New pyrazolo-[3,4-d]-pyrimidine derivative Src kinase inhibitors lead to cell cycle arrest and tumor growth reduction of human medulloblastoma cells.

Medulloblastoma is the most common malignant brain tumor in children, and despite improvements in the overall survival rate, it still lacks an effective treatment. Src plays an important role in cancer, and recently high Src activity was documented in medulloblastoma. In this report, we examined the effects of novel pyrazolo-[3,4-d]-pyrimidine derivative Src inhibitors in medulloblastoma. By MTS assay, we showed that the pyrimidine derivatives indicated as S7, S29, and SI163 greatly reduce the growth rate of medulloblastoma cells by inhibiting Src phosphorylation, compared with HT22 non-neoplastic nerve cells. These compounds also halt cells in the G(2)/M phase, and this effect likely occurs through the regulation of cdc2 and CDC25C phosphorylation, as shown by Western blot. Moreover, the exposure to pyrimidine derivatives induces apoptosis, assayed by the supravital propidium iodide assay, through modulation of the apoptotic proteins Bax and Bcl2, and inhibits tumor growth in vivo in a mouse model. Notably, S7, S29, and SI163 show major inhibitory effects on medulloblastoma cell growth compared with the chemotherapeutic agents cisplatin and etoposide. In conclusion, our results suggest that S7, S29, and SI163 could be novel attractive candidates for the treatment of medulloblastoma or tumors characterized by high Src activity.

The antiretroviral nucleoside analogue Abacavir reduces cell growth and promotes differentiation of human medulloblastoma cells.

Abacavir is one of the most efficacious nucleoside analogues, with a well-characterized inhibitory activity on reverse transcriptase enzymes of retroviral origin, and has been clinically approved for the treatment of AIDS. Recently, Abacavir has been shown to inhibit also the human telomerase activity. Telomerase activity seems to be required in essentially all tumours for the immortalization of a subset of cells, including cancer stem cells. In fact, many cancer cells are dependent on telomerase for their continued replication and therefore telomerase is an attractive target for cancer therapy. Telomerase expression is upregulated in primary primitive neuroectodermal tumours and in the majority of medulloblastomas suggesting that its activation is associated with the development of these diseases. Therefore, we decided to test Abacavir activity on human medulloblastoma cell lines with high telomerase activity. We report that exposure to Abacavir induces a dose-dependent decrease in the proliferation rate of medulloblastoma cells. This is associated with a cell accumulation in the G(2)/M phase of the cell cycle in the Daoy cell line, and with increased cell death in the D283-MED cell line, and is likely to be dependent on the inhibition of telomerase activity. Interestingly, both cell lines showed features of senescence after Abacavir treatment. Moreover, after Abacavir exposure we detected, by immunofluorescence staining, increased protein expression of the glial marker glial fibrillary acidic protein and the neuronal marker synaptophysin in both medulloblastoma cell lines. In conclusion, our results suggest that Abacavir reduces proliferation and induces differentiation of human medulloblastoma cells through the downregulation of telomerase activity. Thus, using Abacavir, alone or in combination with current therapies, might be an effective therapeutic strategy for the treatment of medulloblastoma.

Chaotic dynamic stabilities and instabilities of hematopoietic stem cell growth plasticity.

The hematopoietic system consists of: (1) a network of stem and progenitor cells of varying degrees of maturity interacting with other cells that possess supportive and regulatory capacities and (2) vascular stem cell niches with supporting stem cells in their self-renewal, proliferation, differentiation, and mobilization to the circulation. Recent data suggest that selective expression of organ-specific chemokines promotes the mobilization of bone-marrow-derived pluripotent cells, a process that is essential for tissue vascularization and organ regeneration. Despite intensive investigation, the pathways by which mechanical signals are converted to biochemical responses are not completely understood. Recent studies have suggested that chromatin shifts and cell cycle effects stem cell gene expression, and thus results in changes of its surface receptor expression at different points of the cell cycle machinery, therefore changing cell cycle transit. This review will attempt to discuss new approaches to determine the regulation of stem cell growth and differentiation by underlying the significance of the chaotic dynamics of transcriptional networks within a cell, with a combination of chemokines and cytokines in the environment, and mechanical forces, such as: stretch, strain and laminar flow, all involving both cooperation and competition.

Properties of mechano-transduction via simulated microgravity and its effects on intracellular trafficking of VEGFR's.

This study emphasizes the dynamical properties of mechanical loading via simulated microgravity, its effect on acute myeloid leukemia proliferation and hematopoietic stem cell (HSPC) growth and its implications in the area of tissue regeneration. Data presented illustrates that mechanical transduction changes the expression of humoral factors by facilitating paracrine/autocrine signalling, therefore modulating intracellular trafficking of tyrosine kinase receptors. Understanding mechano-transduction in the context of cell and tissue morphogenesis is the major focus of this study. The effects of external physiological stresses, such as blood flow, on several cellular subtypes seem to produce very intricate cellular responses. It is well accepted that mechanical loading plays an intrinsic and extrinsic influence on cell survival. This study shows how microgravity effects hematopoietic stem cells, and human leukemic cell proliferation and expression of its receptors that control cell survival, such as the tyrosine kinase vascular endothelial growth factor receptor-1, receptor-2 and receptor-3.

Prostaglandin E2: at the crossroads between stem cell development, inflammation and cancer.

Stem cells have tremendous therapeutic potential for a series of pathologies ranging from cancer to genetic diseases. The obstacles to exploiting their potential reside mainly in their limited numbers or potency. Prostaglandins are known to be involved in many physiological and pathological processes. Among these, their importance in stem cell development is just starting to emerge. The recent findings by North and colleagues (Nature, 2007; 447:1007-1011) uncover a crucial role for PGE2 in hematopoietic stem cell growth and development not only in embryonic, but also in adult stem cell homeostasis in both simple and complex vertebrate systems. This new information adds to recent advances in the study of PGE2's role in many diseases and in the reaction to various cellular stress conditions. This is the perfect time to improve our knowledge of stem cell regulation, which hopefully will lead to improved stem cell-based therapeutic options and also to better understand and manage current anti-inflammatory and immuno-suppressive drugs in the therapy of cancer and other diseases.

Effects of a vitamin D3 analog on diabetes in the bio breeding (BB) rat.

Non-hypercalcemic analogs of vitamin D(3) modulate the immune response through antigen-presenting cells (APCs) and activated T-cells. A large population-base case-control showed that vitamin D(3) intake significantly decreases the risk of type 1 diabetes development. The aim of this study was, therefore, to observe the in vivo effects of a vitamin D(3) analog administered to Bio Breeding (BB) rats. 1,25-Dihydroxy-16,23Z-diene-26,27-hexafluoro-19-nor vitamin D(3) (BXL-219, formerly Ro 26-2198) (BioXell, Milan, Italy) was administered in vivo to BB rats from days 42 to 110 of life at 0.2 microg/Kg BW. Control animals received only vehicle (olive oil, 4.8 microl/100 g BW). The animals of these two groups were subjected to insulin treatment as they became diabetic. Insulin (Humulin, 28.6 UI/day) was administered irrespective of diabetes occurrence to another group of rats for comparison. Blood glucose, insulin levels, glycosuria, degree of islet infiltration, and the expression of some antigens were observed. Results showed that the vitamin D(3) analog reduced diabetes incidence, although limitedly, in BB rats while administration of oral insulin increased diabetes incidence. In addition, the vitamin D(3) analog did not stimulate an enhancement in the expression of CD4 and CD25 in BB rats as it does in NOD mice, which may explain the failure of this as well as other antidiabetic treatments in the BB animal model of type 1 diabetes.

MnSOD mimic compounds can counteract mechanical stress and islet beta cell apoptosis, although at appropriate concentration ranges.

Pancreatic islets are commonly isolated for research and transplantation without taking into consideration that they undergo mechanical or chemical stress during this process. In order to counteract both types of injuries, the compound AEOL10150, a novel MnSOD mimic, was added during isolation of islet at concentrations ranging from 18 to 100 microM. Mechanical or chemical stress-related pro-apoptotic signals were then studied. We demonstrate that this MnSOD mimic diminishes the negative effects of mechanical stress by blocking insulin impairment, production of non-specific islet beta-cell proteins, transcription of iNOS and FAS, activation of caspase-3 and -9 and, ultimately, apoptosis. Moreover, the effects of the MnSOD mimic on isolated islets were greatly influenced by dosage: the best dose able to fully counteract mechanical stress was found to be 100 microM; doses > or =150 microM were themselves highly toxic for islet cells. On the other hand, rIL-1beta-induced chemical stress is rather complex, and there was no protection in this scenario. Therefore, contrarily to what has been previously reported, MnSOD mimic administration is only capable of counteracting mechanical stress, and not cytokine-induced cytotoxicity, and that this drug acts within a limited concentration range.

An early but intense cytokine production within the islets may be predictive for type 1 diabetes occurrence in the Bio Breeding (BB) rat.

The Bio Breeding (BB) rat is a useful animal model of type 1 autoimmune diabetes. The aim of this study was to observe and follow the cytokine and antigenic expressions within the islets of Langerhans in young non-diabetic, in pre-diabetic hyperglycemic, and in overtly diabetic animals. BB rats were therefore checked at day 21 up to day 90 of life for blood glucose, insulin levels, degree of islet infiltration, expression of proinflammatory and protective cytokines and antibodies including CD4, CD8, CD25, LFA-1, and ICAM-1. Animals were treated with insulin as they became diabetic. We found that islets of non-diabetic BB rats became positive to both IL-1beta and IL-4 very early on, confirming a local but intense production of both cytokines within the islets during the initial non-diabetic period. In addition, we observed that the production of these interleukins together with the expression levels of CD4 and CD25 are events predictive for type 1 diabetes onset in non-diabetic BB rats, as for non-obese diabetic (NOD) mice. In particular, the production of IL-1beta and IL-4 during the non-diabetic period together with the lack of enhancement of CD4 and CD25, indicating selective recruitment of activated T cells, may explain the failure of anti-diabetic treatments in this animal model of type 1 diabetes.