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Pausch TM, Bartel M, Cui J, Aubert O, Mitzscherling C, Liu X, Gesslein B, Schuisky P, Kommoss FKF, Bruckner T, Golriz M, Mehrabi A, Hackert T. SmartPAN: in vitro and in vivo proof-of-safety assessments for an intra-operative predictive indicator of postoperative pancreatic fistula. Basic Clin Pharmacol Toxicol 2022; 130:542-552. [PMID: 35040273 DOI: 10.1111/bcpt.13708] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2021] [Revised: 12/07/2021] [Accepted: 01/12/2022] [Indexed: 12/01/2022]
Abstract
Pancreatic surgery is complicated by untreated fluid leakage, but no tenable techniques exist to detect and close leakage sites during surgery. A novel hydrogel called SmartPAN has been developed to meet this need and is here assessed for safety before trials on human patients. Firstly, resazurin assays were used to test the cytotoxic effects of SmartPAN's active bromothymol blue (BTB) indicator and its solution of phosphate-buffered saline (PBS) on normal (HPDE: Human Pancreatic Duct Epithelial) or carcinomic (FAMPAC) human pancreatic cells. Cells incubated with BTB showed no significant reduction in cell viability below threshold safety levels. However, PBS had a mild cytotoxic effect on FAMPAC cells. Secondly, SmartPAN's pathological effects were evaluated in vivo by applying 4 mL SmartPAN to a porcine (Sus scrofa domesticus) model of pancreatic resection. There were no significant differences in macroscopic and microscopic pathologies between pigs treated with SmartPAN or saline. Thirdly, measurements using HPLC-MS/MS demonstrate that BTB does not cross into the bloodstream and was eliminated from the body within two days of surgery. Overall, SmartPAN appears safe in the short-term and ready for first-in-human trials because its components are either biocompatible or quickly neutralized by dilution and drainage.
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Affiliation(s)
- Thomas M Pausch
- Department of General, Visceral, and Transplantation Surgery, Heidelberg University Hospital, Germany
| | - Marc Bartel
- Institute of Legal and Traffic Medicine, Heidelberg University Hospital, Heidelberg, Germany
| | - Jiaqu Cui
- Department of General, Visceral, and Transplantation Surgery, Heidelberg University Hospital, Germany
| | - Ophelia Aubert
- Department of General, Visceral, and Transplantation Surgery, Heidelberg University Hospital, Germany
| | - Clara Mitzscherling
- Department of General, Visceral, and Transplantation Surgery, Heidelberg University Hospital, Germany
| | - Xinchun Liu
- Department of General, Visceral, and Transplantation Surgery, Heidelberg University Hospital, Germany
| | | | | | - Felix K F Kommoss
- Institute of Pathology, Heidelberg University Hospital, Heidelberg, Germany
| | - Thomas Bruckner
- Institute of Legal and Traffic Medicine, Heidelberg University Hospital, Heidelberg, Germany
| | - Mohammad Golriz
- Department of General, Visceral, and Transplantation Surgery, Heidelberg University Hospital, Germany
| | - Arianeb Mehrabi
- Department of General, Visceral, and Transplantation Surgery, Heidelberg University Hospital, Germany
| | - Thilo Hackert
- Department of General, Visceral, and Transplantation Surgery, Heidelberg University Hospital, Germany
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2
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Fredebohm J, Boettcher M, Eisen C, Gaida MM, Heller A, Keleg S, Tost J, Greulich-Bode KM, Hotz-Wagenblatt A, Lathrop M, Giese NA, Hoheisel JD. Establishment and characterization of a highly tumourigenic and cancer stem cell enriched pancreatic cancer cell line as a well defined model system. PLoS One 2012; 7:e48503. [PMID: 23152778 PMCID: PMC3495919 DOI: 10.1371/journal.pone.0048503] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2012] [Accepted: 09/26/2012] [Indexed: 12/12/2022] Open
Abstract
Standard cancer cell lines do not model the intratumoural heterogeneity situation sufficiently. Clonal selection leads to a homogeneous population of cells by genetic drift. Heterogeneity of tumour cells, however, is particularly critical for therapeutically relevant studies, since it is a prerequisite for acquiring drug resistance and reoccurrence of tumours. Here, we report the isolation of a highly tumourigenic primary pancreatic cancer cell line, called JoPaca-1 and its detailed characterization at multiple levels. Implantation of as few as 100 JoPaca-1 cells into immunodeficient mice gave rise to tumours that were histologically very similar to the primary tumour. The high heterogeneity of JoPaca-1 was reflected by diverse cell morphology and a substantial number of chromosomal aberrations. Comparative whole-genome sequencing of JoPaca-1 and BxPC-3 revealed mutations in genes frequently altered in pancreatic cancer. Exceptionally high expression of cancer stem cell markers and a high clonogenic potential in vitro and in vivo was observed. All of these attributes make this cell line an extremely valuable model to study the biology of and pharmaceutical effects on pancreatic cancer.
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MESH Headings
- AC133 Antigen
- Aldehyde Dehydrogenase 1 Family
- Alleles
- Animals
- Antigens, CD/genetics
- Antigens, CD/metabolism
- Antimetabolites, Antineoplastic/pharmacology
- Biomarkers, Tumor/genetics
- Biomarkers, Tumor/metabolism
- Carcinoma, Pancreatic Ductal/genetics
- Carcinoma, Pancreatic Ductal/metabolism
- Carcinoma, Pancreatic Ductal/pathology
- Cell Line, Tumor
- Cell Proliferation/drug effects
- Cell Transformation, Neoplastic/genetics
- Cell Transformation, Neoplastic/pathology
- Deoxycytidine/analogs & derivatives
- Deoxycytidine/pharmacology
- Disease Models, Animal
- Drug Resistance, Neoplasm/genetics
- GPI-Linked Proteins/genetics
- GPI-Linked Proteins/metabolism
- Genomic Instability
- Glycoproteins/genetics
- Glycoproteins/metabolism
- Humans
- Isoenzymes/genetics
- Isoenzymes/metabolism
- Keratins/genetics
- Keratins/metabolism
- Male
- Mesothelin
- Mice
- Middle Aged
- Mutation
- Neoplasm Metastasis
- Neoplastic Stem Cells/metabolism
- Pancreatic Neoplasms/genetics
- Pancreatic Neoplasms/metabolism
- Pancreatic Neoplasms/pathology
- Peptides/genetics
- Peptides/metabolism
- Polyploidy
- Retinal Dehydrogenase/genetics
- Retinal Dehydrogenase/metabolism
- Transplantation, Heterologous
- Tumor Microenvironment
- Gemcitabine
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Affiliation(s)
- Johannes Fredebohm
- Functional Genome Analysis, German Cancer Research Center, Heidelberg, Germany.
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Alhamdani MSS, Youns M, Buchholz M, Gress TM, Beckers MC, Maréchal D, Bauer A, Schröder C, Hoheisel JD. Immunoassay-based proteome profiling of 24 pancreatic cancer cell lines. J Proteomics 2012; 75:3747-59. [PMID: 22579748 DOI: 10.1016/j.jprot.2012.04.042] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2012] [Revised: 04/24/2012] [Accepted: 04/27/2012] [Indexed: 01/29/2023]
Abstract
Pancreatic ductal adenocarcinoma is one of the most deadly forms of cancers, with a mortality that is almost identical to incidence. The inability to predict, detect or diagnose the disease early and its resistance to all current treatment modalities but surgery are the prime challenges to changing the devastating prognosis. Also, relatively little is known about pancreatic carcinogenesis. In order to better understand relevant aspects of pathophysiology, differentiation, and transformation, we analysed the cellular proteomes of 24 pancreatic cancer cell lines and two controls using an antibody microarray that targets 741 cancer-related proteins. In this analysis, 72 distinct disease marker proteins were identified that had not been described before. Additionally, categorizing cancer cells in accordance to their original location (primary tumour, liver metastases, or ascites) was made possible. A comparison of the cells' degree of differentiation (well, moderately, or poorly differentiated) resulted in unique marker sets of high relevance. Last, 187 proteins were differentially expressed in primary versus metastatic cancer cells, of which the majority is functionally related to cellular movement.
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Affiliation(s)
- Mohamed Saiel Saeed Alhamdani
- Division of Functional Genome Analysis, Deutsches Krebsforschungszentrum (DKFZ), Im Neuenheimer Feld 580, Heidelberg, Germany.
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4
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Lister A, Nedjadi T, Kitteringham NR, Campbell F, Costello E, Lloyd B, Copple IM, Williams S, Owen A, Neoptolemos JP, Goldring CE, Park BK. Nrf2 is overexpressed in pancreatic cancer: implications for cell proliferation and therapy. Mol Cancer 2011; 10:37. [PMID: 21489257 PMCID: PMC3098205 DOI: 10.1186/1476-4598-10-37] [Citation(s) in RCA: 188] [Impact Index Per Article: 13.4] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2010] [Accepted: 04/13/2011] [Indexed: 12/22/2022] Open
Abstract
BACKGROUND Nrf2 is a key transcriptional regulator of a battery of genes that facilitate phase II/III drug metabolism and defence against oxidative stress. Nrf2 is largely regulated by Keap1, which directs Nrf2 for proteasomal degradation. The Nrf2/Keap1 system is dysregulated in lung, head and neck, and breast cancers and this affects cellular proliferation and response to therapy. Here, we have investigated the integrity of the Nrf2/Keap1 system in pancreatic cancer. RESULTS Keap1, Nrf2 and the Nrf2 target genes AKR1c1 and GCLC were detected in a panel of five pancreatic cancer cell lines. Mutation analysis of NRF2 exon 2 and KEAP1 exons 2-6 in these cell lines identified no mutations in NRF2 and only synonomous mutations in KEAP1. RNAi depletion of Nrf2 caused a decrease in the proliferation of Suit-2, MiaPaca-2 and FAMPAC cells and enhanced sensitivity to gemcitabine (Suit-2), 5-flurouracil (FAMPAC), cisplatin (Suit-2 and FAMPAC) and gamma radiation (Suit-2). The expression of Nrf2 and Keap1 was also analysed in pancreatic ductal adenocarcinomas (n = 66 and 57, respectively) and matching normal benign epithelium (n = 21 cases). Whilst no significant correlation was seen between the expression levels of Keap1 and Nrf2 in the tumors, interestingly, Nrf2 staining was significantly greater in the cytoplasm of tumors compared to benign ducts (P < 0.001). CONCLUSIONS Expression of Nrf2 is up-regulated in pancreatic cancer cell lines and ductal adenocarcinomas. This may reflect a greater intrinsic capacity of these cells to respond to stress signals and resist chemotherapeutic interventions. Nrf2 also appears to support proliferation in certain pancreatic adenocarinomas. Therefore, strategies to pharmacologically manipulate the levels and/or activity of Nrf2 may have the potential to reduce pancreatic tumor growth, and increase sensitivity to therapeutics.
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Affiliation(s)
- Adam Lister
- MRC Centre for Drug Safety Science, Department of Molecular and Clinical Pharmacology, Institute of Translational Medicine, University of Liverpool, UK
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Earl J, Yan L, Vitone LJ, Risk J, Kemp SJ, McFaul C, Neoptolemos JP, Greenhalf W, Kress R, Sina-Frey M, Hahn SA, Rieder H, Bartsch DK. Evaluation of the 4q32-34 locus in European familial pancreatic cancer. Cancer Epidemiol Biomarkers Prev 2007; 15:1948-55. [PMID: 17035404 DOI: 10.1158/1055-9965.epi-06-0376] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023] Open
Abstract
BACKGROUND Familial pancreatic cancer (FPC) describes a group of families where the inheritance of pancreatic cancer is consistent with an autosomal-dominant mode of inheritance. The 4q32-34 region has been previously identified as a potential locus for FPC in a large American family. METHODS The region was allelotyped in 231 individuals from 77 European families using nine microsatellite markers, and haplotyping was possible in 191 individuals from 41 families. Families were selected based on at least two affected first-degree relatives with no other cancer syndromes. RESULTS Linkage to most of the locus was excluded based on LOD scores less than -2.0. Eight families were excluded from linkage to 4q32-34 based on haplotypes not segregating with the disease compared with a predicted six to seven families. Two groups of families were identified, which seem to share common alleles within the minimal disease-associated region of 4q32-34, one group with an apparently earlier age of cancer death than the other pancreatic cancer families. Four genes were identified with potential tumor suppressor roles within the locus in regions that could not be excluded based on the LOD score. These were HMGB2, PPID, MORF4, and SPOCK3. DNA sequence analysis of exons of these genes in affected individuals and in pancreatic cancer cell lines did not reveal any mutations. CONCLUSION This locus is unlikely to harbor a FPC gene in the majority of our European families.
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Affiliation(s)
- Julie Earl
- Division of Surgery and Oncology, University of Liverpool, Liverpool, United Kingdom
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Eisold S, Linnebacher M, Ryschich E, Antolovic D, Hinz U, Klar E, Schmidt J. The effect of adenovirus expressing wild-type p53 on 5-fluorouracil chemosensitivity is related to p53 status in pancreatic cancer cell lines. World J Gastroenterol 2004; 10:3583-9. [PMID: 15534911 PMCID: PMC4611997 DOI: 10.3748/wjg.v10.i24.3583] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
AIM: There are conflicting data about p53 function on cellular sensitivity to the cytotoxic action of 5-fluorouracil (5-FU). Therefore the objective of this study was to determine the combined effects of adenovirus-mediated wild-type (wt) p53 gene transfer and 5-FU chemotherapy on pancreatic cancer cells with different p53 gene status.
METHODS: Human pancreatic cancer cell lines Capan-1p53mut, Capan-2p53wt, FAMPACp53mut, PANC1p53mut, and rat pancreatic cancer cell lines ASp53wt and DSL6Ap53null were used for in vitro studies. Following infection with different ratios of Ad-p53-particles (MOI) in combination with 5-FU, proliferation of tumor cells and apoptosis were quantified by cell proliferation assay (WST-1) and FACS (PI-staining). In addition, DSL6A syngeneic pancreatic tumor cells were inoculated subcutaneously in to Lewis rats for in vivo studies. Tumor size, apoptosis (TUNEL) and survival were determined.
RESULTS: Ad-p53 gene transfer combined with 5-FU significantly inhibited tumor cell proliferation and substantially enhanced apoptosis in all four cell lines with an alteration in the p53 gene compared to those two cell lines containing wt-p53. in vivo experiments showed the most effective tumor regression in animals treated with Ad-p53 plus 5-FU. Both in vitro and in vivo analyses revealed that a sublethal dose of Ad-p53 augmented the apoptotic response induced by 5-FU.
CONCLUSION: Our results suggest that Ad-p53 may synergistically enhance 5-FU-chemosensitivity most strikingly in pancreatic cancer cells lacking p53 function. These findings illustrate that the anticancer efficacy of this combination treatment is dependent on the p53 gene status of the target tumor cells.
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Affiliation(s)
- Sven Eisold
- Department of General Surgery, Thoracic and Vascular Surgery, University of Rostock, Schillingallee 35, D-18057 Rostock, Germany.
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