The selective growth inhibition of oral cancer by iron core-gold shell nanoparticles through mitochondria-mediated autophagy

Abstract

Nanoparticles with an iron core and gold shell (denoted “Fe@AuÓ”) have been reported to limit cancer-cell proliferation and therefore have been proposed as a potential anti-cancer agent. However, the underlying mechanisms are still unknown. In this study, we used flow cytometry, confocal fluorescence microscopy, and transmission electron microscopy to analyse the morphological and functional alterations of mitochondria in cancerous cells and healthy cells when treated with Fe@Au. It was found that Fe@Au caused an irreversible membrane-potential loss in the mitochondria of cancer cells, but only a transitory decrease in membrane potential in healthy control cells. Production of reactive oxygen species (ROS) was observed; however, additions of common ROS scavengers were unable to protect cancerous cells from the Fe@Au-induced cytotoxicity. Furthermore, iron elements, before oxidation, triggered mitochondria-mediated autophagy was shown to be the key factor responsible for the differential cytotoxicity observed between cancerous and healthy cells.

Introduction

In recent years, magnetic nanoparticles have been successfully applied as “theranostic” tools, due to their molecular-level size and their ability to be traced during magnetic resonance imaging (MRI) [1]. In particular, magnetic nanoparticles were highlighted as holding great promise for future anti-cancer therapies [2]. Layered nanoparticles made with an iron core and a gold shell, Fe@Au, were designed a decade ago [3] to take the advantage of the strong magnetic susceptibility of pure iron and the passivating properties of the gold coating; in practice, the gold shell only delays the oxidation, rather than stopping it entirely. Previously, we demonstrated that Fe@Au nanoparticles suppress cancer-cell growth at the S-phase, but spare healthy cells in oral- and colorectal-cancer cells in vitro and in vivo [4]. We found that the key element to suppress the cancer-cell growth is the non-oxidized iron component, and that the nanoparticles gradually lose their cytotoxicity towards cancer cells upon oxidation. To date, however, it remains unclear which molecular mechanisms are responsible for the differential effects of Fe@Au on cancerous and benign cells.

It is known that nanoparticles provoke cell cytotoxicity by inducing the production of reactive oxygen species (ROS) [5], [6]. Mitochondria are the major intracellular source of ROS, which are the primary endogenous agents that damage DNA, lipids and proteins, and so contribute to a variety of diseases. In fact, it has been reported that two ultra-fine metal dusts (Co and Ni) caused glutathione depletion, confirming that oxidative stress was involved in the response to such particles [7]. Although, not all materials have electronic configurations or surface properties that allow spontaneous ROS generation, particle interactions with cellular components, frequently, are capable of generating oxidative stress. Hence, assessment of ROS generation is a valid test paradigm to compare the relative toxicity of nanoparticles [8].

Mitochondria are not only the main source of ROS but also are central in regulating cell death. Loss of mitochondrial membrane-potential and the subsequent release of intra-mitochondria components trigger the apoptotic cascade. It has also been shown that the mitochondrial membrane-potential plays an important role in the onset of autophagy [9]. Autophagy is a lysosome-dependent cellular degradation process that responds to dysfunctional organelles and clears external substances from cells. For example, autophagy occurs in virus-mediated cell infection, in nutrient depletion and in other abnormal physiological conditions. Autophagy is not only a pro-survival process for cells but also a known cell death inducing mechanism [10]. It is noteworthy that nanoparticles have been proposed before as a novel class of autophagy activators [11], with the autophagic response reported to depend on the size and shape of the nanoparticles [12]. Moreover, it has been postulated that autophagy induced by nanoparticles could be a possible mechanism in the observed cytotoxicity caused by the particles [13]. Therefore, in this study, we have investigated the mechanisms of Fe@Au-induced cancer-cell-specific cytotoxicity.