Maria Saveria Gilardini Montani

ATM-depletion in breast cancer cells confers sensitivity to PARP inhibition.

BackgroundMutations in the DNA damage response (DDR) factors, breast cancer 1 (BRCA1) and BRCA2, sensitize tumor cells to poly(ADP-ribose) polymerase (PARP) inhibitors. The ataxia telangiectasia mutated (ATM) kinase is a key DDR protein whose heterozygous germline mutation is a moderate–risk factor for developing breast cancer. In this study, we examined whether ATM inactivation in breast cancer cell lines confers sensitivity to PARP inhibitors.MethodsWild-type BRCA1/2 breast cancer cells (i.e., MCF-7 and ZR-75-1 lines) were genetically manipulated to downregulate ATM expression then assayed for cytostaticity/cytotoxicity upon treatment with PARP inhibitors, olaparib and iniparib.ResultsWhen ATM-depleted cells and their relative controls were treated with olaparib (a competitive PARP-1/2 inhibitor) and iniparib (a molecule originally described as a covalent PARP-1 inhibitor) a different response to the two compounds was observed. ATM-depletion sensitized both MCF-7 and ZR-75-1 cells to olaparib-treatment, as assessed by short and long survival assays and cell cycle profiles. In contrast, iniparib induced only a mild, ATM-dependent cytostatic effect in MCF-7 cells whereas ZR-75-1 cells were sensitive to this drug, independently of ATM inactivation. These latest results might be explained by recent observations indicating that iniparib acts with mechanisms other than PARP inhibition.ConclusionsThese data indicate that ATM-depletion can sensitize breast cancer cells to PARP inhibition, suggesting a potential in the treatment of breast cancers low in ATM protein expression/activity, such as those arising in mutant ATM heterozygous carriers.

Hydroxytyrosol-Derived Compounds: A Basis for the Creation of New Pharmacological Agents for Cancer Prevention and Therapy

Hydroxytyrosol [2-(3,4-dihydroxyphenyl)ethanol, HTyr] is a phenolic compound found in olive leaves and fruits and extra-virgin olive oil, which has well-known strong antioxidant and radical-scavenging properties. Recently, it has received particular attention for its antiproliferative and apoptotic activities and its anti-inflammatory properties. During the past few years, more efforts have been focused on synthesizing HTyr-derived compounds with enhanced biological activities for their potential use in different chronic degenerative diseases. In this paper, we report a dissertation on the current knowledge of selected synthetic HTyr derivatives and analogues and their potential use in cancer prevention and therapy, which are related to their antioxidant, antiproliferative/apoptotic, and anti-inflammatory properties. On the basis of the perspective of using HTyr-derived compounds as anticancer agents, we have taken into account only studies that were performed in experimental cell-based models.

Engagement of CD4 Before TCR Triggering Regulates Both Bax- and Fas (CD95)-Mediated Apoptosis

In the present study, we have aimed at clarifying the CD4-dependent molecular mechanisms that regulate human memory T cell susceptibility to both Fas (CD95)-dependent and Bcl-2-dependent apoptotic pathways following antigenic challenge. To address this issue, we used an experimental system of viral and alloantigen-specific T cell lines and clones and two ligands of CD4 molecules, Leu-3a mAb and HIV gp120. We demonstrate that CD4 engagement before TCR triggering suppresses the TCR-mediated neosynthesis of the Flice-like inhibitory protein and transforms memory T cells from a CD95-resistant to a CD95-susceptible phenotype. Moreover, evidence that the apoptotic programs were executed while Fas ligand mRNA expression was inhibited led us to analyze Bcl-2-dependent pathways. The data show that the engagement of CD4 separately from TCR influences the expression of the proapoptotic protein Bax independently of the anti-apoptotic protein Bcl-2, whereas Ag activation coordinately modulates both Bax and Bcl-2. The increased expression of Bax and the consequent dissipation of the mitochondrial transmembrane potential (ΔΨm) suggest a novel immunoregulatory function of CD4 and demonstrate that both passive cell death and activation-induced cell death are operative in CD4+ memory T cells. Furthermore, analysis of the mechanisms by which IL-2 and IL-4 cytokines exert their protective function on CD4+ T cells in the presence of soluble CD4 ligands shows that they were able to revert susceptibility to Bax-mediated but not to CD95-dependent apoptotic pathways.

Capsaicin-mediated apoptosis of human bladder cancer cells activates dendritic cells via CD91

OBJECTIVES Immunostimulation by anticancer cytotoxic drugs is needed for long-term therapeutic success. Activation of dendritic cells (DCs) is crucial to obtain effective and long-lasting anticancer T-cell mediated immunity. The aim of this study was to explore the effect of capsaicin-mediated cell death of bladder cancer cells on the activation of human monocyte-derived CD1a+ immature DCs. METHODS Immature DCs (generated from human peripheral blood-derived CD14+ monocytes cultured with granulocyte-macrophage colony stimulating factor and interleukin-4) were cocultured with capsaicin (CPS)-induced apoptotic bladder cancer cells. DC activation was investigated using immunofluorescence and flow cytometric analysis for key surface molecules. In some experiments, CD91 was silenced in immature DCs. RESULTS We found that capsaicin-mediated cancer cell apoptosis upregulates CD86 and CD83 expression on DCs, indicating the induction of DC activation. Moreover, silencing of CD91 (a common receptor for damage-associated molecular patterns, such as calreticulin and heat-shock protein-90/70) in immature DCs led to the inhibition of DC activation. CONCLUSIONS Our data show that CPS-mediated cancer cell apoptosis activates DCs via CD91, suggesting CPS as an attractive candidate for cancer therapy.

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

PurposeHistone 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.MethodsCell 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.ResultsWe 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.ConclusionsFrom 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.

Capsaicin triggers immunogenic PEL cell death, stimulates DCs and reverts PEL-induced immune suppression

Capsaicin, the pungent alkaloid of red pepper has been extensively studied for its many properties, especially the anti-inflammatory and anti-oxidant ones. It binds to vanilloid receptor 1, although it has been reported to be able to mediate some effects independently of its receptor. Another important property of Capsaicin is the anticancer activity against highly malignant tumors, alone or in combination with other chemotherapeutic agents. In this study, we found that Capsaicin induced an apoptotic cell death in PEL cells correlated with the inhibition of STAT3. STAT3 pathway, constitutively activated in PEL cells, is essential for their survival. By STAT3 de-phosphorylation, Capsaicin reduced the Mcl-1 expression level and this could represent one of the underlying mechanisms leading to the Capsaicin-mediated cell death and autophagy induction. Next, by pharmacological or genetic inhibition, we found that autophagy played a pro-survival role, suggesting that its inhibition could be exploited to increase the Capsaicin cytotoxic effect against PEL cells. Finally, we show that Capsaicin induced DAMP exposure, as for an immunogenic cell death, directly promoted DC activation and, more importantly, that it counteracted the immune-suppression, in terms of DC differentiation, mediated by the PEL released factors.

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

Limiting dilution analysis of T cell unresponsiveness to mycobacteria in advanced disseminated tuberculosis

Peripheral blood mononuclear cells from patients with advanced disseminated tuberculosis (Dis-TB) do not respond to purified protein derivative (PPD) measured as cell proliferation, lymphokine production and interleukin (IL)-2 receptor (Tac antigen) expression. Limiting dilution analysis revealed “multi-hit” curves and low frequencies of PPD-reactive T cells in cultures of Dis-TB, and “single-hit” curves and high frequencies of PPD-reactive T cells in cultures of patients with localized form of pulmonary tuberculosis. Moreover, a strict relationship between Tac antigen expression and ability of exogenous IL-2 to enhance bulk culture cell proliferation was observed in Dis-TB patients.

A new tripeptide, Pol 509, influences biochemical events associated with antigen presentation efficiency of PPD-specific EBV-B cells

A synthetic tripeptide (pGLU-LEU-TRP-OCH3) Pol 509, derived from snake venom, was studied directly by analyzing the interactions with synthetic lipid bilayers using NMR spectroscopy. Functional studies were also performed by measuring the effects: i), on early biochemical events (adenyl cyclase and phospholipase C activation products), intermediate (surface Ag expression) and late (DNA synthesis) parameters following B-cell activation elicited by PPD-linkage to specific membrane Ig; and ii), on the presentation of PPD to Ag-specific T-cell lines. Comparative experiments using PMA and IFN-gamma were also performed. We found that all parameters studied were affected by Pol 509 treatment. In fact, while PPD linkage to mlg reversed the balance between cAMP and IP3 existing in unstimulated EBV-B cells, Pol 509 reduced the PPD-induced accumulation of cAMP to control values and induced a further decrease of IP3 level. Pol 509-mediated decrease of these second messenger levels was accompanied by a slight increase of HLA-DR molecule expression and DNA synthesis inhibition. Furthermore, Pol 509 enhanced the efficiency of PPD presentation to T-cell lines. Taken together, these observations suggest that Pol 509, which enhances Ag presentation by modifying second messenger levels, may be considered as a new immunomodulatory drug with immunopotentiating activity.

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

BackgroundApigenin 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.MethodsCell 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.ResultsWe 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.ConclusionThe findings of this study demonstrate that apigenin may modulate pro-apoptotic and pro-survival pathways representing a valid therapeutic strategy against PEL.