Nanosecond pulsed current under plasma-producing conditions induces morphological alterations and stress fiber formation in human fibrosarcoma HT-1080 cells
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.
Section snippets
Cell culture
The human sarcoma cell line HT-1080 was obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). Cells were cultured in minimum essential medium eagle (Sigma-Aldrich, MO, USA) containing 10% fetal bovine serum (Thermo Fisher Scientific, MA, USA) and 1% penicillin/streptomycin (Nacalai Tesque, Kyoto, Japan) under humidified conditions with 5% CO2 at 37 °C. For passage, 0.25 w/v % trypsin/1 mM EDTA solution (Fujifilm Wako Pure Chemicals, Osaka, Japan) was used.
For
nsPC stimulation caused elongated cell shape and increased size in HT-1080
In the previous study [16], we found that nsPC promoted the motility of HT-1080 cells, which are human fibrosarcoma cells and widely used for the analysis of cell motility. It has been reported that the activation of cell motility is frequently associated with self-transformation involving morphological alterations, such as pseudopodial protrusion and invadopodia formation [27,37]. Therefore, to obtain insights into nsPC actions on HT-1080 cells, we analyzed morphological characteristics of
Discussion
CAP is increasingly regarded to be a promising method for medical applications, but its precise mechanism of action remains to be fully understood, because of the chemical and electrical complexity of CAP. Currently, it is particularly critical to distinguish biological effects between chemical and electrical factors in CAP. In our previous and present studies, we used human fibrosarcoma HT-1080 cells that are widely utilized for the analysis of cell motility. We applied nsPC to cells under
CRediT authorship contribution statement
Chia-Hsing Chang: Conceptualization, Methodology, Visualization, Investigation, Data curation, Resources, Formal analysis, Writing - original draft. Ken-ichi Yano: Writing - review & editing, Supervision, Funding acquisition. Takehiko Sato: Writing - review & editing, Supervision, Funding acquisition.
Acknowledgments
This study was supported by JSPS KAKENHI Grants No. 16H02311 (TS), 19H00743 (TS), 19H04271 (KY), and the Collaborative Research Project of the Institute of Pulsed Power Science, Kumamoto University.
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