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1.
Dig Dis Sci ; 67(5): 1806-1821, 2022 05.
Article in English | MEDLINE | ID: mdl-33939146

ABSTRACT

BACKGROUND: Toll-like receptors (TLRs) are key players in innate immunity and modulation of TLR signaling has been demonstrated to profoundly affect proliferation and growth in different types of cancer. However, the role of TLRs in human intrahepatic cholangiocarcinoma (ICC) pathogenesis remains largely unexplored. AIMS: We set out to determine if TLRs play any role in ICCs which could potentially make them useful treatment targets. METHODS: Tissue microarrays containing samples from 9 human ICCs and normal livers were examined immunohistochemically for TLR4, TLR7, and TLR9 expression. Proliferation of human ICC cell line HuCCT1 was measured by MTS assay following treatment with CpG-ODN (TLR9 agonist), imiquimod (TLR7 agonist), chloroquine (TLR7 and TLR9 inhibitor) and IRS-954 (TLR7 and TLR9 antagonist). The in vivo effects of CQ and IRS-954 on tumor development were also examined in a NOD-SCID mouse xenograft model of human ICC. RESULTS: TLR4 was expressed in all normal human bile duct epithelium but absent in the majority (60%) of ICCs. TLR7 and TLR9 were expressed in 80% of human ICCs. However, TLR7 was absent in all cases of normal human bile duct epithelium and only one was TLR9 positive. HuCCT1 cell proliferation in vitro significantly increased following IMQ or CpG-ODN treatment (P < 0.03 and P < 0.002, respectively) but decreased with CQ (P < 0.02). In the mouse xenograft model there was significant reduction in size of tumors from CQ and IRS-954 treated mice compared to untreated controls. CONCLUSION: TLR7 and TLR9 should be further explored for their potential as actionable targets in the treatment of ICC.


Subject(s)
Bile Duct Neoplasms , Cholangiocarcinoma , Animals , Bile Duct Neoplasms/drug therapy , Bile Ducts, Intrahepatic/metabolism , Cell Proliferation , Cholangiocarcinoma/drug therapy , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Toll-Like Receptor 4 , Toll-Like Receptor 7/agonists , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 9/agonists , Toll-Like Receptor 9/genetics , Toll-Like Receptors/agonists
2.
Pol J Pathol ; 72(1): 39-47, 2021.
Article in English | MEDLINE | ID: mdl-34060286

ABSTRACT

Colorectal cancer (CRC) is the third most common cancer worldwide and is associated with a high level of mortality and morbidity. In this study we evaluate expression of p-p38 and p-MSK1 in CRC and determine whether there is an association between expression of these markers and any clinicopathologic parameters that could be of prognostic value. Expression of p-p38, p-MSK1 and ki-67 were examined by immunohistochemistry in 135 archival CRC cases and the findings were correlated with the patient clinicopathological data. P-p38 and p-MSK1 were expressed at high level in 58.5 % and 60.7% of CRC cases respectively. A statistically significant negative correlation was found between expression of p-p38 and Ki-67 (p < 0.001, r = -0.63) and between p-MSK1 and Ki-67 expression (p < 0.001, r = -0.61). The majority of CRC cases expressing high levels of p-p38 also expressed high levels of p-MSK1 and this correlation was highly significant (p < 0.001, r = 0.863). The high expression of p-p38 and p-MSK1 was also significantly associated with low Dukes and TNM stage. The elevated expression of p-38 and p-MSK1 in CRC was associated with a good prognosis and prolonged overall survival (p < 0.001, each). Our finding showed that activation of the p38-MSK1 axis determines a good outcome in CRC.


Subject(s)
Adenocarcinoma , Colorectal Neoplasms , Humans , Immunohistochemistry , Prognosis , Ribosomal Protein S6 Kinases, 90-kDa
3.
Liver Int ; 35(3): 1063-76, 2015 Mar.
Article in English | MEDLINE | ID: mdl-24990399

ABSTRACT

BACKGROUND & AIMS: Chronic liver disease is a predisposing factor for development of hepatocellular carcinoma (HCC). Toll-like receptors play a crucial role in immunity against microbial pathogens and recent evidence suggests that they may also be important in pathogenesis of chronic liver disease. The purpose of this study was to determine whether TLR7 and TLR9 are potential targets for prevention and progression of HCC. METHODS: Tissue microarrays containing liver samples from patients with cirrhosis, viral hepatitis and HCC were examined for expression of TLR7 and TLR9 and the data obtained was validated in liver specimens from the hospital archives. Proliferation of human HCC cell lines was studied following stimulation of TLR7 and TLR9 using agonists (imiquimod and CpG-ODN respectively) and inhibition with a specific antagonist (IRS-954) or chloroquine. The effect of these interventions was confirmed in a xenograft model and diethylnitrosamine (DEN)/nitrosomorpholine (NMOR)-induced model of HCC. RESULTS: TLR7 and TLR9 expression was up-regulated in human HCC tissue. Proliferation of HuH7 cells in vitro increased significantly in response to stimulation of TLR7. TLR7 and TLR9 inhibition using IRS-954 or chloroquine significantly reduced HuH7 cell proliferation in vitro and inhibited tumour growth in the mouse xenograft model. HCC development in the DEN/NMOR rat model was also significantly inhibited by chloroquine (P < 0.001). CONCLUSION: The data suggest that inhibiting TLR7 and TLR9 with IRS-954 or chloroquine could potentially be used as a novel therapeutic approach for preventing HCC development and/or progression in susceptible patients.


Subject(s)
Carcinoma, Hepatocellular/metabolism , Liver Neoplasms/metabolism , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 9/metabolism , Animals , Carcinoma, Hepatocellular/prevention & control , Case-Control Studies , Cell Proliferation/drug effects , Chloroquine/pharmacology , Chloroquine/therapeutic use , DNA/pharmacology , DNA/therapeutic use , Hep G2 Cells , Humans , Ki-67 Antigen/metabolism , Liver/metabolism , Liver Neoplasms/prevention & control , Mice, Inbred NOD , Mice, SCID , Molecular Targeted Therapy , NF-kappa B/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Random Allocation , Rats, Inbred F344 , Tissue Array Analysis , Toll-Like Receptor 7/agonists , Toll-Like Receptor 7/antagonists & inhibitors , Toll-Like Receptor 9/agonists , Toll-Like Receptor 9/antagonists & inhibitors , Vascular Endothelial Growth Factor A/metabolism , Xenograft Model Antitumor Assays
4.
Pathol Res Pract ; 216(8): 152980, 2020 Aug.
Article in English | MEDLINE | ID: mdl-32703481

ABSTRACT

AIMS: Unlike other Toll-like receptors (TLRs), the role of toll like receptor 2 (TLR-2) in the pathogenesis of chronic liver disease and hepatocellular carcinoma (HCC) is not well studied. We, therefore, set out to investigate the expression of TLR-2 in different chronic liver disease states along with other markers of cell death, cellular proliferation and tissue vascularisation METHODS AND RESULTS: Immunohistochemistry was performed on liver tissue microarrays comprising hepatitis, cirrhosis and HCC patient samples using antibodies against TLR-2, Ki-67, Caspase-3 and VEGF. This was done in order to characterise receptor expression and translocation, apoptosis, cell proliferation and vascularisation. Cytoplasmic TLR-2 expression was found to be weak in 5/8 normal liver cases, 10/19 hepatitis cases and 8/21 cirrhosis patients. Moderate to strong TLR-2 expression was observed in some cases of hepatitis and cirrhosis. Both, nuclear and cytoplasmic TLR-2 expression was present in HCC with weak intensity in 11/41 cases, and moderate to strong staining in 19/41 cases. Eleven HCC cases were TLR-2 negative. Surprisingly, both cytoplasmic and nuclear TLR-2 expression in HCC were found to significantly correlate with proliferative index (r = 0.24 and 0.37), Caspase-3 expression (r = 0.27 and 0.38) and vascularisation (r = 0.56 and 0.23). Further, nuclear TLR-2 localisation was predominant in HCC, whereas cytoplasmic expression was more prevalent in hepatitis and cirrhosis. Functionally, treatment of HUH7 HCC cells with a TLR-2 agonist induced the expression of cellular proliferation and vascularisation markers CD34 and VEGF. CONCLUSIONS: Our results demonstrate a positive correlation between the expression of TLR-2 and other markers of proliferation and vascularisation in HCC which suggests a possible role for TLR-2 in HCC pathogenesis.


Subject(s)
Biomarkers, Tumor/metabolism , Carcinoma, Hepatocellular/pathology , Liver Neoplasms/pathology , Toll-Like Receptor 2/metabolism , Adult , Carcinoma, Hepatocellular/metabolism , Caspase 3/metabolism , Cell Proliferation/physiology , Female , Humans , Liver Neoplasms/metabolism , Male , Middle Aged , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology
5.
Commun Biol ; 1: 175, 2018.
Article in English | MEDLINE | ID: mdl-30374465

ABSTRACT

Mechanical signaling involved in molecular interactions lies at the heart of materials science and biological systems, but the mechanisms involved are poorly understood. Here we use nanomechanical sensors and intact human cells to provide unique insights into the signaling pathways of connectivity networks, which deliver the ability to probe cells to produce biologically relevant, quantifiable and reproducible signals. We quantify the mechanical signals from malignant cancer cells, with 10 cells per ml in 1000-fold excess of non-neoplastic human epithelial cells. Moreover, we demonstrate that a direct link between cells and molecules creates a continuous connectivity which acts like a percolating network to propagate mechanical forces over both short and long length-scales. The findings provide mechanistic insights into how cancer cells interact with one another and with their microenvironments, enabling them to invade the surrounding tissues. Further, with this system it is possible to understand how cancer clusters are able to co-ordinate their migration through narrow blood capillaries.

6.
Nucleic Acids Res ; 33(19): 6393-404, 2005.
Article in English | MEDLINE | ID: mdl-16282588

ABSTRACT

Gene activation involves protein complexes with diverse enzymatic activities, some of which are involved in chromatin modification. We have shown previously that the base excision repair enzyme thymine DNA glycosylase (TDG) acts as a potent coactivator for estrogen receptor-alpha. To further understand how TDG acts in this context, we studied its interaction with known coactivators of nuclear receptors. We find that TDG interacts in vitro and in vivo with the p160 coactivator SRC1, with the interaction being mediated by a previously undescribed motif encoding four equally spaced tyrosine residues in TDG, each tyrosine being separated by three amino acids. This is found to interact with two motifs in SRC1 also containing tyrosine residues separated by three amino acids. Site-directed mutagenesis shows that the tyrosines encoded in these motifs are critical for the interaction. The related p160 protein TIF2 does not interact with TDG and has the altered sequence, F-X-X-X-Y, at the equivalent positions relative to SRC1. Substitution of the phenylalanines to tyrosines is sufficient to bring about interaction of TIF2 with TDG. These findings highlight a new protein-protein interaction motif based on Y-X-X-X-Y and provide new insight into the interaction of diverse proteins in coactivator complexes.


Subject(s)
Thymine DNA Glycosylase/chemistry , Thymine DNA Glycosylase/metabolism , Trans-Activators/metabolism , Transcription Factors/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , COS Cells , Chlorocebus aethiops , Histone Acetyltransferases , Humans , Molecular Sequence Data , Nuclear Receptor Coactivator 1 , Repetitive Sequences, Amino Acid , Trans-Activators/chemistry , Transcription Factors/chemistry , Tyrosine/analysis
7.
Oncogene ; 23(45): 7561-70, 2004 Sep 30.
Article in English | MEDLINE | ID: mdl-15334066

ABSTRACT

The androgen receptor (AR) is a member of the nuclear receptor superfamily of ligand-activated transcription factors and plays a key role in the development and progression of prostate cancer. Current therapies include the use of antiandrogens aimed at inhibiting the transcriptional activation of AR-regulated genes by AR. Here, we explore a strategy aimed at obtaining silencing of AR-regulated genes, based on the properties of the transcriptional repressor promyelocytic leukamia zinc-finger protein (PLZF). In order to do this, we have made a fusion protein between PLZF and AR, named PLZF-AR, and show that PLZF-AR is able to bring about silencing of genomically encoded AR-regulated genes and inhibit the androgen-regulated growth of LNCaP prostate cancer cells. Together, our results show that this strategy is able to bring about potent repression of AR-regulated responses and, therefore, could be of value in the development of new therapies for prostate cancer.


Subject(s)
Androgens/physiology , DNA-Binding Proteins/genetics , Gene Expression Regulation/physiology , Gene Silencing , Receptors, Androgen/genetics , Recombinant Fusion Proteins/physiology , Transcription Factors/genetics , Animals , Base Sequence , COS Cells , DNA Primers , Fluorescent Antibody Technique, Indirect , Genes, Reporter , Humans , Kruppel-Like Transcription Factors , Promyelocytic Leukemia Zinc Finger Protein , Recombinant Fusion Proteins/genetics
8.
Blood ; 99(2): 627-33, 2002 Jan 15.
Article in English | MEDLINE | ID: mdl-11781247

ABSTRACT

Angioimmunoblastic T-cell lymphoma (AITL) is a systemic disease involving lymph nodes, spleen, and bone marrow. Although the histologic features have been well described, the diagnosis is often challenging, as there are no specific phenotypic or molecular markers available. This study shows that the neoplastic cells of AITL can be identified by aberrant CD10 expression. Archival material from 30 cases of AITL, 10 cases of peripheral T-cell lymphoma unspecified (PTL), and 10 cases of reactive lymphoid hyperplasia were reviewed. Single and double immunostaining for CD3, CD4, CD8, CD20, CD21, CD10, BCL6, Ki67, and LMP-1 in situ hybridization for Epstein-Barr early region and polymerase chain reaction (PCR) for T-cell receptor gamma chain gene and immunoglobulin heavy chain gene were performed. Three overlapping histologic patterns with hyperplastic follicles, depleted follicles, or without follicles were identified in AITL. Of the 30 cases of AITL, 27 contained CD10(+) T cells. No CD10(+) T cells were present in the cases of PTL or reactive hyperplasia. PCR confirmed a monoclonal or oligoclonal T-cell population in 29 of 30 cases of AITL and a monoclonal B-cell population in 6 cases. Analysis of microdissected CD10(+) single cells showed that they belonged to the neoplastic clone. In conclusion CD10 is a phenotypic marker that specifically identifies the tumor cells in 90% of AITL, including the early cases. The presence of these cells distinguishes AITL from other PTLs. This finding provides an objective criterion for accurate and early diagnosis of AITL.


Subject(s)
Biomarkers, Tumor/analysis , Immunoblastic Lymphadenopathy/pathology , Lymphoma, T-Cell/pathology , Neoplasm Proteins/analysis , Neoplastic Stem Cells/metabolism , Neprilysin/analysis , T-Lymphocytes/metabolism , Adaptor Proteins, Signal Transducing , Adult , Aged , Aged, 80 and over , Antigens, CD/analysis , Biopsy , Carrier Proteins/analysis , Cell Separation/methods , Clone Cells/metabolism , Clone Cells/pathology , Cytoskeletal Proteins , DNA, Viral/analysis , DNA-Binding Proteins/analysis , Epstein-Barr Virus Infections/metabolism , Epstein-Barr Virus Infections/pathology , Female , Gene Rearrangement, B-Lymphocyte, Heavy Chain , Gene Rearrangement, T-Lymphocyte , Herpesvirus 4, Human/isolation & purification , Humans , Immunoblastic Lymphadenopathy/metabolism , Immunoblastic Lymphadenopathy/virology , Immunoenzyme Techniques , In Situ Hybridization , Intracellular Signaling Peptides and Proteins , Ki-67 Antigen/analysis , LIM Domain Proteins , Lymph Nodes/chemistry , Lymph Nodes/pathology , Lymphoma, T-Cell/metabolism , Lymphoma, T-Cell, Peripheral/classification , Lymphoma, T-Cell, Peripheral/metabolism , Lymphoma, T-Cell, Peripheral/pathology , Male , Micromanipulation , Middle Aged , Polymerase Chain Reaction , Proto-Oncogene Proteins/analysis , Proto-Oncogene Proteins c-bcl-6 , Pseudolymphoma/metabolism , Pseudolymphoma/pathology , Transcription Factors/analysis
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