metallothionein and Pancreatic-Neoplasms

Copper is one of the indispensable trace metal elements in organisms, but excess copper means cytotoxicity. Cells protect themselves by storing excess copper in copper-binding proteins. Metallothioneins (MTs) are a group of low-molecular-weight, cysteine-rich proteins, which are well known for sensing and binding the overcharged Zn(Ⅱ), Cd(Ⅱ), and Cu(Ⅰ) in cells. However, there are only few reports on MTs that can specifically respond to intracellular copper ions in mammals in real-time. Here, we screened copper-response MTs in pancreatic cancer cells through data-mining, RNA-seq, and qPCR analysis. We found that MT1E, MT1F, and MT1X mRNA were significantly upregulated after exogenous copper ion induction. By constructing the stable cell lines with MT1E, MT1F, or MT1X promoter-driven EGFP as reporters, we found that only PMT1F-EGFP could specifically and stably report the intracellular Cu(Ⅰ) changes in multiple cell lines including Panc-1, 8988T, 293T, HepG2, and normal hepatic cells, indicating that PMT1F-EGFP is an ideal in vivo Cu(Ⅰ) reporter. Using the PMT1F-EGFP reporter, we found that MEK inhibitors (U0126) and Astragaloside IV could significantly increase intracellular copper ions. According to these results, PMT1F-EGFP reporter can sense intracellular copper change and can be used to screen copper-target drugs and study copper-related cellular physiology and pathology.

Topics: Apoptosis; Cell Proliferation; Copper; Green Fluorescent Proteins; Humans; Metallothionein; Pancreatic Neoplasms; Tumor Cells, Cultured

The low survival of patients with pancreatic ductal adenocarcinoma (PDAC) makes the treatment of this disease one of the most challenging task in modern medicine. Here, by mining a large-scale cancer genome atlas data set of pancreatic cancer tissues, we identified 21 long noncoding RNAs (lncRNAs) that significantly associated with overall survival in patients with PDAC (P < .01). Further analysis revealed that 8 lncRNAs turned out to be independently correlated with patients' overall survival, and the risk score could be calculated based on their expression. To obtain a better predicting power, we integrated lncRNA data with a total of 410 differently expressed messenger RNAs (mRNAs) screened from PDAC and normal tissues in gene expression omnibus (GEO) database. The integration resulted in a much better panel including 8 lncRNAs (RP3.470B24.5, CTA.941F9.9, RP11.557H15.3, LINC00960, AP000479.1, LINC00635, LINC00636, and AC073133.1) and 8 mRNAs (DHRS9, ONECUT1, OR8D4, MT1M, TCN1, MMP9, DPYSL3, and TTN) to predict prognosis. A functional evaluation showed that these lncRNAs might play roles in pancreatic secretion, cell adhesion, and proteolysis. Using normal and pancreatic cancer cell lines, we confirmed that a majority of identified lncRNAs and mRNAs showed altered expressions in pancreatic cancer cells. Especially, LINC01589, LINC00960, TCN1, and MT1M showed a profoundly increased expression in pancreatic cancer cells, which suggests their potentially important role in pancreatic cancer. The results of our work indicate that lncRNAs have vital roles in PADC and provide new insights to integrate multiple kinds of markers in clinical practices.

Topics: Biomarkers, Tumor; Carcinoma, Pancreatic Ductal; Data Mining; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Male; Metallothionein; Oligonucleotide Array Sequence Analysis; Pancreatic Neoplasms; Prognosis; RNA, Long Noncoding; RNA, Messenger; Survival Analysis

Lumican, a member of the class II small leucine-rich proteoglycan family, regulates the assembly and diameter of collagen fibers in the extracellular matrix of various tissues. We previously reported that lumican expression in the stromal tissues of pancreatic ductal adenocarcinoma (PDAC) correlates with tumor invasion, and tends to correlate with poor prognosis. Lumican stimulates growth and inhibits the invasion of a PDAC cell line. In the present study, we performed a global shotgun proteomic analysis using lumican-overexpressing PANC‑1 cells and lumican downregulated PANC‑1 cells to identify candidate proteins that are regulated by lumican and related to cell growth and invasion in PDAC cells. A total of 448 proteins were identified from lumican-overexpressing PANC‑1 and control cells. Additionally, 451 proteins were identified from lumican-downregulated PANC‑1 cells and control cells. As a result of semi-quantification based on spectral counting, 174 differentially expressed proteins were identified by lumican upregulation, and 143 differentially expressed proteins were identified by lumican downregulation. The expression levels of 24 proteins, including apoptosis- and invasion-related proteins correlated with lumican expression levels. It is likely that the expression of these proteins is regulated by lumican, and that they are involved in apoptosis and invasion in PDAC. These findings suggest that lumican may be involved in cell growth and invasion through the regulation of these 24 proteins expressed in PDAC.

Topics: Apoptosis; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Proliferation; Chondroitin Sulfate Proteoglycans; Down-Regulation; Gene Expression; Humans; Keratan Sulfate; Lumican; Metallothionein; Neoplasm Invasiveness; Pancreatic Neoplasms; Prognosis; Protein Isoforms; Proteomics; Up-Regulation

To compare the expression of metallothionein (MT) genes and proteins in six human pancreatic cancer cell strains and two human pancreatic cancer drug-resistant cell strains and to explore the relationship between the expression of the MT and pancreatic cancer cell chemo-resistance.. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to determine the MT isoform-specific mRNA, and cadmium/hemoglobin saturation-electrochemistry to determine MT protein levels.. MT protein expression in the pancreatic cancer cell strains was encoded by MT-1A, MT-1B, MT-1E, MT-1F, MT-1G, MT-1X, and MT-2A genes. The expression of MT proteins was upregulated and MT-1B, MT-1E, MT-1X, MT-2A genes overexpressed in human pancreatic cancer drug-resistant cell lines (P < 0.05).. Expressions of MT proteins and genes correlate with the proliferation and chemoresistance of human pancreatic cancer cell strains.

Topics: Cell Division; Drug Resistance, Neoplasm; Humans; Metallothionein; Pancreatic Neoplasms; RNA, Messenger; Tumor Cells, Cultured

The possible involvement of metallothionein (MT) in pancreatic ductal carcinogenesis by N-nitrosobis(2-oxopropyl) amine (BOP) in hamsters was investigated. Hamsters received subcutaneous (s.c.) injections with dissolved BOP to 70 mg/kg body weight (BW) followed 7 days later by 20 mg/kg BW BOP and they were sacrificed at 4, 11, 16 and 27 weeks after the beginning of the experiment. MT expression was studied by immunohistochemistry and MT contents were assayed biochemically. Pancreatic ductal hyperplasias were developed from 11 weeks on and carcinomas from 16 weeks on, the incidence of the latter reaching 73% at the end of experiment. However, while normal appearing proliferating duct cells were sometimes positive, MT expression was not evident in hyperplasia (H), atypical hyperplasia (AH) or carcinoma (C), and MT contents did not significantly differ in pancreas of hamsters receiving saline or BOP at any time point. The results suggest that MT is not involved in pancreatic duct carcinogenesis. However, the presence of MT in proliferating ducts not related to carcinogenesis may suggest some unknown role for MT in cellular homeostasis.

Topics: Animals; Carcinogens; Cell Division; Cricetinae; Epithelial Cells; Female; Immunohistochemistry; Mesocricetus; Metallothionein; Nitrosamines; Pancreatic Ducts; Pancreatic Neoplasms; Time Factors

Metallothioneins (MTs) are intracellular proteins that bind to metal ions and are involved in heavy metal homeostasis and detoxification. Pancreatic islets were shown to be positive for zinc-containing matrix metalloproteinase-2 and -9 by immunocytochemical staining. The immunolocalization of matrix metalloproteinases in pancreatic islets prompted us to study further the link between zinc and MT in 34 cases of pancreatic endocrine neoplasms, including insulinomas, glucagonomas, gastrinomas, pancreatic polypeptide-omas, and non-functioning endocrine neoplasms. Four types of islet cells were found to be positive for MT, whereas pancreatic endocrine neoplasms mostly were either weakly positive or negative for MT. The presence of MT in normal islet cells and pancreatic endocrine neoplasms is consistent with the notion that MTs modulate zinc homeostasis and metabolism in pancreatic islet cells and pancreatic endocrine neoplasms as those tissues contain zinc-containing matrix metalloproteinases.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Chromogranin A; Chromogranins; Endocrine Gland Neoplasms; Female; Gastrins; Glucagon; Humans; Immunohistochemistry; Insulin; Islets of Langerhans; Male; Metallothionein; Middle Aged; Pancreatic Neoplasms

Isolated pancreatic islets from rat and mouse and the insulinoma cell lines, betaHC9 and RINm5F, were investigated to determine the regulation of metallothionein (MT). Dexamethasone (DEX) increased rat and mouse islet and insulinoma cell MT levels in a time- and concentration-dependent manner. Rat islet MT expression was increased with interleukin-1beta (IL-1beta), but not tumor necrosis factor-alpha (TNF). However, MT induction by IL-1beta and TNF was synergistic with DEX in rat islets and insulinoma cells. Mouse islet MT failed to respond to IL-1beta alone, although IL-1beta and TNF were synergistic. IL-1beta and TNF did not synergize with DEX for mouse islet MT induction. Zinc sulfate induced MT in rat islets but not mouse islets. MT messenger RNA levels were significantly increased in rat islets in response to DEX and IL-1beta plus DEX. The inducible nitric oxide synthase inhibitors N(G)-monomethyl-L-arginine and aminoguanidine failed to inhibit IL-1beta induced MT levels in insulinoma cells, and the nitric oxide generating agent sodium nitroprusside failed to significantly affect MT levels. Phorbol dibutyrate increased MT levels in rat islets and betaHC9 cells, but phorbol dibutyrate and IL-1beta effects were not additive. Transgenic MT-null and wild-type mouse islets had similar insulin contents, but basal and glucose-stimulated insulin release from MT-null islets were significantly lower than in wild-type islets. Blood glucose levels in MT-null mice were, however, slightly lower than those in wild-type mice. Thus, MT induction in pancreatic islets and beta-cells is regulated by cytokines and DEX, and protein kinase C activation may play a role. However, regulation of MT induction in mouse and rat islets differs. MT also appears to modulate insulin release from pancreatic islets.

Topics: Animals; Blood Glucose; Dexamethasone; Drug Synergism; Glucocorticoids; In Vitro Techniques; Insulin; Insulin Secretion; Insulinoma; Interleukin-1; Islets of Langerhans; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Knockout; Pancreatic Neoplasms; Phorbol 12,13-Dibutyrate; Protein Kinase C; Rats; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Using a dominant-negative mutant receptor (DNR) approach in transgenic mice, we have functionally inactivated transforming growth factor-beta (TGF-beta) signaling in select epithelial cells. The dominant-negative mutant type II TGF-beta receptor blocked signaling by all three TGF-beta isoforms in primary hepatocyte and pancreatic acinar cell cultures generated from transgenic mice, as demonstrated by the loss of growth inhibitory and gene induction responses. However, it had no effect on signaling by activin, the closest TGF-beta family member. DNR transgenic mice showed increased proliferation of pancreatic acinar cells and severely perturbed acinar differentiation. These results indicate that TGF-beta negatively controls growth of acinar cells and is essential for the maintenance of a differentiated acinar phenotype in the exocrine pancreas in vivo. In contrast, such abnormalities were not observed in the liver. Additional abnormalities in the pancreas included fibrosis, neoangiogenesis and mild macrophage infiltration, and these were associated with a marked up-regulation of TGF-beta expression in transgenic acinar cells. This transgenic model of targeted functional inactivation of TGF-beta signaling provides insights into mechanisms whereby loss of TGF-beta responsiveness might promote the carcinogenic process, both through direct effects on cell proliferation, and indirectly through up-regulation of TGF-betas with associated paracrine effects on stromal compartments.

Topics: Animals; Apoptosis; Cell Differentiation; Cell Division; Fibronectins; Gene Expression; Homeostasis; Immunohistochemistry; Liver; Metallothionein; Mice; Mice, Transgenic; Mutation; Pancreas; Pancreatic Neoplasms; Phenotype; Proliferating Cell Nuclear Antigen; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

Metallothioneins are a family of intracellular metalloproteins that have been thought to be involved in anticancer drug resistance. However, the role of metallothioneins in pancreatic cancer has not been investigated in detail. The immunohistochemical localization of metallothionein was examined in normal human adult pancreas tissue and in 75 pancreatic duct cell carcinomas, using monoclonal anti-metallothionein antibody. Furthermore, in vitro studies on the sensitivity of pancreatic cancer to cisplatin were performed in 10 cases of pancreatic carcinoma. Metallothionein staining was weakly positive in the acinar and islet cells and intralobular ducts but was negative in the large pancreatic ducts. In pancreatic carcinomas, metallothionein staining was diffusely positive in 6 (8%), focally positive in 25 (33%) and negative in 44 (59%) of the 75 pancreatic carcinomas. The expression of metallothioneins in pancreatic tumors was related to metastasis, poor prognosis and poor histological grading (poorer glandular differentiation and nuclear anaplasia). The in vitro study of tumor sensitivity to cisplatin showed no significant correlation between metallothionein expression and resistance to cisplatin. Metallothionein-positive pancreatic carcinoma will be potentially highly malignant or acquire an enhanced ability to produce metallothioneins as the malignant potential increases. The expression of metallothionein could be a prognostic indicator in pancreatic carcinomas.

Topics: Adult; Carcinoma, Ductal, Breast; Humans; Immunohistochemistry; Metallothionein; Pancreas; Pancreatic Neoplasms