Nanotechnology Frontiers in γ-Herpesviruses Treatments.

Epstein-Barr Virus (EBV) and Kaposi's sarcoma associated-herpesvirus (KSHV) are γ-herpesviruses that belong to the Herpesviridae family. EBV infections are linked to the onset and progression of several diseases, such as Burkitt lymphoma (BL), nasopharyngeal carcinoma (NPC), and lymphoproliferative malignancies arising in post-transplanted patients (PTDLs). KSHV, an etiologic agent of Kaposi's sarcoma (KS), displays primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD). Many therapeutics, such as bortezomib, CHOP cocktail medications, and natural compounds (e.g., quercetin or curcumin), are administrated to patients affected by γ-herpesvirus infections. These drugs induce apoptosis and autophagy, inhibiting the proliferative and cell cycle progression in these malignancies. In the last decade, many studies conducted by scientists and clinicians have indicated that nanotechnology and nanomedicine could improve the outcome of several treatments in γ-herpesvirus-associated diseases. Some drugs are entrapped in nanoparticles (NPs) expressed on the surface area of polyethylene glycol (PEG). These NPs move to specific tissues and exert their properties, releasing therapeutics in the cell target. To treat EBV- and KSHV-associated diseases, many studies have been performed in vivo and in vitro using virus-like particles (VPLs) engineered to maximize antigen and epitope presentations during immune response. NPs are designed to improve therapeutic delivery, avoiding dissolving the drugs in toxic solvents. They reduce the dose-limiting toxicity and reach specific tissue areas. Several attempts are ongoing to synthesize and produce EBV vaccines using nanosystems.

KSHV infection skews macrophage polarisation towards M2-like/TAM and activates Ire1 α-XBP1 axis up-regulating pro-tumorigenic cytokine release and PD-L1 expression.

BACKGROUND: Kaposi's Sarcoma Herpesvirus (KSHV) is a gammaherpesvirus strongly linked to human cancer. The virus is also able to induce immune suppression, effect that contributes to onset/progression of the viral-associated malignancies. As KSHV may infect macrophages and these cells abundantly infiltrate Kaposi's sarcoma lesions, in this study we investigated whether KSHV-infection could affect macrophage polarisation to promote tumorigenesis. METHODS: FACS analysis was used to detect macrophage markers and PD-L1 expression. KSHV infection and the molecular pathways activated were investigated by western blot analysis and by qRT-PCR while cytokine release was assessed by Multi-analyte Kit. RESULTS: We found that KSHV infection reduced macrophage survival and skewed their polarisation towards M2 like/TAM cells, based on the expression of CD163, on the activation of STAT3 and STAT6 pathways and the release of pro-tumorigenic cytokines such as IL-10, VEGF, IL-6 and IL-8. We also found that KSHV triggered Ire1 α-XBP1 axis activation in infected macrophages to increase the release of pro-tumorigenic cytokines and to up-regulate PD-L1 surface expression. CONCLUSIONS: The findings that KSHV infection of macrophages skews their polarisation towards M2/TAM and that activate Ire1 α-XBP1 to increase the release of pro-tumorigenic cytokines and the expression of PD-L1, suggest that manipulation of UPR could be exploited to prevent or improve the treatment of KSHV-associated malignancies.

Sourcing the immune system to induce immunogenic cell death in Kras-colorectal cancer cells.

BACKGROUND: Current approaches aimed at inducing immunogenic cell death (ICD) to incite an immune response against cancer neoantigens are based on the use of chemotherapeutics and other agents. Results are hampered by issues of efficacy, combinatorial approaches, dosing and toxicity. Here, we adopted a strategy based on the use of an immunomolecule that overcomes pharmachemical limitations. METHODS: Cytofluorometry, electron microscopy, RT-PCR, western blotting, apotome immunofluorescence, MLR and xenografts. RESULTS: We report that an ICD process can be activated without the use of pharmacological compounds. We show that in Kras-mut/TP53-mut colorectal cancer cells the 15 kDa ßGBP cytokine, a T cell effector with onco-suppressor properties and a potential role in cancer immunosurveillance, induces key canonical events required for ICD induction. We document ER stress, autophagy that extends from cancer cells to the corresponding xenograft tumours, CRT cell surface shifting, ATP release and evidence of dendritic cell activation, a process required for priming cytotoxic T cells into a specific anticancer immunogenic response. CONCLUSIONS: Our findings provide experimental evidence for a rationale to explore a strategy based on the use of an immunomolecule that as a single agent couples oncosuppression with the activation of procedures necessary for the induction of long term response to cancer.

Impact of HHV-6A and HHV-6B lytic infection on autophagy and endoplasmic reticulum stress.

Herpesviruses are known to manipulate autophagy to optimize their replication, counteract immune response and probably to promote tumourigenesis. This study explored, for the first time, the impact of human herpesvirus (HHV)-6 lytic infection on autophagy and demonstrated that HHV-6A and B (viruses sharing more than 80 % homology) differently affected this cellular process. Indeed, while HHV-6A (GS) infection of HSB2 cells promoted autophagy, HHV-6B (Z29) or the virus isolated from the serum of roseola infantum-affected patient-inhibited autophagy in Molt-3 cells or in PBMCs, respectively. Interestingly, the different behaviour of HHV-6A and B on the autophagic process was accompanied by different effects on endoplasmic reticulum stress, unfolded protein response and cell survival that was more strongly reduced by HHV-6B infection. We hypothesize that the ability to inhibit autophagy displayed by HHV-6B could be due to the fact that it contains gene homologues of those encoding for TRS1; the protein responsible for the block of autophagy by human cytomegalovirus. Understanding how HHV-6A/B infection regulates autophagy could be of particular interest, as it has been recently shown that this virus may be involved in Alzheimer's disease in which a dysregulation of autophagy may also play a role.

High glucose and hyperglycemic sera from type 2 diabetic patients impair DC differentiation by inducing ROS and activating Wnt/ß-catenin and p38 MAPK.

Type 2 is the type of diabetes with higher prevalence in contemporary time, representing about 90% of the global cases of diabetes. In the course of diabetes, several complications can occur, mostly due to hyperglycemia and increased reactive oxygen species (ROS) production. One of them is represented by an increased susceptibility to microbial infections and by a reduced capacity to clear them. Therefore, knowing the impact of hyperglycemia on immune system functionality is of utmost importance for the management of the disease. In this study, we show that medium containing high glucose reduced the in-vitro differentiation of monocytes into functional DCs and their activation mediated by PAMPs or DAMPs. Most importantly, the same effects were mediated by the hyperglycemic sera derived by type 2 diabetic patients, mimicking a more physiologic condition. DC dysfunction caused by hyperglycemia may be involved in the inefficient control of infections observed in diabetic patients, given the pivotal role of these cells in both the innate and adaptive immune response. Searching for the molecular mechanisms underlying DC dysfunction, we found that canonical Wnt/ß-catenin and p38 MAPK pathways were activated in the DCs differentiated either in the presence of high glucose or of hyper-glycemic sera. Interestingly, the activation of these pathways and the DC immune dysfunction were partially counteracted by the anti-oxidant quercetin, a flavonoid already known to exert several beneficial effects in diabetes.

PKC theta and p38 MAPK activate the EBV lytic cycle through autophagy induction.

PKC activation by combining TPA with sodium butyrate (T/B) represents the most effective and widely used strategy to induce the Epstein-Barr virus (EBV) lytic cycle. The results obtained in this study show that novel PKCθ is involved in such process and that it acts through the activation of p38 MAPK and autophagy induction. Autophagy, a mechanism of cellular defense in stressful conditions, is manipulated by EBV to enhance viral replication. Besides promoting the EBV lytic cycle, the activation of p38 and autophagy resulted in a pro-survival effect, as indicated by p38 or ATG5 knocking down experiments. However, this pro-survival role was counteracted by a pro-death activity of PKCθ, due to the dephosphorylation of AKT. In conclusion, this study reports, for the first time, that T/B activates a PKCθ-p38 MAPK axis in EBV infected B cells, that promotes the viral lytic cycle and cell survival and dephosphorylates AKT, balancing cell life and cell death.

Hepatitis C virus present in the sera of infected patients interferes with the autophagic process of monocytes impairing their in-vitro differentiation into dendritic cells.

Autophagy has a pivotal role in the in-vitro monocyte differentiation into macrophages and dendritic cells (DCs), the most powerful antigen presenting cells (APC) with the unique capacity to initiate an adaptive immune response. Autophagy is also a mechanism by which these cells of innate immunity may degrade intracellular pathogens and mediate the antigen processing and presentation, essential to clear an infection. For these reasons, pathogens have learned how to manipulate autophagy for their own survival. In this study we found that hepatitis C virus (HCV), derived from sera of infected patients, blocked the autophagic process in differentiating monocytes, seen as LC3 II and p62 expression levels. The suppression of autophagy correlated with a reduction of cathepsins D, B and proteolytic activity, and resulted in impairment of monocyte differentiation into DCs, as indicated by the reduction of CD1a acquirement. These data suggest that the block of autophagy might be one of the underlying mechanisms of the HCV-mediated immune subversion that frequently leads to viral persistence and chronic hepatitis.

Epstein-barr virus blocks the autophagic flux and appropriates the autophagic machinery to enhance viral replication.

UNLABELLED: Autophagy is a catabolic pathway that helps cells to survive under stressful conditions. Cells also use autophagy to clear microbiological infections, but microbes have learned how to manipulate the autophagic pathway for their own benefit. The experimental evidence obtained in this study suggests that the autophagic flux is blocked at the final steps during the reactivation of Epstein-Barr virus (EBV) from latency. This is indicated by the level of the lipidated form of LC3 that does not increase in the presence of bafilomycin and by the lack of colocalization of autophagosomes with lysosomes, which correlates with reduced Rab7 expression. Since the inhibition of the early phases of autophagy impaired EBV replication and viral particles were observed in autophagic vesicles in the cytoplasm of producing cells, we suggest that EBV exploits the autophagic machinery for its transportation in order to enhance viral production. The autophagic block was not mediated by ZEBRA, an immediate-early EBV lytic gene, whose transfection in Ramos, Akata, and 293 cells promoted a complete autophagic flux. The block occurred only when the complete set of EBV lytic genes was expressed. We suggest that the inhibition of the early autophagic steps or finding strategies to overcome the autophagic block, allowing viral degradation into the lysosomes, can be exploited to manipulate EBV replication. IMPORTANCE: This study shows, for the first time, that autophagy is blocked at the final degradative steps during EBV replication in several cell types. Through this block, EBV hijacks the autophagic vesicles for its intracellular transportation and enhances viral production. A better understanding of virus-host interactions could help in the design of new therapeutic approaches against EBV-associated malignancies.

Ultrastructural Changes of Neuroendocrine Pheochromocytoma Cell Line PC-12 Exposed In Vitro to Rotenone.

Rotenone is a pesticide used in research for its ability to induce changes similar, in vivo and in vitro, to those observed in Parkinson's disease (PD). This includes a selective death of dopaminergic neurons in the substantia nigra. Nonetheless, the precise mechanism through which rotenone modifies structure and function of neurons remains unclear. The PC12 cells closely resemble dopamine terminal neurons. This makes it a preferred model for studying the morphology of central dopamine neurons and predicting neurotoxicity. In this paper, we investigated the effects of 0.5 µM rotenone for 24-48 h on PC12 cell viability and ultrastructure (TEM), trying to identify primary and more evident alterations that can be related to neuronal damages similar to that seen in animal PD models. Cell viability decreased after 24 h rotenone treatment, with a further decrease after 48 h. Ultrastructural changes included vacuolar degeneration, mitochondrial mild swelling, decrease in the number of neuropeptide granules, and the loss of cell-to-cell adhesion. These findings are in agreement with previous research suggesting that rotenone, by inhibiting energy production and increasing ROS generation, is responsible for significant alterations of the ultrastructure and cell death of PC12 cells. Our data confirm the link between rotenone exposure, neuronal damage, and changes in dopamine metabolism, suggesting its role in the pathogenesis of PD.

HHV-8 reduces dendritic cell migration through down-regulation of cell-surface CCR6 and CCR7 and cytoskeleton reorganization.

BACKGROUND: For an efficient immune response against viral infection, dendritic cells (DCs) must express a coordinate repertoire of receptors that allow their recruitment to the sites of inflammation and subsequently to the secondary lymphoid organs in response to chemokine gradients.Several pathogens are able to subvert the chemokine receptor expression and alter the migration properties of DCs as strategy to escape from the immune control. FINDINGS: Here we report the inhibitory effect of Human Herpesvirus 8 (HHV-8) on the migratory behavior of immature and mature DCs. We found that the virus altered the DC chemokine receptor expression and chemokine induced migration. Moreover HHV-8 was also able to interfere with basal motility of DCs by inducing cytoskeleton modifications. CONCLUSION: Based on our findings, we suggest that HHV-8 is able to subvert the DC migration capacity and this represents an additional mechanism which interferes with their immune-functions.

Deletion of Epstein-Barr virus BFLF2 leads to impaired viral DNA packaging and primary egress as well as to the production of defective viral particles.

Previous genetic and biochemical studies performed with several members of the Alphaherpesvirus subfamily have shown that the UL31 and UL34 proteins are essential components of the molecular machinery that mediates the primary egress of newly assembled capsids across the nuclear membrane. Further, there is substantial evidence that BFLF2 and BFRF1, the respective positional homologs of UL31 and UL34 in the Epstein-Barr virus (EBV) genome, are also their functional homologs, i.e., that the UL31/UL34 pathway is common to distant herpesviruses. However, the low degree of protein sequence identity between UL31 and BFLF2 would argue against such a hypothesis. To further clarify this issue, we have constructed a recombinant EBV strain devoid of BFLF2 (DeltaBFLF2) and show that BFLF2 is crucial for efficient virus production but not for lytic DNA replication or B-cell transformation. This defective phenotype could be efficiently restored by trans complementation with a BFLF2 expression plasmid. Detailed analysis of replicating cells by electron microscopy revealed that, as expected, DeltaBFLF2 viruses not only failed to egress from the nucleus but also showed defective DNA packaging. Nonfunctional primary egress did not, however, impair the production and extracellular release of enveloped but empty viral particles that comprised L particles containing tegument-like structures and a few virus-like particles carrying empty capsids. The DeltaBFLF2 and DeltaUL31 phenotypes therefore only partly overlap, from which we infer that BFLF2 and UL31 have substantially diverged during evolution to fulfil related but distinct functions.

Autophagy manipulation as a strategy for efficient anticancer therapies: possible consequences.

Autophagy is a catabolic process whose activation may help cancer cells to adapt to cellular stress although, in some instances, it can induce cell death. Autophagy stimulation or inhibition has been considered an opportunity to treat cancer, especially in combination with anticancer therapies, although autophagy manipulation may be viewed as controversial. Thus, whether to induce or to inhibit autophagy may be the best option in the different cancer patients is still matter of debate. Her we will recapitulate the possible advantages or disadvantages of manipulating autophagy in cancer, not only with the aim to obtain cancer cell death and disable oncogenes, but also to evaluate its interplay with the immune response which is fundamental for the success of anticancer therapies.

Kaposi Sarcoma Herpes Virus (KSHV) infection inhibits macrophage formation and survival by counteracting Macrophage Colony-Stimulating Factor (M-CSF)-induced increase of Reactive Oxygen Species (ROS), c-Jun N-terminal kinase (JNK) phosphorylation and autophagy.

Kaposi Sarcoma Herpes Virus (KSHV) is an oncovirus belonging to the human gammaherpesvirus family, able to infect several immune cell types including B cells, dendritic cells (DCs) and monocytes. In this study, we found that KSHV infection of monocytes counteracted the Reactive Oxygen Species (ROS) increase induced by Macrophage Colony-Stimulating Factor (M-CSF), prevented c-Jun N-terminal kinase (JNK) and B-cell lymphoma-2 (Bcl-2) phosphorylation and inhibited autophagy, leading to an impairment of cell survival and differentiation into macrophages. We also show that, to further dysregulate immune response in monocytes, KSHV reduced the production of pro-inflammatory cytokines such as Tumor necrosis factor alpha (TNF α) while increased the release of the immune suppressive cytokine Interleukin-10 (IL-10). These results unveils new strategies put in place by KSHV to induce immune suppression and to persist into the infected host.

Mutant p53, Stabilized by Its Interplay with HSP90, Activates a Positive Feed-Back Loop Between NRF2 and p62 that Induces Chemo-Resistance to Apigenin in Pancreatic Cancer Cells.

Pancreatic cancer is one of the most aggressive cancers whose prognosis is worsened by the poor response to the current chemotherapies. In this study, we investigated the cytotoxic effect of Apigenin, against two pancreatic cell lines, namely Panc1 and PaCa44, harboring different p53 mutations. Apigenin is a flavonoid widely distributed in nature that displays anti-inflammatory and anticancer properties against a variety of cancers. Here we observed that Apigenin exerted a stronger cytotoxic effect against Panc1 cell line in comparison to PaCa44. Searching for mechanisms responsible for such different effect, we found that the higher cytotoxicity of Apigenin correlated with induction of higher level of intracellular ROS, reduction of mutant (mut) p53 and HSP90 expression and mTORC1 inhibition. Interestingly, we found that mutp53 was stabilized by its interplay with HSP90 and activates a positive feed-back loop between NRF2 and p62, up-regulating the antioxidant response and reducing the cytotoxicity of Apigenin. These results suggest that targeting the molecules involved in the mTOR-HSP90-mutp53-p62-NRF2-antioxidant response axis could help to overcome the chemo-resistance of pancreatic cancer to Apigenin.

Quercetin Interrupts the Positive Feedback Loop Between STAT3 and IL-6, Promotes Autophagy, and Reduces ROS, Preventing EBV-Driven B Cell Immortalization.

The oncogenic gammaherpesvirus Epstein-Barr virus (EBV) immortalizes in vitro B lymphocytes into lymphoblastoid cell lines (LCLs), a model that gives the opportunity to explore the molecular mechanisms driving viral tumorigenesis. In this study, we addressed the potential of quercetin, a widely distributed flavonoid displaying antioxidant, anti-inflammatory, and anti-cancer properties, in preventing EBV-driven B cell immortalization. The results obtained indicated that quercetin inhibited thectivation of signal transducer and activator of transcription 3 (STAT3) induced by EBV infection and reduced molecules such as interleukin-6 (IL-6) and reactive oxidative species (ROS) known to be essential for the immortalization process. Moreover, we found that quercetin promoted autophagy and counteracted the accumulation of sequestosome1/p62 (SQSTM1/p62), ultimately leading to the prevention of B cell immortalization. These findings suggest that quercetin may have the potential to be used to counteract EBV-driven lymphomagenesis, especially if its stability is improved.

STAT3 phosphorylation affects p53/p21 axis and KSHV lytic cycle activation.

The Tyr705 STAT3 constitutive activation, besides promoting PEL cell survival, contributes to the maintenance of viral latency. We found indeed that its de-phosphorylation by AG490 induced KSHV lytic cycle. Moreover, Tyr705 STAT3 de-phosphorylation, mediated by the activation of tyrosine phosphatases, together with the increase of Ser727 STAT3 phosphorylation contributed to KSHV lytic cycle induction by TPA. We then observed that p53-p21 axis, essential for the induction of KSHV replication, was activated by the inhibition of Tyr705 and by the increase of Ser727 STAT3 phosphorylation. As a possible link between STAT3, p53-p21 and KSHV lytic cycle, we found that TPA and AG490 reduced the expression of KAP-1, promoting p53 stability, p21 transcription and KSHV lytic cycle activation in PEL cells.

Cytotoxic Drugs Activate KSHV Lytic Cycle in Latently Infected PEL Cells by Inducing a Moderate ROS Increase Controlled by HSF1, NRF2 and p62/SQSTM1.

Previous studies have indicated that cytotoxic treatments may induce or not activate viral lytic cycle activation in cancer cells latently infected by Kaposi's sarcoma-associated herpesvirus (KSHV). To investigate the molecular mechanisms responsible for such an effect, we compared two cytotoxic treatments able to induce the viral lytic cycle, named 12-O-tetradecanoylphorbol 13-acetate (TPA) (T) in combination with sodium butyrate (B) and bortezomib (BZ), with two cytotoxic treatments that did not activate this process, named metformin (MET) and quercetin (Q). Our results indicated that TB and bortezomib increased levels of oxygen reactive species (ROS) while metformin and quercetin reduced them. The finding that N-acetylcysteine (NAC), a reactive oxigen species (ROS) scavenger, counteracted K-bZIP expression induced by TB or bortezomib, confirmed that an ROS increase played a role in KSHV lytic cycle activation. Moreover, we found that TB and bortezomib up-regulated p62/Sequestosome1(p62/SQSTM1) protein, while metformin and quercetin down-regulated it. p62/SQSTM1 silencing or the inhibition of NF-E2-related factor 2 (NRF2) or Heat Shock Factor 1 (HSF1), that mediate p62/SQSTM1 transcription, also reduced KSHV lytic antigen expression induced by TB or bortezomib. Interestingly, such combination treatments further increased intracellular ROS and cytotoxicity induced by the single TB or bortezomib treatment, suggesting that NRF2, HSF1 and p62/SQSTM1 keep the ROS level under control, allowing primary effusion lymphoma (PEL) cells to continue to survive and KSHV to replicate.

EBV up-regulates PD-L1 on the surface of primary monocytes by increasing ROS and activating TLR signaling and STAT3.

Programmed death ligand 1 (PD-L1) (also called B7-H1) is a membrane immune-modulatory protein whose overexpression on the surface of tumor cells as well as APCs impairs T-cell-mediated killing. Viruses that establish chronic infections have developed a number of strategies to escape from immune recognition including the up-regulation of PD-L1. This study shows for the first time that the human oncovirus EBV infects human primary monocytes using HLA-DR and induced a strong up-regulation of PD-L1 expression on their surface. Searching for the underlying mechanism/s leading to this immune suppressive effect, we found that EBV activated TLR signaling, increased intracellular ROS, and phosphorylated STAT3. Targeting these molecules partially reverted PD-L1 up-regulation that correlated with an altered cytokine production and a reduction of monocyte cell survival, strongly impairing the antiviral immune response.

Apigenin, by activating p53 and inhibiting STAT3, modulates the balance between pro-apoptotic and pro-survival pathways to induce PEL cell death.

BACKGROUND: Apigenin is a flavonoid widely distributed in plant kingdom that exerts cytotoxic effects against a variety of solid and haematological cancers. In this study, we investigated the effect of apigenin against primary effusion lymphoma (PEL), a KSHV-associated B cell lymphoma characterized by a very aggressive behavior, displaying constitutive activation of STAT3 as well as of other oncogenic pathways and harboring wtp53. METHODS: Cell death was assessed by trypan blue exclusion assay, FACS analysis as well as by biochemical studies. The latter were also utilized to detect the occurrence of autophagy and the molecular mechanisms leading to the activation of both processes by apigenin. FACS analysis was used to measure the intracellular ROS utilizing DCFDA. RESULTS: We show that apigenin induced PEL cell death and autophagy along with reduction of intracellular ROS. Mechanistically, apigenin activated p53 that induced catalase, a ROS scavenger enzyme, and inhibited STAT3, the most important pro-survival pathway in PEL, as assessed by p53 silencing. On the other hand, STAT3 inhibition by apigenin resulted in p53 activation, since STAT3 negatively influences p53 activity, highlighting a regulatory loop between these two pathways that modulates PEL cell death/survival. CONCLUSION: The findings of this study demonstrate that apigenin may modulate pro-apoptotic and pro-survival pathways representing a valid therapeutic strategy against PEL.

Hyperglycemia triggers HIPK2 protein degradation.

Homeodomain interacting protein kinase-2 (HIPK2) is an evolutionary conserved kinase that modulates several key molecular pathways to restrain tumor growth and induce p53-depending apoptotic cell-death in response to anticancer therapies. HIPK2 silencing in cancer cells leads to chemoresistance and cancer progression, in part due to p53 inhibition. Recently, hyperglycemia has been shown to reduce p53 phosphorylation at serine 46 (Ser46), the target residue of HIPK2, thus impairing p53 apoptotic function. Here we asked whether hyperglycemia could, upstream of p53, target HIPK2. We focused on the effect of high glucose (HG) on HIPK2 protein stability and the underlying mechanisms. We found that HG reduced HIPK2 protein levels, therefore impairing HIPK2-induced p53 apoptotic activity. HG-triggered HIPK2 protein downregulation was rescued by both proteasome inhibitor MG132 and by protein phosphatase inhibitors Calyculin A (CL-A) and Okadaic Acid (OA). Looking for the phosphatase involved, we found that protein phosphatase 2A (PP2A) induced HIPK2 degradation, as evidenced by directly activating PP2A with FTY720 or by silencing PP2A with siRNA in HG condition. The effect of PP2A on HIPK2 protein degradation could be in part due to hypoxia-inducible factor-1 (HIF-1) activity which has been previously shown to induce HIPK2 proteasomal degradation through several ubiquitin ligases. Validation analysed performed with HIF-1α dominant negative or with silencing of Siah2 ubiquitin ligase clearly showed rescue of HG-induced HIPK2 degradation. These findings demonstrate how hyperglycemia, through a complex protein cascade, induced HIPK2 downregulation and consequently impaired p53 apoptotic activity, revealing a novel link between diabetes/obesity and tumor resistance to therapies.