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Zhang L, Lin Y, Hu L, Wang Y, Hu C, Shangguan X, Tang S, Chen J, Hu P, Chen ZS, Ke ZF, Chen Z. Transient intracellular expression of PD-L1 and VEGFR2 bispecific nanobody in cancer cells inspires long-term T cell activation and infiltration to combat tumor and inhibit cancer metastasis. Mol Cancer 2025; 24:119. [PMID: 40253320 PMCID: PMC12008900 DOI: 10.1186/s12943-025-02253-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2024] [Accepted: 01/30/2025] [Indexed: 04/21/2025] Open
Abstract
BACKGROUND PD-L1, an immune checkpoint inhibitor, and VEGFR2, essential for cancer metastasis, play pivotal roles in tumorigenesis. However, their miniature bispecific intracellular nanobodies for combining check-point blockade and anti-metastasis anticancer therapy remain underexplored. METHODS The intrabodies were developed using gene cloning technology. Specificity of the intrabodies was testified using Western blot, co-immunoprecipitation (co-IP) analysis, antibody competitive binding assay, flow cytometry analysis, etc. Checkpoint blockade was demonstrated using antibody-antigen competitive binding assay. Cancer cell migration was determined using scratch assay. Combined anti-cancer therapeutic efficacy of FAP1V2 was determined in vivo of mice models. The PD-1hi immune cells, TCR βhi and CD25hi T-cells were analyzed by flow cytometry, and cancer cell metastasis was performed using immune-fluorescence analysis on lung and liver tissues. Transcriptome analysis was performed to explore signaling pathways associated with the enhanced anticancer efficiency. RESULTS Bispecific intrabody FAP1V2 fused with antibody VH regions, was successfully developed and verified with its ability to target and block human and mouse PD-L1 and VEGFR2, inhibiting cancer cell binding to PD-1 and reducing their migratory capacity. Compared to the other treatment, two-rounds of transient FAP1V2 expression in LLC cells in experimental mice models achieved remarkable tumor inhibition, which brought about complete immune inhibition on growth of secondary-round of LLC tumor in 1/6 of the tested mice, inspired long-term activation of TCR βhi T cells and increased their infiltration to tumors, inhibited the emergence of PD-1hi immune cells, indicating prevented T cell depletion. The elevated CD25 expression also supported the success in enhancing immune response reported by elevated T cell activity in spleen. Transcriptome analysis identified critical intracellular pathways regulated by the concurrent blockade of PD-L1 and VEGFR2. CONCLUSION PD-L1 and VEGFR2- bispecific VH intracellular nanobody was highly biocompatible and showed the potential for combined anti-cancer therapy through long-term immune activation mediated by PD-L1/PD-1 checkpoint blockade and anti-metastasis mediated by VEGFR2 blockade.
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Affiliation(s)
- Lei Zhang
- State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, 350108, China.
- University of Chinese Academy of Sciences, Beijing, 100049, China.
| | - Yunfeng Lin
- State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, 350108, China
- College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
| | - Li Hu
- State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, 350108, China
- College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
| | - Yanan Wang
- State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, 350108, China
- College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
| | - Chaohua Hu
- National Engineering Research Center for Sugarcane, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
| | - Xinyi Shangguan
- State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, 350108, China
- College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
| | - Shuzhi Tang
- State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, 350108, China
| | - Jincan Chen
- State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, 350108, China
| | - Ping Hu
- State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, 350108, China
| | - Zhe-Sheng Chen
- College of Pharmacy and Health Sciences, St. John's University, Queens, NY, 11439, USA
| | - Zun-Fu Ke
- Department of Pathology, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, Guangdong Province, P.R. China.
| | - Zhuo Chen
- State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, 350108, China.
- University of Chinese Academy of Sciences, Beijing, 100049, China.
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Arsalan HM, Mumtaz H, Lagana AS. Biomarkers of endometriosis. Adv Clin Chem 2025; 126:73-120. [PMID: 40185537 DOI: 10.1016/bs.acc.2025.01.004] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/07/2025]
Abstract
Endometriosis represents a diverse disease characterized by three distinct phenotypes: superficial peritoneal lesions, ovarian endometriomas, and deep infiltrating endometriosis. The most widely accepted pathophysiological hypothesis for endometriosis is rooted in retrograde menstruation, a phenomenon observed in most patients. Endometriosis is closely linked to infertility, but having endometriosis does not necessarily imply infertility. The disease can impact fertility through various mechanisms affecting the pelvic cavity, ovaries, and the uterus itself. MicroRNAs (miRNAs) indeed represent a fascinating and essential component of the regulatory machinery within cells. Discovered in the early 1990s, miRNAs have since been identified as critical players in gene expression control. Unfortunately, ovarian endometrioma is a common gynecologic disorder for which specific serum markers are currently lacking. Some have examined urocortin for its ability to differentiate endometriomas from other benign ovarian cysts. Another potential marker, Cancer Antigen 125 (CA-125) is a well-established indicator for epithelial cell ovarian cancer and its levels can be elevated in conditions such as endometriosis. CA-125 is derived from coelomic epithelia, including the endometrium, fallopian tube, ovary, and peritoneum. In this review we examine the pathophysiologic basis for endometriosis and highlight potential markers to more fully characterize the underlying biochemical processes linked to this multifaceted disease state.
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Affiliation(s)
- Hafiz Muhammad Arsalan
- Faculty of General Medicine, Altamimi International Medical University, Bishkek, Kyrgyzstan.
| | - Hina Mumtaz
- Department of Biochemistry, University of Central Punjab, Lahore, Pakistan.
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Guo X, Li Y, Chen X, Sun B, Guo X. Urocortin-1 promotes colorectal cancer cell migration and proliferation and inhibits apoptosis via inhibition of the p53 signaling pathway. J Cancer Res Clin Oncol 2024; 150:163. [PMID: 38546882 PMCID: PMC10978644 DOI: 10.1007/s00432-024-05693-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2023] [Accepted: 03/08/2024] [Indexed: 04/01/2024]
Abstract
PURPOSE To investigate the effect of urocortin-1 (UCN-1) on growth, migration, and apoptosis in colorectal cancer (CRC) in vivo and vitro and the mechanism by which UCN-1 modulates CRC cells in vitro. METHODS The correlation between UCN-1 and CRC was evaluated using The Cancer Genome Atlas (TCGA) database and a tissue microarray. The expression of UCN-1 in CRC cells was assessed using quantitative real-time polymerase chain reaction (RT-qPCR) and western blotting. In vitro, the influence of UCN-1 on the proliferation, apoptosis, and migration of HT-29, HCT-116, and RKO cells was explored using the celigo cell counting assay or cell counting kit-8 (CCK8), flow cytometry, and wound healing or Transwell assays, respectively. In vivo, the effect of UCN-1 on CRC growth and progression was evaluated in nude mice. The downstream pathway underlying UCN-1-mediated regulation of CRC was determined using the phospho-kinase profiler array in RKO cells. Lentiviruses were used to knockdown or upregulate UCN-1 expression in cells. RESULTS Both the TCGA and tissue microarray results showed that UCN-1 was strongly expressed in the tissues of patients with CRC. Furthermore, the tissue microarray results showed that the expression of UCN-1 was higher in male than in female patients, and high expression of UCN-1 was associated with higher risk of lymphatic metastasis and later pathological stage. UCN-1 knockdown caused a reduction in CRC cell proliferation, migration, and colony formation, as well as an increase in apoptosis. In xenograft experiments, tumors generated from RKO cells with UCN-1 knockdown exhibited reduced volumes and weights. A reduction in the expression of Ki-67 in xenograft tumors indicated that UCN-1 knockdown curbed tumor growth. The human phospho-kinase array showed that the p53 signaling pathway participated in UCN-1-mediated CRC development. The suppression in migration and proliferation caused by UCN-1 knockdown was reversed by inhibitors of p53 signal pathway, while the increase in cell apoptosis was suppressed. On the other hand, overexpression of UCN-1 promoted proliferation and migration and inhibited apoptosis in CRC cells. Overexpression of p53 reversed the effect of UCN-1 overexpression on CRC development. CONCLUSION UCN-1 promotes migration and proliferation and inhibits apoptosis via inhibition of the p53 signaling pathway.
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Affiliation(s)
- Xiaolan Guo
- Department of Gastroenterology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China
| | - Ya Li
- Department of Gastroenterology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China
| | - Xiangyu Chen
- Department of Gastroenterology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China
| | - Binghua Sun
- Department of Gastroenterology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China
| | - Xiaolan Guo
- Department of Gastroenterology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China.
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Dimas A, Goussia A, Papoudou-Bai A, Politi A, Paschopoulos M, Navrozoglou I, Makrigiannakis A, Vrekoussis T. The expression of corticotropin-releasing hormone family peptides in premalignant and malignant vulvar lesions. Clin Transl Oncol 2024; 26:260-268. [PMID: 37382757 PMCID: PMC10761541 DOI: 10.1007/s12094-023-03249-8] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2023] [Accepted: 06/07/2023] [Indexed: 06/30/2023]
Abstract
OBJECTIVES To examine the relation of corticotropin-releasing hormone (CRH) family peptides with inflammatory processes and oncogenesis, emphasizing in vulvar inflammatory, premalignant and malignant lesions, as well as to investigate the possibility of lesion cells immunoescaping, utilizing FAS/FAS-L complex. METHODS Immunohistochemical expression of CRH, urocortin (UCN), FasL and their receptors CRHR1, CRHR2 and Fas was studied in vulvar tissue sections obtained from patients with histologically confirmed diagnosis of lichen, vulvar intraepithelial neoplasia (VIN) and vulvar squamous cell carcinoma (VSCC). The patient cohort was selected from a tertiary teaching Hospital in Greece, between 2005 and 2015. For each of the disease categories, immunohistochemical staining was evaluated and the results were statistically compared. RESULTS A progressive increase of the cytoplasmic immunohistochemical expression of CRH and UCN, from precancerous lesions to VSCC was observed. A similar increase was detected for Fas and FasL expression. Nuclear localization of UCN was demonstrated in both premalignant and VSCC lesions, with staining being significantly intensified in carcinomas, particularly in the less differentiated tumor areas or in the areas at invasive tumor front. CONCLUSIONS Stress response system and CRH family peptides seem to have a role in inflammation maintenance and progression of vulvar premalignant lesions to malignancy. It seems that stress peptides may locally modulate the stroma through Fas/FasL upregulation, possibly contributing to vulvar cancer development.
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Affiliation(s)
| | - Anna Goussia
- Department of Pathology, Faculty of Medicine, School of Health Sciences, University of Ioannina, 45110, Ioannina, Greece
- Department of Pathology, University Hospital of Ioannina, 45110, Ioannina, Greece
- Department of Pathology, German Oncology Center, Limassol, Cyprus
| | - Alexandra Papoudou-Bai
- Department of Pathology, Faculty of Medicine, School of Health Sciences, University of Ioannina, 45110, Ioannina, Greece
- Department of Pathology, University Hospital of Ioannina, 45110, Ioannina, Greece
| | - Anastasia Politi
- Department of Dermatology, Venereology, Andreas Syggros Hospital, National and Kapodistrian University of Athens, 16121, Athens, Greece
| | - Minas Paschopoulos
- Department of Obstetrics and Gynecology, Faculty of Medicine, School of Health Sciences, University of Ioannina, 45110, Ioannina, Greece
| | - Iordanis Navrozoglou
- Department of Obstetrics and Gynecology, Faculty of Medicine, School of Health Sciences, University of Ioannina, 45110, Ioannina, Greece
| | - Antonis Makrigiannakis
- Department of Obstetrics and Gynecology, School of Health Science, University of Crete, 71500, Iraklio, Greece
| | - Thomas Vrekoussis
- Department of Obstetrics and Gynecology, School of Health Science, University of Crete, 71500, Iraklio, Greece
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Abramiuk M, Frankowska K, Kułak K, Tarkowski R, Mertowska P, Mertowski S, Grywalska E. Possible Correlation between Urocortin 1 (Ucn1) and Immune Parameters in Patients with Endometriosis. Int J Mol Sci 2023; 24:ijms24097787. [PMID: 37175494 PMCID: PMC10178394 DOI: 10.3390/ijms24097787] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2023] [Revised: 04/20/2023] [Accepted: 04/22/2023] [Indexed: 05/15/2023] Open
Abstract
The etiology of endometriosis (EMS) has not been clearly elucidated yet, and that is probably the reason why its diagnostic process is frequently long-lasting and inefficient. Nowadays, the non-invasive diagnostic methods of EMS are still being sought. Our study aimed to assess the serum and peritoneal fluid levels of urocortin 1 (Ucn1) in patients with EMS and healthy women. Moreover, considering the immune background of the disease, the association between Ucn1 and several immune parameters was studied in both groups. We found that the serum Ucn1 level was significantly upregulated in women with EMS compared to healthy patients. Moreover, higher serum Ucn1 levels tended to correspond with more advanced stages of the disease (p = 0.031). Receiver operating characteristic (ROC) analysis revealed that based on serum Ucn1 levels, it is possible to distinguish deep infiltrating endometriosis (DIE) from among other EMS types. Together, these results indicate Ucn1 as a possible promising biomarker of EMS: however, not in isolation, but rather to enhance the effectiveness of other diagnostic methods.
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Affiliation(s)
- Monika Abramiuk
- Independent Laboratory of Minimally Invasive Gynecology and Gynecological Endocrinology, Department of Oncological Gynaecology and Gynaecology, Medical University of Lublin, Staszica 16 St., 20-081 Lublin, Poland
| | - Karolina Frankowska
- 1st Chair and Department of Oncological Gynaecology and Gynaecology, Student Scientific Association, Medical University of Lublin, Staszica 16 St., 20-081 Lublin, Poland
| | - Krzysztof Kułak
- 1st Chair and Department of Oncological Gynaecology and Gynaecology, Medical University of Lublin, Staszica 16 St., 20-081 Lublin, Poland
| | - Rafał Tarkowski
- 1st Chair and Department of Oncological Gynaecology and Gynaecology, Medical University of Lublin, Staszica 16 St., 20-081 Lublin, Poland
| | - Paulina Mertowska
- Department of Experimental Immunology, Medical University of Lublin, Chodźki 4a St., 20-093 Lublin, Poland
| | - Sebastian Mertowski
- Department of Experimental Immunology, Medical University of Lublin, Chodźki 4a St., 20-093 Lublin, Poland
| | - Ewelina Grywalska
- Department of Experimental Immunology, Medical University of Lublin, Chodźki 4a St., 20-093 Lublin, Poland
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Balogh B, Vecsernyés M, Veres-Székely A, Berta G, Stayer-Harci A, Tarjányi O, Sétáló G. Urocortin stimulates ERK1/2 phosphorylation and proliferation but reduces ATP production of MCF7 breast cancer cells. Mol Cell Endocrinol 2022; 547:111610. [PMID: 35219718 DOI: 10.1016/j.mce.2022.111610] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/23/2021] [Revised: 12/15/2021] [Accepted: 02/22/2022] [Indexed: 11/29/2022]
Abstract
Urocortins are members of the stress-related corticotropin-releasing factor family. Small amounts of them are present in the circulation and they are produced locally in various tissues of higher vertebrates. Aside from regulating circulation, or food uptake they also influence, via auto- and paracrine mechanisms, cell proliferation. In the present study we investigated in MCF7 human breast cancer cells the effect of urocortin onto mitogenic signaling via ERK1/2. Our results revealed that already 10 nM urocortin could stimulate the phosphorylation of these kinases and cell proliferation of MCF7 cells while ATP production was reduced when kept in the presence of the peptide up to two days. We examined the expression and contribution of the specific receptors of urocortin to the activation of ERK1/2 and to cell proliferation, the intracellular distribution of phosphorylated ERK1/2, and the involvement of additional proteins like PKA, PKB/Akt, MEK, p53, Rb and E2F-1 behind the observed phenomena.
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Affiliation(s)
- Bálint Balogh
- Department of Medical Biology and Central Electron Microscope Laboratory, University of Pécs, Medical School, Pécs, H-7643, Pécs, Szigeti út 12, Hungary.
| | - Mónika Vecsernyés
- Department of Medical Biology and Central Electron Microscope Laboratory, University of Pécs, Medical School, Pécs, H-7643, Pécs, Szigeti út 12, Hungary; Signal Transduction Research Group, János Szentágothai Research Centre, Pécs, H-7624, Pécs, Ifjúság útja 20, Hungary.
| | - Apor Veres-Székely
- 1st Department of Pediatrics, Semmelweis University, Budapest, H-1083, Budapest, 53-54. Bókay Street, Hungary; ELKH-SE Pediatrics and Nephrology Research Group, Budapest, Hungary.
| | - Gergely Berta
- Department of Medical Biology and Central Electron Microscope Laboratory, University of Pécs, Medical School, Pécs, H-7643, Pécs, Szigeti út 12, Hungary; Signal Transduction Research Group, János Szentágothai Research Centre, Pécs, H-7624, Pécs, Ifjúság útja 20, Hungary.
| | - Alexandra Stayer-Harci
- Department of Medical Biology and Central Electron Microscope Laboratory, University of Pécs, Medical School, Pécs, H-7643, Pécs, Szigeti út 12, Hungary; Signal Transduction Research Group, János Szentágothai Research Centre, Pécs, H-7624, Pécs, Ifjúság útja 20, Hungary.
| | - Oktávia Tarjányi
- Department of Medical Biology and Central Electron Microscope Laboratory, University of Pécs, Medical School, Pécs, H-7643, Pécs, Szigeti út 12, Hungary; Signal Transduction Research Group, János Szentágothai Research Centre, Pécs, H-7624, Pécs, Ifjúság útja 20, Hungary.
| | - György Sétáló
- Department of Medical Biology and Central Electron Microscope Laboratory, University of Pécs, Medical School, Pécs, H-7643, Pécs, Szigeti út 12, Hungary; Signal Transduction Research Group, János Szentágothai Research Centre, Pécs, H-7624, Pécs, Ifjúság útja 20, Hungary.
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Ramírez-Guerrero AA, González-Villaseñor CO, Leal-Ugarte E, Gutiérrez-Angulo M, Ramírez-Flores M, Delgado-Enciso I, Macías-Gómez NM. Association between genetic variant rs2267716 of CRHR2 gene with colorectal cancer. J Investig Med 2021; 70:947-952. [PMID: 34969780 DOI: 10.1136/jim-2021-002047] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 11/24/2021] [Indexed: 12/12/2022]
Abstract
Colorectal cancer (CRC) is the third most common cancer and one of the main causes of death around the world. Multiple lines of evidence have suggested the role of the corticotropin-releasing hormone (CRH) family in CRC induction, including the low expression of corticotropin-releasing hormone receptor 2 (CRHR2), which is an angiogenesis inhibitor and inflammatory modulator. Previous research suggests that CRHR2 expression in colonic intestinal cells can regulate migration, proliferation and apoptosis through the modulation of several pathways. The aim of this study was to analyze the association of the rs10250835, rs2267716 and rs2267717 variants of CRHR2 gene with CRC in the Mexican population in order to consider its predictive value in CRC. This cross-sectional study included a group of 187 unrelated patients with sporadic CRC and a control group of 191 healthy blood donors. DNA extraction from peripheral blood was carried out using the Miller method. Identification of the rs10250835 variant was performed using PCR-restriction fragment length polymorphism (RFLP) and the rs2267716 and rs2267717 variants using TaqMan allelic discrimination assay. The minor allele homozygous CC of the rs2267716 variant of CRHR2 showed significant difference between CRC and control group (p=0.025), as well as the GCA haplotype (p=0.007), corresponding to the rs10250835, rs2267716 and rs2267717 variants, respectively. Our results suggest that the rs2267716 variant and GCA haplotype of CRHR2 represent a risk factor for CRC development in Mexican patients.
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Affiliation(s)
| | | | - Evelia Leal-Ugarte
- Facultad de Medicina, Universidad Autónoma de Tamaulipas Facultad de Ingeniería y Ciencias, Matamoros, Tamaulipas, Mexico
| | - Melva Gutiérrez-Angulo
- Ciencias de la Salud, Universidad de Guadalajara-Centro Universitario Los Altos, Tepatitlan de Morelos, Mexico
| | | | | | - Nelly Margarita Macías-Gómez
- Laboratorio de Genética Humana, Universidad de Guadalajara Campus Centro Universitario del Sur, Ciudad Guzmán, Mexico
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Ducarouge B, Pelissier-Rota M, Powell R, Buisson A, Bonaz B, Jacquier-Sarlin M. Involvement of CRF2 signaling in enterocyte differentiation. World J Gastroenterol 2017; 23:5127-5145. [PMID: 28811708 PMCID: PMC5537180 DOI: 10.3748/wjg.v23.i28.5127] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/30/2017] [Revised: 05/06/2017] [Accepted: 07/12/2017] [Indexed: 02/06/2023] Open
Abstract
AIM To determine the role of corticotropin releasing factor receptor (CRF2) in epithelial permeability and enterocyte cell differentiation.
METHODS For this purpose, we used rat Sprague Dawley and various colon carcinoma cell lines (SW620, HCT8R, HT-29 and Caco-2 cell lines). Expression of CRF2 protein was analyzed by fluorescent immunolabeling in normal rat colon and then by western blot in dissociated colonic epithelial cells and in the lysates of colon carcinoma cell lines or during the early differentiation of HT-29 cells (ten first days). To assess the impact of CRF2 signaling on colonic cell differentiation, HT-29 and Caco-2 cells were exposed to Urocortin 3 recombinant proteins (Ucn3, 100 nmol/L). In some experiments, cells were pre-exposed to the astressin 2b (A2b) a CRF2 antagonist in order to inhibit the action of Ucn3. Intestinal cell differentiation was first analyzed by functional assays: the trans-cellular permeability and the para-cellular permeability were determined by Dextran-FITC intake and measure of the transepithelial electrical resistance respectively. Morphological modifications associated to epithelial dysfunction were analyzed by confocal microscopy after fluorescent labeling of actin (phaloidin-TRITC) and intercellular adhesion proteins such as E-cadherin, p120ctn, occludin and ZO-1. The establishment of mature adherens junctions (AJ) was monitored by following the distribution of AJ proteins in lipid raft fractions, after separation of cell lysates on sucrose gradients. Finally, the mRNA and the protein expression levels of characteristic markers of intestinal epithelial cell (IEC) differentiation such as the transcriptional factor krüppel-like factor 4 (KLF4) or the dipeptidyl peptidase IV (DPPIV) were performed by RT-PCR and western blot respectively. The specific activities of DPPIV and alkaline phosphatase (AP) enzymes were determined by a colorimetric method.
RESULTS CRF2 protein is preferentially expressed in undifferentiated epithelial cells from the crypts of colon and in human colon carcinoma cell lines. Furthermore, CRF2 expression is down regulated according to the kinetic of HT-29 cell differentiation. By performing functional assays, we found that Ucn3-induced CRF2 signaling alters both para- and trans-cellular permeability of differentiated HT-29 and Caco-2 cells. These effects are partly mediated by Ucn3-induced morphological changes associated with the disruption of mature AJ in HT-29 cells and tight junctions (TJ) in Caco-2 cells. Ucn3-mediated activation of CRF2 decreases mRNA and protein expression levels of KLF4 a transcription factor involved in IEC differentiation. This signaling is correlated to a down-regulation of key IEC markers such as DPPIV and AP, at both transcriptional and post-transcriptional levels.
CONCLUSION Our findings suggest that CRF2 signaling could modulate IEC differentiation. These mechanisms could be relevant to the stress induced epithelial alterations found in inflammatory bowel diseases.
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Owens GL, Lawrence KM, Jackson TR, Crosbie EJ, Sayan BS, Kitchener HC, Townsend PA. Urocortin suppresses endometrial cancer cell migration via CRFR2 and its system components are differentially modulated by estrogen. Cancer Med 2017; 6:408-415. [PMID: 28109061 PMCID: PMC5313640 DOI: 10.1002/cam4.967] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2016] [Revised: 09/20/2016] [Accepted: 09/23/2016] [Indexed: 11/24/2022] Open
Abstract
Urocortin (UCN1) peptide shares structural and functional homology with corticotropin‐releasing factor (CRF). UCN1 is significantly reduced in endometrial adenocarcinoma compared to healthy controls. However, there are no data which evaluate the effects of UCN1 in the endometrium, or how it is modulated. We used proliferation and transwell assays to determine the effect of UCN1 on the proliferation and migration of Ishikawa and HEC1A cells. We also determined the expression levels of UCN1 and its receptors produced by estrogen receptor agonists, and the effect of UCN1 on estrogen receptor expression, using quantitative polymerase chain reaction. UCN1 suppressed migration of endometrial cancer cells in vitro. This effect appears to be specific to CRF receptor 2 (CRFR2), as selective antagonism of CRFR2 but not CRFR1 completely eliminated suppression of migration. Activation of ERA reduced UCN1 expression, but only had a small effect on the expression of CRFR1. However, expression of CRFR2 was more notably reduced at both the mRNA and protein levels by activation of ERB. UCN1 in turn reduced both ERA and ERB expression, as assessed by real‐time quantitative PCR. We demonstrate that UCN1 significantly suppresses the migration of endometrial cancer cells but has no effect on their proliferation. Thus, loss of UCN1 in endometrial cancer may promote invasion and metastatic spread. There is a complex relationship between the UCN1 system and estrogen receptors, which may provide insights into endometrial carcinogenesis, a disease known to be driven by estrogen excess.
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Affiliation(s)
- Gemma L Owens
- Division of Molecular & Clinical Cancer Sciences, School of Medical Sciences, Faculty of Biology, Medicine & Health, Manchester Cancer Research Centre, University of Manchester, Wilmslow Road, Manchester, M20 4QL, United Kingdom
| | - Kevin M Lawrence
- Division of Molecular & Clinical Cancer Sciences, School of Medical Sciences, Faculty of Biology, Medicine & Health, Manchester Cancer Research Centre, University of Manchester, Wilmslow Road, Manchester, M20 4QL, United Kingdom
| | - Tom R Jackson
- Division of Molecular & Clinical Cancer Sciences, School of Medical Sciences, Faculty of Biology, Medicine & Health, Manchester Cancer Research Centre, University of Manchester, Wilmslow Road, Manchester, M20 4QL, United Kingdom
| | - Emma J Crosbie
- Division of Molecular & Clinical Cancer Sciences, School of Medical Sciences, Faculty of Biology, Medicine & Health, St Mary's Hospital, University of Manchester, Oxford Road, Manchester, M13 9WL, United Kingdom
| | - Berna S Sayan
- Division of Molecular & Clinical Cancer Sciences, School of Medical Sciences, Faculty of Biology, Medicine & Health, Manchester Cancer Research Centre, University of Manchester, Wilmslow Road, Manchester, M20 4QL, United Kingdom
| | - Henry C Kitchener
- Division of Molecular & Clinical Cancer Sciences, School of Medical Sciences, Faculty of Biology, Medicine & Health, St Mary's Hospital, University of Manchester, Oxford Road, Manchester, M13 9WL, United Kingdom
| | - Paul A Townsend
- Division of Molecular & Clinical Cancer Sciences, School of Medical Sciences, Faculty of Biology, Medicine & Health, Manchester Cancer Research Centre, University of Manchester, Wilmslow Road, Manchester, M20 4QL, United Kingdom
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Urocortinergic system in the testes of normal and cryptorchid dogs. Ann Anat 2016; 207:91-6. [DOI: 10.1016/j.aanat.2016.05.004] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2015] [Revised: 04/29/2016] [Accepted: 05/04/2016] [Indexed: 01/17/2023]
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11
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Vitale SG, Laganà AS, Rapisarda AMC, Scarale MG, Corrado F, Cignini P, Butticè S, Rossetti D. Role of urocortin in pregnancy: An update and future perspectives. World J Clin Cases 2016; 4:165-171. [PMID: 27458591 PMCID: PMC4945586 DOI: 10.12998/wjcc.v4.i7.165] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/12/2016] [Revised: 04/09/2016] [Accepted: 05/27/2016] [Indexed: 02/05/2023] Open
Abstract
The activities of corticotropin-releasing factor (CRF) and related peptides are mediated a number of receptors with seven transmembrane domains that are coupled to the Gs and Gq proteins. These receptors are known as CRF-Rs. In vitro studies have evidenced that urocortin (UCN) and CRF provoke an increase in the contractility of the uterus which is induced by endometrial prostaglandin F2a. Furthermore, through trophoblasts, it stimulates the secretion of adrenocorticotropic hormone (ACTH) and prostaglandin PGE2 and has a vasodilatory effect on the placenta. While it is well known that the placenta produces considerable quantities of CRF, several studies have, however, excluded that the placenta can generate significant quantities of UCN. In the short term, the human fetal adrenal gland produces more cortisol and dehydroepiandrosterone sulfate. The gestational tissues express UCN3 and UCN2 mRNA in cytotrophoblast and syncytiotrophoblast cells, while UCN2 is only to be found in the maternal and fetal vessels and amniotic cells. Nevertheless, gestational tissues express UCN2 and UCN3 differentially and do not stimulate placental ACTH secretion. In term pregnancies, maternal plasma levels of CRF and UCN are lower than at the beginning of pregnancy and are correlated to labor onset. Conversely, they do not decrease in post-term pregnancies. This evidence would seem to indicate that the fine-regulated expression of these neuropeptides is important in determining the duration of human gestation. In this scenario, low concentrations of UCN in the amniotic fluid at mid-term may be considered a sign of predisposition to preterm birth.
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12
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Immunolocalization of corticotropin-releasing hormone (CRH) and its receptors (CRHR1 and CRHR2) in human endometrial carcinoma: CRHR1 as a potent prognostic factor. Int J Gynecol Cancer 2015; 24:1549-57. [PMID: 25254562 PMCID: PMC4215916 DOI: 10.1097/igc.0000000000000269] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
Supplemental digital content is available in the text. Objective Corticotropin-releasing hormone (CRH), a major regulator of the stress response, regulates various biological functions through its interaction with CRH receptors 1 (CRHR1) and 2 (CRHR2). CRH, CRHR1, and CRHR2 have recently been reported in several types of carcinoma, but the significance of these proteins has remained largely unknown in human endometrial carcinoma. Materials and Methods A total of 87 endometrial carcinoma specimens were obtained from Japanese female patients who underwent surgical treatment, fixed in 10% formalin, and embedded in paraffin wax. Immunohistochemistry for CRH, CRHR1, and CRHR2 was performed, and clinical data were obtained from the medical records. Results Immunopositivity of CRH, CRHR1, and CRHR2 in the specimens was 26%, 15%, and 10%, respectively. Univariate analysis revealed that immunohistochemical CRH status was positively associated with CRHR1 and CRHR2 status and that CRHR1 status was significantly associated with the risk of recurrence and poorer clinical outcome, whereas CRHR2 status was marginally associated with better prognosis for overall survival. Multivariate analysis demonstrated CRHR1 status as an independent prognostic factor for both disease-free and overall survival. Conclusions These results suggest that intratumoral CRH-CRHR1 signaling plays an important role in the progression of endometrial carcinoma and that CRHR1 is a potent prognostic factor in patients with this disease.
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Tsai WC, Hueng DY, Lin CK. Nuclear overexpression of urocortin discriminates primary brain tumors from reactive gliosis. APMIS 2015; 123:465-72. [PMID: 25904177 DOI: 10.1111/apm.12374] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2014] [Accepted: 01/13/2015] [Indexed: 11/26/2022]
Abstract
The role of urocortin (UCN) is still ambiguous in human cancers. We tested the hypothesis that using UCN expression discriminates reactive gliosis from primary brain tumors (PBTs). Immunohistochemical analysis of UCN was performed in six reactive gliosis and 99 PBTs. The immunostain scores of UCN were calculated as the degree of intensity multiplied by the percentage of expressed tumor cells. Nuclear staining of UCN revealed weak intensity and small portion of positively stained cells in reactive gliosis. However, comparing with non-neoplastic tissues, higher immunostain scores of UCN were identified in each WHO grade of astrocytomas and meningiomas. Finally, neither WHO grade nor overall survival rate did not significantly correlate with UCN expression in astrocytomas and meningiomas. Our findings demonstrate for the first time that the application of UCN might be a novel biomarker for not only discriminating reactive gliosis from PBTs, but also deciding where the clear surgical margin was.
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Affiliation(s)
- Wen-Chiuan Tsai
- Department of Pathology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
| | - Dueng-Yuan Hueng
- Department of Neurological Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
| | - Chih-Kung Lin
- Department of Pathology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
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Pelissier-Rota MA, Chartier NT, Jacquier-Sarlin MR. Dynamic Regulation of Adherens Junctions: Implication in Cell Differentiation and Tumor Development. INTERCELLULAR COMMUNICATION IN CANCER 2015:53-149. [DOI: 10.1007/978-94-017-7380-5_4] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/03/2025]
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Ikeda K, Fujioka K, Tachibana T, Kim SU, Tojo K, Manome Y. Secretion of urocortin I by human glioblastoma cell lines, possibly via the constitutive pathway. Peptides 2015; 63:63-70. [PMID: 25239507 DOI: 10.1016/j.peptides.2014.09.007] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/03/2014] [Revised: 09/08/2014] [Accepted: 09/08/2014] [Indexed: 11/24/2022]
Abstract
Corticotropin-releasing factor (CRF) and its family of peptides, i.e., urocortins (UCNs), play a critical role in systemic and peripheral stress-response systems and are widely expressed not only in normal tissues but also in various types of cancer cells. Given limited understanding of the mechanism of UCN I secretion, we investigated the UCN I secretory pathway in human neural stem cells (HNSCs) and in two glioblastoma cell lines, e.g., A172 and U-138 MG. Immunoreactivities for CRF receptors were detected in A172 glioblastoma cells, but not in HNSCs or U-138 glioblastoma cells, while UCN I immunoreactivity was detected in A172 and U-138 MG glioblastoma cell lines by both light field and electron microscopy. Interestingly, electron microscopy revealed UCN I immunoreactivtiy in vesicle-like structures in the plasma membrane of the glioblastoma cells. Tracking of a hybrid fluorescent protein containing a UCN I signal peptide expressed in A172 human glioblastoma cells revealed that fluorescence in secretory granules could be decreased by cycloheximide (100μg/ml), indicating that the forward transport of secretory granules containing fluorescent protein was not altered by the inhibition of protein synthesis by cycloheximide. Retrograde transport and the fusion of fluorescent granules in A172 human glioblastoma cells was induced by brefeldin A (10μg/ml), indicating that UCN I secretory granules may be transported via the constitutive pathway. Based on these results, it appears that UCN I is secreted from human glioblastoma cells by exocytosis through constitutive secretory granules, indicating that transcription of UCN I mRNA may be correlated to secretion of UCN I protein.
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Affiliation(s)
- Keiichi Ikeda
- Division of Molecular Cell Biology, Core Research Facilities for Basic Science, Research Center for Medical Sciences, The Jikei University School of Medicine, Tokyo 105-8461, Japan.
| | - Kouki Fujioka
- Division of Molecular Cell Biology, Core Research Facilities for Basic Science, Research Center for Medical Sciences, The Jikei University School of Medicine, Tokyo 105-8461, Japan
| | - Toshiaki Tachibana
- Division of Molecular Cell Biology, Core Research Facilities for Basic Science, Research Center for Medical Sciences, The Jikei University School of Medicine, Tokyo 105-8461, Japan
| | - Seung U Kim
- Medical Research Institute, Chung-Ang University College of Medicine, Seoul, Republic of Korea; Division of Neurology, Department of Medicine, University of British Columbia, Vancouver, BC, Canada V6T 2B5
| | - Katsuyoshi Tojo
- Division of Diabetes and Endocrinology, Department of Internal Medicine, The Jikei University School of Medicine, Tokyo 105-8461, Japan
| | - Yoshinobu Manome
- Division of Molecular Cell Biology, Core Research Facilities for Basic Science, Research Center for Medical Sciences, The Jikei University School of Medicine, Tokyo 105-8461, Japan
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Song H, Park H, Park G, Kim YS, Lee HK, Jin DH, Kang HS, Cho DH, Hur D. Corticotropin-releasing factor induces immune escape of cervical cancer cells by downregulation of NKG2D. Oncol Rep 2014; 32:425-30. [PMID: 24841552 DOI: 10.3892/or.2014.3191] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2014] [Accepted: 04/23/2014] [Indexed: 11/05/2022] Open
Abstract
Corticotropin-releasing factor (CRF), a coordinator of the body's responses to stress, is found in various cancer tissues and cell lines. However, the exact abilities of CRF to manipulate natural killer (NK) cells during immune response have not been studied. NKG2D is an activating receptor that is expressed on most NK and CD8+ T cells. MHC class I-related chain A (MICA) and UL16-binding protein (ULBP) 1, 2 and 3 are well-known ligands for NKG2D. In the present study, we reported our findings regarding the role of CRF in cervical cancer cell survival. Human cervical cancer cell line, HeLa cells, had significantly higher intracellular expression of UL16-binding protein 2 (ULBP2) following CRF treatment but had only slightly increased surface expression of ULBP2. Notably, MMPi (pan-metalloproteases inhibitor) blocked the release of ULBP2 molecules from the surface of HeLa cells. Furthermore, incubating NK cells with culture supernatants from CRF-treated HeLa cells, which contained soluble NKG2D ligand, reduced NK cell activity by decreasing surface expression of NKG2D. Collectively, downregulation of NKG2D by CRF-induced soluble NKG2D ligand provides a potential mechanism by which cervical cancer cells escape NKG2D-mediated attack under stress conditions.
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Affiliation(s)
- Hyunkeun Song
- Department of Anatomy and Research Center for Tumor Immunology, Inje University College of Medicine, Busan 614-735, Republic of Korea
| | - Hyunjin Park
- Department of Anatomy and Research Center for Tumor Immunology, Inje University College of Medicine, Busan 614-735, Republic of Korea
| | - Gabin Park
- Department of Anatomy and Research Center for Tumor Immunology, Inje University College of Medicine, Busan 614-735, Republic of Korea
| | - Yeong Seok Kim
- Department of Anatomy and Research Center for Tumor Immunology, Inje University College of Medicine, Busan 614-735, Republic of Korea
| | - Hyun-Kyung Lee
- Department of Internal Medicine, Inje University Busan Paik Hospital, Busan 614-735, Republic of Korea
| | - Dong-Hoon Jin
- Institute for Innovative Cancer Research, College of Medicine, University of Ulsan, Asan Medical Center, Seoul 138-736, Republic of Korea
| | - Hyung-Sik Kang
- School of Biological Sciences and Technology, Chonnam National University, Buk-gu, Gwangju 500-757, Republic of Korea
| | - Dae-Ho Cho
- Department of Life Science, Sookmyung Women's University, Yongsan-ku, Seoul 140-742, Republic of Korea
| | - Daeyoung Hur
- Department of Anatomy and Research Center for Tumor Immunology, Inje University College of Medicine, Busan 614-735, Republic of Korea
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Urocortin affects migration of hepatic cancer cell lines via differential regulation of cPLA2 and iPLA2. Cell Signal 2014; 26:1125-34. [PMID: 24518041 DOI: 10.1016/j.cellsig.2014.02.002] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2014] [Accepted: 02/01/2014] [Indexed: 12/14/2022]
Abstract
Urocortin (UCN) is a member of corticotrophin-releasing factor (CRF) family, which has been reported to play a role in many biological processes, including inflammation and cancer development. Growing evidence shows that PLA2 (phospholipase A2) enzymes also participate in inflammation and tumor development. The primary aim of the present study was to identify a novel signaling pathway of CRF receptor activation leading to migration of two kinds of hepatoma carcinoma cell lines, HepG2 and SMMC-7721, linking the stimulation of PLA2 expression by UCN to UCN-induced tumor cell migration. Pharmacological inhibitors and genetic approaches (such as stable transfection and siRNAs) were used in this study. Unlike HepG2 cells which express both CRF receptors themselves, SMMC-7721 cells which hardly express these two CRF receptors needed stable transfection with CRFR1 or CRFR2 to observe the effect of UCN. Two types of PLA2 enzymes, cPLA2 and iPLA2, were found to be regulated by UCN. Our data showed that UCN raised cPLA2 expression but lowered iPLA2 expression. Moreover, UCN was found to act on the certain region of iPLA2 promoter to reduce its transcription. UCN promoted tumor cell migration by up-regulating cPLA2 expression via CRFR1 whereas it suppressed tumor cell migration by down-regulating iPLA2 expression via CRFR2. These results indicate the dual roles for UCN in the hepatoma carcinoma cell migration, which involve the regulation of both cPLA2and iPLA2.
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Cheng MF, Tsai WC, Hsia KT, Yang YS, Jin JS. Expression of urocortin in pancreatic ductal adenocarcinoma and pancreatic intraepithelial neoplasia. APMIS 2013; 122:147-54. [PMID: 23755913 DOI: 10.1111/apm.12117] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2012] [Accepted: 04/02/2013] [Indexed: 01/09/2023]
Abstract
Urocortin (UCN) is a 40-amino acid neuropeptide that regulates angiogenesis and inhibits cell proliferation. Our aim was to examine the relationship of UCN expression to the clinicopathological parameters of pancreatic ductal adenocarcinoma (PDAC) and histological grade of pancreatic intraepithelial neoplasia (PanIN). Tissue microarray was used to analyze UCN protein expression in 89 surgical specimens including 21 PanIN, 3 PDAC arising from PanIN, and 65 PDAC without PanIN. UCN immunoscores ranging from 0 to 12 were obtained by multiplying intensity (scored on a 3-point scale) by the percentage of stained cells (scored on a 4-point scale). Strong expression of UCN was detected in 5 specimens of non-neoplastic pancreatic ductal epithelia. UCN immunoscore was significantly higher in PanIN-1 than in PanIN-2 and PanIN-3 (p = 0.038) and significantly higher in well-differentiated PDAC or early American Joint Committee on Cancer (AJCC) stage PDAC than in poorly differentiated or advanced stage PDAC (p = 0.025, p = 0.018). Higher expression of UCN correlates with PDAC tumor grade and AJCC pathologic stage as well as PanIN grade. Immunohistochemical assessment of UCN may help clinicians predict tumor recurrence rate and help pathologists make a proper diagnosis.
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Affiliation(s)
- Ming-Fang Cheng
- Division of Histological and Clinical Pathology, Hualian Army Forces General Hospital, Hualien, Taiwan; Department of Pathology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan; Institute of Oral Biology, School of Dentistry, National Yang-Ming University, Taipei, Taiwan
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Tezval H, Jurk S, Atschekzei F, Becker JU, Jahn O, Serth J, Kuczyk MA. Urocortin and corticotropin-releasing factor receptor 2 in human renal cell carcinoma: disruption of an endogenous inhibitor of angiogenesis and proliferation. World J Urol 2011; 27:825-30. [PMID: 19437022 PMCID: PMC2780655 DOI: 10.1007/s00345-009-0417-x] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2009] [Accepted: 04/24/2009] [Indexed: 02/04/2023] Open
Abstract
Purpose Urocortin (Ucn) exerts its actions through activation of two corticotropin-releasing factor receptors (CRFRs), CRFR1 and CRFR2. Involvement of Ucn/CRFR2 system in pathophysiological conditions such as the regulation of angiogenesis and inhibition of proliferation has been already reported. Suppression of neovascularization through reduction of vascular endothelial growth factor and inhibition of tumor cell cycling is modulated mainly through activation of CRFR2. To find out a possible involvement of Ucn/CRFR2 in kidney tumor, we examined the expression of Ucn and CRFR2 in normal and tumoral kidney specimens. Methods We applied reverse transcriptase PCR (n = 14), immunofluorescence (IF) on tissue microarrays (n = 25) and confocal microscopy to examine the mRNA expression and peptide/protein localization of Ucn and CRFR2 in normal kidney versus clear cell renal cell carcinoma, respectively. Results Ucn and CRFR2 mRNAs are expressed in normal and tumor specimens. In normal tissue, IF showed a cytoplasmic staining of Ucn mainly in proximal tubules, whereas a diffuse nuclear staining with diverse intensity was observed in tumoral tissues. CRFR2 was detected in proximal tubules and vasculature of normal specimens. Intriguingly, an almost complete loss of CRFR2 was observed in epithelial cells and microvessels within tumor tissues. Conclusions Here, and for the first time, we show the expression of Ucn and CRFR2 in human kidney and renal cell carcinoma. We propose that the nuclear translocation of Ucn along with the loss of CRFR2 in epithelial cells and microvasculature of tumoral specimens may be involved in the pathobiology of renal cell carcinoma.
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Affiliation(s)
- Hossein Tezval
- Department of Urology and Urological Oncology, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
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Kaprara A, Pazaitou-Panayiotou K, Chemonidou MC, Constantinidis TC, Lambropoulou M, Koffa M, Kiziridou A, Kakolyris S, Kortsaris A, Chatzaki E. Distinct distribution of corticotropin releasing factor receptors in human breast cancer. Neuropeptides 2010; 44:355-61. [PMID: 20630588 DOI: 10.1016/j.npep.2010.06.005] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/30/2010] [Revised: 06/15/2010] [Accepted: 06/17/2010] [Indexed: 11/17/2022]
Abstract
The hypothalamic neuropeptide corticotropin releasing factor (CRF) has been found in several types of human cancer, where its biological role is not clarified. In experimental models of breast cancer CRF has been shown to exert anti-proliferative and other actions. Aim of the present study was to describe the expression of the two types of CRF receptors CRF(1) and CRF(2) in human breast tumors. Receptor expression was studied in breast biopsies from patients diagnosed for primary breast adenocarcinoma, obtained from the tumor and the adjacent benign tissue. Gene expression levels were evaluated by real-time PCR following reverse transcription of total RNA extracts. CRF(1) transcripts were found in 23.1% of benign and in 23.1% of malignant biopsies. CRF(2(a)) was found in 22.2% of benign and 36.0% of malignant biopsies. Transcript levels of both receptors did not differ significantly between cancer and benign biopsies from the same tumor. No correlation was found between CRF receptor expression and patient histo/clinicopathological characteristics. Histological mapping using immunohistochemistry revealed positive CRF(1) immunostaining in the cancerous implants and breast ducts, whereas CRF(2) immunoreactivity was localized mainly in the perineural invasions. In conclusion, both CRF receptors were found in breast cancer and the respective benign adjacent tissue. The two CRF receptor proteins presented distinct distribution and subcellular localization, pointing into differing biological roles. CRF receptors could serve as targets of endogenous ligands expressed in the tumor microenvironment, regulating cancer growth.
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Affiliation(s)
- A Kaprara
- Anticancer Hospital 'Theagenio', Simeonidi 2, 54639 Thessaloniki, Greece
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Kaprara A, Pazaitou-Panayiotou K, Kortsaris A, Chatzaki E. The corticotropin releasing factor system in cancer: expression and pathophysiological implications. Cell Mol Life Sci 2010; 67:1293-306. [PMID: 20143250 PMCID: PMC11115652 DOI: 10.1007/s00018-010-0265-2] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2009] [Revised: 12/30/2009] [Accepted: 01/08/2010] [Indexed: 12/15/2022]
Abstract
Malignant tumors express multiple factors that have some role in the regulating networks supporting their ectopic growth. Recently, increased interest has been developing in the expression and biological role of the neuropeptides and receptors of the corticotropin releasing factor (CRF) system, the principal neuroendocrine mediator of the stress response, especially in the light of several R&D programs for small molecule antagonists that could present some anticancer therapeutic benefit. In the present article, we review the literature suggesting that the CRF system could be involved in the regulation of human cancer development. Potential implication in growth, metastasis, angiogenesis, or immune parameters via activation of locally expressed receptors could be clinically exploited by presenting targets of new therapeutic approaches.
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Affiliation(s)
| | | | - Alexandros Kortsaris
- Laboratory of Biochemistry, Democritus University of Thrace, Alexandroupolis, Thrace Greece
| | - Ekaterini Chatzaki
- Department of Pharmacology, Faculty of Medicine, Democritus University of Thrace, Dragana, 681 00 Alexandroupolis, Greece
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Wang J, Jin L, Chen J, Li S. Activation of corticotropin-releasing factor receptor 2 inhibits the growth of human small cell lung carcinoma cells. Cancer Invest 2010; 28:146-55. [PMID: 19968503 DOI: 10.3109/07357900903179617] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
Previously, we found the activation of corticotropin-releasing factor receptor 2 (CRFR2) could inhibit tumor growth via an anti-angiogenic pathway, implying CRFR2 may be a tumor therapeutic target. Here, CRFR2 expression in human neuroendocrine small cell lung carcinoma (SCLC) tissues and cell lines NCI-H446 and NCI-H1688 were detected. Meanwhile, UCNs could directly inhibit the proliferation and promote the apoptosis of SCLC cells via CRFR2. It was also shown that the activation of CRFR2 could inhibit p38 and Akt phosphorylation to suppress the secretion of VEGF in SCLC cells. These observations implied CRFR2 might be a therapeutic target in human SCLC.
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Affiliation(s)
- Juejin Wang
- Department of Pharmacology, Nanjing Medical University, Nanjing, China
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Gu X, Qi P, Zhou F, Ji Q, Wang H, Dou T, Zhao Y, Gao C. An intronic polymorphism in the corticotropin-releasing hormone receptor 2 gene increases susceptibility to HBV-related hepatocellular carcinoma in Chinese population. Hum Genet 2009; 127:75-81. [PMID: 19813023 DOI: 10.1007/s00439-009-0750-6] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2009] [Accepted: 09/17/2009] [Indexed: 01/26/2023]
Abstract
Corticotropin-releasing hormone receptor 2 (CRHR2) plays a role in both the central nervous system (CNS) and the peripheral nervous system. CRHR2 together with its ligands, urocortins (Ucns) and corticotropin-releasing hormone (CRH), functions as a mediator of inflammatory response and inhibitor of angiogenesis. Recently, it has been reported to be expressed in many human cancers. An association between rs2267716 polymorphism in the CRHR2 gene and susceptibility to hepatocellular carcinoma (HCC) was found in patients with chronic hepatitis C virus (HCV) infection. In the present study we analyzed, using a polymerase chain reaction-ligation detection reaction (PCR-LDR), the rs2267716 polymorphism in 364 hepatitis B virus (HBV)-related HCC patients, 196 non-HCC patients with HBV infection, and 404 healthy controls. The aim was to detect the possible association of this single-nucleotide polymorphism (SNP) with susceptibility to HBV-related HCC. Significant differences of rs2267716 allele were detected between HBV-related HCC patients and healthy controls (OR = 1.55, 95% CI 1.13-2.15, P = 0.007) or non-HCC patients with HBV infection (OR = 1.61, 95% CI 1.13-2.31, P = 0.009). These results suggest that the rs2267716 polymorphism in the CRHR2 gene might influence the risk of developing HCC in patients with HBV infection in Chinese population.
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Affiliation(s)
- Xing Gu
- Department of Laboratory Medicine, Eastern Hepatobiliary Hospital, Second Military Medical University, 200438 Shanghai, People's Republic of China
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Miceli F, Ranelletti FO, Martinelli E, Petrillo M, Scambia G, Navarra P, Ferrandina G. Expression and subcellular localization of CRH and its receptors in human endometrial cancer. Mol Cell Endocrinol 2009; 305:6-11. [PMID: 19433256 DOI: 10.1016/j.mce.2009.02.013] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/06/2008] [Revised: 01/07/2009] [Accepted: 02/19/2009] [Indexed: 12/14/2022]
Abstract
CRH and its receptors are expressed in human normal endometrial cells, where they are associated to anti-proliferative progesterone-like activity. We aimed to investigate CRH, CRH-R1 and CRH-R2 expression and intracellular localization in human endometrial cancers and their relationships with tumor biological parameters. Surgical specimens were obtained from 51 untreated endometrial cancer patients and immunohistochemistry for CRH, CRH receptors, ER, PR and Ki-67 was performed. We found a diffuse cytoplasmic staining in 100%, 92 % and 60.7 % of tumor specimens for CRH, CRH-R1 and CRH-R2, respectively. At variance with tumor tissues, the surrounding normal endometrial glands exhibit a typical paranuclear/apical pattern for CRH and stained for CRH-R2 at the nuclear level, whereas CRH-R1 staining was similar to that observed in tumor area. Positive correlations were found between CRH-R1 and PR expression, as well as between CRH-R2 cytoplasmic pattern and more advanced FIGO stage disease, respectively.
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Affiliation(s)
- Fiorella Miceli
- Institute of Pharmacology, Catholic University School of Medicine, Largo Francesco Vito 1, 00168 Rome, Italy
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Tezval H, Merseburger AS, Serth J, Herrmann TW, Becker JU, Jahn O, Kuczyk MA. Differential Expression of Urocortin in Human Testicular Germ Cells in Course of Spermatogenesis: Role for Urocortin in Male Fertility? Urology 2009; 73:901-5. [DOI: 10.1016/j.urology.2008.12.029] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2008] [Revised: 11/27/2008] [Accepted: 12/09/2008] [Indexed: 01/20/2023]
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The role of urocortin in gynecological and obstetrical conditions. Arch Gynecol Obstet 2008; 279:613-9. [DOI: 10.1007/s00404-008-0782-5] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2008] [Accepted: 08/21/2008] [Indexed: 11/30/2022]
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Wang J, Xu Y, Xu Y, Zhu H, Zhang R, Zhang G, Li S. Urocortin's inhibition of tumor growth and angiogenesis in hepatocellular carcinoma via corticotrophin-releasing factor receptor 2. Cancer Invest 2008; 26:359-68. [PMID: 18443956 DOI: 10.1080/07357900701788106] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/22/2022]
Abstract
Urocortin (UCN) functions via corticotrophin-releasing factor receptors (CRFRs), CRFR1 & 2. CRFR2 is reported to be a tonic suppressor of vascularization, implying its role in tumor angiogenesis. Here, it was found that UCN inhibited the growth of hepatocellular carcinoma (HCC) and reduced tumor microvessel density in nude mice. Hepatoma cells didn't express CRFRs whereas vessels expressed CRFRs, mainly CRFR2. In vitro three-dimensional culture assay showed UCN inhibited angiogenesis, this effect was abolished by CRFR2 antagonist, anti-sauvagine-30, demonstrating involvement of CRFR2. Furthermore, UCN inhibited the proliferation and promoted the apoptosis of endothelial cells and down-regulated VEGF expression in vivo via CRFR2.
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Affiliation(s)
- Juejin Wang
- Department of Pharmacology, Nanjing Medical University, Nanjing, China
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Florio P, Bruni L, De Falco C, Filardi G, Torricelli M, Reis FM, Galleri L, Voltolini C, Bocchi C, De Leo V, Petraglia F. Evaluation of Endometrial Urocortin Secretion for Prediction of Pregnancy after Intrauterine Insemination. Clin Chem 2008; 54:350-5. [DOI: 10.1373/clinchem.2007.094987] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
Abstract
Background: Urocortin is a neuropeptide produced by the human endometrium and has biological effects putatively important for promoting blastocyst implantation. We measured urocortin concentrations in samples of endometrial wash fluid collected from women with unexplained infertility who underwent intrauterine insemination (IUI).
Methods: Patients 28–42 years of age (n = 71) were consecutively enrolled after a complete clinical evaluation. Endometrial wash fluid was retrieved before IUI, at the time of ultrasound evaluation of endometrial thickness. Urocortin concentrations were assayed with a specific ELISA.
Results: After IUI, 28 patients (39%) became pregnant. Urocortin concentrations were significantly higher in women who became pregnant than in those who did not (0.38 μg/L vs 0.13 μg/L, P <0.0001). At a cutoff of 0.321 μg/L, urocortin results were positive in 61% [95% confidence interval (CI), 41%–78%] of women who had successful implantation and negative in 98% (95% CI, 88%–99.6%) of those who did not. The pregnancy rate for women with urocortin concentrations >0.32 μg/L was 94%, which differed significantly (P <0.05) from the overall pregnancy rate of 39% in the study population.
Conclusions: Urocortin is measurable in endometrial wash fluid, and its concentrations before IUI are higher in women who subsequently achieve pregnancy. These data suggest that the probability of having a successful pregnancy-producing IUI may be better estimated by measuring urocortin in endometrial wash fluid.
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Affiliation(s)
| | - Luca Bruni
- Department of Pediatrics, Obstetrics and Reproductive Medicine, Section of Obstetrics and Gynecology, University of Siena, Siena, Italy
| | - Carmen De Falco
- Department of Pediatrics, Obstetrics and Reproductive Medicine, Section of Obstetrics and Gynecology, University of Siena, Siena, Italy
| | - Gilda Filardi
- Department of Pediatrics, Obstetrics and Reproductive Medicine, Section of Obstetrics and Gynecology, University of Siena, Siena, Italy
| | - Michela Torricelli
- Department of Pediatrics, Obstetrics and Reproductive Medicine, Section of Obstetrics and Gynecology, University of Siena, Siena, Italy
| | - Fernando M Reis
- Department of Pediatrics, Obstetrics and Reproductive Medicine, Section of Obstetrics and Gynecology, University of Siena, Siena, Italy
| | - Letizia Galleri
- Department of Pediatrics, Obstetrics and Reproductive Medicine, Section of Obstetrics and Gynecology, University of Siena, Siena, Italy
| | - Chiara Voltolini
- Department of Pediatrics, Obstetrics and Reproductive Medicine, Section of Obstetrics and Gynecology, University of Siena, Siena, Italy
| | - Caterina Bocchi
- Department of Pediatrics, Obstetrics and Reproductive Medicine, Section of Obstetrics and Gynecology, University of Siena, Siena, Italy
| | - Vincenzo De Leo
- Department of Pediatrics, Obstetrics and Reproductive Medicine, Section of Obstetrics and Gynecology, University of Siena, Siena, Italy
| | - Felice Petraglia
- Department of Pediatrics, Obstetrics and Reproductive Medicine, Section of Obstetrics and Gynecology, University of Siena, Siena, Italy
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Florio P, Reis FM, Torres PB, Calonaci F, Toti P, Bocchi C, Linton EA, Petraglia F. Plasma urocortin levels in the diagnosis of ovarian endometriosis. Obstet Gynecol 2007; 110:594-600. [PMID: 17766605 DOI: 10.1097/01.aog.0000278572.86019.ae] [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: 12/18/2022]
Abstract
OBJECTIVE Urocortin is a neuropeptide, member of the corticotropin-releasing hormone family, that is produced by the human endometrium. Ovarian endometrioma is a prevalent gynecologic disorder still lacking specific serum markers. In the present study we measured systemic levels of urocortin to assess the diagnostic performance of its determination in distinguishing endometriomas from other benign ovarian cysts. METHODS Plasma urocortin was measured by radioimmunoassay in women with ovarian endometrioma (n=40) and in women with benign, nonendometriotic ovarian cysts (n=40). The diagnostic accuracy of urocortin measurement was evaluated by receiver operating characteristic curve and compared with the standard marker, CA 125. To support the local origin of the peptide, we also evaluated its localization in endometriomas by immunohistochemistry and its concentrations in cyst fluid and peritoneal fluid of 12 women with endometrioma. RESULTS Plasma urocortin levels were twice as high in women with endometrioma (median 49 pg/mL, interquartile range 41-63 pg/mL) than in the control group (19 [15-23] pg/mL, P<.001) and significantly higher in the cystic content of endometriomas than in the peritoneal fluid and plasma (P<.05). The peptide was immunolocalized in endometrioma glands and stromal capillary vessels. Elevated plasma urocortin levels detected 88% of the cases of endometrioma with 90% specificity, whereas CA 125 detected only 65% of the cases with the same specificity. CONCLUSION Plasma urocortin is increased in women with endometriomas, and its measurement may be useful for the differential diagnosis of endometrioma compared with other benign ovarian cysts. LEVEL OF EVIDENCE II.
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Affiliation(s)
- Pasquale Florio
- Department of Pediatrics, Obstetrics and Reproductive Medicine, Section of Obstetrics and Gynecology, University of Siena, Siena, Italy.
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Wang J, Li S. Corticotropin-releasing factor family and its receptors: tumor therapeutic targets? Biochem Biophys Res Commun 2007; 362:785-8. [PMID: 17822675 DOI: 10.1016/j.bbrc.2007.08.014] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2007] [Accepted: 08/03/2007] [Indexed: 12/19/2022]
Abstract
Urocortin (UCN) and corticotropin-releasing factor (CRF) are members of CRF family. Though CRF is mainly distributed in central nervous system (CNS), UCN has been reported to play biologically diverse roles in several systems such as cardiovascular, respiratory, digestive, reproductive, stress, immunologic system, etc. UCN and CRF bind to two known receptors, CRFR1 and CRFR2, to function. Both CRF receptors are distributed in CNS and periphery tissues, and their expression in cancer tissues has been reported. Now there are many documents indicating UCN/CRF play an important role in the regulation of carcinogenesis. There is also evidence indicating UCN/CRF have anticancer effects via CRFRs. This paper will review the effects of CRF family in cancers.
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Affiliation(s)
- Juejin Wang
- Department of Pharmacology, Nanjing Medical University, Nanjing 210029, PR China
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