Nanosecond pulsed current under plasma-producing conditions induces morphological alterations and stress fiber formation in human fibrosarcoma HT-1080 cells

Highlights

• Cold atmospheric plasma (CAP) is a novel promising method for medicine.

• Cellular effects of nanosecond pulsed current (nsPC) from CAP were analyzed.

• nsPC elicited various morphological changes in human HT-1080 cells.

• nsPC appeared to exert its biological effects through actin dynamics.

• This study provides a novel insight into the electrical aspect of CAP action.

Abstract

Cold atmospheric plasma (CAP) is a promising means for various biomedical applications, including cancer therapy. Although the biological action of CAP is considered to be brought about by synergistic effects of reactive species and electrical factors of CAP, limited information is currently available on the contribution of electrical factors to CAP-induced cell responses. We have previously demonstrated that nanosecond pulsed current (nsPC) under CAP-producing conditions significantly promoted the motility of human HT-1080 cells. In this study, we explored the effects of nsPC on cell morphology associated with cell motility. We observed that nsPC stimulation caused extended cell shape, membrane protrusion formation, and increased cell surface area, but not cell death induction. nsPC stimulation also caused elevated intracellular ROS and Ca²⁺. HT-1080 cells can undergo two modes of cell motility, namely mesenchymal and ameboid motility, and we found that morphological features of mesenchymal motility was partly shared with nsPC-stimulated cells. Furthermore, nsPC-stimulated cells had extended stress fibers composed of filamentous actin. Taken together, this study provides a novel insight into the electrical aspect of CAP action, and we speculate that nsPC activates a certain mechanism involving intracellular signaling for stress fiber formation, leading to altered cell morphology and increased cell motility.

Introduction

Cold atmospheric plasma (CAP) is an ionized gas that is generated under atmospheric pressure and does not cause excessive heating. CAP has recently received considerable attention because of its potential for various medical applications, such as cancer therapy, wound healing, and skin regeneration [[1], [2], [3], [4], [5], [6]]. Currently, biological actions of CAP are considered to be brought about by the synergistic effects of chemical and electrical factors of CAP. As for chemical factors, CAP contains multiple forms of reactive species, such as radicals and ions [7]. Cytotoxicity of CAP is mainly attributed to the action of reactive species [8,9]. Whereas the biological significance of reactive species in CAP has been well documented so far, limited information is currently available on the contribution of electrical factors to CAP-induced biological responses. Two major obstacles hampered the effort to understand the biological influence of electrical factors and their transmission process. First, reactive species in CAP exert strong cytotoxic effects that easily overwhelm the biological effects of electrical factors. Second, electrical stimulation itself frequently induces cell death, and careful examination of stimulation conditions is required to avoid gross induction of cell death. For these reasons, many previous studies have been performed from the viewpoints of reactive species and cell death induction [[10], [11], [12], [13], [14], [15]].

In our previous study [16], we attempted to analyze the significance of electrical factors of CAP for a better understanding of CAP actions on cells. We developed a novel device, in which cells were indirectly stimulated with CAP through salt bridges. The use of this device markedly reduced the contact of CAP-derived reactive species with cells and allowed us to analyze biological effects of the electrical factors under CAP-producing conditions. We next carefully optimized the electrical conditions for cell stimulation to avoid cell death induction. Using this device and optimized electrical conditions, we electrically stimulated the human fibrosarcoma HT-1080 cells, which are widely utilized for the analysis of cell motility. We applied pulsed current in duration of nanoseconds (nsPC) to cells under the CAP-producing conditions and observed that cell motility was significantly increased. Interestingly, the direction of increased cell movement was random and was not associated with the direction of electric current. This observation suggested that nsPC under the CAP-producing conditions stimulated a certain mechanism for cell motility and led to the activation of cell movement in random directions, instead of one-directional movement guided by electric current [17,18].

Based on these observations in our previous study, we undertook further exploration of the biological action of nsPC under CAP producing conditions in this study. Here, we analyzed morphological features of nsPC-stimulated HT-1080 cells, because it is well established that the status of cell motility frequently manifests as characteristic morphological features [19]. HT-1080 cells are known to show two distinct modes of cell motility [20]. Cell motility is generally modulated by extracellular stimuli, such as growth factors and mechanical contact to extracellular matrices. Rho activity is increased by contact with extracellular matrices mediated by integrin [21] and N-cadherin [22], leading to stress fiber formation. Rac is stimulated by growth factors, including PDGF and EGF, and facilitates membrane protrusion formation [[23], [24], [25]]. Cells in mesenchymal motility exhibit fibroblast-like extended cell shape with membrane protrusions that undergo integrin-mediated adhesion [[26], [27], [28]]. Actin-rich protrusions play a critical role in mesenchymal motility and called pseudopodia. The process in which a cell gains the ability for mesenchymal motility is called an epithelial-to-mesenchymal transition and is a critical step for the malignant transformation [29]. Another form of cell motility is ameboid motility that is accompanied with round cell shape and relatively weak adhesion [26]. In the human body, cancer cells undergo invasion and metastasis by switching these modes of cell motility [19,30,31]. Both modes of cell motility are driven by actin polymerization, cell adhesion, and act-myosin contraction, which are controlled by intracellular signaling, particularly by Rho family small GTPase members, such as Rho, Rac, and Cdc42. Rho GTPases are key players to regulate the actin cytoskeleton and gene transcription to promote coordinated changes in cell behavior: Rac controls cell protrusion, and Cdc42 modulates cell polarity [26,[32], [33], [34], [35]]. The Rho family members play critical roles in cell morphology and motility: they regulate the formation of filopodia and lamellipodia through the control of actin polymerization by switching on/off their activities [36,37]. Of note, two modes of cell motility can be induced in HT-1080 cells by use of inhibitors for ROCK, a downstream factor of Rho which suppresses Rho-mediated signal transduction, and Rac1. Intracellular signaling for cell motility can be modulated by other factors, such as reactive oxygen species. For example, superoxide anion derived from the mitochondrial respiratory chain can affect cytoskeleton formation through modulating ERK activity [38,39].

In this study, we stimulated HT-1080 cells with nsPC under the CAP-producing conditions and analyzed their morphological alterations. Furthermore, we compared the morphological features of nsPC-stimulated cells with those of two modes of cell motility. We observed that nsPC induced morphological alterations in HT-1080 cells. Common morphological features between nsPC-stimulated cells and those in mesenchymal motility were observed, suggesting the presence of a shared mechanism. Our observations provide further insights into the biological actions of CAP and are important for better medical applications for CAP.