Mass spectrometric analysis reveals O-methylation of pyruvate kinase from pancreatic cancer cells.

Pyruvate kinase (PK) is an important glycolytic enzyme that catalyzes the dephosphorylation of phosphoenolpyruvate to pyruvate. Human PK isozyme M2 (PKM2), a splice variant of M1, is overexpressed in many cancer cells, and PKM2 has been investigated as a potential tumor marker for diagnostic assays and as a target for cancer therapy. To facilitate identification and characterization of PK, we studied the enzyme from pancreatic cancer cells and normal pancreatic duct cells by electrophoresis and mass spectrometry, and identified multiple O-methylated residues from PK. These findings advance our knowledge of the biochemical properties of PK and will be important in understanding its biological function in cells.

Human pancreatic islet preparations release HMGB1: (ir)relevance for graft engraftment.

High levels of donor-derived high-mobility group box 1 (HMGB1) protein have been associated with poor islet graft outcome in mouse models. The aim of our work was to determine whether HMGB1 released by human islets had independent proinflammatory effects that influence engraftment in humans. Human islet preparations contained and released HMGB1 in different amounts, as determined by Western blot and ELISA (median 17 pg/ml/IEQ/24 h; min-max 0-211, n = 74). HMGB1 release directly correlated with brain death, donor hyperamilasemia, and factors related to the pancreas digestion procedure (collagenase and digestion time). HMGB1 release was significantly positively associated with the release of other cytokines/chemokines, particularly with the highly released "proinflammatory" CXCL8/IL-8, CXCL1/GRO-α, and the IFN-γ-inducible chemokines CXCL10/IP-10 and CXCL9/MIG. HMGB1 release was not modulated by Toll-like receptor 2, 3, 4, 5, and 9 agonists or by exposure to IL-1ß. When evaluated after islet transplantation, pretransplant HMGB1 release was weakly associated with the activation of the coagulation cascade (evaluated as serum cross-linked fibrin products), but not with the immediate posttransplant inflammatory response. Concordantly, HMGB1 did not affect short-term human islet function. Our data show that human islet HMGB1 release is a sign of "damaged" islets, although without any independent direct role in graft failure.

MS analysis reveals O-methylation of L-lactate dehydrogenase from pancreatic ductal adenocarcinoma cells.

L-lactate dehydrogenase (LDH) converts pyruvate to lactate when oxygen is absent or in short supply, and the enzyme plays a crucial role in cancer metabolism. The functions of many mammalian proteins are modulated by posttranslational modifications (PTMs), and it has been reported that LDH was subjected to several PTMs, including phosphorylation, acetylation, and methylation. In this present work, we characterized the PTMs of LDH from pancreatic ductal adenocarcinoma (PDAC) cells by electrophoresis and mass spectrometry, and identified 13 O-methylated residues from the enzyme. In addition, our qualitative analysis revealed differential methylation of LDH from normal duct cells. The preliminary findings from this study provide important biochemical information toward further understanding of the LDH modifications and their functional significance in pathophysiological processes of pancreatic cancer.

Proteomic analysis reveals Warburg effect and anomalous metabolism of glutamine in pancreatic cancer cells.

In this present work, we characterized the proteomes of pancreatic ductal adenocarcinoma (PDAC) cell line PANC-1 and normal pancreatic duct cells by mass spectrometry using LTQ-Orbitrap and identified more than 1700 proteins from each sample. On the basis of the spectra count label-free quantification approach, we identified a large number of differentially expressed metabolic enzymes and proteins involved in cytoskeleton, cell adhesion, transport, transcription, translation, and cell proliferation as well. The data demonstrated that metabolic pathways were altered in PANC-1, consistent with the Warburg effect. In addition, the comparative MS analysis unveiled anomalous metabolism of glutamine, suggesting that glutamine was largely consumed as a nitrogen donor in nucleotide and amino acid biosynthesis in PANC-1. Our analysis provides a potentially comprehensive picture of metabolism in PANC-1, which may serve as the basis of new diagnostics and treatment of PDAC.

Gemcitabine-loaded PEGylated unilamellar liposomes vs GEMZAR: biodistribution, pharmacokinetic features and in vivo antitumor activity.

The systemic efficacy of the chemotherapeutic agents presently used to treat solid tumors is limited by their low therapeutic index. Previously, our research group improved the in vitro antitumoral activity of gemcitabine, an anticancer agent rapidly deaminated to the inactive metabolite 2',2'-difluorodeoxyuridine, entrapping it into unilamellar pegylated liposomes made up of 1,2-dipalmitoyl-snglycero-3-phosphocholine monohydrate/cholesterol/N-(carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (6:3:1 molar ratio). In this work, we investigated the in vivo efficiency of the gemcitabine liposomal formulation (5mg/kg) with respect to the antitumoral commercial product GEMZAR (50mg/kg) on an anaplastic thyroid carcinoma xenograft model obtaining similar effects in terms of inhibition of tumor mass proliferation after 4weeks of treatment. The investigation of the carrier biodistribution and the drug pharmacokinetic profile furnished the rationalization of the efficacy of the vesicular system containing the active compound 10-fold less concentrated; in fact, liposomes promoted the concentration of the drug inside the tumor and they increased its plasmatic half-life. In addition, no signs of blood toxicity were observed when vesicular devices of effective doses of the drug were used.

Effect of all trans retinoic acid on lysosomal alpha-D-mannosidase activity in HL-60 cell: correlation with HL-60 cells differentiation.

Human promyelocytic leukemia HL-60 cells represent an in vitro model of acute promyelocytic leukemia (APL), and are inducible to terminally differentiate into morphologically mature granulocytes by incubation with all trans retinoic acid (ATRA). Lysosomal glycohydrolases are involved in the changes of the membrane surface proteins' glycosylation, linked to the metastatic progression potential of neoplastic cells. In particular, it has been demonstrated that the Asn-linked glucidic residues were directly responsible for the metastatic potential, and it is known that the glycohydrolase alpha-D-mannosidase specifically hydrolyze the Asn-linked oligosaccharides. In this report, we present an in vitro study on the ATRA effects on lysosomal glycohydrolases expression and the eventual relationship with the retinoic acid-induced differentiation of HL-60 cells. We have investigated two highly expressed lysosomal glycohydrolases, namely beta-D-hexosaminidase and alpha-D-mannosidase, and showed that they were differently affected by ATRA differentiating action. In particular, due to the specific action on Asn-linked oligosaccharides, we tested alpha-D: -mannosidase enzymatic activity and observed that it was dramatically decreased after ATRA incubation, indicating a relationship with the differentiation state of the cells. These observations may directly be linked with the loss of metastatic progession of differentiated HL-60.

Neonatal pig liver-derived progenitors for insulin-producing cells: an in vitro study.

Beta (beta)-cell replacement represents an attractive approach for the possible cure of type 1 insulin-dependent diabetes mellitus (IDDM). In a search for potential sources of insulin-secreting cells for IDDM substitution therapy, we have focused on the neonatal pig liver, which is putatively enriched in multipotent stem cells. We then isolated cells measuring 10 to 15 microm in diameter, identified as small cells, characterized by a high proliferation rate and positive staining for immature liver and pancreatic endocrine cell markers (i.e., insulin and pancreatic duodenal homeobox). The ability of these cells to transdifferentiate into pancreatic beta-like cells under culture conditions with exendin-4 (Ex-4) or high glucose concentration was examined. We observed that insulin secretion was not physiological in basal conditions, although it became responsive to glucose after 5 days of exposure to Ex-4. This beta-cell-like phenotype remained physiologically stable, even after stimulus withdrawal. Based on these observations, we contend that the proposed cell and tissue model might offer several advantages as a candidate for substitution cell therapy in IDDM, because the neonatal pig liver seems enriched in cells, with a mixed pancreas-liver phenotype, that are easier to purify and grow in culture and are more functional than other beta-like cells upon in vitro single short-term stimulation challenge.

Long-term cultured neonatal porcine islet cell monolayers: a potential tissue source for transplant in diabetes.

BACKGROUND: The restricted availability of cadaveric human donor pancreases mandates validation of possibly inexhaustible, alternative sources of insulin secretory cells in order to expand islet transplant for the therapy of insulin dependent diabetes mellitus (T1DM). METHODS: Neonatal pig pancreatic islets (NPI), isolated and purified by our method, were specially cultured until confluent cell monolayers were obtained. Expression of several beta-cell phenotype transcriptional factors, under glucose and other stimuli, were examined throughout 90 days of culture. RESULTS: High glucose concentration and glucagon-like peptide 1 (GLP-1) were associated with maintenance either of insulin secretory patterns from the incubated cell monolayers, or expression of transcriptional markers associated with beta-cell like phenotypes. CONCLUSION: Morphological and molecular expression of beta-cell markers and products from NPI cell monolayers seem to identify a novel and potentially powerful source of insulin producing cells that might fulfill transplant needs for insulin substitution therapy.

Grafts of microencapsulated pancreatic islet cells for the therapy of diabetes mellitus in non-immunosuppressed animals.

Pancreatic-islet-cell transplantation may reverse hyperglycaemia in diabetic recipients that undertake general pharmacological immunosuppression. A major challenge that remains is the need to avoid immunosuppression associated with the use of allogeneic or heterologous islet cells. In the present study we demonstrate the use of microencapsulation of cells using artificial biocompatible and permselective membranes prepared with alginic acid derivatives and polyamino acids. While characterization of the microcapsule constituent polymers continues to progress, other technical issues such as definition of the immunobarrier capacity, biocompatibility, size, shape and graft site have come into sharper focus. Assessment of microcapsules properties, in order to establish possible guidelines for fabrication of reproducible membranes, and results from both in vitro functional testing, and in vivo encapsulated-islet-transplant outcome in several animal models of diabetes are reported.

Up-regulation of glycohydrolases in Alzheimer's Disease fibroblasts correlates with Ras activation.

The lysosomal system is up-regulated in the brain of patients with Alzheimer's Disease (AD), as demonstrated by previous experiments carried out in postmortem samples of brain patients. In this paper we provide evidence that an up-regulation of lysosomal glycohydrolases (alpha-D-mannosidase, beta-D-hexosaminidase, and beta-D-galactosidase) takes place in skin fibroblasts from AD patients affected either by sporadic or familial forms and is detectable also in presymptomatic subjects carrying the above mutations but healthy at the time of skin biopsy. This increase of enzyme activity is consequent to a transcriptional up-regulation. The oncogene Ras appears to be involved in the regulation of enzymatic activity. A parallel increase of Ras transcript and Ras protein, without an increase of p44/p42 MAPK activation was revealed in the same AD fibroblasts. An activation of p38 MAPK already described to occur in neurodegenerative diseases such as Alzheimer's, was also found in fibroblasts derived from AD patients. High levels of expression of the constitutively active form of Ras in normal or AD fibroblasts induced glycohydrolases up-regulation. Overall results demonstrated that glycohydrolases up-regulation, as well as Ras up-regulation, are early markers of AD, detectable at peripheral level, and good candidates to be exploited for diagnostic purposes. These data also provide the first proof for a role of Ras in regulating lysosomal glycohydrolases expression.