The molecular mechanisms underlying the development of pancreatic neuroendocrine tumors (PanNETs) have not been well defined. We report here that the genomic region of the PHLDA3 gene undergoes loss of heterozygosity (LOH) at a remarkably high frequency in human PanNETs, and this genetic change is correlated with disease progression and poor prognosis. We also show that the PHLDA3 locus undergoes methylation in addition to LOH, suggesting that a two-hit inactivation of the PHLDA3 gene is required for PanNET development. We demonstrate that PHLDA3 represses Akt activity and Akt-regulated biological processes in pancreatic endocrine tissues, and that PHLDA3-deficient mice develop islet hyperplasia. In addition, we show that the tumor-suppressing pathway mediated by MEN1, a well-known tumor suppressor of PanNETs, is dependent on the pathway mediated by PHLDA3, and inactivation of PHLDA3 and MEN1 cooperatively contribute to PanNET development. Collectively, these results indicate the existence of a novel PHLDA3-mediated pathway of tumor suppression that is important in the development of PanNETs.p53 | PH domain | everolimus | p53 target gene | mTOR N euroendocrine tumors (NETs) arise from cells of the endocrine and nervous systems, and are found in tissues such as lung, pancreas and pituitary (1-3). NETs often produce, store and release biogenic amines and polypeptide hormones, and secretary granules containing these products provide a diagnostic marker for NETs. The mechanisms underlying the development of NETs remain unclear to date, due to the low incidence of these tumors and due to the lack of suitable experimental model systems, including genetically engineered mouse models. Pancreatic NET (PanNET), which is probably the best-studied NET, is the second-most common pancreatic tumor, having an incidence of ∼1 per 100,000 individuals. Patients having late-stage PanNET often harbor tumors that are unresectable or metastatic and face limited treatment options. Accordingly, the prognosis of patients having metastatic PanNET is the worst among the NET subtypes, with a 5-y survival rate of 27-43% (1). Recently, the drug Everolimus has shown promise in the treatment of PanNETs (4), providing a significant improvement in progression-free survival. Everolimus is an inhibitor of mammalian target of rapamycin (mTOR), a downstream mediator of the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway. The striking efficacy of Everolimus demonstrates the importance of the PI3K/Akt pathway in the pathology of PanNETs.In agreement with these clinical results, studies on pancreatic endocrine cell lines have identified the PI3K/Akt signaling pathway as a major proliferation and survival pathway in these cells (5). Activated Akt phosphorylates substrates such as mTOR and controls various biological processes, including protein synthesis, proliferation, cell growth, and survival. Regulation of pancreatic islet β-cell proliferation, cell size, and apoptosis by Akt has been demonstrated using various mouse models. For examp...

Islet transplantation is a minimally invasive treatment for severe diabetes. However, it often requires multiple donors to accomplish insulin-independence and the long-term results are not yet satisfying. Therefore, novel ways to overcome these problems have been explored. Isolated islets are fragile and susceptible to pro-apoptotic factors and poorly proliferative. In contrast, mesenchymal stem cells (MSCs) are highly proliferative, anti-apoptotic and pluripotent to differentiate toward various cell types, promote angiogenesis and modulate inflammation, thereby studied as an enhancer of islet function and engraftment. Electrofusion is an efficient method of cell fusion and nuclear reprogramming occurs in hybrid cells between different cell types. Therefore, we hypothesized that electrofusion between MSC and islet cells may yield robust islet cells for diabetes therapy. We establish a method of electrofusion between dispersed islet cells and MSCs in rats. The fusion cells maintained glucose-responsive insulin release for 20 days in vitro. Renal subcapsular transplantation of fusion cells prepared from suboptimal islet mass (1,000 islets) that did not correct hyperglycemia even if co-transplanted with MSCs, caused slow but consistent lowering of blood glucose with significant weight gain within the observation period in streptozotocin-induced diabetic rats. In the fusion cells between rat islet cells and mouse MSCs, RT-PCR showed new expression of both rat MSC-related genes and mouse β-cell-related genes, indicating bidirectional reprogramming of both β-cell and MSCs nuclei. Moreover, decreased caspase3 expression and new expression of Ki-67 in the islet cell nuclei suggested alleviated apoptosis and gain of proliferative capability, respectively. These results show that electrofusion between MSCs and islet cells yield special cells with β-cell function and robustness of MSCs and seems feasible for novel therapeutic strategy for diabetes mellitus.

Islet transplantation is a method for the treatment of type 1 diabetes mellitus (DM) and has been widely performed around the world. The long-term cryopreservation of islets shows many advantages in the field of islet transplantation. Previous studies have described the development of sheet-type polyvinyl alcohol (PVA) macro-encapsulated islets (MEI) to treat type 1 DM without any immunotherapy. The present study examined their beneficial effects on islet cryopreservation. PVA MEI of Wistar rats were divided into three groups of 1-day, 7-day and 30-day cryopreservation at -80 degrees C. The 30-day group showed a lower recovery rate of the islet number and impaired insulin release in comparison to the 1-day group, whereas no significant differences of the in vitro results were observed between the 1-day and 7-day groups. The MEI transplantation recipient mice in the 1-day and 7-day groups reached normoglycemia for a 4-week observation period, and the recipients in 30-day group also showed a significant decrease followed by a slightly higher non-fasting blood glucose level. These results suggest that the PVA MEI are useful for islet long-term cryopreservation, and that the use of cryopreserved PVA MEI may, therefore, be a promising modality for performing DM therapy.

Immune rejection and scarcity of donor tissues are the restrictions of islets transplantation. In this study, the cytoprotection of chitosan hydrogels in xenogeneic islet transplantation was demonstrated. Wistar rat islets encapsulated in chitosan hydrogels were performed glucose challenge test and live/dead cell staining in vitro. Islets/chitosan hydrogels were transplanted into the renal subcapsular space of diabetic C57BL/6 mice. Non-fasting blood glucose level (NFBG), body weight, intraperitoneal glucose tolerance test (IPGTT), and glucose disappearance rate were determined perioperatively. The serum insulin level was analyzed, and the kidney transplanted with islets/chitosan hydrogels were retrieved for histological examination after sacrifice. The present results showed that islets encapsulated in chitosan hydrogels secreted insulin in response to the glucose stimulation as naked islets with higher cell survival. The NFBG of diabetic mice transplanted with islets/chitosan hydrogels decreased from 487+/-46 to 148+/-32 at one day postoperation and maintained in the range of 201+/-36 mg/dl for four weeks with an increase in body weight. IPGTT showed the glucose disappearance rate of mice transplanted with islets/chitosan hydrogels was significant faster than that of mice transplanted with naked islets; the serum insulin level increased from 0.29+/-0.06 to 1.69+/-0.65 microg/dl postoperatively. Histological examination revealed that the islets successfully engrafted at renal subcapsular space with positive insulin staining. The immunostain was negative for neither the T-cell lineages nor the monocyte/macrophages. This study indicates that the chitosan hydrogels deliver and protect encapsulated islets successfully in xenotransplantation.

Islet transplantation has shown great success in the treatment of type 1 diabetes since the Edmonton protocol was established. However, it still has two major problems to overcome: the lack of organ donors and the side effects of immunosuppression. Encapsulated islets have emerged as a potential option for islet transplantation because it can, at least partly, overcome these two problems. Wistar rat islets suspended in 3% polyvinyl alcohol (PVA) hydrogel were frozen-thawed to make macroencapsulated islets (MEIs). The recovery rate, insulin content, and morphological change in culture medium with/without fresh human plasma (FHP) were measured in MEIs and free islets in vitro. In vivo, MEIs of either Wistar or Lewis rats were transplanted into the peritoneal cavity of streptozotocin (STZ)-induced diabetic Lewis rats and nonfasting blood glucose (NFBG), body weight, and histological evaluations were processed. FHP destroyed rat free islets but did not affect the islet morphology, islet recovery rate, or insulin content of rat MEIs. The transplantation of MEIs decreased the NFBG level and prevented body weight loss without a significant difference between the donor strains. Insulin-positive islets were observed in PVA MEIs 24 weeks after allotransplantation. These results suggest that PVA MEIs may be used as a cure for type 1 diabetes.