The Involvement of Xanthohumol in the Expression of Annexin in Human Malignant Glioblastoma Cells

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. Recently, we have shown that the potential chemotherapeutic polyphenol xanthohumol (XH), isolated from Humulus Lupulus, induces apoptosis of human T98G glioblastoma cells by increasing reactive oxygen species and activating MAPK pathways. Then we have found, by western blotting and microscopic analysis, that XH up-regulates cytosolic levels of ANXA1 and induces translocation of the protein on the cell membrane of T98G cells in a time-dependent manner with significant effects observed 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 GBM cells. RT-PCR analysis has shown that XH up-regulates mRNA levels of ANXA1 after 16 h treatment. To demonstrate the involvement of ANXA1 in apoptosis of GBM cells we down-regulated ANXA1 expression with small interfering RNA (siRNA) and then analysed apoptosis in the presence and absence of apoptotic stimuli. Importantly, apoptosis induced by XH was reduced in siRNA-ANXA1 transfected cells where western blot analysis shows a significant reduction of ANXA1 protein levels. To investigate the role of ANXA1 expression on the 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 human blood monocyte derived macrophages (M=). After co-incubation period we analysed the percentage of M= phagocytosing the apoptotic cells by cytofluorimetric FACS analysis and by confocal microscopy. Our results show that XH induces phagocytosis of apoptotic T98G cells by human M= in a concentration-effect manner, a processes that is dependent on caspase mediated apoptosis. ANXA1 acts as an “eat-me” signal on the cell membrane of T98G cells, and interestingly, apoptotic siRNA-ANXA1 transfected cells are not completely ingested by M=. These results were confirmed by incubating apoptotic cells with a neutralizing anti-ANXA1 antiboby and ANXA1 membrane depletion by EDTA washing. ANXA1 was also detected in supernatants of apoptotic cells and the incubation of enriched supernatants enhanced the percentage of phagocytosis by M=. These results demonstrated that ANXA1 is involved both in the apoptosis and phagocytosis of glioblastoma cells. This study shows a possible role of ANXA1 in maintenance of brain homeostasis and may lead to novel therapeutic approaches for neuro-inflammatory diseases and chemotherapy targets in the treatment of glioblastoma multiforme.