Livia Di Renzo

Primary Effusion Lymphoma Cell Death Induced by Bortezomib and AG 490 Activates Dendritic Cells through CD91

To understand how cytotoxic agent-induced cancer cell death affects the immune system is of fundamental importance to stimulate immune response to counteract the high mortality due to cancer. Here we compared the immunogenicity of Primary Effusion Lymphoma (PEL) cell death induced by anticancer drug Bortezomib (Velcade) and Tyrphostin AG 490, a Janus Activated Kinase 2/signal trasducer and activator of transcription-3 (JAK2/STAT3) inhibitor. We show that both treatments were able to induce PEL apoptosis with similar kinetics and promote dendritic cells (DC) maturation. The surface expression of molecules involved in immune activation, namely calreticulin (CRT), heat shock proteins (HSP) 90 and 70 increased in dying cells. This was correlated with DC activation. We found that PEL cell death induced by Bortezomib was more effective in inducing uptake by DC compared to AG 490 or combination of both drugs. However the DC activation induced by all treatments was completely inhibited when these cells were pretreated with a neutralizing antiboby directed against the HSP90/70 and CRT common receptor, CD91. The activation of DC by Bortezomib and AG 490 treated PEL cells, as seen in the present study, might have important implications for a combined chemo and immunotherapy in such patients.

JNK and Macroautophagy Activation by Bortezomib Has a Pro-Survival Effect in Primary Effusion Lymphoma Cells

Understanding the mechanisms of autophagy induction and its role during chemotherapeutic treatments is of fundamental importance in order to manipulate it to improve the outcome of chemotherapy. In particular whether the bortezomib-induced autophagy plays a pro-survival or pro-death role is still controversial. In this study we investigated if bortezomib induced endoplasmic reticulum (ER) stress and activated autophagy in Primary Effusion Lymphoma (PEL) cells and how they influenced cell survival. We found that bortezomib induced up-regulation of the pro-survival and pro-death ER stress molecules BIP and CHOP and activated c-Jun NH2-terminal kinase (JNK), resulting in Bcl-2 phosphorylation and induction of autophagy. JNK and autophagy activation played a pro-survival role in this setting, thus their inhibition increased the bortezomib cytotoxic effect and PARP cleavage in PEL cells. Based on our results we suggest that the combination of bortezomib with JNK or autophagy inhibitors could be exploited to improve the outcome of therapy of this aggressive B cell lymphoma.

Zinc supplementation is required for the cytotoxic and immunogenic effects of chemotherapy in chemoresistant p53-functionally deficient cells

Optimal tumor eradication often results from the death of malignant cells, as induced by chemotherapeutic agents, coupled to the induction of antitumor immune responses. However, cancer cells frequently become resistant to the cytotoxic activity of chemotherapy. The aim of the present study was to evaluate whether zinc dichloride (ZnCl2), which was known to re-establish the chemosensitivity of cancer cells by reactivating p53, promotes immunogenic instances of cell death. We found that ZnCl2, in combination with chemotherapeutic agents such as cisplatin and adriamycin (ADR), favors the apoptotic demise of chemoresistant cells, while cisplatin and ADR alone fail to do so. The co-culture of immature dendritic cells (DCs) with cancer cells succumbing to the co-administration of chemotherapy and ZnCl2 led to DC activation, as indicated by the upregulation of the activation markers CD83 and CD86. In part, such process depended on cell death, as it was limited (but not abrogated) by the pan-caspase inhibitor Z-VAD-fmk. Moreover, DC activation relied on the ZnCl2-induced exposure of calreticulin (CRT) on the surface of cancer cells, correlating with the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), a marker of endoplasmic reticulum stress. The siRNA-mediated knockdown of CRT as well as the inhibition of CRT exposure with brefeldin A strongly impaired DC maturation, indicating CRT translocation as induced by that ZnCl2 is a key event in this setting. Altogether, these results suggest that ZnCl2, has the potential to enhance the therapeutic effects of antineoplastic agents not only by improving their cytotoxic activity but also by promoting CRT exposure.

Targeting COX-2/PGE2 Pathway in HIPK2 Knockdown Cancer Cells: Impact on Dendritic Cell Maturation

Background Homeodomain-interacting protein kinase 2 (HIPK2) is a multifunctional protein that exploits its kinase activity to modulate key molecular pathways in cancer to restrain tumor growth and induce response to therapies. For instance, HIPK2 knockdown induces upregulation of oncogenic hypoxia-inducible factor-1 (HIF-1) activity leading to a constitutive hypoxic and angiogenic phenotype with increased tumor growth in vivo. HIPK2 inhibition, therefore, releases pathways leading to production of pro-inflammatory molecules such as vascular endothelial growth factor (VEGF) or prostaglandin E2 (PGE2). Tumor-produced inflammatory mediators other than promote tumour growth and vascular development may permit evasion of anti-tumour immune responses. Thus, dendritic cells (DCs) dysfunction induced by tumor-produced molecules, may allow tumor cells to escape immunosurveillance. Here we evaluated the molecular mechanism of PGE2 production after HIPK2 depletion and how to modulate it. Methodology/Principal findings We show that HIPK2 knockdown in colon cancer cells resulted in cyclooxygenase-2 (COX-2) upregulation and COX-2-derived PGE2 generation. At molecular level, COX-2 upregulation depended on HIF-1 activity. We previously reported that zinc treatment inhibits HIF-1 activity. Here, zinc supplementation to HIPK2 depleted cells inhibited HIF-1-induced COX-2 expression and PGE2/VEGF production. At translational level, while conditioned media of both siRNA control and HIPK2 depleted cells inhibited DCs maturation, conditioned media of only zinc-treated HIPK2 depleted cells efficiently restored DCs maturation, seen as the expression of co-stimulatory molecules CD80 and CD86, cytokine IL-10 release, and STAT3 phosphorylation. Conclusion/Significance These findings show that: 1) HIPK2 knockdown induced COX-2 upregulation, mostly depending on HIF-1 activity; 2) zinc treatment downregulated HIF-1-induced COX-2 and inhibited PGE2/VEGF production; and 3) zinc treatment of HIPK2 depleted cells restored DCs maturation.

Activation of dendritic cells by tumor cell death

A growing number of studies indicate that cell death can be either immunogenic or not, depending on its modalities, the type and the activation state of the cells, and finally, the environment where it happens. Increased understanding of the immunogenicity of cancer cell death will significantly improve the outcome of chemotherapeutic treatments.

Inhibition of p38 MAP kinase pathway induces apoptosis and prevents Epstein Barr virus reactivation in Raji cells exposed to lytic cycle inducing compounds

BackgroundEBV lytic cycle activators, such as phorbol esters, anti-immunoglobulin, transforming growth factor β (TGFβ), sodium butyrate, induce apoptosis in EBV-negative but not in EBV-positive Burkitts lymphoma (BL) cells. To investigate the molecular mechanisms allowing EBV-infected cells to be protected, we examined the expression of viral and cellular antiapoptotic proteins as well as the activation of signal transduction pathways in BL-derived Raji cells exposed to lytic cycle inducing agents.ResultsOur data show that, following EBV activation, the latent membrane protein 1 (LMP1) and the cellular anti-apoptotic proteins MCL-1 and BCL-2 were quickly up-regulated and that Raji cells remained viable even when exposed simultaneously to P(BU)2, sodium butyrate and TGFβ. We report here that inhibition of p38 pathway, during EBV activation, led to a three fold increment of apoptosis and largely prevented lytic gene expression.ConclusionThese findings indicate that, during the switch from the latent to the lytic phase of EBV infection, p38 MAPK phosphorylation plays a key role both for protecting the host cells from apoptosis as well as for inducing viral reactivation. Because Raji cells are defective for late antigens expression, we hypothesize that the increment of LMP1 gene expression in the early phases of EBV lytic cycle might contribute to the survival of the EBV-positive cells.

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.

Reactive oxygen and nitrogen species are involved in sorbitol-induced apoptosis of human erithroleukaemia cells K562

In this study, we found that production of both reactive oxygen (ROS) and nitrogen (RNS) species is a very early event related to treatment with hyperosmotic concentration of sorbitol. The production of nitric oxide (NO) was paralleled by the increase of the mRNA and protein level of the inducible form of the nitric oxide synthase (iNOS). ROS and RNS enhancement, process concomitant to the failure of mitochondrial trans-membrane potential (ΔΨ), was necessary for the induction of apoptosis as demonstrated by the protection against sorbitol-mediated toxicity observed after treatment with ROS scavengers or NOS inhibitors. The synergistic action of ROS and RNS was finally demonstrated by pre-treatment with rosmarinic acid that, by powerfully buffering both these species, prevents impairment of ΔΨ and cell death. Overall results suggest that the occurrence of apoptosis upon sorbitol treatment is an event mediated by oxidative/nitrosative stress rather than a canonical hyperosmotic shock.

Quercetin Affects Hsp70/IRE1α Mediated Protection from Death Induced by Endoplasmic Reticulum Stress

Relative to their normal counterparts, tumor cells generally exhibit a greater “stress phenotype” and express heat shock proteins (Hsp) that represent candidate targets for anticancer therapy. Here we investigated the role of Hsp70 in survival induced by endoplasmic reticulum (ER) stressors in human leukemia U937 cells. Quercetin, a major dietary flavonoid, or specific silencing affected the expression level of Hsp70 and did not allow the upregulation of inositol-requiring kinase 1α (IRE1α), the prototype ER stress sensor regulating the unfolded protein response (UPR), that protects the cells against the stress of misfolded proteins in the ER. The reduction of Hsp70 prevented the upregulation of immunoglobulin heavy-chain binding protein (BiP), but not of CCAAT/enhancer-binding protein-homologous protein (CHOP), and induced apoptosis. Also specific silencing of IRE1α or inhibition of its endoribonuclease activity by 4μ8c hampered the upregulation of BiP, but not of CHOP, and induced apoptosis. These results suggest that drugs affecting the Hsp70-IRE1α axis, like quercetin, or affecting directly IRE1α may represent an effective adjuvant antileukemia therapy.

Lack of biological relevance of platelet cyclooxygenase-2 dependent thromboxane A2 production

INTRODUCTION There is emerging evidence of a considerable variability of the impact of aspirin on clinical outcome and laboratory findings. Persistent TxA2 production seems to be the most likely reason. Aim of this study was to determine whether the mechanism responsible for TxA2 persistent production is, at least partially, dependent upon aspirin-insensitive platelet COX-2 enzymatic pathway. METHODS AND RESULTS In 100 consecutive patients, under chronic aspirin anti-platelet treatment (100-160 mg/day) selected on the basis of detectable plasma salicylate levels, serum and Arachidonic Acid (AA)-induced platelet TxA2 production, immunoblot analysis of platelet COX-1/COX-2 expression and COX-2 activity were studied. Immunoblot revealed COX-2 expression in 46% patients, in an amount that was markedly lower than COX-1. In 10 COX-2 positive patients with TxA2 levels over the median, AA-induced TxA2 production performed in vitro in the presence of the COX-2 inhibitor CAY10404 and aspirin demonstrated that COX-2 dependent TxA2 production is less than 2%. CONCLUSION Our data demonstrate that the inter-individual variability of platelet sensitivity to aspirin is due to a reduced efficacy of aspirin on platelet COX-1 despite ascertained patient compliance. We suggest that serum TxA2 assay might be performed in future clinical studies to improve our knowledge on the residual TxA2 production in aspirin-treated patients.