Non-thermal atmospheric gas plasma (AGP) exhibits cytotoxicity against malignant cells with minimal cytotoxicity toward normal cells. mROS accumulation induced by AGP-activated medium or H2O2. The present findings expand our previous observations on death receptor-mediated tumor-selective cell killing and reinforce the importance of mitochondrial network remodeling as a powerful target for tumor-selective cancer treatment. xenograft model [5, 9, 10]. AGP generated from a variety of gas types is capable of killing tumor cells. AGP has been shown to cause cell cycle arrest and DNA damage checkpoint responses and to alter gene expression profiles [10C13]. Reactive oxygen/nitrogen species (ROS/RNS) generation and/or reductions in antioxidant systems are associated with most tumor cell killing by AGP, and therefore oxidative stress is suggested to play a key role in the antitumor activity [5, 14C17]. Recently, culture with AGP-activated medium was Pirinixil demonstrated to be effective for killing of various tumor cells, such as glioblastoma, chemoresistant ovarian, gastric, and pancreatic cancer cells, and while exhibiting minimal cytotoxicity toward normal cells [18C21]. Indirect Spry3 AGP treatment appears to share many biological activities with direct AGP irradiation, including apoptosis induction and ROS generation [20, 21]. However, compared with direct AGP irradiation, little is known about the mechanisms for the antitumor activity of indirect AGP treatment, and the molecular basis of its tumor-selectivity remains unclear. Mitochondria are highly dynamic organelles with a reticular network that is delicately balanced between two antagonistic machineries responsible for fission and fusion of the mitochondrial membrane. The mitochondrial network is critical for cell function and apoptosis [22, 23], because a defect in either fission or fusion Pirinixil causes severe mitochondrial and cellular dysfunctions. Mitochondrial fission helps to eliminate damaged mitochondria through mitophagy [24], such that disruption of mitochondrial fission leads to an extensively interconnected and collapsed mitochondrial network, and defects in mitochondrial quality control. Meanwhile, mitochondrial fusion facilitates the exchange of mitochondrial DNA and metabolites required for mitochondrial function [25]. Consequently, defects in mitochondrial fusion lead to mitochondrial fragmentation and loss of mitochondrial DNA [26], reduced growth, decreased mitochondrial membrane potential (m), and defective respiration [27]. In mammalian cells, mitochondrial fusion and fission are controlled by dynamin-related proteins with GTPase activity, namely mitofusin 1/2 (Mfn1/2), optic atrophy 1 (OPA1), and dynamin-related protein 1 (Drp1). Mfn1/2, and OPA1 act in concert to regulate mitochondrial fusion and cristae organization, while Drp1 regulates mitochondrial fission [22, 23]. We previously demonstrated that TNF-related apoptosis-inducing ligand (TRAIL), a highly tumor-selective anticancer drug, induces aberrant mitochondrial network changes in cancer cells, but not in non-transformed cells [28]. The mitochondria within tumor cells specifically undergo excessive mitochondrial fragmentation followed by clustering. This mitochondrial network collapse is paralleled by apoptosis and mitochondrial ROS (mROS) accumulation stimulated it. By analogy with TRAIL in terms of the tumor-selective cytotoxicity and involvement of ROS, we hypothesized Pirinixil that AGP also targets mitochondrial network remodeling for its cytotoxicity. We developed a non-thermal AGP jet, established an model to examine the antitumor activity of AGP-activated medium, and elucidated the mechanisms of action within the context of tumor-selectivity. Here we show that AGP-activated medium exhibits cytotoxicity toward chemoresistant cancer cells such as malignant melanoma, non-small cell lung cancer (NSCLC), and osteosarcoma cells while sparing non-transformed cells. We also demonstrate that indirect AGP treatment preferentially stimulates mitochondrial network collapse in tumor cells compared with non-transformed cells through their vulnerability to mitochondrial mROS accumulation and ROS-mediated mitochondrial network remodeling. RESULTS AGP-activated medium exhibits cytotoxicity against a panel of human cancer cell lines, but not non-transformed cells AGP was generated at room temperature using a low-frequency (LF) plasma jet device by discharging helium gas under atmospheric conditions (Figure 1AC1D) and used to irradiate Dulbecco’s minimum essential medium (DMEM). The target Pirinixil cells had been cultured in the AGP-activated moderate for 24 or 72 h after that, and assessed because of their cell development. When AGP-activated moderate was ready using different amounts of DMEM (1C5 ml), the cytotoxicity from the ensuing medium against individual malignant melanoma A375 cells reduced as the quantity increased (Body ?(Figure2A).2A). AGP-activated moderate ready with DMEM at amounts of 4 ml exhibited no significant cytotoxicity at 72 h. In the meantime, AGP irradiation to at least one 1 ml of DMEM for 5 min, however, not 1 min, created extremely poisonous moderate reproducibly, which almost totally (optimum of 90%) reduced the cell viability at 24 h. As a result, this protocol was applied by us through the entire present study. Lifestyle in AGP-activated moderate for.