnson Space Center, Houston, Texas
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Bensaadi N, Clemente F, Vaysse N. Modulation of enzymatic activities during spontaneous and induced differentiation in a human pancreatic adenocarcinoma cell line CAPAN-1. INTERNATIONAL JOURNAL OF PANCREATOLOGY : OFFICIAL JOURNAL OF THE INTERNATIONAL ASSOCIATION OF PANCREATOLOGY 1989; 4:391-406. [PMID: 2543714 DOI: 10.1007/bf02938475] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
Different enzymatic activities were studied in the human pancreatic cancer cell line CAPAN-1 in order to analyze their relation to differentiation. Alkaline phosphatase (Alk Ph), acid phosphatase, aminopeptidase, dipeptidyl peptidase IV, acid and neutral alpha-glucosidases, and acid beta-galactosidase were present. Especially alkaline phosphatase, which we have found to be of the placental type isoenzyme, is being highly expressed. Spontaneous cell differentiation at confluence as well as differentiating agents: sodium butyrate and DMSO, modulated the levels of three enzymes: Alk. Ph., aminopeptidase, and acid alpha-glucosidase. The exposure of the cells to the differentiating agents amplified the modulations occurring during the spontaneous differentiation. Aminopeptidase and acid alpha-glucosidase were found to be induced by differentiation. Alk Ph specific activity was significantly increased by the spontaneous and the butyrate-induced differentiations; whereas DMSO exerted an opposite effect, probably related to its biphasic action on cell proliferation.
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Affiliation(s)
- N Bensaadi
- Inserm U 151, CHU Rangueil, Toulouse, France
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