Amphotericin-A21 produces antiproliferative effects through modulating EGFR expression in glioblastoma multiforme cells

Abstract ID 131002 Poster Board 059 Glioblastoma multiforme (GBM) is the most frequent malignant astrocytic tumor of the central nervous system (CNS). The survival time of patients with GBM is 8 to 15 months. Our research group has developed a semi-synthetic compound derived from amphotericin B, called Amphotericin-A21 (AmB-A21), which has shown to have a better biosafety profile in animal models and in non-tumor cells (HEK-293) compared to its precursor. Recently, AmB-A21 was shown to induce cytotoxicity in breast cancer cell lines (MDA-MB-231 and MCF-7). In addition, AmB-A21 has been shown to have a synergistic effect with carboplatin inducing apoptosis and inhibiting proliferation and cell invasion of lung adenocarcinoma cells (A549). The aim of this work was to determine the effect of AmB-A21 on EGFR and it signaling pathways involved in tumor proliferation and invasion in GBM cells. Cell viability and cell proliferation were determined using MTS (CellTiter 96® AQueous One Solution de Promega). Briefly, for cell viability, U-87 MG (HTB-14 TM; ATCC) cells were seeded into a 96-well plate (10,000/well) and incubated for 24 h; for cell proliferation cells were incubated during 24, 48 and 72 h at 37°C and 5% CO₂. The culture medium was replaced by a fresh one supplemented with different concentrations of AmB-A21 (1 to 300 μM) and incubated for 24 h. After treatment, MTS-PMS was added and incubated for 1 h. The optical density was determined on a microplate reader (Bio-Rad) at a wavelength of 490 nm and we calculated inhibitory concentration 25 (IC₂₅) and the effect on proliferation rates. Morphological analysis was performed by using hemotoxylin and eosin (H&E) staining of U-87 MG cells. Cells were seeded into a 24-well plate (50,000/well) and treated for 24 h with AmB-A21, as described above. Finally, microphotographs were taken in a light microscopy (Olympus Microscope). We evaluated cell migration with the wound-healing assay after 24h of treatment. Microphotographs were taken and migrating cells were counted. We analyze the expression of phosphosite pY1173-EGFR and PLCγ1 by immunocytochemistry. AmB-A21 had cytotoxic effect and inhibited the proliferation of U-87MG cells in a concentration- and time-dependent manner, respectively (p<0.05). the IC25 for AmB-A21 was 139.18 μM. AmB-A21 induced morphological changes in cytoplasmic due to presence of macropinocytosis and loss of cell adhesion.AmB-A21 had a greater antimigratory effect on U-87MG cells than AmB and cisplatin. Immunofluorescence detection for pY1173-EGFR in AmB-A21 group cells showed lower expression and was located at membranal level. Its localization was also observed in membranes of macropinosome. Therefore, it is suggested to be related to receptor endocytosis and different dynamics of EGFR perinuclear trafficking. Immunostaining of PLCγ1 cells treated with AmB-A21 showed a decrease in the expression of protein. AmB-A21 has a cytotoxic effect in a concentration-time-dependent manner and inhibits cell proliferation and cell migration. AmB-A21 induced morphological changes typical of macropinocytosis in U-87MG cells. In addition, pY1173-EGFR expression was increased and decreased expression of PLCγ1 after treatment with AmB-A21. Therefore, it is suggested that AmB-A21 modulates the oncogenic EGFR signaling pathway associated with proliferation and invasion in an in vitro model of GBM. This work was funded by CONAHCyT via project Fronteras 74884 and DGAPA-PAPIIT via project IG100920