Rita Reitano

Parkin modulates expression of HIF-1α and HIF-3α during hypoxia in gliobastoma-derived cell lines in vitro

Mutation of the Parkin gene causes an autosomal recessive juvenile-onset form of Parkinson’s disease. However, recently, it has been also linked to a wide variety of malignancies, including glioblastoma multiforme (GBM). In this pathology, Parkin exhibits a tumor suppressor role by mitigating the proliferation rate in both in vitro and in vivo models. However, Parkin involvement in the hypoxic process has not as yet been investigated. GBM is the most common and aggressive primary brain tumor in adults and is characterized by hypoxic areas. The low oxygen supply causes the expression of hypoxia-inducible factors (HIFs) leading to an accumulation of pro-angiogenic factors and tumoral invasiveness. We assess the relationship between Parkin and two HIFs expressed during hypoxic conditions, namely HIF-1α and HIF-3α. Our data show that Parkin is downregulated under hypoxia and that it interferes with HIF expression based on cellular oxygen tension. These results suggest a role for the involvement of Parkin in GBM, although further studies will be needed to understand the mechanism by which it modulates HIF-1α and HIF-3α expression.

PACAP and VIP Inhibit the Invasiveness of Glioblastoma Cells Exposed to Hypoxia through the Regulation of HIFs and EGFR Expression

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) through the binding of vasoactive intestinal peptide receptors (VIPRs), perform a wide variety of effects in human cancers, including glioblastoma multiforme (GBM). This tumor is characterized by extensive areas of hypoxia, which triggers the expression of hypoxia-inducible factors (HIFs). HIFs not only mediate angiogenesis but also tumor cell migration and invasion. Furthermore, HIFs activation is linked to epidermal growth factor receptor (EGFR) overexpression. Previous studies have shown that VIP interferes with the invasive nature of gliomas by regulating cell migration. However, the role of VIP family members in GBM infiltration under low oxygen tension has not been clarified yet. Therefore, in the present study we have investigated, for the first time, the molecular mechanisms involved in the anti-invasive effect of PACAP or VIP in U87MG glioblastoma cells exposed to hypoxia induced by treatment with desferrioxamine (DFX). The results suggest that either PACAP or VIP exert an anti-infiltrative effect under low oxygen tension by modulating HIFs and EGFR expression, key elements involved in cell migration and angiogenesis. These peptides act through the inhibition of PI3K/Akt and MAPK/ERK signaling pathways, which are known to have a crucial role in HIFs regulation.

Proteomic Analysis of Parkin Isoforms Expression in Different Rat Brain Areas

PARK2 gene’s mutations are related to the familial form of juvenile Parkinsonism, also known as the autosomic recessive juvenile Parkinsonism. This gene encodes for parkin, a 465-amino acid protein. To date, a large number of parkin isoforms, generated by an alternative splicing mechanism, have been described. Currently, Gene Bank lists 27 rat PARK2 transcripts, which matches to 20 exclusive parkin alternative splice variants. Despite the existence of these isoforms, most of the studies carried out so far, have been focused only on the originally cloned parkin. In this work we have analyzed the expression profile of parkin isoforms in some rat brain areas including prefrontal cortex, hippocampus, substantia nigra and cerebellum. To discriminate among these isoforms, we detected their localization through the use of two antibodies that are able to identify different domains of the parkin canonical sequence. Our analysis has revealed that at least fourteen parkin isoforms are expressed in rat brain with a various distribution in the regions analyzed. Our study might help to elucidate the pathophysiological role of these proteins in the central nervous system.

Correlation between expression profile of Wilms tumor 1 gene isoforms and neuroblastoma grade malignancy

Wilms tumor 1 gene (WT1) is expressed in neuroblastoma (NB) which represents the most aggressive extracranial pediatric tumor. This latter may transform into a more benign form such as ganglioneuroblastoma and ganglioneuroma or progress into a highly aggressive metastatic cancer with a poor survival rate. WT1 acts as tumor suppressor gene in NB by inducing the maturation in a less invasive mass. To date, it has been identified 13 mRNA WT1 variants encoding 13 proteins, however, most of the studies have focused their attention exclusively on isoform of ~49 kDa molecular weight (1, 2). In the present study, we have analyzed, the expression profile of WT1 isoforms, in undifferentiated and all-trans retinoic acid (RA) differentiated NB cells in order to evaluate their involvement in tumor malignancy. Results have shown that different isoforms are expressed both in untreated and RA treated NB cells. Their expression is significantly increased in RA treated cells, suggesting that WT1 isoforms are inversely related to NB malignancy grade. In accord to this hypothesis, WT1 isoforms and nestin expression are inversely related in undifferentiated and RA treated cells. Furthermore, the inhibition of the two signalling pathways specifically involved in differentiation of NB, PI3K/Akt and MAPK/ERK respectively, trigger an overexpression of all WT1 isoforms. In conclusion, these data suggest that overexpression of WT1 isoforms might promote trans-differentiation of NB into a more benign tumor such as ganglioneuroblastoma or ganglioneuroma.

PACAP and VIP counteract glioblastoma cells invasiveness

Glioblastoma multiforme (GBM) is an aggressive brain tumor characterized by hypoxic areas. The low oxygen supply induces expression of hypoxia-inducible factors (HIFs) leading to overexpression of epidermal growth factor receptor (EGFR) [1]. It is well known that pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are involved in human tumors, however, their role on GBM invasiveness has not been well elucidated [2]. To this end, we have investigated, for the first time, the anti-invasive effect of PACAP or VIP on GBM cells following exposure to hypoxia, by using desferrioxamine mesylate salt (DFX), an hypoxia-mimetic agent.The results have shown that under low oxygen tension, both PACAP and VIP reduce cells migration by modulating HIFs and EGFR expression. This effect is mediated through the inhibition of phosphoinositide 3 kinase (PI3K)/Akt and mammalian mitogen activated protein kinase/Erk kinase (MAPK/ERK) signaling cascades, which, as previously demonstrated, interfere with HIF-1 α and HIF-2α expression. In conclusion, our data suggest that PACAP and VIP might be good candidates to modulate GBM invasiveness exacerbated by hypoxic microenvironment.This work was supported by grants from MIUR FIRB 2010 and MIUR PRIN-2009.

Ameliorative effect of VIP family members on blood retinal barrier breakdown in diabetic macular edema

Diabetic macular edema (DME) is one of the main complications of diabetic retinopathy [1]. This pathology is owed to impairment of the blood-retinal barrier (BRB) [2]. Many factors, such as hypoxia, contribute to barrier dysfunction and progression of the disease. Low oxygen tension is one of the main events involved in the formation of new blood vessels that characterize the typical uncontrolled angiogenesis in proliferative stage of diabetic retinopathy. In the last decades, various studies have focused their attention on the role of pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) in the pathophysiology of DME. However, the effects of these peptides in maintaining the integrity of the BRB exposed to hypoxia remains to be elucidated. In the present work we have studied, for the first time, the effect of these peptides on outer BRB integrity following hypoxic insult in an experimental model of DME. To this end, we have used the human retinal pigment epithelial cells (ARPE-19) to test the effect of both peptides on cellular permeability, transepithelial electrical resistance, tight junctions expression and hypoxia-induced apoptosis. Results have demonstrated that both PACAP and VIP are able to rescue the integrity of cell monolayer during the hypoxic event, minimizing apoptotic damages induced by low tissue oxygen tension through the activation of phosphoinositide 3 kinase /Akt and mammalian mitogen activated protein kinase/Erk kinase signaling pathways. Furthermore, these peptides modulate the expression of vascular endothelial growth factor which is one of the downstream transcription factor activated during the hypoxic process. In conclusion, we have demonstrated that PACAP and VIP are able to counteract the damage induced by hypoxia on BRB through the modulation of hypoxia inducible factors expression.

Parkin interferes with hypoxia-inducible factors expression in glioblastoma cells

Parkin, also known as PARK2, is one of the largest genes in the human genome encoding for an E3 ubiquitin ligase. Its mutation cause a form of autosomal recessive juvenile-onset of Parkinson’s disease, but recently it has been linked in a wide variety of malignancies, including glioblastoma multiforme (GBM) (1). This is the most common and aggressive primary brain tumor in adults, characterized by hypoxic areas. The insufficient oxygen supply causes expression of hypoxia-inducible factors (HIF) which induce tumor growth and vascularization. In particular, in this condition increases expression of HIF-1α which has been correlated with angiogenesis, glucose metabolism and poor prognosis in glioblastoma. Instead HIF-3α is a negative regulator of HIF-1 α. In the present study we investigated if parkin interferes with such HIFs expression during hypoxic event. Parkin knockdown in glioblastoma cells induces a significant increase of HIF-1α expression in normoxia, whereas during hypoxia the absence of parkin abolishes its expression. HIF-3α expression significantly decreases both in normoxic or hypoxic condition following parkin silencing. These data have also been confirmed by immunofluorescence analysis. These results suggest that parkin is implicated in HIF regulation, therefore its modulation might be considered in GBM progression

Hypoxia inducible factors expression in lung adenocarcinoma cells

Lung adenocarcinoma is one of the most deadly malignancies with a low sur- vival rate.A typical characteristic of this tumor is angiogenesis which stimulates its growth. It is generate following hypoxia that induces activation of the hypoxia-inducible factors (HIFs) including HIF-1α, HIF-1β, HIF-2α, HIF-2β and HIF-3α. Previous studies have demonstrated the expression of these factors in lung adenocarcinomas [1-3]. In the present work we have analyzed their temporal expression profile in lung adenocarcinomas cells A549 by comparing it to that of normal bronchial epithelial cell lines BEAS-2B, during hypoxia with deferoxamine (DFX). This stressor induces a significant, time dependent, reduction of viabilityin both cell lines but more evident in BEAS-2B as shown by MTT analysis. Expression profile of HIFs members was assessed by Western blot analysis. During hypoxia HIF-1α expression increased in both cell lines, with a peak after 6h to 48h and then decreased significantly at 72h following treatment with DFX. HIF-1β levels reached a peak after 72h of treatment in both A549 and BEAS-2B cells, whereas HIF-2βsignificantly increases at 6h in A549 and at 72h in BEAS-2B of hypoxia. HIF-3α expression levels were inversely linked to those of HIF-1α in A549 while this correlation was absent in BEAS-2B. These data were also visualized by immunofluoerescence analysis. The present results have confirmed the involvement of HIFs members in lung cancer.