Title

 

Authors

M. Festa¹, C. W. D’Acunto ¹, A. G. Rossi ², M. Caputo ¹,   M. F. Tecce ¹, C. Pizza ¹,  S. Piacente ¹, A. Capasso ¹.

¹ Department of Biochemical and Pharmaceutical Sciences , University of Salerno, Italy
² MRC Centre for Inflammation Research, The Queen’s Medical Research Institute, University of Edinburgh Medical School, Edinburgh, UK

 

Abstract

Annexin A1 (ANXA1) is an endogenous protein of annexins superfamily with well-described  anti-inflammatory properties in the peripheral system. It has also been detected in the brain, constitutively expressed in glia cells, but its function is still ambiguous (Solito et al, 2008). ANXA1 has been known to be involved in apoptosis and differentiation of several cell lines (Parente & Solito, 2004). More recently, a new role for ANXA1 in phagocytosis and engulfment and/or clearance of apoptotic cells has emerged.Moreover, apoptotic cells release annexin A1 and related peptides which stimulate phagocytosis of apoptotic neutrophils by macrophages (Scannell et al, 2007).Glioblastoma multiforme (GBM) is the most common malignant and resistant tumor of the central nervous system in humans and new therapeutic strategies are urgently required (Terzis et al, 2006). We have been shown that xanthohumol (XH), a prenylated chalcone isolated from Humulus lupulus L. with promising chemotherapy activities (Zanoli & Zavatti, 2008), induces apoptosis of human T98G glioblastoma cells by increasing reactive oxygen species and activating MAPK pathways. We have found, by western blotting and microscopy analysis, that XH is able up-regulate cytosolic levels of ANXA1 and to induce translocation of the protein on cell membrane of T98G cells in time-dependent manner with significant effect after 24 h.On the basis of the above evidence the aim of this work was to investigate the role of intracellular and cell membrane localized ANXA1 in glioblastoma multiforme. To demonstrate the involvement of ANXA1 in apoptosis of GBM cells we down-regulated ANXA1 expression with small interfering RNA (siRNA) and then we analysed apoptosis in presence and absence of apoptotic stimuli. Apoptosis induced by XH was importantly reduced in siRNA-ANXA1 transfected cells where western blot analysis shows a significant reduction of protein levels. To investigate the role of ANXA1 expression on cell membrane of T98G cells as potential “eat-me” signal we studied phagocytosis of apoptotic cells by human macrophages. We incubated apoptotic T98G cells with monocytes isolated from human blood and then differentiated to mature macrophages (Mɸ). After co-incubation period we analysed percentage of eating macrophages by cytofluorimetric FACS analysis and by confocal microscopy. Our results show that XH is able to induce phagocytosis of apoptotic T98G cells by human Mɸ in a concentration-effect manner and with a caspase-dependent mechanism. ANXA1 can act as an “eat-me” signal on cell membrane of T98G cells, indeed apoptotic siRNA-ANXA1 transfected cells are not completely ingested by Mɸ. The same result was obtained incubating apoptotic cell with a neutralizing antiboby anti-ANXA1 and ANXA1 membrane depletion by EDTA washing. ANXA1 was also detected in supernatant of apoptotic cells and the incubation  with clarified supernatant of apoptotic cells enhanced the percentage of phagocytosis by Mɸ. ANXA1 could acts by triggering FPR receptors on Mɸ cell membrane, indeed pre-incubation of macrophages with FPR antagonists blocks phagocytosis of apoptotic cells induced by XH. These results demonstrated that ANXA1 is involved both in the apoptosis and phagocytosis of glioblastoma cells. These results may contribute to clarify the role of ANXA1 in maintenance of brain homeostasis andmay lead to novel therapeutic approaches for neuro-inflammatory diseases and chemotherapy targets in the treatment of glioblastoma multiforme.

References

1.      Solito et al. (2008).Trends Pharmacol Sci. 185:6317-28
2.      Parente & Solito (2004) Inflamm Res. 53:125-32
3.      Scannell et al. (2007). J. Immunol. 178: 4595-4605
4.      Terzis et al (2006). Expert Opin Biol Ther. 6: 739-749
5.      Zanoli & Zavatti  (2008). J  Ethnopharmacol.116:383-96.