Oxidative Stress in Canine Histiocytic Sarcoma Cells Induced by an Infection with Canine Distemper Virus Led to a Dysregulation of HIF-1α Downstream Pathway Resulting in a Reduced Expression of VEGF-B In Vitro

Abstract

Histiocytic sarcomas represent malignant tumors which require new treatment strategies. Canine distemper virus (CDV) is a promising candidate due to its oncolytic features reported in a canine histiocytic sarcoma cell line (DH82 cells). Interestingly, the underlying mechanism might include a dysregulation of angiogenesis. Based on these findings, the aim of the present study was to investigate the impact of a persistent CDV-infection on oxidative stress mediated changes in the expression of hypoxia-inducible factor (HIF)-1α and its angiogenic downstream pathway in DH82 cells in vitro. Microarray data analysis, immunofluorescence for 8-hydroxyguanosine, superoxide dismutase 2 and catalase, and flow cytometry for oxidative burst displayed an increased oxidative stress in persistently CDV-infected DH82 cells (DH82Ond pi) compared to controls. The HIF-1α expression in DH82Ond pi increased, as demonstrated by Western blot, and showed an unexpected, often sub-membranous distribution, as shown by immunofluorescence and immunoelectron microscopy. Furthermore, microarray data analysis and immunofluorescence confirmed a reduced expression of VEGF-B in DH82Ond pi compared to controls. In summary, these results suggest a reduced activation of the HIF-1α angiogenic downstream pathway in DH82Ond pi cells in vitro, most likely due to an excessive, unusually localized, and non-functional expression of HIF-1α triggered by a CDV-induced increased oxidative stress.

P2Y2 and P2X4 Receptors Mediate Ca2+ Mobilization in DH82 Canine Macrophage Cells

“…To date, the DH82 canine macrophage cell line has primarily been utilized as a model of viral and protozoan infection [23][24][25], such as for the study of canine distemper and its oncolytic potential [55][56][57]. Although studies have described the expression of functional P2 receptors in human or rodent macrophages and macrophage cell lines [8,10,11,34], studies directly investigating P2 receptors in canine macrophages have been absent.…”

“…Studies have demonstrated that DH82 cells express a number of macrophage markers, such as CD11c and CD18 [ 21 ], and can secrete tumour necrosis factor (TNF)-α and interleukin (IL)-6 similar to that observed in lipopolysaccharide (LPS)-stimulated canine monocytes [ 22 ]. Despite its use as an in vitro model of viral and protozoan infection [ 23 , 24 , 25 ], knowledge regarding purinergic signalling in DH82 cells is limited to a single report describing ATP- and adenosine-induced cytokine release [ 26 ]. Although this study did not investigate any purinergic receptor per se, DH82 cells represent a possible model to study endogenous P2 receptors in canine macrophages for a number of reasons.…”

Attenuation of the Hypoxia Inducible Factor Pathway after Oncolytic Adenovirus Infection Coincides with Decreased Vessel Perfusion

“…Adenoviruses appear to only suppress HIF-1α at the transcriptional level, in contrast to other oncolytic viruses that alter the proteasomal pathway to destabilise HIF-1α [18,31], or, as recently described, inhibit HIF-1α activity by preventing its nuclear localisation [32]. Even though HIF activity is mainly controlled by post-translational regulation, several studies highlight the contribution of HIF-1α mRNA expression towards hypoxia-specific gene expression [33][34][35].…”

Intratumoral Canine Distemper Virus Infection Inhibits Tumor Growth by Modulation of the Tumor Microenvironment in a Murine Xenograft Model of Canine Histiocytic Sarcoma

“…The less pronounced effect in the present study lacking complete regression after intratumoral CDV infection might result from an inhibition of virus spread within the neoplastic tissue (<0.5% of tumor area infected) compared to typically high numbers of infected cells (>85%) in persistently infected cultures [ 34 , 45 ]. The mechanism of reduced angiogenesis might be addressed to an impairment of the hypoxia-inducible factor-1 α (HIF-1α) downstream pathway that resulted in a decreased vascular endothelial growth factor-B (VEGF-B) expression in persistently CDV-infected DH82 cells in vitro [ 46 ].…”

“…94062922). Cultivation of DH82 cells for grafting was carried out in standard conditions as previously described [ 33 , 34 , 45 , 46 ]. To ensure maximum viability for grafting, cells were harvested at 70% to 80% confluence of the monolayer.…”