In recent years, the antitumoral activity of antimicrobial peptides (AMPs) has been the goal of many research studies. Among AMPs, gomesin (Gm) displays antitumor activity by unknown mechanisms. Herein, we studied the cytotoxicity of Gm in the Chinese hamster ovary (CHO) cell line. Furthermore, we investigated the temporal ordering of organelle changes and the dynamics of Ca 2+ signaling during Gm-induced cell death. The results indicated that Gm binds to the plasma membrane and rapidly translocates into the cytoplasm. Moreover, 20 μM Gm increases the cytosolic Ca 2+ and induces membrane permeabilization after 30 min of treatment. Direct Ca 2+ measurements in CHO cells transfected with the genetically encoded D1-cameleon to the endoplasmic reticulum (ER) revealed that Gm induces ER Ca 2+ depletion, which in turn resulted in oscillatory mitochondrial Ca 2+ signal, as measured in cells expressing the genetically encoded probe to the mitochondrial matrix mit Pericam. This leads to mitochondria disruption, loss of mitochondrial membrane potential and increased reactive oxygen species prior to membrane permeabilization. Gm-induced membrane permeabilization by a Ca 2+ -dependent pathway involving Gm translocation into the cell, ER Ca 2+ depletion and disruption, mitochondrial Ca 2+ overload and oxidative stress.

Many reports have shown that antimicrobial peptides exhibit anticancer abilities. Gomesin (Gm) exhibits potent cytotoxic activity against cancer cells by a membrane pore formation induced after well-orchestrated intracellular mechanisms. In this report, the replacements of the Cys by Ser or Thr, and the use D-amino acids in the Gm structure were done to investigate the importance of the resistance to degradation of the molecule with its cytotoxicity. [Thr2,6,11,15]-Gm, and [Ser2,6,11,15]-Gm exhibits low cytotoxicity, and low resistance to degradation, and after 24 h are present in localized area near to the membrane. Conversely, the use of D-amino acids in the analogue [D-Thr2,6,11,15]-D-Gm confers resistance to degradation, increases its potency, and maintained this peptide spread in the cytosol similarly to what happens with Gm. Replacements of Cys by Thr and Gln by L- or D-Pro ([D-Thr2,6,11,15, Pro9]-D-Gm, and [Thr2,6,11,15, D-Pro9]-Gm), which induced a similar β-hairpin conformation, also increase their resistance to degradation, and cytotoxicity, but after 24 h they are not present spread in the cytosol, exhibiting lower cytotoxicity in comparison to Gm. Additionally, chloroquine, a lysosomal enzyme inhibitor potentiated the effect of the peptides. Furthermore, the binding and internalization of peptides was determined, but a direct correlation among these factors was not observed. However, cholesterol ablation, which increase fluidity of cellular membrane, also increase cytotoxicity and internalization of peptides. β-hairpin spatial conformation, and intracellular localization/target, and the capability of entry are important properties of gomesin cytotoxicity.

1871 Introduction: PI3K/AKT pathway is involved in cell growth, proliferation and apoptosis. A key downstream effector is the phosphorylated serine-threonine Akt (p-AKT). Constitutive activation of PI3K/AKT has been observed in solid tumours and leukemic cells. Inhibition of PI3K/AKT activity, results in apoptosis in cell lines (CL) after treatment with different compounds, e.g. deguelin, a natural product from the leguminous Mundulea sericea, with antitumour effects. Aims: To evaluate PI3K/AKT activation in MDS patients and its therapeutic potential in MDS. Methods: PI3K/AKT activation was evaluated by flow cytometry (FC) using an alexa-fluor 488-antibody Ser 473 p-AKT (Cell Signalling Technology). A triple immunostaining procedure using CD45-PerCP and CD34-PE was used for p-AKT expression in CD34+ primary samples. The p-AKT activity was determined using Kolmogorov-Smirnov test (D). CD34+ cells from healthy donors and Jurkat cells were used as negative and positive controls respectively. Apoptosis (determined by Annexin V and PI/7AAD) and cell cycle arrest (using RNAse and PI) were determined following treatments with LY294002 (50uM), and deguelin (100-500nM) in P-39 myeloid leukemia cell line, with constitutive PI3K/AKT activation. Apoptosis was determined in bone marrow mononuclear cells and CD34+ cells from MDS patients with the same treatments. To evaluate in vivo activity of deguelin, we used a xenotransplant model. Briefly, NODSCID mice were injected intrafemurally with P-39 CL and 12 days post transplant a three week-course of treatment, every other day, was started (deguelin 4mg/Kg, n=3 vs vehicle, n=3). Results: P-39 CL showed constitutive PI3K/AKT activation with levels significantly higher than in CD34+cells from controls (median±SD= 0.73. Disclosures: No relevant conflicts of interest to declare.