Silvana Morello

Inhibition of CD73 Improves B Cell-Mediated Anti-Tumor Immunity in a Mouse Model of Melanoma

CD73 is a cell surface enzyme that suppresses T cell-mediated immune responses by producing extracellular adenosine. Growing evidence suggests that targeting CD73 in cancer may be useful for an effective therapeutic outcome. In this study, we demonstrate that administration of a specific CD73 inhibitor, adenosine 5′-(α,β-methylene)diphosphate (APCP), to melanoma-bearing mice induced a significant tumor regression by promoting the release of Th1- and Th17-associated cytokines in the tumor microenvironment. CD8+ T cells were increased in melanoma tissue of APCP-treated mice. Accordingly, in nude mice APCP failed to reduce tumor growth. Importantly, we observed that after APCP administration, the presence of B cells in the melanoma tissue was greater than that observed in control mice. This was associated with production of IgG2b within the melanoma. Depletion of CD20+ B cells partially blocked the anti-tumor effect of APCP and significantly reduced the production of IgG2b induced by APCP, implying a critical role for B cells in the anti-tumor activity of APCP. Our results also suggest that APCP could influence B cell activity to produce IgG through IL-17A, which significantly increased in the tumor tissue of APCP-treated mice. In support of this, we found that in melanoma-bearing mice receiving anti–IL-17A mAb, the anti-tumor effect of APCP was ablated. This correlated with a reduced capacity of APCP-treated mice to mount an effective immune response against melanoma, as neutralization of this cytokine significantly affected both the CD8+ T cell- and B cell-mediated responses. In conclusion, we demonstrate that both T cells and B cells play a pivotal role in the APCP-induced anti-tumor immune response.

Lung cancer and Toll-like receptors

Lung carcinoma is one of the leading causes of death worldwide. It is a non-immunogenic cancer, resistant to immune surveillance. Toll-like receptors (TLRs) connect the innate to the adaptive immune system. Given that cancerous cells evade the immune system, the activation of TLRs could represent a potential target for cancer therapy. The induction of Th1-like and cytotoxic immunity by TLR signalling could lead to tumour cell death, resulting in tumour regression or arrest. However, basic research and clinical trials revealed that the activation of specific TLRs, such as TLR2, TLR4 and TLR9, do not have any anti-tumour activity in lung carcinoma. Increasing evidence suggests that TLRs are important regulators of tumour biology; however, little is known about their function in lung cancer. Thus, in order to develop new therapeutic approaches, further studies are needed to understand the connection between TLRs and lung cancer progression. This review focuses on the potential mechanisms by which TLR ligands can facilitate or not lung cancer and lung metastases establishment/progression.

Cl-IB-MECA inhibits human thyroid cancer cell proliferation independently of A3 adenosine receptor activation

A3 adenosine receptor (A3AR) agonists have been reported to modulate cellular proliferation. This work was aimed to investigate the expression and the possible action of A3 receptor in the human thyroid carcinomas. Normal thyroid tissue samples did not express A3 adenosine receptor, while primary thyroid cancer tissues expressed high level of A3AR, as determined by immunohistochemistry analysis. In human papillary thyroid carcinoma cell line, NPA, at concentrations ≥10µM, the A3AR-selective agonist 2-chloro-N6-(3-iodobenzyl)adenosine-5’-N-methylcarboxamide (Cl-IB-MECA) produced inhibition of cell growth, by blocking the G1 cell cycle phase in a concentration- and time-dependent manner. This effect was well correlated with a reduction of protein expression of cyclins D1 and E2 after 24 hours of Cl-IB-MECA treatment. Moreover Cl-IB-MECA induced dephosphorylation of ERK1/2 in a time- and concentration-dependent manner, which in turn inhibits cell proliferation. The effect of Cl-IB-MECA was not prevented by A3AR antagonists, MRS1191 or MRS1523 or FA385. Furthermore, neither nucleoside transporter inhibitors, Dypiridamole and NBTI, nor the A1, A2A and A2B receptors antagonists were able to block the response to Cl-IB-MECA. Although Cl-IB-MECA has been shown to influence cell death and survival in other systems through an A3AR-mediated mechanism, in NPA cells the growth inhibition induced by micromolar concentrations of Cl-IB-MECA appeared not mediated through activation of this receptor and hence that its effects on human papillary carcinoma cell line seem to be independent of the presence of this receptor subtype.

Vascular effects of caffeic acid phenethyl ester (CAPE) on isolated rat thoracic aorta

This study was aimed to investigate the vascular activity of caffeic acid phenethylester (CAPE), one of the major components of honeybee propolis. Experiments were performed on rat thoracic aortic rings, mounted in an isolated organ bath and connected to an isometric force transducer. The effect of CAPE (0.1-300 microM) was evaluated on tissue pre-contracted with phenylephrine (PE, 1 microM) or with KCl (100 mM). In another set of experiments, tissue was incubated with CAPE (1-100 microM) and responses to PE (0.01-3 microM) or KCl (60 mM) were evaluated. The effect of CAPE on cytosolic Ca(2+) concentration in aortic smooth muscle cells stimulated with PE or KCl was also evaluated. CAPE (0.1-300 microM) caused a concentration-dependent relaxation (pEC(50) 4.99 +/- 0.19; Emax 100.75 +/- 1.65%; n = 4) of tissue pre-contracted with PE that was reduced by endothelium removal or by incubation with N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 microM). CAPE also relaxed KCl-precontracted tissue (pEC(50) 4.40 +/- 0.08; n = 4). CAPE inhibited contractile responses to PE or to KCl, and also inhibited the contractile response to PE obtained in a Ca(2+)-free medium. In addition, CAPE inhibited the increase in cytosolic Ca(2+) concentration triggered by stimulation of aortic smooth muscle cells with PE or KCl. Our results demonstrate a vascular activity for CAPE, that is only partially dependent on nitric oxide. Indeed, at high concentrations, CAPE vasorelaxant effect occurs also in absence of endothelium and it is likely due to an inhibitory effect on calcium movements through cell membranes.

Polyinosinic-Polycytidylic Acid Limits Tumor Outgrowth in a Mouse Model of Metastatic Lung Cancer

Polyinosinic-polycytidylic acid (poly I:C), a TLR3 ligand, is currently being tested in human clinical trials as an adjuvant to anti-cancer vaccines and in combination with other therapies. However, little is known about its activity in established pulmonary metastasis. The aim of our study was to elucidate the effect of poly I:C (1, 10, or 100 μg/mouse) in a mouse model of B16-F10–induced metastatic lung cancer. Lung tumor growth was arrested after a single administration of poly I:C. This was associated with higher influx of mature dendritic cells (DCs), which drove toward a Th1-like, Th17-like, and cytotoxic immune environment. The interference with IFN type I receptor signaling by means of a specific mAb reversed poly I:C-mediated tumor regression due to lower presence of myeloid DCs, cytotoxic DCs (CD11c+CD8+), NKT cells, CD8+ T cells, and Th1-like cytokines. Moreover, the adoptive transfer of poly I:C-activated bone marrow-derived DCs into tumor-bearing mice resulted in activities similar to those of the systemic administration of poly I:C on lung tumor burden. In conclusion, our data prove that poly I:C has potential anti-tumor activity in a mouse model of established pulmonary metastasis. The activation of DCs and the production of IFN type I are responsible for an effective T cytotoxic immune response against metastatic lung cancer progression after poly I:C treatment.

B Cells Contribute to the Antitumor Activity of CpG-Oligodeoxynucleotide in a Mouse Model of Metastatic Lung Carcinoma

RATIONALE CpG-oligodeoxynucleotide (CpG-ODN; CpG), a Toll-like receptor-9 ligand, has been widely studied as a potential antitumor adjuvant. Toll-like receptor-9 is highly expressed on lung carcinoma tissues and some immune cells, such as plasmacytoid dendritic cells and B cells. OBJECTIVES The aim of our study was to elucidate the effect of CpG on B cells in a mouse model of lung carcinoma. METHODS C57Bl/6j, B cell-deficient, and Nude mice were intravenously implanted with the lung metastatic B16-F10 melanoma cells and killed 3 and 7 days after CpG administration. MEASUREMENTS AND MAIN RESULTS Administration of CpG increased lung tumor growth in B16-F10-implanted C57BL/6J mice. The genetic absence of B cells strongly facilitated CpG-induced tumor progression. In contrast, the adoptive transfer of CpG-activated B cells induced tumor arrest, associated with a reduced suppressive immune environment due to the lower recruitment of regulatory T cells, myeloid-derived suppressor cells, and CD8(+) regulatory T cells along with the reduced expression of suppressive cytokines such as IL-10 and transforming growth factor-β. Furthermore, concomitant with higher production of IFN-γ, the apoptosis rate in the lungs of mice adoptively transferred with CpG-activated B cells was increased. Depletion of mature CD20(+) B cells increased the lung tumor burden in CpG-treated C57BL/6J mice and nude mice. Moreover, nude mice had the same lung tumor burden as B cell-deficient mice when mature CD20(+) B cells were depleted. CONCLUSIONS Our data demonstrate the protective antitumor activity of CpG-activated B cells and shed light on CpG as an antitumor adjuvant for lung cancer therapy.

Cl‐IB‐MECA enhances TRAIL‐induced apoptosis via the modulation of NF‐κB signalling pathway in thyroid cancer cells

Apoptosis is an endogenous process that can be a useful anti‐cancer tool. This study aimed to investigate the effect of Cl‐IB‐MECA, adenosine receptor A3 agonist, on TRAIL‐induced apoptosis of thyroid carcinoma cells. Cl‐IB‐MECA enhanced TRAIL‐mediated apoptosis in FRO but not in ARO cells. This effect was correlated to higher expression levels of DR5 on FRO than ARO cells, that instead presented higher levels of decoy receptors, DcR1 and DcR2. To understand the cross‐talk between the effect of Cl‐IB‐MECA and TRAIL, we evaluated the nuclear translocation of p65 and c‐Rel. Since the dependency by NF‐κB, TRAIL promoted the nuclear translocation of both p65 and c‐Rel subunits. However, the addition of Cl‐IB‐MECA led to the predominant translocation of c‐Rel after TRAIL addition. Furthermore, Bcl‐2, cFLIP and pAkt were lower induced than caspase‐3 and ‐9 in FRO cells. To discriminate a specific effect of TRAIL, we used tumour necrosis factor‐alpha (TNF‐α) with Cl‐IB‐MECA. In this case, no synergism was observed. In addition, the effect of Cl‐IB‐MECA was not A3 receptor‐dependent since its antagonists, MRS1191 and FA385, failed to block Cl‐IB‐MECA activity on TRAIL‐treated FRO cells. In conclusion, Cl‐IB‐MECA enhanced TRAIL‐mediated apoptosis via NF‐κB/c‐Rel activation and DR5‐dependent manner. This study may shed light on a potential drug cocktail that may prove useful as anti‐cancer in an in vivo animal model. J. Cell. Physiol. 221: 378–386, 2009.

Pharmacological dissection of vascular effects caused by activation of protease-activated receptors 1 and 2 in anesthetized rats

To clarify the role of protease‐activated receptor 1 and 2 (PAR‐1 and PAR‐2) in controlling blood pressure, we evaluated changes in blood pressure induced by a peptide that activates the receptor PAR‐1 (PAR‐1AP, SFLLRNPND) and a peptide that activates the receptor PAR‐2 (PAR‐2AP, SLIGRL) in naive and ganglion‐blocked anesthetized rats. The role of nitric oxide on the effects observed was also investigated. Intravenous injection of PAR‐1AP induced a biphasic change in mean arterial blood pressure (MABP) characterized by hypotension followed by hypertension, the latter was enhanced strongly by ganglion‐blockade. After L‐NAME infusion in ganglion‐blocked rats, hypertension induced by PAR‐1AP was still increased, which returned to control value after L‐arginine infusion. L‐NAME did not inhibit PAR‐1AP–induced hypotension. Intravenous injection of PAR‐2AP induced a biphasic change in MABP, characterized by hypotension followed by a hypertensive phase that reached the maximum value in 5–6 min. Hypertension was abolished by ganglion‐blockade and was restored by an infusion of L‐NAME. This effect was reverted by L‐arginine. L‐NAME reduced the duration of hypotension induced by PAR‐2AP. In conclusion, we define that PAR‐1 mainly mediates hypertension, whereas PAR‐2 mainly is responsible for hypotension. Furthermore, we give evidence for a hypertensive effect of PAR‐2AP linked to a reflex mechanism that is modulated by nitric oxide.

Myeloid-derived suppressor cells contribute to A2B adenosine receptor-induced VEGF production and angiogenesis in a mouse melanoma model.

Vascular endothelial growth factor (VEGF) is an angiogenic factor critically involved in tumor progression. Adenosine A2B receptor plays a pivotal role in promoting tumor growth. The aim of this study was to investigate the role of myeloid-derived suppressor cells (MDSCs) in the pro-angiogenic effects of A2B and to determine whether A2B blockade could enhance the effectiveness of anti-VEGF treatment. Mice treated with Bay60-6583, a selective A2B receptor agonist, showed enhanced tumor VEGF-A expression and vessel density. This effect was associated with accelerated tumor growth, which could be reversed with anti-VEGF treatment. Bay60-6583 increased the accumulation of tumor CD11b+Gr1+ cells. Depletion of MDSCs in mice significantly reduced A2B-induced VEGF production. However, A2B receptor stimulation did not directly regulate VEGF expression in isolated tumor myeloid cells. Mechanistically, Bay60-6583-treated melanoma tissues showed increased STAT3 activation. Inhibition of STAT3 significantly decreased the pro-tumor activity of Bay60-6583 and reduced tumor VEGF expression. Pharmacological blockade of A2B receptor with PSB1115 significantly reduced tumor growth by inhibiting tumor angiogenesis and increasing T cells numbers within the tumor microenvironment. These effects are, at least in part, dependent on impaired tumor accumulation of Gr1+ cells upon A2B receptor blockade. PSB1115 increased the effectiveness of anti-VEGF treatment.

Therapeutic potential of a pyridoxal‐based vanadium(IV) complex showing selective cytotoxicity for cancer versus healthy cells

Vanadium compounds can exert anticancer effects, partly due to inhibition of tyrosine phosphatases. Here, we report the effect of N,N′‐ethylenebis (pyridoxylideneiminato) vanadium (IV) complex (Pyr2enV(IV)), that induced 93% and 57% of cell mortality in A375 (human melanoma) and A549 (human lung carcinoma) cells, respectively; the mortality was <24% in other cancer cell lines and in human normal epidermal keratinocytes, lung cells and peripheral blood mononuclear cells. The mechanism of Pyr2enV(IV) effect relied on apoptosis induction; this was triggered by ROS increase, followed by mitochondrial membrane depolarization. Indeed, the addition of N‐acetyl cysteine to cell cultures abated Pyr2enV(IV)‐induced apoptosis. These results disclose the pro‐apoptotic activity of Pyr2enV(IV) and its mechanism, relying on intracellular ROS increase. J. Cell. Physiol. 228: 2202–2209, 2013.