Cytotoxic effects of a novel pyrazolopyrimidine derivative entrapped in liposomes in anaplastic thyroid cancer cells in vitro and in xenograft tumors in vivo.

In this study, we evaluated the activity of two novel pyrazolopyrimidine derivatives (Si 34 and Si 35) against ARO cells, a human anaplastic thyroid cancer cell line. ARO cells exposed to different concentrations of the drugs showed a reduced growth rate and an increase of mortality. After 72 h incubation, doses of 5 and 10 microM Si 34 determined a decrease of cell counts by approximately 25% and approximately 75% compared with those of control cells respectively. Similar findings were observed using Si 35. Treatment with both Si 34 and Si 35 at 10 microM increased cell mortality also ( approximately 29% and approximately 18% respectively). At these concentrations, a decrease in cyclin D1 levels was observed. To improve the biopharmaceutical properties, a liposome formulation was prepared. When entrapped in unilamellar liposomes, Si 34 exerted its cytotoxic effects even at lower doses (maximal inhibition at 5 microM) and after shorter incubation time (48 h) either in ARO or other thyroid cancer cell lines. The effects were associated with weak apoptotic death. Inhibition of epidermal growth factor-stimulated src and ERK phosphorylation, as well as reduction of migration properties of ARO cells was also observed. Moreover, the growth of tumor xenografts induced in severe combined immunodeficiency (SCID) mice was inhibited by i.v. administration of 25-50 mg/kg of the drug liposomal formulation. In conclusion, the liposomal preparation of this novel pyrazolopyrimidine derivative appears to be a promising tool for the treatment of anaplasic thyroid cancer.

Effects of poly-L-lysine and collagen on FH-B-TPN cell differentiation into endocrine cell phenotype.

Pdx-1 genetically engineered FH-B-TPN cells might represent a source for insulin-secreting cells. We then have tested whether poly-L-lysine (PLL) and collagen (C) exposure in vitro promote three-dimensional particle formation and differentiation toward an endocrine cell phenotype. On these matrices, we observed that FH-B-TPN cells showed a tendency to either aggregate when seeded on PLL or to form uniform cell monolayers, but not to aggregate on C. While insulin was released in any condition, GSIR was only associated with PLL mainly at 24 and 72 hours of culture. Various culture matrices influenced the expression of glucose transporter type 2 and gluco kinase, being they expressed more intensively on PLL rather than C or in controls. mRNA expression for NeuroD/Beta2, Isl-1, Ras, Metalloproteinase-2 (MMP-2), -9 and -7 also were affected, with PLL inducing increased expression of NeuroD/Beta2 of Isl-1, and no difference between C and control. PLL, unlike C, strongly increased Ras through observation times. MPP-2 and -9 were decreased by both PLL and C, whereas MMP-7 was increased by PLL. PLL, usually employed to promote culture cell adhesion, has been proven capable to stimulate pancreatic endocrine function and cell aggregation and to stimulate gene expression of key markers for either insulin transcription or MMP-7.

Effects of streptozotocin on in vitro long-term cultured human islet cell monolayers.

Replacement beta cells may be generated from stem/progenitor cells, possibly residing within the pancreatic islet cells. We sought to investigate the possible use of human islet (HI)-derived cell monolayers as a possible source for beta cells. These cells could be propagated in vitro and potentially induced to acquire glucose-stimulated insulin release (GSIR) ability. Loss of three-dimensional architecture, following monolayer establishment, resulted in either rearrangement of both pancreatic hormone expression and key transcription factors or decrease in insulin production or GSIR disappearance. We showed that cell deregulation/dedifferentiation was reversible by treating the cell monolayers, after five passages with streptozotocin (STZ), a well-known beta-cell toxin. When used at subtoxic concentrations, STZ promoted differentiation of HI-derived cell monolayers and GSIR recovery. This effect allowed production of insulin-secreting cells starting from cell monolayers doubled five times in vitro, meaning a 400-fold increase in the cellular starting material. Such islet cell expansion capability in vitro might elucidate a new source of insulin-secreting cells thereby possibly overcoming the problem posed by searching for human organ donation.

Standard technical procedures for microencapsulation of human islets for graft into nonimmunosuppressed patients with type 1 diabetes mellitus.

To comply with regulatory restrictions, with regard to graft of human islets immunoprotected within artificial microcapsules, into patients with type 1 diabetes mellitus (T1DM) with no recipient immunosuppression, we have prepared standard protocols on: (1) sodium alginate purification (clinical grade) for microcapsule fabrication; (2) preparation of biocompatible and permselective microcapsules containing human islets; and (3) minimally invasive techniques for grafting of the encapsulated human islets into the recipients' peritoneal cavity. As to no. 1, starting from pharmaceutical grade, raw sodium alginate powder, we prepared a pyrogen- and endotoxin-free 1.6% alginate solution by means of dialysis, multiple filtrations, and dilution/osmolality adjustments. As to no. 2, we have selected human islet preparations associated with >80% purity/viability, which underwent careful functional quality control testing prior to encapsulation; namely, most capsules contained one islet. As for no. 3, we have devised a simple intraperitoneal injection method under abdominal echography guidance with only local anesthesia to deposit the encapsulated islets in saline within the peritoneal leaflets. These technical protocols were officially approved by the Italian Ministry of Health which has released permission to conduct a phase I, closed human trial in 10 patients using encapsulated human islet grafts into nonimmunosuppressed patients with T1DM.

Short-term stimulation studies on neonatal pig pancreatic duct-derived cell monolayers.

Short-term stimulation with insulinotropic factors may induce morphologic and functional changes in primary ductal cell cultures as a potential source of stem cells. We sought to assess the capacity of hepatocyte growth factor (HGF) to induce expression and maturation of proteins--PDX-1 and GLUT-2--and the subsequent beta-cell secretory profiles. HGF, which is involved in pancreatic development, may induce islet beta-cell neogenesis. Primary ductal cell monolayers were cultured in Click's + FBS 10% at 37 degrees C until tissue confluence. The medium was enriched with HGF (10 ng/mL for different periods); controls were treated for similar times with normal culture medium. At the end of the study, three-dimensional islet-like cell aggregates were observed in both conditions. In all conditions immunostaining studies showed positivity for the major endocrine-phenotype cell markers: insulin, PDX-1, glucokinase, and GLUT-2. Furthermore, treatment with HGF for short periods induced the expression of a functionally active, phosphorylated isoform of PDX-1. Finally, we observed that under basal conditions the cells initially and progressively released proinsulin throughout 5 days in all settings. Thereafter proinsulin was gradually replaced by insulin in the culture medium, reflecting a maturation progress. This pattern of insulin maturation and release was more evident when the cells were continuously stimulated with HGF for 12 days. The employed stimuli seemed to differentiate the original ductal cell layers toward endocrine cell phenotypes that synthesize and release proinsulin and subsequently insulin. HGF seems to provide a more efficient differentiation.

Transdifferentiation molecular pathways of neonatal pig pancreatic duct cells into endocrine cell phenotypes.

Restrictions in availability of cadaveric human donor pancreata have intensified the search for alternate sources of pancreatic endocrine tissue. We have undertaken to assess whether nonendocrine pancreatic tissue, with special regard to ducts, including epithelial cells, and retrieved from neonatal pig pancreata that are used for islet isolation, may under special in vitro culture conditions generate endocrine cell phenotypes. Special care was taken to identify the time-related appearance of molecular and biochemical markers associated with beta-cell specificity, in terms of glucose-sensing apparatus and insulin secretion. For this purpose, established ductal origin monolayer cell cultures were incubated with a battery of mono- or polyvalent growth factors. Morphological, immunocytochemical, molecular, and functional assays indicated that under special culture conditions ductal origin cells acquired an endocrine identity, based upon expression of key gene transcripts that govern the stimulus-coupled insulin secretory activity. Among factors eliciting transdifferentiation of ductal epithelial into endocrine cells, Sertoli cell (SC)-conditioned medium seemed to be the most powerful inducer of this process. In fact, the resulting cultures not only expressed beta-cell-oriented metabolic markers but also were associated with insulin and C-peptide output at equimolar ratios. This finding indicates that SC coincubation, more than other conditions, caused originally ductal cell cultures to gradually differentiate and mature into beta-cell-like elements. In vivo studies with this early cell differentiation product will test whether our approach may be suitable for correction of hyperglycemia in diabetic animal models.

Optimized parameters for microencapsulation of pancreatic islet cells: an in vitro study clueing on islet graft immunoprotection in type 1 diabetes mellitus.

Alginate (AG)-based microcapsules may provide a selective permeable and biocompatible physical barrier to prevent islet graft (TX)-directed immune destruction. However, extent of the achieved immunoprotection will continue to be variable and unpredictable until the role of the individual mechanisms involved with TX-related inflammatory cell and immune reactivity are clarified. Macrophages (M) are believed to play a pivotal role in controlling the host/TX interaction and its consequences. We then have studied the effects of isolated rat M and their secretory products on allogeneic islets enveloped in variably sized and configured microcapsules, within in vitro mixed islet-M cocultures. In particular, we aimed to determine the sequence of immune or not immune specific cascade of early events that derive from such on interaction. One of the specific aims was to assess whether the membrane's physical intactness and conversely its even minimal rupture, along with the microcapsules' size (i.e., large vs. small) would significantly impact M reactivity and, thereby, the encapsulated islet viability and function. Special care was taken to evaluate extent of the elicited reactivity by meticulously monitoring cytokine, N2 derivative, and other proinflammatory protein curve profiles during the early M activation process. The study has preliminarily shown that, for equally formulated microcapsules, the capsular size and membrane's morphologic thoroughness are key to prevent M reactivity and possibly avoid the intracapsular islet cell damage. While elucidation of pathways involved with the encapsulated islet TX-directed host's responsiveness actually is in progress, it has clearly emerged that microcapsules should comply with well-defined physical properties and formulation specifications in order to obviate the primum movens of the inflammatory reaction process.

Neonatal pig pancreatic duct-derived insulin-producing cells: preliminary in vitro studies.

Neonatal pig pancreata could represent an ideal tissue resource for donor islets for transplantation trials. Because functional islet beta-cells could derive from precursors situated in the ductal system, and neonatal animals are better suitable than adults for recovering such elements, we have examined whether isolated neonatal pancreatic ducts (NPD) could form insulin-producing cells. NPD, retrieved from the pancreas by collagenase digestion, were cultured for 2 weeks. A compact tissue monolayer detached by trypsin was re-incubated to form upon culture. The primary tissue monolayer was plated, yielding secondary monolayers that were supplemented in culture with the following factors: insulin transferrin selenium, niacinamide, keratinocyte growth factor, and high glucose, which promoted formation of islet cell-like clusters during 30 days of culture. Upon reaching 50 to 100 microm in diameter, the cell clusters were subjected to morphologic examination (assessment of viability by staining with ethidium bromide+fluorescein diacetate [EB+FD]; staining for insulin with diphenylthiocarbazone [DTZ]); DNA assay; insulin radioimmunoassay both in the basal state and after in vitro static incubation with high glucose; immunolabeling with anti-insulin fluorescent antibodies. Of the cell clusters, 80% were composed of viable cells that faintly showed DTZ staining. Basal insulin was 16.7 microU/mL, but no insulin response was elicited by stimulation with high glucose. Acid-ethanol extraction showed high insulin levels in the clusters. Finally, immunofluorescence for insulin was positive, indicating the presence of beta-cell-like committed elements. In conclusion, NPD may differentiate into insulin-producing cells, which are at a very early stage when the glucose-sensing apparatus is still immature.

Calcium and fos involvement in brain-derived Ca(2+)-binding protein (S100)-dependent apoptosis in rat phaeochromocytoma cells.

Brain-derived calcium-binding protein S100 induces apoptosis in a significant fraction of rat phaeochromocytoma (PC12) cells. We used single cell techniques (patch clamp, videomicroscopy and immunocytochemistry) to clarify some of the specific aspects of S100-induced apoptosis, the modality(ies) of early intracellular Ca2+ concentration increase and the expression of some classes of genes (c-fos, c-jun, bax, bcl-x, p-15, p-21) known to be implicated in apoptosis of different cells. The results show that S100: (1) causes an increase of [Ca2+]i due to an increased conductance of L-type Ca2+ channels; (2) induces a sustained increase of the Fos levels which is evident since the first time point tested (3 h) and remains elevated until to the last time point (72 h). All these data suggest that S100-derived apoptosis in PC12 cells may be the consequence of a system involving an increase in L-type Ca2+ channel conductance with consequent [Ca2+]i increase which up-regulates, directly or indirectly, the expression of Fos.