Extracellular ATP formation from ADP and inorganic phosphate, attributed to the activity of a cell surface ATP synthase, has so far only been reported in cultures of some proliferating and tumoral cell lines. We now provide evidence showing the presence of a functionally active ecto-F(o)F(1)-ATP synthase on the plasma membrane of normal tissue cells, i.e. isolated rat hepatocytes. Both confocal microscopy and flow cytometry analysis show the presence of subunits of F(1) (alpha/beta and gamma) and F(o) (F(o)I-PVP(b) and OSCP) moieties of ATP synthase at the surface of rat hepatocytes. This finding is confirmed by immunoblotting analysis of the hepatocyte plasma membrane fraction. The presence of the inhibitor protein IF(1) is also detected on the hepatocyte surface. Activity assays show that the ectopic-ATP synthase can work both in the direction of ATP synthesis and hydrolysis. A proton translocation assay shows that both these mechanisms are accompanied by a transient flux of H(+) and are inhibited by F(1) and F(o)-targeting inhibitors. We hypothesise that ecto-F(o)F(1)-ATP synthase may control the extracellular ADP/ATP ratio, thus contributing to intracellular pH homeostasis.

Inhibition of multiple myeloma (MM) plasma cells in their permissive bone marrow microenvironment represents an attractive strategy for blocking the tumor/ vessel growth associated with the disease progression. However, target speci-ficity is an essential aim of this approach. Here, we identified platelet-derived growth factor (PDGF)-receptor beta (PDGFR) and pp60c-Src as shared constitutively activated tyrosine-kinases (TKs) in plasma cells and endothelial cells (ECs) isolated from MM patients (MMECs). Our cellular and molecular dissection showed that the PDGF-BB/PDGFR kinase axis promoted MM tumor growth and vessel sprouting by activating ERK1/2, AKT, and the transcription of MMEC-released proangio-genic factors, such as vascular endothe-lial growth factor (VEGF) and interleukin-8 (IL-8). Interestingly, pp60c-Src TK-activity was selectively induced by VEGF in MM tumor and ECs, and the use of small-interfering (si)RNAs validated pp60c-Src as a key signaling effector of VEGF loop required for MMEC survival, migration, and angiogenesis. We also assessed the antitumor/vessel activity of dasatinib, a novel orally bioactive PDGFR/Src TK-inhibitor that significantly delayed MM tumor growth and angiogenesis in vivo, showing a synergistic cytotoxicity with conventional and novel antimyeloma drugs (ie, melphalan, prednisone, bor-tezomib, and thalidomide). Overall data highlight the biologic and therapeutic relevance of the combined targeting of PDGFR/c-Src TKs in MM, providing a framework for future clinical trials. (Blood. 2008;112:1346-1356) Introduction Multiple myeloma (MM) is characterized by a clonal proliferation of immunoglobulin-secreting plasma cells in the bone marrow (BM), with clinical manifestations including lytic bone lesions, anemia, renal failure, immunodeficiency, and hypercalcemia. 1 For patients treated with conventional and high-dose chemotherapies, the median survival time from diagnosis is 3 to 4 years. 2 Approximately one-half of patients with newly diagnosed MM achieve remission from these therapies, but options are more limited for primary resistant or relapsing disease. Partial remission occurs in only 20% of resistant patients and in 45% of relapsing patients. Survival time improves in approximately 50% of those younger than 60 who are able to undergo high-dose chemotherapy followed by autologous stem cell transplantation, but approximately 30% of them are late in the graft intake or even develop myelodysplasia that prevents additional chemotherapy while relapse or resistant disease occurs. Induction of MM plasma cells is thought to involve genetic lesions (ie, translocations between immunoglobulin enhancers and oncogenes) and secondary events underlying the activation of bidirectional MM tumor-microenvironment interactions. 3-5 Indeed, homing and survival of MM plasma cells are sustained by overangiogenic sprouting of microvascular endothelial cells (ECs), 6,7 as well as by osteoclasts, fibroblasts, monocytes, macrophages, and mast cells, 4,8,9 thus resulting in a multiplicity of autocrine/...

Dasatinib (BMS-354825, Sprycel®) is an oral, multitargeted inhibitor of receptor tyrosine kinases (RTKs), including BCR-ABL fusion protein, stem cell factor receptor (c-KIT), platelet-derived growth factor receptor (PDGFR), and Src family kinases (SFKs). Several early- and late-phase clinical trials for chronic myelogeneous leukaemia (CML) have demonstrated the direct inhibition of BCR-ABL fusion protein and SFKs, which led to dasatinib approval by the Food and Drug Administration (FDA) and the European Union for the treatment of imatinib-resistant or -intolerant CML, and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). Phase III dose-optimization study was performed to compare different regimens, stating that dasatinib 100 mg once daily is now the recommended schedule for patients with chronic CML, and 140 mg once daily for patients with accelerated phase or myeloid or lymphoid blast phase CML, and for patients with Ph+ ALL until progression. Because of the myriad of critical roles of SFKs in biological processes, SFKs inhibition could induce numerous biological responses. Ongoing clinical trials evaluate dasatinib in the treatment of several solid tumours, including gastrointestinal stromal tumours (GIST), prostate cancer, malignant pleural mesothelioma, sarcomas, NSCLC, colorectal cancer, glioblastoma and other haematologic malignancies as multiple myeloma. Ongoing pre-clinical studies assess the therapeutic potential of dasatinib in other solid tumours, including melanoma, head and neck cancer, breast cancer and ovarian cancer. Dasatinib is generally well tolerated. Myelosuppression is the common adverse event which is, however, reversible by dose reduction, discontinuation, or interruption. Thrombocytopenia is more significant than neutropenia and associated to gastrointestinal bleeding and CNS haemorrhage. The most common non-haematologic adverse events include gastrointestinal symptoms (diarrhoea, nausea, vomiting, abdominal pain and anorexia), headache, peripheral edema, and pleural effusion. In respect of these encouraging studies investigating dasatinib in the treatment of patients with GIST, prostate cancer, multiple myeloma and sarcomas, ongoing phase III clinical trials warrant the drug evaluation as recommended agent for the treatment of these diseases, also in association with chemotherapy or other targeted therapies.

Pancreatic ductal adenocarcinoma (PDAC) occurs in the majority of cases with early loco-regional spread and distant metastases at diagnosis, leading to dismal prognosis with a 5-year overall survival rate moderately over than 5%. This malignancy is largely resistant to chemotherapy and radiation, but the reasons of the refractoriness to the therapies is still unknown. Evidence is accumulating to indicate that the PDAC microenvironment and vascularity strongly contribute to the clinical features of this disease. In particular, PDAC is characterized by excessive dense extracellular matrix deposition associated to vasculature collapse and hypoxia with low drug delivery, explaining at least partly the low efficacy of antiangiogenic drugs in this cancer. Strategies aimed to modulate tumor stroma favoring vasculature perfusion and chemotherapeutics delivery are under investigation.

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