Tiziana Annese

Effect of resveratrol on mitochondrial function: implications in parkin-associated familiar Parkinson's disease

Mitochondrial dysfunction and oxidative stress occur in Parkinsons disease (PD), but the molecular mechanisms controlling these events are not completely understood. Peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) is a transcriptional coactivator known as master regulator of mitochondrial functions and oxidative metabolism. Recent studies, including one from our group, have highlighted altered PGC-1α activity and transcriptional deregulation of its target genes in PD pathogenesis suggesting it as a new potential therapeutic target. Resveratrol, a natural polyphenolic compound proved to improve mitochondrial activity through the activation of several metabolic sensors resulting in PGC-1α activation. Here we have tested in vitro the effect of resveratrol treatment on primary fibroblast cultures from two patients with early-onset PD linked to different Park2 mutations. We show that resveratrol regulates energy homeostasis through activation of AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1) and raise of mRNA expression of a number of PGC-1αs target genes resulting in enhanced mitochondrial oxidative function, likely related to a decrease of oxidative stress and to an increase of mitochondrial biogenesis. The functional impact of resveratrol treatment encompassed an increase of complex I and citrate synthase activities, basal oxygen consumption, and mitochondrial ATP production and a decrease in lactate content, thus supporting a switch from glycolytic to oxidative metabolism. Moreover, resveratrol treatment caused an enhanced macro-autophagic flux through activation of an LC3-independent pathway. Our results, obtained in early-onset PD fibroblasts, suggest that resveratrol may have potential clinical application in selected cases of PD-affected patients.

Mast cells and angiogenesis in gastric carcinoma

Previous studies have shown that increased vascularity is associated with haematogenous metastasis and poor prognosis in gastric cancer. The role of mast cells in gastric cancer angiogenesis has not been clarified completely. In this study, we correlated microvascular density and tryptase‐ and chymase‐positive mast cells with histopathological type in gastric cancer. Specimens of primary gastric adenocarcinomas obtained from 30 patients who had undergone curative gastrectomy were investigated immunohistochemically by using anti‐CD31 antibody to stain endothelial cells and anti‐tryptase and anti‐chymase antibodies to stain mast cells. The results showed that stage IV gastric carcinoma has a higher degree of vascularization than other stages and that both tryptase‐ and chymase‐positive mast cells increase in parallel with malignancy grade even if the density of chymase‐positive mast cells was significantly lower than the density of tryptase‐positive mast cells and is highly correlated with the extent of angiogenesis. This study has demonstrated that mast cell density correlates with angiogenesis and progression of patients with gastric carcinoma. Understanding the mechanisms of gastric cancer angiogenesis provides a basis for a rational approach to the development of an antiangiogenic therapy in patients with this malignancy.

Aquaporins in cancer.

BACKGROUND The aquaporins (AQPs) are a family of 13 small hydrophobic integral transmembrane water channel proteins involved in transcellular and transepithelial water movement, transport of fluid and cell migration. SCOPE OF THE REVIEW This review article summarizes our knowledge concerning the involvement of AQPs in tumor growth, angiogenesis and metastatic process. MAJOR CONCLUSIONS Tumor cells types express AQPs and a positive correlation exists between histological tumor grade and the AQP expression. Moreover, AQPs are involved also in tumor edema formation and angiogenesis in several solid and hematological tumors. GENERAL SIGNIFICANCE AQPs inhibition in endothelial and tumor cells might limit tumor growth and spread, suggesting a potential therapeutic use in the treatment of tumors. This article is part of a Special Issue entitled Aquaporins.

Intussusceptive microvascular growth in human glioma

Intussusceptive microvascular growth (IMG), which occurs by splitting of the existing vasculature by transluminal pillars or transendothelial bridges, has been demonstrated in several tumors such as colon and mammary carcinomas, melanoma and B-cell non-Hodgkin’s lymphomas. In this study, we have correlated in human glioma the extent of angiogenesis, evaluated as microvascular density, the immunoreactivity of tumor cells to vascular endothelial growth factor (VEGF), vessel diameter and IMG to the tumor stage. Results demonstrate for the first time a relationship in human glioma progression between angiogenesis, VEGF immunoreactivity of tumor cells, vessel diameter and the number of connections of intraluminal tissue folds with the opposite vascular wall, expression of IMG and suggest that IMG could be a mechanism of compensatory vascular growth occurring in human glioma. The advantages are that (1) blood vessels are generated more rapidly; (2) it is energetically and metabolically more economic; (3) the capillaries thereby formed are less leaky.

Epo is involved in angiogenesis in human glioma

In this study, the extent of angiogenesis, evaluated as microvascular density, and the immunoreactivity of tumor cells to erythropoietin (Epo) and of endothelial cells to Epo receptor (EpoR) have been correlated in human glioma specimens, and the effect of anti-Epo antibody on glioma-induced angiogenesis in vivo in the chick embryo chorioallantoic membrane (CAM) has been investigated. Results show that: (1) Epo/EpoR expression correlates with angiogenesis, (2) in the CAM assay, tumor bioptic specimens induce a strong angiogenic response, comparable to that induced by VEGF, and (3) an anti-Epo antibody co-administered with tumor bioptic specimens significantly inhibits the angiogenic response. These findings suggest the presence of a loop in the Epo/EpoR system, i.e. Epo is secreted by glioma tumor cells and it affects glioma vascular endothelial cells via its receptor and promotes angiogenesis in a paracrine manner. Moreover, as demonstrated by in vivo experiments, Epo is responsible for the strong angiogenic response induced by human glioma bioptic specimens, because an anti-Epo antibody is able to significantly inhibit this response.

Aquaporin-4 contributes to the resolution of peritumoural brain oedema in human glioblastoma multiforme after combined chemotherapy and radiotherapy

Brain tumour oedema is coupled with blood-brain barrier damage and alteration in water flow. Aquaporin-4 (AQP4) is involved in the development and resolution of brain oedema, and it is strongly upregulated in glioblastoma multiforme (GBM). Here, we evaluated AQP4 expression and content in GBM and correlated with VEGF-VEGFR-2 expression. In the relapse after chemotherapy and radiotherapy, AQP4 content reduced in parallel with VEGF-VEGFR-2, as compared with primary tumours, and in the peripheral areas of relapsed tumours AQP4 mimicked normal findings of perivascular rearrangement. After immunogold electron microscopy, gold particles were attached on the glial membrane facing the perivascular side, likewise AQP4 gold labelling of the vessels of the control areas. In primary tumours the peripheral vessels appeared faintly marked by AQP4, while the perivascular tumour cells showed a strong expression. The vasculature of the inner tumour areas was unlabelled by AQP4, while tumour cells were labelled, in both primary and relapsing tumours. Relapsed tumours after radiotherapy alone showed slight AQP4 reduction and perivascular restoring in the peripheral areas of the tumour. These data indicate that in GBM chemotherapy and radiotherapy induce a down-regulation in AQP4 expression restoring its perivascular rearrangement suggesting its potential role in the resolution of brain oedema.

Glial dystrophin-associated proteins, laminin and agrin, are downregulated in the brain of mdx mouse

In this study, we investigated the involvement of dystrophin-associated proteins (DAPs) and their relationship with the perivascular basement membrane in the brains of mdx mice and controls at the age of 2 months. We analyzed (1) the expression of glial DAPs α–β-dystroglycan (DG), α-syntrophin, aquaporin-4 (AQP4) water channel, Kir 4.1 and dystrophin isoform (Dp71) by immunocytochemistry, laser confocal microscopy, immunogold electron microscopy, immunoblotting and RT-PCR; (2) the ultrastructure of the basement membrane and expression of laminin and agrin; and (3) the dual immunofluorescence colocalization of AQP4/α–β-DG, and of Kir 4.1/agrin. The following results were observed in mdx brain as compared with controls: (1) a significant reduction in protein content and mRNA expression of DAPs; (2) ultrastructurally, a thickened and discontinuous appearance of the basement membrane and a significant reduction in laminin and agrin; and (3) a molecular rearrangment of α–β-DG, coupled with a parallel loss of agrin and Kir 4.1 on basement membrane and glial endfeet. These data indicate that in mdx brain the deficiency in dystrophin and dystrophin isoform (Dp71) is coupled with a reduction of DAP components, coupled with an altered anchoring to the basement membrane.

HIF-1α of bone marrow endothelial cells implies relapse and drug resistance in patients with multiple myeloma and may act as a therapeutic target

Purpose: To investigate the role of hypoxia-inducible factor-1α (HIF-1α) in angiogenesis and drug resistance of bone marrow endothelial cells of patients with multiple myeloma. Experimental Design: HIF-1α mRNA and protein were evaluated in patients with multiple myeloma endothelial cells (MMEC) at diagnosis, at relapse after bortezomib- or lenalidomide-based therapies or on refractory phase to these drugs, at remission; in endothelial cells of patients with monoclonal gammapathies of undetermined significance (MGUS; MGECs), and of those with benign anemia (controls). The effects of HIF-1α inhibition by siRNA or panobinostat (an indirect HIF-1α inhibitor) on the expression of HIF-1α proangiogenic targets, on MMEC angiogenic activities in vitro and in vivo, and on overcoming MMEC resistance to bortezomib and lenalidomide were studied. The overall survival of the patients was also observed. Results: Compared with the other endothelial cell types, only MMECs from 45% of relapsed/refractory patients showed a normoxic HIF-1α protein stabilization and activation that were induced by reactive oxygen species (ROS). The HIF-1α protein correlated with the expression of its proangiogenic targets. The HIF-1α inhibition by either siRNA or panobinostat impaired the MMECs angiogenesis–related functions both in vitro and in vivo and restored MMEC sensitivity to bortezomib and lenalidomide. Patients with MMECs expressing the HIF-1α protein had shorter overall survival. Conclusions: The HIF-1α protein in MMECs may induce angiogenesis and resistance to bortezomib and lenalidomide and may be a plausible target for the antiangiogenic management of patients with well-defined relapsed/refractory multiple myeloma. It may also have prognostic significance. Clin Cancer Res; 20(4); 847–58. ©2013 AACR.

Microenvironment drug resistance in multiple myeloma: emerging new players

Multiple myeloma (MM) drug resistance (DR) is a multistep transformation process based on a powerful interplay between bone marrow stromal cells and MM cells that allows the latter to escape anti-myeloma therapies. Here we present an overview of the role of the bone marrow microenvironment in both soluble factors-mediated drug resistance (SFM-DR) and cell adhesion-mediated drug resistance (CAM-DR), focusing on the role of new players, namely miRNAs, exosomes and cancer-associated fibroblasts.

Halting pro-survival autophagy by TGFβ inhibition in bone marrow fibroblasts overcomes bortezomib resistance in multiple myeloma patients

Bortezomib (bort) has improved overall survival in patients with multiple myeloma (MM), but the majority of them develop drug resistance. In this study, we demonstrate that bone marrow (BM) fibroblasts (cancer-associated fibroblasts; CAFs) from bort-resistant patients are insensitive to bort and protect the RPMI8226 and patients’ plasma cells against bort-induced apoptosis. Bort triggers CAFs to produce high levels of interleukin (IL)-6, IL-8, insulin-like growth factor (IGF)-1 and transforming growth factor (TGF) β. Proteomic studies on CAFs demonstrate that bort resistance parallels activation of oxidative stress and pro-survival autophagy. Indeed, bort induces reactive oxygen species in bort-resistant CAFs and activates autophagy by increasing light chain 3 protein (LC3)-II and inhibiting p62 and phospho-mammalian target of rapamycin. The small-interfering RNA knockdown of Atg7, and treatment with 3-methyladenine, restores bort sensitivity in bort-resistant CAFs and produces cytotoxicity in plasma cells co-cultured with CAFs. In the syngeneic 5T33 MM model, bort-treatment induces the expansion of LC3-II+ CAFs. TGFβ mediates bort-induced autophagy, and its blockade by LY2109761, a selective TβRI/II inhibitor, reduces the expression of p-Smad2/3 and LC3-II and induces apoptosis in bort-resistant CAFs. A combination of bort and LY2109761 synergistically induces apoptosis of RPMI8226 co-cultured with bort-resistant CAFs. These data define a key role for CAFs in bort resistance of plasma cells and provide the basis for a novel targeted therapeutic approach.