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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Metformin triggers apoptosis in PEL cells and alters bortezomib-induced Unfolded Protein Response increasing its cytotoxicity and inhibiting KSHV lytic cycle activation.

Metformin, the most used drug for the treatment of diabetes type 2 patients, has been shown to have anti-cancer properties. In this study, we found that metformin induced apoptosis in Primary Effusion Lymphoma (PEL) cells, an aggressive B cell lymphoma associated with KSHV against which the conventional therapies usually fail. The cytotoxic effect of metformin correlated with intracellular reactive oxygen species reduction, activation of AMPK, the inhibition of pro-survival pathways such as mTOR and STAT3 and the down-regulation of v-FLIP, a latent viral antigen that also plays a pivotal role in PEL cell survival. Interestingly, we found that metformin could be used to potentiate the bortezomib-mediated cytotoxicity against PEL cells and to inhibit the activation of KSHV lytic cycle, a side effect of this treatment that resulted in a block of autophagy in these cells. Mechanistically, metformin altered UPR activated by bortezomib, leading to a reduced expression of BiP, up-regulation of CHOP and down-regulation of Bcl-2. In summary, this study suggests that metformin could represent a promising strategy for the treatment of PEL alone or in combination with bortezomib. In the latter case, besides exerting a stronger cytotoxic effect, it might be used to restrain bortezomib-induced viral replication that is involved in the maintenance and progression of KSHV-associated malignancies.

Bortezomib promotes KHSV and EBV lytic cycle by activating JNK and autophagy.

KSHV and EBV are gammaherpesviruses strictly linked to human cancers. Even if the majority of cancer cells harbor a latent infection, the few cells that undergo viral replication may contribute to the pathogenesis and maintenance of the virus-associated malignancies. Cytotoxic drugs used for the therapies of cancers harboring virus-infection often have, as side effect, the activation of viral lytic cycle. Therefore it is important to investigate whether they affect viral reactivation and understand the underlying mechanisms involved. In this study, we found that proteasome inhibitor bortezomib, a cytotoxic drug that efficiently target gammaherpesvirus-associated B cell lymphomas, triggered KSHV or EBV viral lytic cycle by activating JNK, in the course of ER stress, and inducing autophagy. These results suggest that the manipulation of these pathways could limit viral spread and improve the outcome of bortezomib treatment in patients affected by gammaherpesvirus-associated lymphomas.

Oxidant species are involved in T/B-mediated ERK1/2 phosphorylation that activates p53-p21 axis to promote KSHV lytic cycle in PEL cells.

KSHV is a gammaherpesvirus strongly associated to human cancers such as Primary Effusion Lymphoma (PEL) and Kaposi's Sarcoma. The naturally virus-infected tumor cells usually display latent infection since a minority of cells undergoes spontaneous viral replication. The lytic cycle can be induced in vitro upon appropriate stimuli such as TPA (T), alone or in combination with butyrate (B), (T/B). In previous studies, Protein Kinase C (PKC) δ, Extracellular Signal-regulated Kinase1/2 (ERK1/2) and p53-p21 axis have been separately reported to play a role in KSHV reactivation from latency. Here, we found that these pathways were interconnected to induce KSHV lytic cycle in PEL cells treated with T/B. T/B also increased H2O2 that played an important role in the activation of these pathways. Oxidant specie production correlated with PKC δ activation, as the PKC δ inhibitor rottlerin reduced both H2O2 and KSHV lytic antigen expression. H2O2 contributed to T/B-mediated ERK1/2 activation that mediated p53 phosphorylation at serine 15 (Ser15) and increased p21 expression. Oxidant specie inhibition by quercetin indeed strongly reduced the activation of these pathways, lytic antigen expression and interestingly it also increased T/B-induced cell death. The use of ERK inhibitor PD98059 or p53 silencing demonstrated the importance of p53Ser15 phosphorylation and of p53-p21 axis in KSHV lytic cycle activation. Understanding the role of oxidant species and the molecular mechanisms involved in KSHV lytic cycle induction is particularly important since oxidant species represent the most physiological stimulus for viral reactivation in vivo and it is known that viral production contributes to the maintenance/progression of KSHV associated malignancies.

Histone deacetylase inhibitors VPA and TSA induce apoptosis and autophagy in pancreatic cancer cells.

PURPOSE: Histone deacetylase inhibitors (HDACi) are anti-neoplastic agents that are known to affect the growth of different cancer types, but their underlying mechanisms are still incompletely understood. Here, we compared the effects of two HDACi, i.e., Trichostatin A (TSA) and Valproic Acid (VPA), on the induction of cell death and autophagy in pancreatic cancer-derived cells that exhibit a high metastatic capacity and carry KRAS/p53 double mutations. METHODS: Cell viability and proliferation tests were carried out using Trypan blue dye exclusion, MTT and BrdU assays. FACS analyses were carried out to assess cell cycle progression, apoptosis, reactive oxygen species (ROS) production and mitochondrial depolarization, while Western blot and immunoprecipitation analyses were employed to detect proteins involved in apoptosis and autophagy. RESULTS: We found that both VPA and TSA can induce apoptosis in Panc1 and PaCa44 pancreatic cancer-derived cells by triggering mitochondrial membrane depolarization, Cytochrome c release and Caspase 3 activation, although VPA was more effective than TSA, especially in Panc1 cells. As underlying molecular events, we found that ERK1/2 was de-phosphorylated and that the c-Myc and mutant p53 protein levels were reduced after VPA and, to a lesser extent, after TSA treatment. Up-regulation of p21 and Puma was also observed, concomitantly with mutant p53 degradation. In addition, we found that in both cell lines VPA increased the pro-apoptotic Bim level, reduced the anti-apoptotic Mcl-1 level and increased ROS production and autophagy, while TSA was able to induce these effects only in PaCA44 cells. CONCLUSIONS: From our results we conclude that both VPA and TSA can induce pancreatic cancer cell apoptosis and autophagy. VPA appears have a stronger and broader cytotoxic effect than TSA and, thus, may represent a better choice for anti-pancreatic cancer therapy.

Quercetin induces apoptosis and autophagy in primary effusion lymphoma cells by inhibiting PI3K/AKT/mTOR and STAT3 signaling pathways.

Quercetin, a bioflavonoid contained in several vegetables daily consumed, has been studied for long time for its antiinflammatory and anticancer properties. Quercetin interacts with multiple cancer-related pathways such as PI3K/AKT, Wnt/ß-catenin and STAT3. These pathways are hyperactivated in primary effusion lymphoma (PEL), an aggressive B cell lymphoma whose pathogenesis is strictly linked to the oncogenic virus Kaposis' Sarcoma-associated Herpesvirus (KSHV). In this study, we found that quercetin inhibited PI3K/AKT/mTOR and STAT3 pathways in PEL cells, and as a consequence, it down-regulated the expression of the prosurvival cellular proteins such as c-FLIP, cyclin D1 and cMyc. It also reduced the release of IL-6 and IL-10 cytokines, leading to PEL cell death. Moreover, quercetin induced a prosurvival autophagy in these cells and increased the cytotoxic effect of bortezomib, a proteasomal inhibitor, against them. Interestingly, quercetin decreased also the expression of latent and lytic KSHV proteins involved in PEL tumorigenesis and up-regulated the surface expression of HLA-DR and calreticulin, rendering the dying cells more likely detectable by the immune system. The results obtained in this study indicate that quercetin, which does not exert any cytotoxicity against normal B cells, may represent a good candidate for the treatment of this aggressive B cell lymphoma, especially in combination with autophagy inhibitors or with bortezomib.

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.

Concomitant reduction of c-Myc expression and PI3K/AKT/mTOR signaling by quercetin induces a strong cytotoxic effect against Burkitt's lymphoma.

Burkitt's lymphoma is an aggressive B cell lymphoma whose pathogenesis involves mainly c-Myc translocation and hyperexpression, in addition to antigen-independent BCR signaling and, in some cases, EBV infection. As result of BCR signaling activation, the PI3K/AKT/mTOR pathway results constitutively activated also in the absence of EBV, promoting cell survival and counterbalancing the pro-apoptotic function that c-Myc may also exert. In this study we found that quercetin, a bioflavonoid widely distributed in plant kingdom, reduced c-Myc expression and inhibited the PI3K/AKT/mTOR activity in BL, leading to an apoptotic cell death. We observed a higher cytotoxic effect against the EBV-negative BL cells in comparison with the positive ones, suggesting that this oncogenic gammaherpesvirus confers an additional resistance to the quercetin treatment. Besides cell survival, PI3K/AKT/mTOR pathway also regulates autophagy: we found that quercetin induced a complete autophagic flux in BL cells, that contributes to c-Myc reduction in some of these cells. Indeed, autophagy inhibition by chloroquine partially restored c-Myc expression in EBV-positive (Akata) and EBV-negative (2A8) cells that harbor c-Myc mutation. Interestingly, chloroquine did not affect the quercetin-mediated reduction of c-Myc expression in Ramos cells, that have no c-Myc mutation in the coding region, although autophagy was induced. These results suggest that mutant c-Myc could be partially degraded through autophagy in BL cells, as previously reported for other mutant oncogenic proteins.