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// Elena Ciaglia 1, 2 , Giovanni Torelli 3, 4 , Simona Pisanti 1, 2 , Paola Picardi 1, 2 , Alba D’Alessandro 2 , Chiara Laezza 5, 6 , Anna Maria Malfitano 1, 2 , Donatella Fiore 2 , Antonio Christian Pagano Zottola 2 , Maria Chiara Proto 2 , Giuseppe Catapano 3 , Patrizia Gazzerro 2 , Maurizio Bifulco 1, 2 1 Department of Medicine and Surgery, University of Salerno, Baronissi Salerno, Italy 2 Department of Pharmacy, University of Salerno, Fisciano, Salerno, Italy 3 ”G.Rummo” Medical Hospital, Department of Neurosurgery, Benevento, Italy 4 Neurosurgery Unit A.O. San Giovanni di Dio e Ruggi d’ Aragona - Salerno’s School of Medicine, Largo Citta di Ippocrate, Salerno, Italy 5 Institute of Endocrinology and Experimental Oncology, IEOS CNR, Naples, Italy 6 Department of Biology and Cellular and Molecular Pathology, University of Naples Federico II, Naples, Italy Correspondence to: Maurizio Bifulco, e-mail: maubiful@unisa.it Elena Ciaglia, e-mail: eciaglia@unisa.it Keywords: STAT3, CB1, MICA, NK cells, gliomas Received: February 18, 2015 Accepted: April 29, 2015 Published: May 11, 2015 ABSTRACT Herein we show that a majority of human brain tumor samples and cell lines over-expressed cannabinoid receptor CB1 as compared to normal human astrocytes (NHA), while uniformly expressed low levels of CB2. This finding prompted us to investigate the therapeutic exploitation of CB1 inactivation by SR141716 treatment, with regard to its direct and indirect cell-mediated effects against gliomas. Functional studies, using U251MG glioma cells and primary tumor cell lines derived from glioma patients expressing different levels of CB1, highlighted SR141716 efficacy in inducing apoptosis via G1 phase stasis and block of TGF-β1 secretion through a mechanism that involves STAT3 inhibition. According to the multivariate role of STAT3 in the immune escape too, interestingly SR141716 lead also to the functional and selective expression of MICA/B on the surface of responsive malignant glioma cells, but not on NHA. This makes SR141716 treated-glioma cells potent targets for allogeneic NK cell-mediated recognition through a NKG2D restricted mechanism, thus priming them for NK cell antitumor reactivity. These results indicate that CB1 and STAT3 participate in a new oncogenic network in the complex biology of glioma and their expression levels in patients dictate the efficacy of the CB1 antagonist SR141716 in multimodal glioma destruction. SIGNIFICANCE CB1 is implicated in the regulation of cellular processes linked to survival, proliferation, invasion and angiogenesis in several physio-pathological conditions. We shed light on previously unrecognized molecular mechanism of CB1-mediated modulation of human glioma progression and provide the first and original demonstration of CB1-STAT3 axis as a new target and predictor biomarkers of the benefit from specific therapies. Indeed CB1 antagonism capable of tumoral cell division’ control while making the glioma immunovisible and engaging the immune system to fight it may represent a hopeful alternative to other established chemotherapeutics. Because different aspects of glioma biology have been separately targeted with very limited success, we speculate that CB1 inhibitors which enclose in the same molecule cytotoxic potential and high activity to boost competent immune surveillance mechanisms, at a degree that seems to be correlated to the levels of CB1 immunoreactivity, might have profound implications for exploring new therapeutic anti-glioma actions.

Cancer cell stress induced by cytotoxic agents promotes antitumor immune response. Here, we observed that N6-isopentenyladenosine (iPA), an isoprenoid modified adenosine with a well established anticancer activity, was able to induce a significant upregulation of cell surface expression of natural killer (NK) cell activating receptor NK Group 2 member D (NKG2D) ligands on glioma cells in vitro and xenografted in vivo. Specifically suboptimal doses of iPA (0.1 and 1 µM) control the selective upregulation of UL16-binding protein 2 on p53wt-expressing U343MG and that of MICA/B on p53mut-expressing U251MG cells. This event made the glioblastoma cells a potent target for NK cell-mediated recognition through a NKG2D restricted mechanism. p53 siRNA-mediated knock-down and pharmacological inhibition (pifithrin-α), profoundly prevented the iPA action in restoring the immunogenicity of U343MG cells through a mechanism that is dependent upon p53 status of malignancy. Furthermore, accordingly to the preferential recognition of senescent cells by NK cells, we found that iPA treatment was critical for glioma cells entry in premature senescence through the induction of S and G2/M phase arrest. Collectively, our results indicate that behind the well established cytotoxic and antiangiogenic effects, iPA can also display an immune-mediated antitumor activity. The indirect engagement of the innate immune system and its additional activity in primary derived patient's glioma cell model (GBM17 and GBM37), fully increase its translational relevance and led to the exploitation of the isoprenoid pathway for a valid therapeutic intervention in antiglioma research.

We previously showed that methyl-F-anandamide, a stable analogue of the ananda- mide, inhibited the growth and the progression of cultured human breast cancer cells. As accu- mulating evidences indicate that the constitutive activation of the canonical Wnt pathway in human breast cancer may highlight a key role for aberrant activation of the b-catenin-TCF cascade and tumour progression, we studied the anandamide effect on the key elements of Wnt pathway in breast cancer cells. In this study we described that the treatment of human breast cancer cells, MDA MB 231 cells, with methyl-F-anandamide reduced protein levels of b-catenin in the cytoplasmic and nuclear fractions inhibiting the transcriptional activation of T Cell Factor (TCF) responsive element (marker for b-catenin signalling). The anandamide treatment resulted in up-regulation of epithelial markers, like E-cadherin with a concomitant decrease in protein levels of mesenchymal markers, including vimentin and Snail1. We, fur- thermore, observed that the induction of experimental epithelial-mesenchymal transition by exposure to adriamycin in MCF7 human breast cancer cell line was inhibited by anandamide treatment. In the present study we reported a novel anticancer effect of anandamide involving the inhibition of epithelial-mesenchymal transition, a process triggered during progression of cancer to invasive state. 2012 Elsevier Ltd. All rights reserved.

Emerging findings suggested the efficacy of the cannabinoid CB1 receptor antagonist rimonabant (SR141716) in several pathological conditions included tumours. In this study we investigated in vitro the effects of SR141716 on viability and the molecular pathways of methylcholanthrene-induced fibrosarcoma (Meth-A) cells and in vivo its anti-tumour properties in Meth-A-bearing mice. We evaluated in vitro the effect of SR141716 on Meth-A cell viability by trypan blue staining assay. Cell cycle progression and apoptosis were assessed by flow cytometry. Protein expression was investigated by Western blot. The anti-turnout efficacy of SR141716 was evaluated in vivo monitoring weight increase and survival of Meth-A injected mice. SR141716 affects Meth-A cell viability inducing apoptosis and controls cell cycle progression by modulation of the levels of the cell cycle inhibitor p21waf, cyclins E. D1 and NF-kB molecules. Importantly, SR141716 affects AKT/pFoxO1 pathway which promotes cell survival and regulates the cell cycle. The molecular effects observed are accompanied by reduced COX2 expression and induction of the CBI receptor expression. Finally, SR141716 was able to reduce the tumour size and prolong animal survival, when administered in vivo during tumour growth. Our findings shed light on a novel molecular pathway associated with control of tumour growth by SR141716 and confirm the anti-cancer and anti-inflammatory properties of this drug suggesting its potential applications in the treatment of cancer.