cyclin-d1 and Cholangiocarcinoma

Intraductal papillary neoplasm of the bile duct (IPNB) is characterized by exophytic proliferation of neoplastic epithelial cells with fibrovascular stalks in bile duct lumen, mucin hypersecretion, and considerable dilatation or multilocular changes of the affected bile ducts. A mucin-producing bile duct tumor is an IPNB with excessive mucin production and clinical symptoms. Herein, the phenotypes as well as the tumorigenesis and progression of IPNB are reviewed with immunohistochemical assistance. The tumors are subdivided into three phenotypes: pancreatobiliary, intestinal, and gastric. About half of IPNB cases are of the pancreatobiliary type, and the remaining half are of the intestinal type. Aberrant expression of CDX2 with MUC2 and CK20 is related to the development of intestinal metaplasia. Inactivation of P16INK4a and nuclear expression of beta-catenin are related to the development of IPNB. Decreased expression of membranous beta-catenin and E-cadherin and aberrant expression of MMP-7 and -9 and of MUC1 are related to invasion of IPNB with tubular adenocarcinoma, whereas MUC2 is involved in the invasion of IPNB with mucinous carcinoma. IPNB can be regarded as a counterpart of intraductal papillary mucinous neoplasm (IPMN) of the pancreas, particularly the main duct type. More comparative studies between IPNB and pancreatic IPMN are recommended for further analysis of these papillary neoplasms.

Topics: Adenocarcinoma, Mucinous; beta Catenin; Bile Duct Neoplasms; Cadherins; Carcinoma, Ductal; Carcinoma, Papillary; CDX2 Transcription Factor; Cholangiocarcinoma; Cyclin D1; Disease Progression; Homeodomain Proteins; Humans; Immunohistochemistry; Keratin-20; Matrix Metalloproteinase 7; Matrix Metalloproteinase 9; Mucin-1; Mucin-2; Mucins; Neoplasm Invasiveness; Pancreatic Neoplasms; Phenotype

Cholangiocarcinoma (CCA) is a highly aggressive tumor with a greater risk of distant metastasis. The promising anti-CCA activity and safety profile of Atractylodes lancea (AL) have previously been reported in a series of in vitro, in vivo and clinical studies. The present study investigated the effect of AL extract on apoptosis and metastasis signaling pathways in the Opisthorchis viverrini/dimethylnitrosamine (OV/DMN)-induced CCA hamster model.. Hamster liver tissues were obtained from the four groups (n = 5 per group), i.e., (i) 5-FU treated CCA (40 µg/mL); (ii) CCA; (iii) AL-treated CCA (5,000 mg/kg), and (iv) normal hamsters. Total RNA was isolated, and the expression levels of apoptosis-related and metastasis-related genes were determined by qRT-PCR analysis.. The expression levels of p16, caspase-3, caspase-8, caspase-9, Apaf-1, p53 and Eef1a1 were downregulated, while that of the remaining genes were upregulated in CCA hamsters compared with normal hamsters. AL treatment increased the expression of p16, caspase-9, caspase-3, Apaf-1, p53 and E-cadherin and decreased the expression of cyclin D1, cdk4, Bax, Akt/PKB, Bcl-2, Mfge-8, Lass4, S100A6, TGF-β, Smad-2, Smad-3, Smad-4, MMP-9, and N-cadherin. The expression of Eef1a1 was unchanged.. The anti-CCA activity of AL in OV/DMN-induced CCA hamsters could be due to the induction of cell cycle arrest at the G1 phase and activation of the apoptosis pathway, resulting in cancer cell death. The activation of the apoptosis pathway mainly involved the intrinsic pathway (activation of caspase-3 and caspase-9 through p53 and Mfge-8 modulation and downregulation of anti-apoptotic genes Akt and Bcl-2). In addition, AL could also inhibit the canonical TGF-β signaling pathway, MMP-9 and N-cadherin to suppress tumor metastasis.

Topics: Animals; Atractylodes; bcl-2-Associated X Protein; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Cadherins; Caspase 3; Caspase 8; Caspase 9; Cholangiocarcinoma; Cricetinae; Cyclin D1; Dimethylnitrosamine; Fluorouracil; Humans; Matrix Metalloproteinase 9; Mesocricetus; Opisthorchiasis; Opisthorchis; Plant Extracts; Proto-Oncogene Proteins c-akt; RNA; Transforming Growth Factor beta; Tumor Suppressor Protein p53

Cholangiocarcinoma (CCA) is a group of tumors that arise along the human biliary duct tree, ranking second in primary hepatic malignancies. Intrahepatic CCA (iCCA) represents about 10%-20% of CCAs. There is an increasing body of evidence suggesting that iCCAs' incidence and mortality have been increasing globally over the past few decades. In this study, we found that the EIF3H expression level in iCCA tissues was significantly increased compared to the adjacent non-cancerous tissues by immunohistochemistry analysis (IHC). A similar tendency of EIF3H mRNA and protein level was confirmed in iCCA cell lines using RT-qPCR and Western blot. EIF3H has been identified as a critical molecule that plays a pro-neoplasmic role in iCCA both in vivo and in vitro, such as proliferation, migration, and anti-apoptosis. Mechanistically, we found that EIF3H knockdown can promote the degradation of CCND1 and the proteolysis of CCND1 is mediated by ubiquitin-proteasome system (UPS). Thus, we come to the conclusion that EIF3H promotes proliferation and migration of iCCAs, inhibiting apoptosis of iCCA cells at the same time by stabilizing the CCND1 protein structure. Our findings provide insights into the mechanism of tumorigenesis role of EIF3H in iCCAs and a potential therapeutic target for iCCA treatment.

Topics: beta Catenin; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Cholangiocarcinoma; Cyclin D1; Humans

Cholangiocarcinoma is the most common biliary duct malignancy and the second most common primary liver cancer, accounting for 10‑20% of hepatic malignancies. With high mortality and poor prognosis, the 5‑year survival rate of cholangiocarcinoma is only 10%. A previous study demonstrated a significant association between aspirin use and a decreased risk of cholangiocarcinoma. However, the effect of aspirin on cholangiocarcinoma remains unknown. Therefore, the aim of the present study was to investigate the effects of aspirin on cholangiocarcinoma in vitro and in vivo. Three cholangiocarcinoma cell lines were used to analyze the effect of aspirin on cell proliferation, cell cycle progression, apoptosis, and the regulation of microRNAs. MicroRNAs are known to regulate the development and progression of various types of cancer. An HuCCT‑1 xenograft model was used for the in vivo study. It was determined that aspirin inhibited the proliferation of human cholangiocarcinoma cells (except TKKK cells). Aspirin induced cell cycle arrest in the G0/G1 phase and regulated cell‑cycle related proteins in cholangiocarcinoma cells (HuCCT‑1 cells) but did not induce apoptosis. The expression of miR‑340‑5p was significantly upregulated after treatment, and overexpression of miR‑340‑5p inhibited the proliferation of HuCCT‑1 cells and decreased the levels of cyclin D1. TKKK cells had low miR‑340‑5p expression, which may explain why aspirin had no effect on their proliferation. In vivo, aspirin reduced the growth of xenografted tumors. In conclusion, the present study indicated that aspirin partially inhibited cholangiocarcinoma cell proliferation and tumor growth by inducing G0/G1 phase cell cycle arrest, potentially through the miR‑340‑5p/cyclin D1 axis.

Topics: Animals; Apoptosis; Aspirin; Bile Duct Neoplasms; Cell Line, Tumor; Cell Proliferation; Cholangiocarcinoma; Computational Biology; Cyclin D1; Female; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; Mice; MicroRNAs; Xenograft Model Antitumor Assays

Accumulating reports have demonstrated that long non-coding RNAs (lncRNAs) play critical roles in the occurrence and metastasis of cholangiocarcinoma (CCA). LncRNA myocardial infarction associated transcript (MIAT) has been widely reported in hepatocellular carcinoma, pancreatic cancer and colorectal cancer, but the relationship between MIAT and CCA progression has not yet been investigated. In the present study, we found that the expression of MIAT in CCA tissues was prominently higher than that in normal bile duct tissues. Moreover, TCGA-CHOL data in the GEPIA platform further revealed the upregulated expression of MIAT in CCA tissues. Additionally, quantitative real-time PCR results showed that MIAT expression was increased in CCA cell lines compared to the human intrahepatic biliary epithelial cell line. Functionally, MIAT knockdown significantly inhibited cell proliferation and induced G0/G1 phase arrest as well as apoptosis in HuCCT-1 and QBC939 cells. Conversely, ectopic expression of MIAT obviously facilitated the proliferation, cell cycle progression and apoptosis resistance of RBE cells. Mechanistically, MIAT directly interacted with miR-551b-3p and inversely modulated miR-551-3p level in CCA cells. Furthermore, MIAT knockdown reduced the expression of cyclin D1 (CCND1), which was rescued by miR-551b-3p silencing in HuCCT-1 cells. Importantly, CCND1 restoration partially reversed MIAT knockdown-induced proliferation inhibition, G0/G1 phase arrest and apoptosis in HuCCT-1 cells. In conclusion, MIAT was frequently overexpressed in CCA. MIAT contributed to the growth of CCA cells by targeting miR-551b-3p/CCND1 axis.

Topics: Apoptosis; Bile Duct Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cholangiocarcinoma; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; RNA, Long Noncoding; Signal Transduction

Gemcitabine is clinically used to treat certain types of cancers, including pancreatic and biliary cancer. We investigated the signal transduction pathways underlying the local antitumor effects of gemcitabine-eluting membranes (GEMs) implanted in pancreatic/biliary tumor-bearing nude mice. Here, we report that GEMs increased the E3 ubiquitin ligase c-CBL protein level, leading to degradation of epidermal growth factor receptor (EGFR) in SCK and PANC-1 cells. GEMs decreased the RAS and PI3K protein levels, leading to a reduction in the protein levels of active forms of downstream signaling molecules, including PDK, AKT, and GSK3β. GEM reduced proliferation of cancer cells by upregulating cell cycle arrest proteins, particularly p53 and p21, and downregulating cyclin D1 and cyclin B. Moreover, GEMs reduced the levels of proangiogenic factors, including VEGF, VEGFR2, CD31, and HIF-1α, and inhibited tumor cell migration and invasion by inducing the expression of E-cadherin and reducing that of N-cadherin, snail, and vimentin. We demonstrated that local delivery of gemcitabine using GEM implants inhibited tumor cell growth by promoting c-CBL-mediated degradation of EGFR and inhibiting the proliferation, angiogenesis, and epithelial-mesenchymal transition of pancreatic/biliary tumors. Use of gemcitabine-eluting stents can improve stent patency by inhibiting the ingrowth of malignant biliary obstructions.

Topics: Animals; Antigens, CD; Cadherins; Cell Cycle; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cholangiocarcinoma; Cyclin B; Cyclin D1; Deoxycytidine; Epithelial-Mesenchymal Transition; ErbB Receptors; Female; Gemcitabine; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Mice; Mice, Nude; Pancreas; Pancreatic Neoplasms; Platelet Endothelial Cell Adhesion Molecule-1; Proto-Oncogene Proteins c-cbl; Signal Transduction; Ubiquitin-Protein Ligases; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2; Xenograft Model Antitumor Assays

5-Florouracil (5-FU) is a commonly used chemotherapeutic drug for cholangiocarcinoma, whereas it has unsatisfactory effect, and patients often have chemo-resistance to it. The combination of chemotherapeutic agents and traditional Chinese medicine has already exhibited a promising application in oncotherapy. Huaier extract (Huaier) has been used in clinical practice widely, exhibiting good anti-tumor effect. This paper aims to investigate the possibility of combination 5-FU and Huaier as a treatment for cholangiocarcinoma.. A series of experiments were performed on the Huh28 cells in vitro, which involved cell proliferation, colony formation, apoptosis, cell cycle, migratory and invasive tests. Besides, western blots were also performed to examine the potential mechanism of 5-FU.. The combination effect (antagonism, synergy or additive) was assessed using Chou-Talalay method. Using the CCK-8 and Colony formation assay, the anti-proliferation effect of 5-FU combined with Huaier was observed. Apoptosis inducing and cell cycle arrest effect of the combination of two drugs were assessed by flow cytometry. To determine the combined treatment on cell immigration and invasion ability, wound healing and Transwell assay were performed. The above experiment results suggest that the combined 5-FU and Huaier, compared with treatment using either drug alone, exhibited stronger effects in anti-proliferation, cycle arrest, apoptosis-induced and anti-metastasis. Further, western blot results reveal that the inhibition of STAT3 and its target genes (e.g. Ki67, Cyclin D1, Bcl-2 and MMP-2) might be set as the potential therapeutic targets. Besides, the inhibition of combination treatment in proteins expression associated with proliferation, apoptosis, cell cycle and metastasis was consistent with that of previous phenotypic experiments.. Huaier combined with 5-FU exhibited a synergistic anti-tumor effect in Huh28 cell. Furthermore, the mechanisms might be associated with the activation and translocation of STAT3, as well as its downstream genes.

Topics: Antineoplastic Agents; Apoptosis; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cholangiocarcinoma; Complex Mixtures; Cyclin D1; Drug Synergism; Fluorouracil; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Signal Transduction; STAT3 Transcription Factor; Trametes

CyclinD1 is crucial for cell cycling and can regulate the expression of Dicer, a crucial regulator of microRNA maturation. However, little is known on how CyclinD1 regulates Dicer and miRNA expression, and the progression of intrahepatic cholangiocarcinoma (ICC).. The expression of CyclinD1 and Dicer in non-tumor cholangiocytes, ICC cells and tissues as well as their association with clinicopathological characteristics and survival were examined. The potential mechanisms by which CyclinD1 regulates Dicer and relative miRNA expression were determined by immunoprecipitation, ChIP sequence, BSP and luciferase reporter assays following induction of CyclinD1 over-expression or silencing and Dicer silencing. The impact of CyclinD1 and/or Dicer silencing on the growth of ICC was tested in vivo.. Up-regulated CyclinD1 was associated with down-regulated Dicer expression in ICC tissues and poorer overall survival in patients with ICC. CyclinD1 interacted with the nuclear H3K9me3 and SUV39H1 and bound to the Dicer promoter to increase its CpG island methylation in ICC cells. Functionally, CyclinD1 silencing inhibited the malignancy of ICC cells, which were mitigated partially by Dicer silencing in ICC cells. Dicer silencing down-regulated miR-1914-5p and miR-541-5p expression, which targeted and promoted CyclinD1 and CDK6 expression in ICC cells.. Our findings uncover that CyclinD1 inhibits Dicer expression by chromatin modification to reduce miR-1914-5p/miR-541-5p expression, which positively-feedback enhances CyclinD1 and CDK6 expression and progression of ICC.

Topics: Animals; Bile Duct Neoplasms; Cholangiocarcinoma; Chromatin; Cyclin D1; DEAD-box RNA Helicases; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Middle Aged; Neoplasm Invasiveness; Prognosis; Ribonuclease III; Survival Rate; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Cholangiocarcinoma (CCA) is a malignant tumor originating from cholangiocyte. Prolonged alcohol consumption has been suggested as a possible risk factor for CCA, but there is no information about alcohol's mechanisms in cholangiocyte. This study was designed to investigate global transcriptional alterations through RNA-sequencing by using chronic alcohol exposure (20 mM for 2 months) in normal human cholangiocyte MMNK-1 cells. To observe the association of alcohol induced CCA pathogenesis, we combined differentially expressed genes (DEGs) with computational bioinformatics of CCA by using publicly gene expression omnibus (GEO) datasets. For biological function analysis, Gene ontology (GO) analysis showed biological process and molecular function related to regulation of transcription from RNA polymerase II promoter, while cellular component linked to the nucleoplasm. KEGG pathway presented pathways in cancer that were significantly enriched. From KEGG result, we further examined the oncogenic features resulting in chronic alcohol exposure, enhanced proliferation, and migration through CCND-1 and MMP-2 up-regulation, respectively. Finally, combined DEGs were validated in clinical data including TCGA and immunohistochemistry from HPA database, demonstrating that

Topics: Bile Duct Neoplasms; Cell Line; Cell Movement; Cell Proliferation; Cholangiocarcinoma; Cyclin D1; Epithelial Cells; Ethanol; Humans; Matrix Metalloproteinase 2; Oncogene Proteins v-fos; Transcriptome

MicroRNAs (miRNAs) are powerful regulators in the tumorigenesis of cholangiocarcinoma (CCA). Previous studies report that miR-551b-3p acts as an oncogenic factor in ovarian cancer, but plays a tumour suppressive role in gastric cancer. However, the expression pattern and potential function of miR-551b-3p were still unclear in CCA. Therefore, this study aimed to explore the expression of miR-551b-3p and its role as well as molecular mechanism in CCA. Analysis of TCGA dataset suggested that miR-551b-3p was under-expressed in CCA tissues compared to normal bile duct tissues. Furthermore, our data confirmed the decreased levels of miR-551b-3p in CCA samples and cell lines. Interestingly, TCGA data suggested that low miR-551b-3p level indicated reduced overall survival of CCA patients. Gain- and loss-of-function experiments found that miR-551b-3p inhibited the proliferation, G1-S phase transition and induced apoptosis of CCA cells. In vivo experiments revealed that ectopic expression of miR-551b-3p inhibited tumour growth of CCA in mice. Further investigation demonstrated that miR-551b-3p directly bond to the 3'-UTR of Cyclin D1 (CCND1) mRNA and negatively regulated the abundance of CCND1 in CCA cells. An inverse correlation between miR-551b-3p expression and the level of CCND1 mRNA was detected in CCA tissues from TCGA dataset. Notably, CCND1 knockdown showed similar effects to miR-551b-3p overexpression in HuCCT-1 cells. CCND1 restoration rescued miR-551b-3p-induced inhibition of proliferation, G1 phase arrest and apoptosis in HuCCT-1 cells. In summary, miR-551b-3p inhibits the expression of CCND1 to suppress CCA cell proliferation and induce apoptosis, which may provide a theoretical basis for improving CCA treatment.

Topics: 3' Untranslated Regions; Animals; Apoptosis; Bile Duct Neoplasms; Cell Line, Tumor; Cell Proliferation; Cholangiocarcinoma; Cyclin D1; Databases, Genetic; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Transplantation, Heterologous

Berberine is a natural compound found in several herbs. Anticancer activity of berberine was reported in several cancers, however, little is known regarding the effects of berberine against cholangiocarcinoma (CCA). In this study, the growth inhibitory effects of berberine on CCA cell lines and its molecular mechanisms were explored. Cell growth and cell cycle distribution were examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry. The expression levels of cell cycle regulatory proteins were determined by Western blot analysis. Berberine significantly inhibited growth of CCA cell lines in a dose and time dependent fashion. The inhibition was largely attributed to cell cycle arrest at the G1 phase through the reduction of cyclin D1, and cyclin E. Moreover, berberine could reduce the expression and activation of signal transducers and activator of transcription 3 (STAT3) and probably nuclear factor-kappaB (NF-κB) via suppression of extracellular signal-regulated kinase (ERK) 1/2 action. These results highlight the potential of berberine to be a multi-target agent for CCA treatment.

Topics: Antineoplastic Agents; Berberine; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cholangiocarcinoma; Cyclin D1; Cyclin E; Humans; MAP Kinase Signaling System; NF-kappa B; STAT3 Transcription Factor

Intrahepatic cholangiocarcinoma (ICC) is an aggressive tumor with a high fatality rate. It was recently found that parathyroid hormone-like hormone (PTHLH) was frequently overexpressed in ICC compared with non-tumor tissue. This study aimed to elucidate the underlying mechanisms of PTHLH in ICC development.. The CCK-8 assay, colony formation assays, flow cytometry and a xenograft model were used to examine the role of PTHLH in ICC cells proliferation. Immunohistochemistry (IHC) and western blot assays were used to detect target proteins. Luciferase reporter, chromatin immunoprecipitation (ChIP) and DNA pull-down assays were used to verify the transcription regulation of activating transcription factor-2 (ATF2).. PTHLH was significantly upregulated in ICC compared with adjacent and normal tissues. Upregulation of PTHLH indicated a poor pathological differentiation and intrahepatic metastasis. Functional study demonstrated that PTHLH silencing markedly suppressed ICC cells growth, while specific overexpression of PTHLH has the opposite effect. Mechanistically, secreted PTHLH could promote ICC cell growth by activating extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling pathways, and subsequently upregulated ATF2 and cyclinD1 expression. Further study found that the promoter activity of PTHLH were negatively regulated by ATF2, indicating that a negative feedback loop exists.. Our findings demonstrated that the ICC-secreted PTHLH plays a characteristic growth-promoting role through activating the canonical ERK/JNK-ATF2-cyclinD1 signaling pathways in ICC development. We identified a negative feedback loop formed by ATF2 and PTHLH. In this study, we explored the therapeutic implication for ICC patients.

Topics: Activating Transcription Factor 2; Adult; Aged; Aged, 80 and over; Animals; Autocrine Communication; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Cell Line, Tumor; Cell Proliferation; Cholangiocarcinoma; Cyclin D1; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation, Neoplastic; Humans; JNK Mitogen-Activated Protein Kinases; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Paraneoplastic Endocrine Syndromes; Parathyroid Hormone-Related Protein; Signal Transduction

Epidemiological studies have indicated diabetes mellitus (DM) as a risk of cholangiocarcinoma (CCA), however, the effects and mechanisms of high glucose on progression of CCA remain unclear. This study reports for the first time of the enhancing effects of high glucose on aggressive phenotypes of CCA cells via STAT3 activation. CCA cells cultured in high glucose media exerted significantly higher rates of cell proliferation, adhesion, migration and invasion than those cultured in normal glucose. The phosphokinase array revealed STAT3 as the dominant signal activated in response to high glucose. Increased nuclear STAT3, p-STAT3 and its downstream target proteins, cyclin D1, vimentin and MMP2, were shown to be underling mechanisms of high glucose stimulation. The link of high glucose and STAT3 activation was confirmed in tumor tissues from CCA patients with DM that exhibited higher STAT3 activation than those without DM. Moreover, the levels of STAT3 activation were correlated with the levels of blood glucose. Finally, decreasing the level of glucose or using a STAT3 inhibitor could reduce the effects of high glucose. These findings suggest that controlling blood glucose or using a STAT3 inhibitor as an alternative approach may improve the therapeutic outcome of CCA patients with DM.

Topics: Adult; Aged; Bile Duct Neoplasms; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cholangiocarcinoma; Cyclin D1; Diabetes Complications; Diabetes Mellitus; Female; Gene Expression Regulation, Neoplastic; Glucose; Humans; Male; Matrix Metalloproteinase 2; Microtomy; Middle Aged; Signal Transduction; STAT3 Transcription Factor; Tissue Embedding; Vimentin

The role of transforming growth factor beta 1 (TGFβ1) in cholangiocarcinoma (CCA) initiation and growth requires further definition.. We employed pharmacological and genetic approaches to inhibit or enhance TGFβ1 signaling, respectively, and determine the cellular mechanisms involved.. It was observed that inhibiting TGFβ1 activity with short hairpin RNA (shRNA) or pharmaceutical agents suppressed CCA development and growth, whereas overexpression of TGFβ1 enhanced CCA tumor size and promoted intrahepatic metastasis in a rat model. Suppression of TGFβ1 activity inhibits downstream target gene expression mediated by miR-34a that includes cyclin D1, CDK6, and c-Met. In addition, "knockdown" of TGFβ1 expression revealed a miR-34a positive feedback mechanism for enhanced p21 expression in CCAs. A miR-34a inhibitor reversed the effects of "knocking down" TGFβ1 on cell growth, migration, cyclin D1, CDK6 and c-Met expression, suggesting that TGFβ1 mediated effects occur, in part, through this miR-34a signaling pathway. Overexpression of TGFβ1 was associated with CCA tumor progression.. This study suggests that TGFβ1 is involved in CCA tumor progression and participates through miR-34a mediated downstream cascades, and is a target to inhibit CCA development and growth.

Topics: Animals; Bile Duct Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cholangiocarcinoma; Cyclin D1; Cyclin-Dependent Kinase 6; Disease Progression; Gene Expression Regulation, Neoplastic; Humans; Male; MicroRNAs; Neoplasms, Experimental; Proto-Oncogene Proteins c-met; Rats; Rats, Inbred F344; RNA Interference; Signal Transduction; Transfection; Transforming Growth Factor beta1; Up-Regulation

Cholangiocarcinoma (CCA) is a malignant cancer of the biliary tract and its occurrence is associated with chronic cholestasis which causes an elevation of bile acids in the liver and bile duct. The present study aimed to investigate the role and mechanistic effect of bile acids on the CCA cell growth. Intrahepatic CCA cell lines, RMCCA-1 and HuCCA-1, were treated with bile acids and their metabolites to determine the growth promoting effect. Cell viability, cell cycle analysis, EdU incorporation assays were conducted. Intracellular signaling proteins were detected by western immunoblotting. Among eleven forms of bile acids and their metabolites, only taurolithocholic acid (TLCA) concentration dependently (1-40 µM) increased the cell viability of RMCCA-1, but not HuCCA-1 cells. The cell cycle analysis showed induction of cells in the S phase and the EdU incorporation assay revealed induction of DNA synthesis in the TLCA-treated RMCCA-1 cells. Moreover, TLCA increased the phosphorylation of EGFR, ERK 1/2 and also increased the expression of cyclin D1 in RMCCA-1 cells. Furthermore, TLCA-induced RMCCA-1 cell growth could be inhibited by atropine, a non-selective muscarinic acetylcholine receptor (mAChR) antagonist, AG 1478, a specific EGFR inhibitor, or U 0126, a specific MEK 1/2 inhibitor. These results suggest that TLCA induces CCA cell growth via mAChR and EGFR/EKR1/2 signaling pathway. Moreover, the functional presence of cholinergic system plays a certain role in TLCA-induced CCA cell growth.

Topics: Bile Acids and Salts; Bile Duct Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Survival; Cholangiocarcinoma; Cyclin D1; ErbB Receptors; Humans; MAP Kinase Signaling System; Phosphorylation; Receptors, Muscarinic; Taurolithocholic Acid

Cholangiocarcinoma is a crucial health problem in northeast Thailand. Although rare, it is a highly fatal disease and the prognosis of CCA patients is very poor. To determine if expression of specific genes is useful for diagnosis and prognosis for CCA. We examined the expression of HSP70, HSP90, RB1, cyclin D1, and HDAC6 in 50 resections of human CCA tissues by quantitative real-time PCR. The expression of HSP70, RB1, and HDAC6 was "dominant down-regulation," while the expression of cyclin D1 and HSP90 was "dominant up-regulation." There were no correlations between RB1, cyclin D1, HSP90, and clinicopathological parameters such as status, histology type, histological grading, stage of CCA, and metastasis. A significant association was found between HDAC6 and CCA staging (p = 0.000), CCA gross type and HSP70 (p = 0.046) as well as RB1 expression (p = 0.046). Patients with down-regulation of HSP70 had significantly poorer prognosis than those in the up-regulation group (p = 0.002). Expression of HSP70 may be useful as a new prognostic marker for CCA.

Topics: Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Biomarkers, Tumor; Cholangiocarcinoma; Cyclin D1; Down-Regulation; Female; Histone Deacetylase 6; Histone Deacetylases; HSP70 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Humans; Male; Middle Aged; Neoplasm Metastasis; Real-Time Polymerase Chain Reaction; Retinoblastoma Protein; RNA, Messenger

The aim of this study was to determine the anticancer effects of lobaplatin in cholangiocarcinoma (CCA) RBE cells. We also explored the mechanism of action of lobaplatin by analyzing its influence on apoptosis and cell cycle. Our findings have shown that lobaplatin inhibits cell proliferations in human CCA cells with an IC50 value of approximately 5.26±0.63 μg/mL. Flowcytometry assay confirmed that lobaplatin affected CCA cell survival by blocking cell cycle progression and inducing apoptosis. Lobaplatin treatment reduced Cyclin D1, CDK4, and CDK6 expression, which led to the blocking of G0/G1 transition. In addition, lobaplatin increased p53, Bax expression, caspase-3 and poly (ADP-ribose) polymerase (PARP) cleavage, and reduced Bcl-2 expression, which contributed to the apoptosis of CCA cells. Lobaplatin showed a good anti-tumour activity in in vitro models of human CCA cells. These results indicate that Lobaplatin, as the third-generation platinum antineoplastic agent, could be an effective chemotherapeutic agent in human CCA treatment through induction apoptosis and cell cycle arrest.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Caspase 3; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cholangiocarcinoma; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclobutanes; Down-Regulation; G1 Phase; Humans; Inhibitory Concentration 50; Organoplatinum Compounds; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Resting Phase, Cell Cycle; Tumor Suppressor Protein p53

The mechanisms that mediate the initiation and development of intrahepatic cholangiocarcinoma (ICC) associated with hepatitis B and C virus (HBV and HCV, respectively) infection remain largely unclear. In this study we conditionally coexpressed hepatitis B virus X (HBx) and hepatitis C virus core (HCP) proteins in zebrafish livers, which caused fibrosis and consequently contributed to ICC formation at the age of 3 months. Suppressing the transgene expression by doxycycline (Dox) treatment resulted in the loss of ICC formation. The biomarker networks of zebrafish ICC identified by transcriptome sequencing and analysis were also frequently involved in the development of human neoplasms. The profiles of potential biomarker genes of zebrafish ICC were similar to those of human cholangiocarcinoma. Our data also showed that the pSmad3L oncogenic pathway was activated in HBx and HCP-induced ICC and included phosphorylation of p38 mitogen-activated proteinbase (MAPK) and p44/42 mitogen-activated protein kinase (ERK1/2), indicating the association with transforming growth factor beta 1 (TGF-β1) signaling pathway in ICC. Bile duct proliferation, fibrosis, and ICC were markedly reduced by knockdown of TGF-β1 by in vivo morpholinos injections.. These results reveal that TGF-β1 plays an important role in HBx- and HCP-induced ICC development. This in vivo model is a potential approach to study the molecular events of fibrosis and ICC occurring in HBV and HCV infection.

Topics: Animals; Animals, Genetically Modified; Anti-Bacterial Agents; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Cholangiocarcinoma; Connective Tissue Growth Factor; Cyclin D1; Disease Models, Animal; Doxycycline; Gene Expression; Hepacivirus; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 3; p38 Mitogen-Activated Protein Kinases; Smad2 Protein; Smad3 Protein; Trans-Activators; Transforming Growth Factor beta1; Up-Regulation; Vascular Endothelial Growth Factor A; Viral Core Proteins; Viral Regulatory and Accessory Proteins; Zebrafish; Zebrafish Proteins

Cholestasis contributes to hepatocellular injury and promotes liver carcinogenesis. We created a mouse model of chronic cholestasis to study its effects on progression of cholangiocarcinoma and the oncogenes involved.. To induce chronic cholestasis, Balb/c mice were given 2 weekly intraperitoneal injections of diethylnitrosamine (DEN); 2 weeks later, some mice also received left and median bile duct ligation (LMBDL) and, then 1 week later, were fed DEN, in corn oil, weekly by oral gavage (DLD). Liver samples were analyzed by immunohistochemical and biochemical assays; expression of Mnt and c-Myc was reduced by injection of small inhibitor RNAs.. Chronic cholestasis was induced by DLD and accelerated progression of cholangiocarcinoma, compared with mice given only DEN. Cystic hyperplasias, cystic atypical hyperplasias, cholangiomas, and cholangiocarcinoma developed in the DLD group at weeks 8, 12, 16, and 28, respectively. LMBDL repressed expression of microRNA (miR)-34a and let-7a, up-regulating Lin-28B, hypoxia-inducible factor (HIF)-1α, HIF-2α, and miR-210. Up-regulation of Lin-28B might inhibit let-7a, which is associated with development of cystic hyperplasias, cystic atypical hyperplasias, cholangiomas, and cholangiocarcinoma. Knockdown of c-Myc reduced progression of cholangiocarcinoma, whereas knockdown of Mnt accelerated its progression. Down-regulation of miR-34a expression might up-regulate c-Myc. The up-regulation of miR-210 via HIF-2α was involved in down-regulation of Mnt. Activation of the miR-34a-c-Myc and HIF-2α-miR-210-Mnt pathways caused c-Myc to bind the E-box element of cyclin D1, instead of Mnt, resulting in cyclin D1 up-regulation.. DLD induction of chronic cholestasis accelerated progression of cholangiocarcinoma, which is mediated by down-regulation of miR-34a, up-regulation miR-210, and replacement of Mnt by c-Myc in binding to cyclin D1.

Topics: Animals; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Basic Helix-Loop-Helix Transcription Factors; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Cholangiocarcinoma; Cholestasis; Cyclin D1; Diethylnitrosamine; Disease Models, Animal; Disease Progression; Gene Expression Regulation, Neoplastic; Hyperplasia; Hypoxia-Inducible Factor 1, alpha Subunit; Ligation; Liver; Male; Mice; Mice, Inbred BALB C; MicroRNAs; Proto-Oncogene Proteins c-myc; Repressor Proteins; RNA Interference; RNA-Binding Proteins; Signal Transduction; Time Factors; Transcription Factors

Opisthorchiasis has the significant relationship with the high prevalence of cholangiocarcinoma (CCA; a bile duct cancer) in the endemic areas in Southeast Asia. To reveal the molecular mechanism of the tumorigenesis induced by Opisthorchis viverrini infection, the present study investigated the kinetic expression of RB pathway genes, including RB1, p16(INK4), cyclin D1, and CDK4, during the development of opisthorchiasis-associated CCA in hamster model. The results of quantitative real-time polymerase chain reaction indicated that the expressions of RB1 and p16(INK4) were down-regulated during the development of CCA induced by infection plus N-nitrosodimethylamine treatment in a time-dependent manner. On the other hand, the expressions of cyclin D1 and CDK4 were up-regulated. The expression kinetics was corresponding to the pathological progression of the opisthorchiasis-associated CCA, revealed by histopathological observation. Moreover, the analysis of the expression of these genes in human opisthorchiasis-associated CCA cases showed the decreased expression of RB1 and p16(INK4) in 50% and 82.7% cases and overexpression of cyclin D1 and CDK4 in half cases, respectively. The results suggested that RB pathway is likely involved in the tumorigenesis of opisthorchiasis-induced CCA and proposed the potential application of some of these genes as biomarkers in predispose and molecular therapy of the parasite-associated cancer.

Topics: Animals; Cholangiocarcinoma; Clonorchiasis; Cricetinae; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p16; Gene Expression Regulation; Humans; Molecular Sequence Data; Opisthorchis; Retinoblastoma Protein

This is the first study to explore the relationship between the expression of fragile histidine triad, FHIT and cyclin D1 proteins, and the clinicopathological significance of the two proteins in Chinese patients with cholangiocarcinoma.. Immunohistochemistry was used to study 53 cases of cholangiocarcinoma, 30 para-neoplastic and 20 normal bile ducts for their expression status of FHIT and cyclin D1 and then the results were analyzed with the patient's age, sex, tumour site, histological grade and clinical stage as well as overall median survival time.. Compared with the para-neoplastic and normal cholangiocytes, the expression of FHIT was obviously decreased (p=0.0001), whereas that of cyclin D1 was significantly increased (p=0.0001) in carcinoma cells. The expression of FHIT was found to be correlated with the histological grade (p=0.007) and the clinical stage (p=0.004), but not with age (p=0.776), sex (p=0.246) or tumour site (p=0.347). The expression of cyclin D1 was also showed statistically associated with the histological grade (p=0.043) and clinical stage (p=0.047), but not with age (p=0.965), sex (p=0.751) or tumour site (p=0.948). Further, the expression of FHIT was found to be inversely correlated with the expression of cyclin D1 (p=0.0001). The loss of expression of FHIT and the expression of cyclin D1 were significantly related to the cancers with shorter median survival time (p=0.0001, p=0.0081). The expression of FHIT was an independent prognostic factor (p=0.005).. The expression of FHIT may be inversely correlated with the expression of cyclin D1. It is suggested that the loss of FHIT protein and overexpression of cyclin D1 protein may play an important role in carcinogenesis and prognosis of cholangiocarcinoma.

Topics: Acid Anhydride Hydrolases; Adolescent; Adult; Aged; Bile Duct Neoplasms; Bile Ducts; Bile Ducts, Intrahepatic; Biomarkers, Tumor; Cholangiocarcinoma; Cohort Studies; Cyclin D1; Female; Humans; Immunoenzyme Techniques; Male; Middle Aged; Neoplasm Proteins; Neoplasm Staging; Prognosis; Survival Rate; Young Adult

Cholangiocarcinoma of intrahepatic and extrahepatic bile ducts has a multistep carcinogenesis. Two premalignant lesions have been suggested for invasive cholangiocarcinoma: biliary intraepithelial neoplasia and intraductal papillary neoplasm of the bile duct. How the carcinogenetic process differs between biliary intraepithelial neoplasia and intraductal papillary neoplasm of the bile duct is not clear. In this study, we performed a pathological study to reveal the expression of key molecules related to the cell cycle during 2 carcinogenetic lineages. We immunohistochemically examined the expression of p21, p53, cyclin D1, and Dpc4 in a total of 89 cases: nonneoplastic biliary epithelium, biliary intraepithelial neoplasia, intraductal papillary neoplasm of the bile duct, and invasive cholangiocarcinoma. The expression of p21, p53, and cyclin D1 was up-regulated with histological progression in both biliary intraepithelial neoplasia and intraductal papillary neoplasm of the bile duct, whereas Dpc4 expression was down-regulated in these 2 lineages. In biliary intraepithelial neoplasia, p21 expression was significantly up-regulated early on. In contrast, levels of all molecules changed gradually in intraductal papillary neoplasm of the bile duct. Changes in p53 expression during histological progression differed significantly between biliary intraepithelial neoplasia and intraductal papillary neoplasm of the bile duct. p53 expression was dramatically up-regulated at the invasive stage of biliary intraepithelial neoplasia, whereas it was quite low in noninvasive biliary intraepithelial neoplasia. In contrast, p53 expression was already up-regulated in low-grade intraductal papillary neoplasm and reached a plateau in high-grade intraductal papillary neoplasm and invasive cholangiocarcinoma. This study suggested p21, p53, cyclin D1, and Dpc4 to be involved in the carcinogenesis of both biliary intraepithelial neoplasia and intraductal papillary neoplasm of the bile duct. p53 expression was regulated differently in biliary intraepithelial neoplasia compared with intraductal papillary neoplasm of the bile duct.

Topics: Adult; Aged; Aged, 80 and over; Bile Duct Neoplasms; Cell Cycle Proteins; Cholangiocarcinoma; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Female; Humans; Immunohistochemistry; Male; Middle Aged; Mucin-1; Mucin-2; Mucins; Precancerous Conditions; Smad4 Protein; Tumor Suppressor Protein p53

The Hedgehog (Hh) and pERK1/2 pathways participate in the tumorigenesis of various tissues, but there has been no report on the involvement of these two pathways in cholangiocarcinoma (CCA). The aim of this study was to evaluate the effects of the Hh pathway inhibitor, cyclopamine, and MEK inhibitor, U0126, as a single agent or in combination on CCA cell proliferation and survival.. Seven CCA cell lines were treated with cyclopamine and/or U0126, and cell proliferation was determined by WST-1 assay. The cell cycle was investigated by fluorescence-activated cell sorter analysis. The expression levels of several cell cycle-related genes were determined by western blot analyses.. Cyclopamine decreased cell proliferation and arrested the cell cycle at the G1 phase, while U0126 decreased the proliferation of CCA cells with KRAS mutation stronger than with wild-type KRAS. The combination of both inhibitors had an additive antiproliferative effect, particularly in cells with KRAS mutation, and induced caspase-dependent apoptosis in the CCA cells. The expression levels of cell cycle-related proteins that are targets of the two pathways, such as cyclin D1 and cyclin B1, were strongly decreased in some CCA cell lines after combined inhibitor treatment.. Our results suggest that the Hedgehog and ERK1/2 pathways are important for CCA cell proliferation, and simultaneous inhibition of the two pathways may lead to stronger decreases in cell growth and viability in a subset of CCA cases.

Topics: Apoptosis; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Butadienes; Cell Cycle; Cell Division; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cholangiocarcinoma; Cyclin D1; Drug Synergism; Enzyme Inhibitors; Flow Cytometry; G1 Phase; Gene Expression Regulation, Neoplastic; Hedgehog Proteins; Humans; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Veratrum Alkaloids

Biliary tract adenocarcinomas (BTAs), although anatomically related, arise through ill-defined and possibly different location-related pathogenetic pathways. This clinicopathologic study characterizes differences in cell cycle-regulatory protein expression across the spectrum of BTA.. Tissue microarrays were prepared from paraffin-embedded surgical specimens with triplicate cores of BTA and benign tissue. Immunohistochemical expression of p53, cyclin D1, p21, Bcl2, p27, Mdm2, and Ki-67 was assessed, and the results were correlated with pathologic variables and survival. Hierarchical clustering was used to partition the data based on protein expression, and then the data were analyzed according to anatomic location.. Tissue from 128 surgical patients (1992 to 2002) was obtained. Tumor sites of origin were intrahepatic cholangiocarcinoma (IH; n = 23), hilar cholangiocarcinoma (Hilar; n = 54), gallbladder (GB; n = 32), and distal bile duct (Distal; n = 19). p27 expression decreased progressively from proximal to distal in the biliary tree and correlated with location-related differences in outcome; cyclin D1 and Bcl2 overexpression also varied according to anatomic site. Aberrant p53 staining and cyclin D1 overexpression were lower in papillary tumors compared with the more common sclerosing tumors. The expression profiles of GB and Hilar were more similar to each other than either was to IH or Distal (86% clustering in the first partition). After an R0 resection, overexpression of Mdm2 (P = .0062) and absent p27 expression (P = .0165) independently predicted poor outcome.. BTAs differentially express cell cycle-regulatory proteins based on tumor location and morphology. Prognostic roles were identified for Mdm2 and p27. Overlap in the pathogenesis of GB and Hilar tumors was suggested.

Topics: Aged; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Biomarkers, Tumor; Cell Cycle Proteins; Cholangiocarcinoma; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Female; Gallbladder Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Ki-67 Antigen; Male; Middle Aged; Neoplasm Recurrence, Local; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-mdm2; Survival Analysis; Tumor Suppressor Protein p53; Up-Regulation

The mechanisms of carcinogenesis in intrahepatic cholangiocarcinoma (ICC) are not well characterized although alterations in several oncogenes and onco-suppressor genes have been reported to occur in ICC. In the present study, we focused on alterations in the Wnt signaling components and target genes by analyzing 24 surgically resected samples of ICC. Immunohistochemical analysis of beta-catenin showed positive staining in cytoplasm and/or nucleus in 58.3% of the samples, indicating the presence of alterations in the Wnt signaling pathway in these samples. In sequencing analyses, mutations in the beta-catenin, adenomatous polyposis coli and Axin 1 genes were observed in 8.3, 12.5 and 41.7%, respectively, of the 24 ICC samples; however, the functional significance of these mutated genes is controversial. Furthermore, cyclin D1, c-myc and urinary-type plasminogen activator receptor, which are the downstream target genes in the Wnt signaling pathway, were overexpressed in 41.7, 41.7 and 58.3%, respectively, of the 24 ICC samples. The overexpression of cyclin D1 was statistically correlated with that of beta-catenin. Based on these results, we speculated that the Wnt signaling pathway plays an important role in carcinogenesis in ICC through overexpression of its target genes, particularly cyclin D1.

Topics: Aged; Animals; Axin Protein; beta Catenin; Cell Nucleus; Cholangiocarcinoma; Cyclin D1; Cytoplasm; DNA Mutational Analysis; Female; Humans; Immunohistochemistry; Liver Neoplasms; Male; Middle Aged; Mutation; Proto-Oncogene Proteins c-myc; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Repressor Proteins; Sequence Analysis, DNA; Signal Transduction; Urokinase-Type Plasminogen Activator; Wnt Proteins

We studied the cytotoxic effects of recombinant TNF-alpha and supernate of phytohemagglutinin stimulated peripheral blood mononuclear cells individually and in combination against a cholangiocarcinoma cell line. Levels of cyclins D1, E and A in the cell line were detected by immunoblotting, and the cell cycle stage was assayed by propidium iodide staining followed by flow cytometry analysis. Viable and apoptotic cells were assessed by trypan blue dye exclusion, DAPI staining, agarose DNA laddering and propidium iodide staining. At the beginning of each experiment, the majority of cholangiocarcinoma cells expressed cyclin A and were in S phase as determined by propidium iodide staining. Treatment of such cells with recombinant TNF-alpha resulted in cytotoxic effects clearly evident at 36 hours post exposure. There was a synergistic killing effect when recombinant TNF-alpha was combined with PHA supernate and this effect could be partly neutralized by monoclonal anti TNF-alpha, interleukin (IL)-2, IL-12 and IFN-gamma.

Topics: Adult; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Biomarkers, Tumor; Cholangiocarcinoma; Cyclin A; Cyclin D1; Cyclin E; Cytotoxicity, Immunologic; Disease Susceptibility; Drug Therapy, Combination; Female; Humans; Immunoblotting; Male; Phytohemagglutinins; Recombinant Proteins; Time Factors; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Aberrations of G1-S cell cycle arrest and TGF-beta/Smad pathway are critical events in human carcinogenesis. We studied alterations of both pathways by immunohistochemical staining for p53, p16, p27, cyclin D1, Rb and Smad4/Dpc4 in 42 intrahepatic cholangiocarcinomas (ICCs). Abnormal nuclear overexpression of p53 and cyclin D1 was noted in 15 (35.7%) and 26 (61.9%) cases, respectively. Total loss of p16, p27, Rb and Smad4 was detected in 15 (35.7%), 13 (31.0%), 5 (11.9%) and 19 (45.2%) cases, respectively. Forty cases (95.2%) showed aberrations of at least one of the pathways, of which 21 (50%) revealed abnormality in G1-S pathway only, 17 (40.5%) had abnormalities in both pathways and 2 (4.8%) had an abnormality in TGF-beta/Smad pathway only. Among the examined genes, loss of Smad4 was found to have a positive relationship with the pTNM stage (P < 0.05). The overall stage of the high-altered group (alterations in 2 to 5 of the genes, n = 29) was significantly higher than that of the low-altered group (alteration of one or no gene, n = 13) (P < 0.01). We also examined the expression of above genes in the accompanying biliary dysplasia and found out abnormal expression of p53, cyclin D1 or p16 in 7 out of 13 dysplastic lesions. Our data suggest that abnormal G1-S cell cycle and altered TGF-beta/Smad pathway are major events in cholangiocarcinogenesis. Moreover, there might be a possible cumulative effect of the alterations in the examined genes upon the clinical outcome of patients with resectable ICCs.

Topics: Adult; Aged; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Biomarkers, Tumor; Cell Cycle Proteins; Cell Nucleus; Cholangiocarcinoma; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; DNA-Binding Proteins; Female; Genes, Tumor Suppressor; Humans; Immunoenzyme Techniques; Male; Microfilament Proteins; Middle Aged; Muscle Proteins; Neoplasm Proteins; Neoplasm Staging; Retinoblastoma Protein; Smad4 Protein; Trans-Activators; Tumor Suppressor Protein p53

Evaluation of the biological character of carcinomas requires understanding of cell cycle regulators. P57 (Kip2) belongs to the Cip/Kip family and is known to be one of the universal negative regulators of cell cycle.. In the present study, therefore, we investigated p57 expression in 37 extrahepatic bile duct carcinomas (BDC) and 28 intrahepatic cholangiocellular carcinomas (CCC).. The average p57 labeling index (LI) in BDC and CCC were 60.8 +/- 7.9 and 58.6 +/- 18.6, respectively, which were significantly lower (p = 0.0008 and p = 0.0408, respectively) than those in normal duct epithelia (73.1 +/- 7.9, 70.4 +/- 8.2). p57 LI was significantly lower in BDC and CCC cases with biological aggressive phenotypes such as poor differentiation (p = 0.0260 and p = 0.0069), lymph node metastasis (p = 0.0274 and p = 0.0214), high Ki-67 LI (p = 0.0164 and p = 0.0343) and cyclin D1 overexpression (p = 0.0359 and p = 0.0255).. These findings suggest that decreased p57 expression is related to the increased activity of cell proliferation and also the progression of these carcinomas.

Topics: Adult; Aged; Aged, 80 and over; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Cell Division; Cholangiocarcinoma; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p57; Enzyme Inhibitors; Female; Humans; Immunoenzyme Techniques; Ki-67 Antigen; Lymph Nodes; Lymphatic Metastasis; Male; Middle Aged; Nuclear Proteins

To elucidate the clinical roles of G1-S modulators in cholangiocellular carcinoma (CCC).. We performed immunohistochemistry using antibodies against the retinoblastoma gene product (pRb), p16, p21, p27, p53 and cyclin D1 for 41 cases of CCC as well as normal bile ducts.. The p27 labeling index (LI) was significantly higher in cases without lymph node metastasis than in normal bile ducts, but it decreased greatly in cases with lymph node metastasis. It was inversely related to the Ki-67 LI. The p16 LI also showed a relationship with lymph node metastasis, but not with the Ki-67 LI. The p21 LI was even higher in poorly differentiated cases and showed a direct relationship with the Ki-67 LI, although it is a negative regulator of the cell cycle. pRb expression did not correlate with any clinicopathological features. Cyclin D1 overexpression was more frequently observed in cases with poor or moderate differentiation and with lymph node metastasis. Cyclin D1 overexpression and aberrant p53 expression showed direct relationships with the Ki-67 LI.. These results suggest that in CCC: (1) p27 expression reflects the biological character of the carcinoma and may regulate its progression; (2) cyclin D1 plays a crucial role in cell cycle progression, and (3) aberrant p53 expression has some effect on CCC cell proliferating activity.

Topics: Adult; Aged; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Cholangiocarcinoma; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Female; G1 Phase; Genes, p16; Humans; Immunohistochemistry; Male; Middle Aged; Retinoblastoma Protein; S Phase; Tumor Suppressor Protein p53

Intrahepatic cholangiocarcinoma (ICC) is a primary liver malignant tumor with an extremely poor prognosis, but less attention has been directed to factors related to molecular carcinogenesis, including cell cycle proteins. We examined the expression and gene amplification of cyclin D1, the cell cycle regulating protein. Our objective was to evaluate correlations with clinicopathological factors in ICC.. Cyclin D1 overexpression and cellular proliferative activity (Ki-67 labeling index) were investigated immunohistochemically, and 20 cases were further investigated for cyclin D1 gene amplification, using differential PCR. We examined the correlation between the expression and gene amplification of cyclin D1 and clinicopathological factors, including overall survival in patients with ICC.. Immunohistochemical analysis revealed an overexpression of cyclin D1 protein in 28 of 66 subjects with ICCs (42%). The cyclin D1 overexpression was associated with poor histological differentiation (P = 0.04), high cellular proliferative activity (P < 0.01), and a poor prognosis (P = 0.02) by univariate analysis, although it is not an independent prognostic factor by multivariate analysis. Cyclin D1 gene amplification was confirmed in five of the 20 patients. Of those five cases of ICC, all had poor histological differentiation, and four of the five ICCs (80%) showed evidence of cyclin D1 immunoreactivity.. Overexpression and gene amplification of cyclin D1 are frequent and contribute to dedifferentiation and cellular proliferative activity of ICCs, and overexpression also indicates a poor prognosis for patients with ICC.

Topics: Adult; Aged; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Cell Division; Cholangiocarcinoma; Cyclin D1; Female; Gene Amplification; Humans; Immunohistochemistry; Ki-67 Antigen; Liver Neoplasms; Male; Middle Aged; Polymerase Chain Reaction; Prognosis