Efficaciousness of Low Affinity Compared to High Affinity TSPO Ligands in the Inhibition of Hypoxic Mitochondrial Cellular Damage Induced by Cobalt Chloride in Human Lung H1299 Cells.

Nidal Zeineh,Massimo Franco,Nunzio Denora,Abraham Weizman,Valentino Laquintana,Moshe Gavish

doi:10.3390/biomedicines8050106

Abstract

The 18 kDa translocator protein (TSPO) plays an important role in apoptotic cell death, including apoptosis induced by the hypoxia mimicking agent cobalt chloride (CoCl2). In this study, the protective effects of a high (CB86; Ki = 1.6 nM) and a low (CB204; Ki = 117.7 nM) affinity TSPO ligands were investigated in H1299 lung cancer cell line exposed to CoCl2. The lung cell line H1299 was chosen in the present study since they express TSPO and able to undergo programmed cell death. The examined cell death markers included: ATP synthase reversal, reactive oxygen species (ROS) generation, mitochondrial membrane potential (Δψm) depolarization, cellular toxicity, and cellular viability. Pretreatment of the cells with the low affinity ligand CB204 at a concentration of 100 µM suppressed significantly (p < 0.05 for all) CoCl2-induced cellular cytotoxicity (100%), ATP synthase reversal (67%), ROS generation (82%), Δψm depolarization (100%), reduction in cellular density (97%), and also increased cell viability (85%). Furthermore, the low affinity TSPO ligand CB204, was harmless when given by itself at 100 µM. In contrast, the high affinity ligand (CB86) was significantly effective only in the prevention of CoCl2–induced ROS generation (39%, p < 0.001), and showed significant cytotoxic effects when given alone at 100 µM, as reflected in alterations in ADP/ATP ratio, oxidative stress, mitochondrial membrane potential depolarization and cell death. It appears that similar to previous studies on brain-derived cells, the relatively low affinity for the TSPO target enhances the potency of TSPO ligands in the protection from hypoxic cell death. Moreover, the high affinity TSPO ligand CB86, but not the low affinity ligand CB204, was lethal to the lung cells at high concentration (100 µM). The low affinity TSPO ligand CB204 may be a candidate for the treatment of pulmonary diseases related to hypoxia, such as pulmonary ischemia and chronic obstructive pulmonary disease COPD.

Highlights

In this study the hypoxic toxic agent cobalt chloride (CoCl2 ) was used to induce hypoxic death inH1299 lung cancer cells
It was demonstrated that the high affinity CB86 ligand and the high affinity alpidem, did not protect against the cytotoxicity caused by CoCl2 (0.5 mM), while the low affinity CB204 achieved significant protective capacity by 76% at a concentration of 25 μM (Figure 1C) and by 85% at 100 μM
61% and by 59%, respectively, against CoCl2 cytotoxicity only at a concentration of 100 μM (Figure 1E). According to these data (Figure 1), the high affinity ligand CB86 and the low affinity ligand CB204 at a concentration of 100 μM were chosen for further investigation of the potential effects of the affinity of the translocator protein (TSPO) ligands in the protection from CoCl2 -induced cellular damage

Summary

Introduction

In this study the hypoxic toxic agent cobalt chloride (CoCl2 ) was used to induce hypoxic death inH1299 lung cancer cells. Decreased oxygen levels or tension in a tissue results in hypoxic damage due to altered cellular signaling networks. Hypoxia may occur both in physiological conditions such as climbing to high altitudes, as well as in pathological conditions such as myocardial ischemia, chronic obstructive pulmonary disease (COPD), and obstructive sleep apnea [2]. Hypoxia affects cellular homeostasis, which may lead to reactive oxygen species (ROS) production, elevated vasoconstrictive substances, increased expression of pro-inflammatory cytokines, and increased intercellular and vascular cell adhesion molecules [3,4,5,6,7,8,9]. Impaired protein homeostasis in the muscle system is a well-known condition in COPD patients [10,11,12]

Objectives

Results

Discussion

Conclusion