Cell-Permeable Gomesin Peptide Promotes Cell Death by Intracellular Ca²⁺ Overload

Abstract

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.

Resistance to Degradation and Cellular Distribution are Important Features for the Antitumor Activity of Gomesin

“…To identify the type of cell death promoted by Gm and its analogues we used annexin-V, which binds to phosphatidylserine that become exposed to extracellular cell membrane during apoptosis, and 7-AAD, an membrane-impermeant nuclear stain. Gm promotes primarily An + 7-AAD + label (Figure 1B) that correspond to necrosis-like mechanism by membrane permeabilization observed previously in B16 cells [6], and others tumor cell lines [12], [17]. Similar results were obtained with Gm analogues (Figure S1) .…”

“…It is possible to observe the internal presence of Gm, [D-Thr 2,6,11,15 ]-D-Gm, [D-Thr 2,6,11,15 , Pro 9 ]-D-Gm, and [Thr 2,6,11,15 , D-Pro 9 ]-Gm after 24 h. On the other hand, the peptides that present lower cytotoxicity were not retained after 24 h (Figure 3A-F). However, a direct relation between binding, peptide entrance, and cytotoxicity effect was not full observed, the entrance into cells seem to be important to the action of several AMPs [12]. To verify the participation of the entry of peptide with cytotoxicity cholesterol level cholesterol level was reduced incubating the cells with MβCD for 1 h, which increase the membrane fluidity [22].…”

“…Gm and its analogues induce cell death with necrosis-like features at IC 50 , and apoptosis (Z-VAD) or necroptosis (Necrostatin) inhibitors (Figure 1) did not inhibit their actions. These mechanism seem to be dependent of cell type [6], [8],[12],[19]. Although, to cell death occur the first step is the AMPs interaction with the cell membrane.…”

“…The effectiveness of Gm had been shown as a topical agent against B16 melanoma tumor cells [6], neuroblastoma SH-SY5Y, and pheochromocytoma PC12 cells [17]. We recently explore the intracellular mechanisms that promote cell death by Gm in tumor cells demonstrating that the membrane permeabilization induced by Gm is preceded by specific intracellular events such as endoplasmic reticulum disturbance, cytosolic Ca 2+ increase, followed by an accumulation of Ca 2+ in organelles, which induces loss of mitochondria potential leading to collapse of mitochondria, which culminates in the disruption of cellular membrane [12], [17].…”

Anticancer Peptides: Prospective Innovation in Cancer Therapy

“…This peptide has the ability to form pores and is active as a topical agent against melanoma, breast, and colon carcinomas neuroblastomas and pheochromocytomas (Rodrigues et al 2008). Gomesin induces membrane permeabilization through a Ca 2+ dependent pathway which involves particular intracellular events: perturbation of the endoplasmic reticulum, accumulation of Ca 2+ in organelles, of mitochondrial potential and oxidative stress (Paredes-Gamero et al 2012). Mastoparan is a 14-amino acid α-helical cell penetrating peptide from the venom of Vespula lewisii wasp that has nocive effects on cell membranes (Saar et al 2005).…”