metallothionein and Colorectal-Neoplasms

We aimed to understand the molecular mechanism underlying the incidence of Oxaliplatin resistance in colorectal cancer. The Oxaliplatin-resistant (OR) HT29 colorectal cell line was established by long-term exposure to Oxaliplatin. Cell viability and proliferation were determined by the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide and direct counting assays, respectively. Transcript level of metallothionein 2A (MT2A) was measured by real-time polymerase chain reaction. Protein levels of MT2A, BRCA1-associated RING domain 1 (BARD1), BRCA1, and β-actin were quantified by immunoblotting. Direct interaction between MT2A with BARD1 and BRCA1 was analyzed by co-immunoprecipitation. Colocalization between of MT2A and BARD1 was determined by immunofluorescence. MT2A was upregulated in OR cells at both transcript and protein levels. Knockdown of MT2A in HT29 OR cells improved sensitivity to Oxaliplatin, while ectopic overexpression of MT2A conferred HT29 cells relative resistance to Oxaliplatin. We further demonstrated that MT2A interacted with and positively regulated BARD1/BRCA1 in colorectal cancer cells. BARD1 overexpression partially restored the compromised Oxaliplatin resistance elicited by MT2A deficiency in terms of both cell proliferation and viability. Our data highlighted the critical contributions of MT2A-BARD1/BRCA1 in Oxaliplatin resistance in colorectal cancer cells.

Topics: Colorectal Neoplasms; Drug Resistance, Neoplasm; Gene Knockdown Techniques; HCT116 Cells; HT29 Cells; Humans; Metallothionein; Oxaliplatin; Signal Transduction; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases; Up-Regulation

Colorectal cancer (CRC) is a heterogeneous disease resulting from the combined influence of many genetic factors. This complexity has caused the molecular characterization of CRC to remain uncharacterized, with a lack of clear gene markers associated with CRC and the prognosis of this disease. Thus, highly sensitive tumor markers for the detection of CRC are the most essential determinants of survival. In this study, we examined the simultaneous downregulation of the mRNA levels of six metallothionein (MT) genes in CRC cell lines and public CRC datasets for the first time. In addition, we detected downregulation of these six MT mRNAs' levels in 30 pairs of tumor (T) and adjacent non-tumor (N) CRC specimens. In order to understand the potential prognostic relevance of these six MT genes and CRC, we presented a four-gene signature to evaluate the prognosis of CRC patients. Further discovery suggested that the four-gene signature (

Topics: Biomarkers, Tumor; Colorectal Neoplasms; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Kaplan-Meier Estimate; Metallothionein; Prognosis; RNA, Messenger; Transcriptome

Colorectal cancer (CRC) is the third most frequent malignancy worldwide. Coffee is the second most consumed drink in the globe and suggested to decrease the CRC risk. Here, we explored whether coffee, decaffeinated coffee, or caffeine impact on the development of colorectal carcinogenesis induced by the direct carcinogen N-methyl-N-nitro-N-nitrosoguanidine (MNNG) in rats. To this end, sixty-four young male Wistar rats were divided into eight groups of eight animals each. We analyzed the frequency of dysplastic crypts and expression of metallothionein as a biomarker of the cancer risk, as well the expression of phosphorylated H2A histone family/member X (γH2AX) for DNA damage and cyclooxygenase-2 (COX-2) for inflammatory response. We also studied the oxidative stress profile in hepatic and colonic frozen samples (malondialdehyde [MDA], glutathione [GSH], and α-tocopherol). We found that coffee but neither decaffeinated coffee nor caffeine decreased the development of dysplastic crypts in MNNG-exposed rats. All treatments reduced DNA damage intensity in colonocytes. Only decaffeinated coffee increased the numbers of metallothionein positive crypts in comparison with coffee-treated rats. Coffee and caffeine inhibited COX-2 expression in the colon. Both decaffeinated coffee and caffeine decreased hepatic α-tocopherol levels. We suggest that coffee may have other compounds that elicit greater chemoprotective effects than caffeine reducing the CRC risk.

Topics: alpha-Tocopherol; Animals; Anticarcinogenic Agents; Caffeine; Carcinogens; Coffee; Colon; Colorectal Neoplasms; Cyclooxygenase 2; DNA Damage; Histones; Male; Metallothionein; Methylnitronitrosoguanidine; Oxidative Stress; Rats, Wistar

The rapid increase in the incidence rate of colorectal cancer has led to the search and identification of biomarkers that can predict risk for and future behavior of this malignancy and management. To study the biological role of the phosphorylated Fas associated death domain (pFADD) gene in colorectal cancer, we performed a GAL4-based yeast two-hybrid screening of a human heart cDNA library.. A series of two yeast hybrid method was used to identification of protein-protein interaction. It was confirmed by glutathione S-transferase (GST) pull down assay and co-immunoprecipitation (co-IP). Three channeled fluorescence microscopy further confirmed the interaction in cellular level. Xenograft in vivo model was developed and knockdown relevant genes by RNAi techniques and confirmed the relationship which leads to colorectal cancer.. Using the FADD cDNA as bait, we identified six putative clones as associated proteins. The interaction of pFADD and metallothionein 2A (MT2A) was confirmed by GST pull-down assays in vitro and co-IP experiments in vivo. FADD co-localized with MT2A mostly to nuclei and slightly to cytoplasm, as shown by three channel fluorescence microscopy. Co-transfection of pFADD with MT2A gene inhibited cell apoptosis and induced cell proliferation in colorectal cancer cells compared with control groups. When we used antisense MT2A and pFADD which is serine 194 in the C terminal of FADD gene that has been reported to be phosphorylated to interdict the effect of respective genes the inhibition of cell proliferation and induction of apoptosis were significantly enhanced in animal model.. Further in this study we identify non-canonical nuclear factor-κB (NF-κB) signaling up regulated and it was directly linked with the tumor necrosis with MT2A and pFADD genes. pFADD with MT2A can inhibit the apoptosis and promote proliferation, of colorectal cancer cells, and antisense sequence of MT2A and pFADD approaches which might swell the combination of deregulated proliferation and suppressed apoptosis.

Topics: Biomarkers, Tumor; Cell Line, Tumor; Colorectal Neoplasms; Fas-Associated Death Domain Protein; Fluorescence Resonance Energy Transfer; HeLa Cells; Humans; Metallothionein; Microscopy, Fluorescence; NF-kappa B; Phosphorylation

Metallothioneins (MT) are a family of low molecular weight proteins that are silenced during colorectal cancer progression, mainly through epigenetic mechanisms, and this loss is associated with poor survival. In this article, we show that overexpression of the MT1G isoform sensitizes colorectal cell lines to the chemotherapeutic agents oxaliplatin (OXA) and 5-fluorouracil (5-FU), in part through enhancing p53 and repressing NF-κB activity. Despite being silenced, MTs can be reinduced by histone deacetylase inhibitors such as trichostatin A and sodium butyrate. In fact, this induction contributes to the cytotoxicity of these agents, given that silencing of MTs by siRNAs reduces their growth-inhibitory activities. Zinc ions also potently enhance MT expression and are cytotoxic to cancer cells. We show for the first time that OXA and 5-FU induce higher levels of intracellular labile zinc, as measured using the fluorescent probe FLUOZIN-3, and that such zinc contributes to the activation of p53 and repression of NF-κB. Addition of zinc enhanced growth inhibition by OXA and 5-FU, and was also capable of resensitizing 5-FU-resistant cell lines to levels comparable with sensitive cell lines. This effect was MT independent because silencing MTs did not affect zinc cytotoxicity. In conclusion, we show that MT induction and zinc administration are novel strategies to sensitize colorectal cancer cells to presently utilized chemotherapeutic agents.

Topics: Animals; Antineoplastic Agents; Cell Death; Cell Line, Tumor; Colorectal Neoplasms; Disease Models, Animal; Drug Resistance, Neoplasm; Gene Expression; Gene Expression Regulation, Neoplastic; HCT116 Cells; Histone Deacetylase Inhibitors; HT29 Cells; Humans; Male; Metallothionein; NF-kappa B; Protein Transport; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays; Zinc

Metallothioneins are a family of small, cysteine-rich proteins with many functions. Immunohistochemical evaluation of all metallothionein 1 + 2 isoforms in colorectal tumors has demonstrated an important down-regulation compared with normal tissue, although its prognostic significance is unclear. Moreover, the contribution of individual isoforms to overall metallothionein down-regulation is not known. To address these important issues, we analyzed the messenger RNA expression levels of all functional metallothionein 1 + 2 isoforms by quantitative reverse transcription polymerase chain reaction in 22 pairs of normal and tumor-microdissected epithelia and correlated these to the overall immunohistochemical protein expression. Our results showed that 5 isoforms (MT1G, 1E, 1F, 1H, and 1M) were lost during the transition from normal mucosa to tumor, whereas MT1X and MT2A were less down-regulated, and their expression was correlated with overall protein positivity. Second, we showed that MT1G hypermethylation occurred in cell lines and in 29% of tumor samples, whereas histone deacetylase inhibitors are able to induce most isoforms. Furthermore, we analyzed by immunohistochemistry 107 normal mucosae, 25 adenomas, 81 carcinomas, and 19 lymph node metastases to evaluate metallothionein expression during different stages of cancer development and to assess its relationship to patient survival. A lower immunohistochemical expression was associated with poorer survival, although it was not an independent predictor. Overall, this study identifies for the first time the relevant metallothionein isoforms for colorectal cancer progression, supports the concept that their loss is associated with worse prognosis, and suggests 2 mechanisms for epigenetic repression of metallothionein expression in colorectal tumors.

Topics: Adenocarcinoma; Adenoma; Aged; Aged, 80 and over; Argentina; Biomarkers, Tumor; Colorectal Neoplasms; Disease Progression; DNA Methylation; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Male; Metallothionein; Middle Aged; Protein Isoforms; RNA, Messenger; Survival Rate

Stratifying patients defective in mismatch repair (dMMR) with high microsatellite instability (MSI-H) in colorectal cancer (CRC) is of increasing relevance and may provide a more tailored approach to CRC adjuvant therapy. Here, we describe the discovery of a new MSI marker for colorectal cancer located in the 3'-untranslated region (3'UTR, T20 mononucleotide repeat) of the metallothionein 1X gene (MT1XT20).. We studied 340 consecutive CRCs using three multiplexed polymerase chain reactions amplifying BAT25, BAT26, TGFBR2, MybT22, BAT40, MT1XT20, NR21, NR24, CAT25, D2S123, D5S346, D17S250, D18S58, CSF1PO, D7S820, and D18S51. Fragments length was evaluated by automated capillary electrophoresis.. Based on the NCI/ICG-HNPCC criteria for MSI classification, 40 CRCs were found to be MSI-high (11.8%), 46 (13.5%) CRCs were MSI-low, and 254 CRCs (74.7%) were stable (MSS). MT1XT20 showed very high sensitivity (97.3%) comparable to BAT26 (97.5%) and CAT25 (97.1%) and the best specificity (100%) as well as MybT22 and CAT25. Indeed, MT1XT20 instability was detected in 36 out of 37 cases (97.3%) of MSI-high colorectal cancers, whereas no MT1XT20 alterations were observed in 254 MSS or in 46 MSI-low cases. On the contrary, BAT40 was found to be unstable in 8/46 MSI-low cases, BAT25 in 6/46, BAT26 4/46, NR21 1/46, and NR24 in 1/45.. Our results suggest that MT1XT20 represents a sensitive and specific marker for MSI testing and could be included in a complete set of MSI markers for the confident identification of familial or sporadic dMMR patients in CRCs.

Topics: 3' Untranslated Regions; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Colorectal Neoplasms; DNA Mismatch Repair; Female; Genetic Markers; Humans; Male; Metallothionein; Microsatellite Instability; Polymerase Chain Reaction; Sensitivity and Specificity; Tandem Repeat Sequences

Our study aimed to evaluate metallothionein and p53 expression in colorectal cancer and to correlate their combined expression with selected clinical and pathologic variables of the disease, to define their prognostic significance. Colorectal cancer specimens from 99 patients were retrospectively analyzed by immunohistochemistry for metallothionein and p53 expression. Survival curves were generated according to the Kaplan-Meier method, and univariate survival distributions were compared with the use of the log-rank test. Multivariate models were computed using Cox proportional hazards regression. This research was approved by the institutional review boards of all centers. Tumors showing concomitant high metallothionein expression and negative p53 (metallothionein(H)/p53(-)) were significantly inversely related to depth of invasion, frequency of nodal metastasis, and Dukes stage (P < .01). In univariate analysis, patients with metallothionein(H)/p53(-) phenotype showed a better overall survival (hazard ratio [HR], 2.83; P < .05) and disease-free survival (HR, 2.03; P < .05). In multivariate analysis, considering staging, metallothionein, and metallothionein + p53 variables, in 83 patients with Dukes stages B and C, metallothionein(H)/p53(-) combination was the sole factor showing an independent prognostic value for overall survival (HR, 3.88; P < .1) and disease-free survival (HR, 2.56; P < .1). In conclusion, the combined analysis of metallothionein and p53 may enhance the prognostic power of each individual marker by predicting the progression of the disease and contributing to a better identification of patients at low risk for mortality, especially for those with Dukes stage B and C colorectal cancer.

Topics: Biomarkers, Tumor; Colorectal Neoplasms; Disease-Free Survival; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Metallothionein; Neoplasm Staging; Prognosis; Proportional Hazards Models; Retrospective Studies; Tumor Suppressor Protein p53

Tea polyphenol has been shown to have anti-colorectal cancer and anti-gene mutation effects, although the mechanism of inhibition of microsatellite instability (MSI) colorectal cancer is not known.. Using LoVo, HCT-116, HT-29, and SW480 cells treated with an aqueous solution of tea polyphenol, cell proliferation was detected by the methyl thiazolyl tetrazolium method, changes in microsatellite sequences by the Genescan method and changes in the gene expression of LoVo cells using Illumina expression arrays.. The proliferation inhibition rate of LoVo, HCT-116, HT-29, and SW480 cells treated with tea polyphenol increased with increasing drug concentration and showed an increasing tendency with time. The proliferation inhibition rate of LoVo and HCT-116 cells with tea polyphenols was higher than that of HT-29 and SW480 cells, and there was a significant difference in the proliferation inhibition rate at 24, 72 h and 1 week. The microsatellite sequence of LoVo cells treated with tea polyphenols remained stable.. The gene expression arrays and quantitative RT-PCR suggested that tea polyphenol inhibited the gene expression of metallothionein 2A (MT2A), transcription factor (MAFA), hairy and enhancer of split 1 (HES1), and jagged1 (JAG1) nearly twofold over controls. It was also found that tea polyphenol inhibited the BAX and p38 genes with a more than twofold difference but did not significantly inhibited the NFκB pathway.. Tea polyphenol significantly inhibited the proliferation of MSI colorectal cancer signals maintained stable at the microsatellite state in MSI colorectal cancer. Tea polyphenol inhibited the gene expression of HES1, JAG1, MT2A, and MAFA but upregulated the gene expression of BAX and downregulated that of (P)38. Further research is required to investigate how these pathways are interrelated.

Topics: Basic Helix-Loop-Helix Transcription Factors; bcl-2-Associated X Protein; Calcium-Binding Proteins; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Dose-Response Relationship, Drug; Flavonoids; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Intercellular Signaling Peptides and Proteins; Jagged-1 Protein; Maf Transcription Factors, Large; Membrane Proteins; Metallothionein; Microsatellite Instability; p38 Mitogen-Activated Protein Kinases; Phenols; Polyphenols; Serrate-Jagged Proteins; Tea; Transcription Factor HES-1

Hypoxia-inducible factor 1 (HIF-1) is a hypoxia-induced transcription factor that regulates gene expression in critical pathways involved in tumour growth and metastasis. Metallothionein (MT) is a group of small molecular weight cysteine-rich proteins with a broad variety of functions. The present study aimed to analyse the prognostic impact of HIF-1alpha and MT expression in colorectal cancer and to evaluate a possible link of combined HIF-1alpha and MT expression with colorectal cancer progression.. We investigated the relationship of HIF-1alpha and MT with each other and clinicopathological parameters including proliferative activity (Ki67) and apoptosis (terminal desoxyribonucleotide transferase-mediated dUTP nick-end labelling) using immunohistochemistry.. HIF-1alpha expression was identified as an independent prognostic parameter in multivariate survival analysis and characterised an aggressive cancer phenotype. In addition, HIF-1alpha was significantly linked to an increased expression of MT.. HIF-1alpha expression qualified as an independent prognostic and characterised an aggressive cancer phenotype associated with an increased expression of MT. Our study suggests that MT can be added to the complex biological pathways induced by hypoxia in human cancer tissue.

Topics: Amino Acids, Dicarboxylic; Apoptosis; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Epithelial Cells; Female; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; Kaplan-Meier Estimate; Male; Metallothionein; Phenotype; Reactive Oxygen Species; Staining and Labeling

Trypanosoma cruzi infection and nonsteroidal anti-inflammatory drugs inhibit colorectal carcinogenesis by mechanisms not completely known and metallothionein proteins (MTs) may be involved in this process. Sixty-six male Wistar rats weighing 90 to 120 g were randomly divided into seven groups (GI to GVII). GI, GII and GIII animals were subcutaneously infected with 200,000 trypomastigote forms of the Y strain of T. cruzi. After 8 weeks, GI, GII, GIV, and GVI were injected with one weekly subcutaneous dose of 12 mg/kg dimethylhydrazine for 4 weeks. In sequence, GI, GIV and GV were treated with nimesulide (10 mg/kg per dose, five times per week for 8 weeks). Groups I, III, IV, and VI had 12 animals, and each of the other groups had 6 animals. All the animals were euthanized 8 weeks after the last dimethylhydrazine injection. The colons were fixed and processed for MT immunohistochemistry. The index of MT-overexpressing colonic crypts (MTEC) was estimated as the percentage of MT-stained crypts in relation to the total number of crypts scored. Five hundred crypts per animal were scored. Data were analyzed by the Kruskal-Wallis test followed by the Dunn test. There was an increase in MTEC index in the groups either infected with T. cruzi or treated with nimesulide or both infected and treated when compared to control (401, 809, and 1011%, respectively). We suggest that the increased formation of MTEC may be related to the protection against carcinogenesis provided both by T. cruzi infection and nimesulide.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Carcinogens; Chagas Disease; Colorectal Neoplasms; Dimethylhydrazines; Disease Models, Animal; Immunohistochemistry; Male; Metallothionein; Random Allocation; Rats; Rats, Wistar; Sulfonamides; Trypanosoma cruzi

To study the expression of metallothionein (MT) and FasL in colorectal cancer and their relation to lymph node and liver metastasis.. Immunohistochemistry and quantitative RT-PCR were used to detect expression of MT and FasL in protein and mRNA levels in 93 cases of colorectal cancer.. The rates of MT expression in primary foci, non-cancerous colon mucosa, lymph node metastasis and liver metastasis were 58.1%, 32.3%, 81.1%, 64.3% respectively. And the rates of FasL expression were 41.9%, 19.4%, 62.3%, and 92.9% respectively. The positive rates of MT and FasL in primary foci, liver and lymph node metastasis were higher than that in non-cancerous mucosa (chi(2) = 35.2421, 57.5152, P < 0.01). MT expression rate in lymph node metastasis was higher than that in primary foci (chi(2) = 8.0565, P < 0.01). In liver metastasis, FasL expression rate was higher than in lymph node metastasis and primary foci (chi(2) = 8.6674, 22.4455, P < 0.01). The positive rates of MT and FasL in Dukes stage C and D were higher than that in Dukes stage A and B (chi(2) = 18.8871, 25.1650, P < 0.01). And higher rates of MT and FasL expression were observed in low differentiation adenocarcinoma and mucus adenocarcinoma than in middle-high differentiation adenocarcinoma (chi(2) = 11.1546, 9.2239, P < 0.05). High MT mRNA level was found in lymph node metastasis and high FasL mRNA level in liver metastasis.. Enhanced expression of MT and FasL was associated significantly with lymph node and liver metastasis of colorectal cancer. Assay of MT and FasL expression has prognostic values for colorectal cancer patients.

Topics: Adult; Aged; Aged, 80 and over; Colorectal Neoplasms; Fas Ligand Protein; Female; Humans; Immunohistochemistry; Liver Neoplasms; Lymph Nodes; Lymphatic Metastasis; Male; Metallothionein; Middle Aged; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

To construct the yeast two-hybrid system, and screen the proteins which interact with FasL, and investigate the relationship of FasL and hepatic metastasis of colorectal carcinoma.. We have cloned the FasL gene into the pGBKT7 vector as the bait, then screened the fetal liver cDNA library, and have got a series of specific proteins that interact with FasL protein. Using the bioinformatics, we analyzed the interacting proteins in the mechanism of hepatic metastasis of colorectal carcinoma.. We have screened several proteins that interaction with FasL protein, including metallothionein 1K, 1G, 2A, cathepsin B, fatty acid synthase, interferon alpha-inducible protein 27, phospholipid scramblase, Ser/Thr-like kinase, anchor attachment protein, fibulin-5.. We have successfully constructed the yeast two-hybrid system, and preliminary identified that the interaction between FasL, metallothionein, cathepsin and anchor attachment protein is radically related to the hepatic metastasis of colorectal carcinoma.

Topics: Cathepsin B; Cloning, Molecular; Colorectal Neoplasms; Fas Ligand Protein; Gene Library; Humans; In Vitro Techniques; Liver Neoplasms; Membrane Glycoproteins; Metallothionein; Protein Binding; Tumor Necrosis Factors; Two-Hybrid System Techniques; Yeasts

The present study aimed at analysing the intensity of apoptosis, as related to the expression of pro- and anti-apoptotic cellular markers (cas-3, MT, Ki-67 antigen), and at evaluating their expression in relation to the survival time of patients with colorectal adenocarcinoma. Material for the studies was obtained from 40 patients with primary colorectal adenocarcinomas (G2, T3N0M0), treated at the Lower Silesia Centre of Oncology in Wrocław. Tumour samples were fixed in 4% buffered formalin and embedded in paraffin blocks. In obtained paraffin sections, TUNEL reaction (detection of apoptosis) and immunocytochemical reactions were performed (detection of cas-3, MT and Ki-67 antigen expression). The results disclosed a weak correlation between the intensity of apoptosis and the expression of MT, cas-3 and Ki-67 antigen (r = 0.18; r = 0.33; r = 0.15, respectively) in cells of colorectal adenocarcinomas. The survival time of the patients was shorter, when the apoptosis and expression of Ki-67 antigen were highly pronounced. The time periods of the patients' survival showed no correlation with the expression of cas-3 or MT. The obtained results point to the key role of apoptosis and proliferation processes in the clinical course of colorectal adenocarcinomas.

Topics: Adenocarcinoma; Apoptosis; Caspase 3; Caspases; Colorectal Neoplasms; Humans; Ki-67 Antigen; Metallothionein; Survival Analysis

Metallothionein (MT) expression is observed in various carcinomas, but its role is not fully understood. To clarify the clinicopathological significance of MT, 87 colorectal adenomas and 128 early-stage carcinomas were immunohistochemically analysed for MT expression. The degree of MT immunostaining of a specimen was graded according to the proportion of MT-positive cells; negative (<5%) and positive (focally 5-50%, diffusely >50%). MT expression significantly decreased with tumour development. For carcinomas, MT-positivity was significantly associated with depth of invasion (T1 60% versus T2 33%; P<0.01), vascular involvement (positive 35% versus negative 61%; P<0.01) and morphology (polypoid 62% versus depressed 26%; P<0.01). Regarding MT-positive distribution, the diffuse-positive rate in MT-positive polypoid lesions was 28%, while MT-positive depressed lesions were all diffusely stained (P<0.01). In conclusion, our results suggested that decreasing MT expression is an early event in colorectal carcinogenesis and may reflect local invasion. Furthermore, MT-positive distribution may reflect genetic differences between the polypoid and depressed-type.

Topics: Adult; Aged; Aged, 80 and over; Colonic Polyps; Colorectal Neoplasms; Female; Humans; Immunohistochemistry; Intestinal Mucosa; Male; Metallothionein; Middle Aged; Neoplasm Proteins; Proliferating Cell Nuclear Antigen

Metallothioneins (MTs) are zinc-binding proteins whose overexpression may lead to sequestration of zinc ions and consequently to functional inactivation of the p53 tumor suppressor gene. The aim of the study was to investigate the potential role of MTs in the carcinogenesis of ulcerative colitis (UC) as well as possible effects on p53 function. The monoclonal antibodies E9 (anti-MT), DO-7, and 1801 (anti-p53) and the polyclonal antibody CM-1 (anti-p53) were used to stain formalin-fixed, paraffin-embedded colon specimens obtained from 14 patients with UC-associated colorectal carcinoma (CAC), 13 with high-grade dysplasia (HGD), 10 with low-grade dysplasia (LGD), and 30 with UC without dysplasia or carcinoma. Statistical significance (p <0.05) was assessed using Fisher's exact test. Positive MT staining (> 20% of tumor, dysplastic, or epithelial cells) was found in most UC and LGD but in only a small percentage of HGD and CAC (p <0.01 for CAC vs. UC and LGD vs. HGD). Positive p53 immunoreactivity was observed predominantly in HGD and CAC but not in LGD and UC (p <0.01 for CAC vs. UC and HGD vs. LGD). In histologically normal tissue neighboring CAC, significant MT expression was found in six of seven specimens with simultaneous lack of p53 expression. MT overexpression may represent an important early step in the development of CAC independent of p53 expression and should be investigated in the long term as an independent cancer risk factor in UC.

Topics: Adenocarcinoma; Adult; Aged; Biomarkers; Cell Transformation, Neoplastic; Colitis, Ulcerative; Colorectal Neoplasms; Humans; Immunohistochemistry; Metallothionein; Middle Aged; Precancerous Conditions; Risk Factors; Tumor Suppressor Protein p53

Metallothionein (MT) is a small protein with a high affinity for divalent heavy metal ions. This metalloproteinis involved in many (patho)physiological processes, like metal homeostasis and detoxification, cell proliferation, apoptosis, therapy resistance, and protection against oxidative damage. Alterations in the immunohistochemical expression of MT have been reported for various human tumors, and a high expression has been found to be associated with a poor clinical outcome. We showed previously that gastrointestinal cancer is accompanied by a decrease in MT expression, but the most malignant phenotypes had the highest MT levels. The purpose of the present study was to assess the clinical relevance of MT in gastrointestinal cancer.. In this study, we determined the MT levels, by radioimmunoassay, in intestinal tissue of 251 patients with colorectal cancer and 81 patients with gastric cancer and assessed the relation with the overall survival of these patients.. More than 74% of the carcinomas were found to have a lower MT level than their corresponding normal mucosa. In colorectal cancer patients, but not in gastric cancer patients, a high MT level in both the carcinomas and normal mucosa was, however, significantly associated with a poor overall survival, independently from clinicopathological features.. Overexpression of MT in intestinal tissue of colorectal cancer patients is a prognostic marker for a poor overall survival. In gastric cancer, however, MT expression in the gastric mucosa is not of prognostic significance. This observation emphasizes the clinical relevance of this multifunctional metalloprotein in colorectal carcinogenesis and therapy.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Case-Control Studies; Colorectal Neoplasms; Female; Gastrointestinal Neoplasms; Humans; Male; Metallothionein; Middle Aged; Mucous Membrane; Prognosis; Prospective Studies; Radioimmunoassay; Survival Rate

The aim of this study is to clarify whether the expression of metallothionein (MT) is related with the malignant potential in primary colorectal cancer and/or synchronous liver metastasis. Immunohistochemical staining for MT was performed on the specimens of adenocarcinoma of the colon and rectum and its liver metastases in 34 patients treated with curative surgery, respectively. Expression of MT was compared with clinicopathological variables and patient survival. In patients with primary colorectal cancer, positive expression was found in 7 of 34 (20.6%) patients, but MT was not detected in any of the cases of liver metastases (0%; p = 0.0111). In the primary tumor, positive MT expression was significantly associated with a higher degree of lymph node involvement (mean +/- SD: 48.4 +/- 33.8 vs. 18.6 +/- 24.4% in MT-positive and MT-negative tumors, respectively; p = 0.0122). The survival rate in the patients with MT-negative tumors was significantly better than that in those with MT-positive tumors as primary sites (p = 0.0198). MT expression in colorectal cancer may be a potential marker affecting lymph node metastases and may be a predictor of a poor prognosis, particularly in patients with synchronous liver metastases.

Topics: Adenocarcinoma; Aged; Antineoplastic Combined Chemotherapy Protocols; Chemotherapy, Adjuvant; Cisplatin; Colorectal Neoplasms; Combined Modality Therapy; Doxorubicin; Drug Resistance, Neoplasm; Female; Fluorouracil; Humans; Life Tables; Liver Neoplasms; Male; Metallothionein; Middle Aged; Mitomycin; Multivariate Analysis; Neoplasm Proteins; Prognosis; Survival Analysis

Because the status of the inherent drug-resistance of colorectal cancers remains obscure, human colorectal cancers with no neoadjuvant therapy were retrospectively investigated regarding the expression of three drug-resistant proteins: metallothionein, glutathione S-transferase-pi, and P-glycoprotein.. Paraffin-embedded tissues of 130 colorectal cancers (Dukes A, 20; B, 49; C, 41; D, 20) obtained by surgical resections from 1982 to 1989 were used. The three proteins were immunostained by the streptavidin-biotin complex method. The immunostaining was judged to be positive if more than 5 percent of cells showed positive staining by use of cell analysis system. The data were compared with clinicopathologic features (Dukes A-D) and patients' prognosis (Dukes AC).. Metallothionein, glutathione S-transferase-pi, and P-glycoprotein were positively expressed in 91 (70 percent), 30 (23 percent), and 98 (75 percent), respectively. A total of 120 (86 percent) expressed at least one drug-resistant protein. No intergroup differences were observed between positive and negative expressions of the proteins and their clinicopathologic features except tumor location. Rectal cancers positively expressed P-glycoprotein and three proteins more frequently. Twenty-six (20 percent), 65 (50 percent), and 21 (16 percent) cancers positively expressed one, two, and three proteins, respectively. The disease-free survival rates of patients with Dukes A through C cancer with positive staining for one, two, and three proteins were 100, 94, and 83 percent (at 1 year); 100, 72, and 51 percent (at 3 years); and 94, 66, and 38 percent (at 5 years), respectively (Kaplan-Meier with log-rank test; P = 0.016). In the multivariate Cox analysis, age, Dukes stage, tumor size, and glutathione S-transferase-pi were independent prognostic factors.. The patients with concurrent expression of drug-resistant proteins in their cancers had worse prognoses. Examining drug-resistant proteins in colorectal cancers may be useful in selecting adjuvant chemotherapy and in predicting prognosis more accurately.

Topics: Adenocarcinoma; Aged; ATP Binding Cassette Transporter, Subfamily B, Member 1; Biomarkers, Tumor; Colorectal Neoplasms; Combined Modality Therapy; Disease-Free Survival; Female; Glutathione S-Transferase pi; Glutathione Transferase; Humans; Immunoenzyme Techniques; Isoenzymes; Male; Metallothionein; Neoplasm Invasiveness; Retrospective Studies

Metallothionein (MT) is a small thiol-rich metalloprotein with antioxidant properties, involved in tumour pathophysiology and therapy resistance. In order to assess the contribution of MT in gastrointestinal carcinogenesis, this study examined both the MT content by radioimmunoassay and the MT localization by immunohistochemistry in pairs of neoplastic and normal-appearing human gastrointestinal tissues. In addition, the relationship between MT expression and major clinicopathological parameters was assessed. The MT concentration of gastric carcinomas and of colorectal adenomas, carcinomas, and liver metastases was found to be significantly lower than that of corresponding normal-appearing tissue. A relatively high MT content, however, was found to be associated with the villous character of colorectal adenomas and with the Dukes' stage of colorectal carcinomas, indicating a relationship between MT level and malignant potential. Immunohistochemical evaluation showed a fairly good correlation with these quantitative data. MT was found to be expressed at a low level and in a patchy pattern in the gastrointestinal neoplastic and metastatic tissues, whereas in normal-appearing gastrointestinal mucosa MT was uniformly distributed in the cytoplasm and/or nucleus of apical cells. Although in the gastric cancer patients no association was found between the MT concentration and the clinicopathological parameters, the strong MT expression in areas with intestinal metaplasia, known to have neoplastic potential, further points to a relationship between this antioxidant metalloprotein and the malignant character of cells. Gastrointestinal neoplasms are apparently accompanied by a low level and decreased expression of MT, but those with a relatively high level seem to have an increased malignant potential. Further studies will be required to determine the clinical relevance of these observations.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Colorectal Neoplasms; Female; Fluorescent Antibody Technique, Indirect; Humans; Liver Neoplasms; Male; Metallothionein; Middle Aged; Radioimmunoassay; Stomach Neoplasms

Metallothionein is a protein with affinity for metals and is present in several tumors. We examined its immunohistochemical expression in 37 resected primary liver tumors and 117 colorectal metastases. The reaction was intense in the two fibrolamellar hepatocellular carcinomas and in many of the hepatocytes of the pseudotumor case of focal nodular hyperplasia. The reaction was low or moderate in 5 of 17 ordinary hepatocellular carcinomas and in 4 of 14 cholangiocellular carcinomas. There was no reaction in one case each of spindle cell hepatocellular carcinoma, oncocytic adenoma and hemangioendothelial sarcoma. In the metastases, the reaction was low or moderate in 14 cases and negative in 103. Surrounding hepatocytes and stromal cells were more or less positive in all cases.

Topics: Carcinoma, Hepatocellular; Colorectal Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Liver Neoplasms; Metallothionein

The insulin-like growth factor II receptor (IGFIIR) has been implicated as a tumor suppressor gene in human malignancy. Frequent mutation, loss of heterozygosity, and microsatellite instability (MSI) directly affecting the IGFIIR gene have been reported in several primary human tumor types. However, to our knowledge, dynamic functional evidence of a growth-suppressive role for IGFIIR has not yet been provided. We identified one MSI-positive colorectal carcinoma cell line, SW48, with monoallelic mutation in IGFIIR identical to that seen in primary colorectal carcinomas. A zinc-inducible construct containing the wild-type IGFIIR cDNA was stably transfected into SW48 cells. Growth rate and apoptosis were compared between zinc-treated, untreated, and untransfected cells. A twofold increase in IGFIIR protein expression was detected after zinc treatment in discrete clonal isolates of transfected SW48 cells. Moreover, zinc induction of exogenous wild-type IGFIIR expression reproducibly decreased growth rate and increased apoptosis. These data prove that wild-type IGFIIR functions as a growth suppressor gene in colorectal cancer cells and provide dynamic in vitro functional support for the hypothesis that IGFIIR is a human growth suppressor gene.

Topics: Adenocarcinoma; Apoptosis; Cell Division; Colorectal Neoplasms; DNA, Complementary; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor II; Loss of Heterozygosity; Metallothionein; Microsatellite Repeats; Neoplasm Proteins; Polymerase Chain Reaction; Promoter Regions, Genetic; Receptor, IGF Type 2; Tumor Cells, Cultured; Zinc

To investigate the role of metallothionein in colorectal tumours and the possible relation with other factors associated with tumour progression: expression of cathepsin D (CD), CD44, p53, Rb, bcl-2, c-erbB-2, epidermal growth factor receptor (EGFR), proliferation indices (Ki-67, proliferating cell nuclear antigen (PCNA)), and conventional clinicopathological variables.. The immunohistochemical expression of metallothionein was investigated in 23 cases of colorectal adenoma and 94 adenocarcinomas. Metallothionein expression was examined by the avidinbiotin peroxidase immunoperoxidase (ABC) using the monoclonal mouse antibody E9, on formalin fixed, paraffin embedded tissue.. Positive metallothionein expression (> 5% of neoplastic cells) was observed in 30.4% of adenomas and 25.5% of adenocarcinomas, while 8.7% of adenomas and 14.9% carcinomas showed focal metallothionein positivity. In contrast, 60.9% of adenomas and 59.6% of carcinomas almost completely lacked metallothionein expression. In the series of adenocarcinomas, metallothionein expression was inversely correlated with CD44 in neoplastic cells (p = 0.01). There was no statistically significant difference of metallothionein expression, or the other variables examined, between adenocarcinomas and adenomas.. Metallothionein expression does not seem to indicate aggressive biological behaviour in colorectal adenocarcinomas, in comparison with the other types of carcinoma. The inverse correlation with CD44 could suggest that the decreased metallothionein expression may contribute to the metastatic spread of the lymph node involvement in colorectal cancer. Metallothionein expression does not seem to represent an independent prognostic marker in colorectal cancer.

Topics: Adenocarcinoma; Biomarkers, Tumor; Cathepsin D; Colorectal Neoplasms; Female; Humans; Hyaluronan Receptors; Male; Metallothionein; Middle Aged; Prognosis

We describe the engineering of a novel single-chain fragment (scFv) metallothionein (MET) containing anti-carcinoembryonic antigen (CEA) antibody (referred to as MET-scFv) for use as a diagnostic imaging agent in colorectal cancer.. Site-directed cloning of annealed oligonucleotides, containing both the MET and a c-myc tag sequence, into a pUC19-based expression vector enabled soluble secreted protein expression from Escherichia coli. Affinity purification was used to purify the protein using an anti-c-myc affinity column. The specificity of both the unlabeled and labeled MET-scFv for CEA was demonstrated by solid-phase enzyme-linked immunosorbent assay and radioimmunoassay and by fluorescence-activated cell sorting analysis on CEA-expressing human colorectal LS-174T cells. Technetium-99m labeling was achieved using a Zn2+ transchelation step, enabling direct 99mTc transfer without separate reduction of MET. In vitro stability was demonstrated by fast protein liquid chromatography analysis of labeled MET-scFv, incubated with bovine serum albumin (BSA), transferrin and mouse serum. Last, in vivo pharmacokinetics, biodistribution and imaging were performed.. Yields of 6 mg/liter induced culture purified protein were achieved. Successful site-specific labeling was demonstrated using a Zn2+ transchelation modification of a pretinning method, which also enabled lower amounts of the reducing agent to be used. The specificity for CEA was retained after labeling. Despite a rapid serum clearance (t(1/2alpha) = 2.8 min), adequate localization to tumor of 5.37% injected dose/g at 4 hr was demonstrated. Moreover, the short-lived t(1/2alpha) of scFv, its early tumor targeting and rapid blood-pool clearance gave tumor-to-blood ratios of 2.07 by 4 hr, enabling early gamma camera imaging. Successful and specific imaging was achieved using LS-174T xenografts in nude mice by 3-6 hr.. A recombinant MET containing scFv was successfully expressed, purified and labeled with 99Tc. The stable site-specific labeling of 99Tc, combined with the rapid plasma clearance of the scFv, led to successful early in vivo imaging of xenografted mice.

Topics: Animals; Antibodies, Monoclonal; Carcinoembryonic Antigen; Colorectal Neoplasms; Female; Genetic Engineering; Humans; Isotope Labeling; Metallothionein; Mice; Mice, Inbred BALB C; Mice, Nude; Mice, SCID; Neoplasm Transplantation; Radioimmunodetection; Recombinant Proteins; Technetium; Tissue Distribution

Metallothionein (MT) overexpression occurs frequently in human tumours but the underlying mechanism is unknown. Morphologically normal-appearing mucosa from human colorectal carcinoma resection specimens and of the colons of ageing laboratory mice contains scattered single crypts whose cells show uniformly increased MT immunostaining, suggesting that MT overexpression arises directly from random crypt stem cell somatic mutation, followed by colonization of the clonal unit by the mutated progeny. This hypothesis has now been tested by quantifying the frequency of immunocytochemically detectable monocryptal colorectal MT overexpression, 5 and 30 days after injection of 8-week-old mice with a single dose of the mutagen dimethylhydrazine (DMH, 30 mg/kg subcutaneous). Otherwise normal-appearing MT-positive crypts were recorded as either wholly or partially involved by the overexpressing phenotype. Five days after DMH injection, the median frequency of partially involved MT-positive crypts was 11.7 x 10(-4), declining significantly to 1.8 x 10(-4) at 30 days (Mann-Whitney U, P < 0.01). In contrast, the median frequency of wholly involved crypts was 0.2 x 10(-4) at 5 days, increasing significantly (P < 0.005) to 12.9 x 10(-4) at 30 days. The frequency of MT-positive crypts and the time course of evolution of partially involved to wholly involved forms were similar to those described for mutation-induced crypt-restricted loss of glucose-6-phosphate dehydrogenase activity in mice treated with an identical DMH regimen. The findings indicate that cellular MT overexpression can occur as a direct consequence of somatic mutation, either cis-activating mutation(s) of the MT gene itself, or trans-activating mutation(s) of other genes involved in controlling MT expression. This is likely to be an important mechanism underlying MT overexpression in neoplasia. Such mutation-induced aberrant MT expression may be involved in the acquisition of selective cellular growth of survival advantage during tumour progression.

Topics: 1,2-Dimethylhydrazine; Animals; Clone Cells; Colon; Colorectal Neoplasms; Female; Gene Expression; Intestinal Mucosa; Male; Metallothionein; Mice; Mice, Inbred BALB C; Mutagens; Mutation; Neoplasm Proteins

Topics: Adenocarcinoma; Colorectal Neoplasms; Humans; Immunohistochemistry; Metallothionein

The immunohistochemical distribution pattern of metallothionein, a low molecular weight protein with strong affinity for divalent heavy metal ions, has been investigated in normal and neoplastic conditions of the large bowel. Utilizing a monoclonal mouse antibody the following formalin-fixed paraffin-embedded surgical or biopsy samples were studied: tubulo-villous adenomas (8 cases); adenocarcinomas with various degree of differentiation (85), nine of which were mucinous-type; synchronous tubular or tubulo-villous adenomas separate from carcinomas (30); transitional mucosa (45); metastases in lymph nodes (43); and distant metastases (45). Twenty biopsies from the right and left colon of 10 patients affected by irritable bowel syndrome were also analyzed. Normal colonic mucosa as well as transitional mucosa showed metallothionein immunopositivity in enterocytes at the luminal surface and crypts. Evident nuclear and cytoplasmic staining was encountered in tubulo-villous adenomas; the same reactivity was noted in the basal glandular component of colorectal carcinomas-synchronous adenomas, while less intense staining was noted in the apical villous portions. A variable metallothionein immunostaining was observed in adenocarcinomas (62.3%), in lymph node (55.8%) and distant hepatic (17.2%) and omental (43.8%) metastases, although it was not always concordant with that reported in the corresponding primary tumour. Whether the metallothionein positivity observed in normal and neoplastic cells is the result of expression of a stable form of the protein or an accumulation in the nucleus and cytoplasm remains to be clarified.

Topics: Adenocarcinoma; Adenoma; Colon; Colorectal Neoplasms; Humans; Immunohistochemistry; Intestinal Mucosa; Metallothionein; Rectum

Metallothioneins (MTs), a set of ubiquitous low-molecular-weight proteins essential for the protection of cells against heavy metal ion toxicity, were demonstrated immunohistochemically using a monoclonal antibody (E9) against a conserved epitope of I and II isoforms in a series of 109 colorectal adenocarcinomas. In a semiquantitative analysis strong MT expression in the majority of tumour cells was observed in 34 (31%) cases, 24 (22%) tumours showed a focal MT positivity, and 51 (47%) almost completely lacked MT expression. These differences in MT expression were statistically significantly (P < 0.05) associated with the tumour stage (Dukes classification) and the lymph node involvement at the time of operation (pN stages). However, in contrast to previous findings obtained on a variety of tumours, MT positivity was associated with a favourable clinical outcome in colonic carcinoma, which may indicate their different biological behaviour. Survival curves of cases with MT-positive and MT-negative status differed from each other in a univariate analysis (Mantel-Haenszel: 8.9, P < 0.05) but lost significance when a multivariate analysis was carried out by means of the Cox proportional regression model with Dukes' stages as a stratification factor. It is concluded that immunohistochemically demonstrated MT expression is significantly associated with tumour stages but does not represent an independent prognostic variable in colorectal cancer. However, it may provide important information about some of the biological mechanisms underlying progression in colorectal cancer.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Colorectal Neoplasms; Female; Humans; Immunohistochemistry; Male; Metallothionein; Middle Aged; Neoplasm Staging; Prognosis; Survival Analysis

In patients with primary biliary cirrhosis and Wilson's disease liver copper concentrations become elevated during the evolution of the disorder. The accumulated copper is thought to be detoxified by metallothionein, a protein which binds copper and zinc. In liver metastasis of colorectal cancer, copper and zinc concentrations are usually decreased compared to normal liver tissue, but little is known about the concomitant metallothionein levels. In the present study metallothionein concentrations were determined in archival liver samples from patients with primary biliary cirrhosis and Wilson's disease, and in both normal and malignancy-containing liver samples from patients with metastasis from a colorectal adenocarcinoma. Twenty-seven control liver samples contained 3.98 +/- 1.55 mg metallothionein/g protein. From the 21 liver samples of patients with primary biliary cirrhosis, which had a mean metallothionein concentration of 6.06 +/- 5.03 mg/g protein, 6 were above the highest control level. Liver metallothionein concentrations for the 8 patients with Wilson's disease were significantly elevated (10.98 +/- 6.93 mg/g protein, p < 0.005 vs. controls and p < 0.05 vs. primary biliary cirrhosis). In the 11 liver metastases from colorectal adenocarcinomas metallothionein concentrations (1.17 +/- 0.90 mg/g protein) were significantly (p < 0.005) lower than surrounding normal liver tissue (4.25 +/- 1.75 mg/g protein). We conclude that in primary biliary cirrhosis and Wilson's disease increased liver metallothionein concentrations may detoxify the accumulated copper. Furthermore, liver metastasis of colorectal cancer contains less metallothionein than the surrounding normal liver tissue.

Topics: Adenocarcinoma; Adult; Aged; Colorectal Neoplasms; Female; Hepatolenticular Degeneration; Humans; Liver; Liver Cirrhosis, Biliary; Liver Neoplasms; Male; Metallothionein; Middle Aged; Radioimmunoassay

Alterations in gene expression associated with colorectal cancer have been difficult to study because mucosal cell progenitors are not available in culture. We therefore examined specific genes in approximately 100 human colon cancer cell lines using complementary DNA probes and found profound alterations and heterogenity of gene expression in human colorectal carcinoma. Our data imply that understanding human colorectal cancer will not be accomplished by studying one or two oncogenes in a limited number of cell lines or fresh human tissue. More appropriate postulates of transformation to dictate experimental design may include the investigation of proposed three-dimensional structural changes of interface chromatin or other generalized structural relationships that could predispose to an aberrant gene expression program during transformation. Furthermore, focusing on mechanisms of initiation and defining the molecular genetic markers of gastrointestinal mucosal initiation should lead to a more focused set of genetic, rather than epigenetic, mechanisms that underlie oncogenic transformation.

Topics: Blotting, Northern; Carrier Proteins; Cell Line; Colorectal Neoplasms; ErbB Receptors; Gene Expression; Humans; Laminin; Metallothionein; Microfilament Proteins; Proto-Oncogenes; RNA, Messenger; RNA, Neoplasm