Plasma-activated Medium Research Articles

Ozone mediates the anticancer effect of air plasma by triggering oxidative cell death caused by H2O2 and iron

Cold atmospheric plasmas and plasma-treated solutions (PTSs) have emerged as promising approaches in cancer treatment because of their tumor-selective actions. While oxidative stress is critical for their effects, the precise mechanisms, including chemical mediators, remain obscure. Previously, we reported that air plasma-activated medium (APAM) exhibited tumor-selective anticancer activity. The fragmentation of mitochondria and their asymmetrical assembly around the peripheral regions of the damaged nucleus, namely, monopolar perinuclear mitochondrial clustering (MPMC), proceed to the effect. Subsequently, we found that APAM had a substantial amount of O3 in addition to hydrogen peroxide (H2O2), nitrile (NO2-), and nitrate (NO3-). In the present study, we investigated the possible role of O3 in the anticancer effect. For this purpose, we created a nitrogen oxide-free ozonated medium ODM. ODM exhibited potent cytotoxicity against various cancer but not nonmalignant cells. ODM also increased MPMC, hydroxyl radicals, lipid peroxides, and their shifts to perinuclear sites in cancer cells. Catalase and iron chelation prevented these events and cytotoxicity. ODM also decreases the intracellular labile irons while increasing those within mitochondria. ODM had substantial H2O2, but this oxidant failed to cause MPMC and cytotoxicity. These results show that ODM can mimic the effects of APAM, including MPMC and tumor-selective anticancer effects. The findings suggest that O3 is critical in mediating the anticancer effects of APAM by triggering oxidative cell death caused by H2O2 and iron.

Low-Temperature Plasma-Activated Medium Inhibits the Migration of Non-Small Cell Lung Cancer Cells via the Wnt/β-Catenin Pathway.

This study explored the molecular mechanism of the plasma activation medium (PAM) inhibiting the migration ability of NSCLC (non-small cell lung cancer) cells. The effect of PAM incubation on the cell viability of NSCLC was detected through a cell viability experiment. Transwell cells and microfluidic chips were used to investigate the effects of PAM on the migration capacity of NSCLC cells, and the latter was used for the first time to observe the changes in the migration capacity of cancer cells treated with PAM. Moreover, the molecular mechanisms of PAM affecting the migration ability of NSCLC cells were investigated through intracellular and extracellular ROS detection, mitochondrial membrane potential, and Western blot experiments. The results showed that after long-term treatment with PAM, the high level of ROS produced by PAM reduced the level of the mitochondrial membrane potential of cells and blocked the cell division cycle in the G2/M phase. At the same time, the EMT process was reversed by inhibiting the Wnt/β-catenin signaling pathway. These results suggested that the high ROS levels generated by the PAM treatment reversed the EMT process by inhibiting the WNT/β-catenin pathway in NSCLC cells and thus inhibited the migration of NSCLC cells. Therefore, these results provide good theoretical support for the clinical treatment of NSCLC with PAM.

Optimization of Indirect CAP Exposure as an Effective Osteosarcoma Cells Treatment with Cytotoxic Effects

Over the past decade, cold atmospheric plasma (CAP) has undergone extensive research as a promising therapeutic approach in oncology, with different treatment methods and exposure configurations being investigated and resulting in various biological effects, most of them after long exposure or treatment durations. This study aimed to evaluate the potential of a custom-made CAP generation source to produce plasma-activated medium (PAM) with cytotoxic effects and subsequently to establish the optimal exposure and treatment parameters. The exposure’s electrical parameters, as well as pH and NO2− content of PAM were analyzed. The cytotoxic potential and optimal parameters of the treatment were established by evaluating the viability of human osteosarcoma cells (HOS cell line) and human osteoblasts (HOB cell line) treated with PAM under different conditions. Our results showed that indirect treatment with CAP presents selective dose-dependent cytotoxic effects, while the cell viability decrease was not found to be correlated with the PAM acidification due to CAP exposure. The Griess assay revealed very high and long-term stable concentrations of NO2− in PAM. Overall, this study presents a simple and faster method of producing PAM treatment with cytotoxic effects on HOS cells, by using a custom-built CAP source.

Plasma-activated medium triggers immunomodulation and autophagic activity for periodontal regeneration.

Periodontitis is an infection-induced inflammation, evidenced by an increase in inflammatory macrophage infiltration. Recent research has highlighted the role of plasma-activated medium (PAM) as a regulator of the innate immune system, where macrophages are the main effector cells. This study therefore aims to investigate the immunomodulatory effects of PAM on macrophages and its potential applications for periodontitis management. PAM was generated using an argon jet and applied to culture macrophages. Proinflammatory macrophage markers were significantly reduced after PAM stimulation, and this was correlated with the activation of autophagy via the Akt signaling pathway. Further investigations on the proregenerative effects of PAM-treated macrophages on periodontal ligament cells (PDLCs) revealed a significant increase in the expression of osteogeneis/cementogenesis-associated markers as well as mineralization nodule formation. Our findings suggest that PAM is an excellent candidate for periodontal therapeutic applications.

765 Cold atmospheric plasma-activated medium promotes wound healing by stimulation of human keratinocyte migration

765 Cold atmospheric plasma-activated medium promotes wound healing by stimulation of human keratinocyte migration

Abstract 1388: Non-thermal plasma-activated medium induces ferroptotic cell death by intracellular ferrous iron overload in head and neck cancer

Abstract The notoriously high proliferation rate of cancer cells requires them to constantly deal with the balance adjustment between oxidative stress overload and upregulated protective antioxidant pathways. It puts them in a vulnerable state whenever one piece of this puzzle went out of control. Non-thermal plasma (NTP) is partially ionized gas operated at or around body temperature and has emerged as a new potential anti-cancer therapy due to its ability to release and induce ROS, RNS, and other free radicals as well as generate UV- photon and electromagnetic field; but the specific mechanism and its specificity to cancer cell are still controversial. Using transcriptomic analysis, we previously reported the link between NTP-activated media (NTPAM)and apoptosis; but the sequencing data also revealed a strong Ferroptotic response in all the 6Head and Neck cancer cell lines we checked. Ferroptosis is a newly identified oxidative-regulated cell death, characterized by the iron-dependent generation of lipid peroxidation. In this study, we found the prolonged treatment of NTPAM-induced cell death via the non-canonical ferroptosis pathway by directly increasing intracellular toxic ferrous Iron (Fe2+). We found that the excessive overexpression of HMOX1 under prolonged NTPAM-induced oxidative stress was actually responsible for the surge of intracellular Fe2+ due to the increase of heme degradation. Pharmacological inhibition or RNA silencing of HMOX1 was able to rescue 2 HNC cell lines from NTPAM-induced increased Iron concentration as well as ferroptosis cell death, both in vivo and in vitro. These results suggest NTP directly induces ferroptosis via an increase of Fe2+ concentration by triggering the over-responding of the NRF2 - HMOX1 antioxidant system. This finding could be exploited to develop future NTP-based cancer therapy as well as in combination with other Iron sensitive anti-neoplastic agents; and could be a potential method of overcoming conventional cancer treatment resistance. Citation Format: QuocKhanh Nguyen, Chan Oh, Seung-Nam Jung, Yudan Piao, Mi Ae Lim, Young Il Kim, Yanli Jin, Hae Jong Kim, Bon Seok Koo. Non-thermal plasma-activated medium induces ferroptotic cell death by intracellular ferrous iron overload in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1388.

Induction of Apoptosis of Melanoma Skin Cancer Cells by Atmospheric Plasma Jet

The apoptotic effect is an important issue in cancer treatment. To achieve this goal, an atmospheric plasma jet (APJ) was set up for use on cultured cells in a temperature-controlled environment. Melanoma skin cancer and normal skin cells were targeted with this device. Following a 5 s plasma exposure, there was a 67% cell death in melanoma skin cancer cells compared with 5% in normal skin cells as measured after 24 h. When the treatment time was increased to 15, a 98% cell death was reported for melanoma skin cancer cells, which was 80% greater than the cell death in normal skin cells. Our observations further indicate that this preferential cell death is largely due to apoptosis. It shows that an APJ is a selective device in the induction of apoptosis in cancer and normal cells. APJ was shown to affect cells directly and indirectly through a plasma-activated medium (PAM). In direct treatment, cells were exposed to plasma while suspended in a culture medium, and in indirect treatment, cells were added to a culture medium previously activated by plasma treatment. PAM was able to induce cell death 29% higher than direct treatment as measured after 48 h. The depth of the growth medium is also one of the factors in the induction of apoptosis of cancer cells. The growth medium protected the cells from plasma exposure. The result shows that the low level (0 mm) of growth medium will cause more cell death as compared with the high level (2 mm) of growth medium. Apoptotic behavior of skin cancer cells was deduced from the fact that treated cells initially grew and died 12 h following the treatment, while untreated cells continued to grow and proliferate.

Properties and anticancer effects of plasma-activated medium stored at different temperatures

Plasma-activated solution (PAS) has attracted wide attention in cancer treatment because it can treat deep tumors and offer storability. The changes in reactive species and physicochemical properties of PAS during storage can affect its anticancer effect. In this study, the plasma-activated medium (PAM) was prepared by treating RPMI 1640 medium with afterglow gas generated by a custom-built air surface dielectric barrier discharge device. PAM was stored at four common temperature conditions (25 °C, 4 °C, −20 °C, and −80 °C) for 1 day, 4 days, and 7 days, and then, the physicochemical properties, reactive oxygen and nitrogen species (RONS), and the anti-cancer effect on A549 cells under different storage conditions were compared. The results showed that PAM exhibited different anticancer effects at different storage temperatures over a 7 day storage period. The anticancer ability of PAM under 80 °C storage remained stable after 7 days of storage and decreased at all other temperatures. These results were also verified by apoptosis results, and the differences were mainly related to the concentration of H2O2 and NO2−, and oxido reduction potential. Our results provided a theoretical basis for the storage of PAM and its application in anticancer therapy.

Comment “On the dose of plasma medicine: Plasma-activated medium (PAM) and its effect on cell viability” [Phys. Plasmas 29, 063506 (2022)

Comment “On the dose of plasma medicine: Plasma-activated medium (PAM) and its effect on cell viability” [Phys. Plasmas <b>29</b>, 063506 (2022)

Response to “Comment on ‘On the dose of plasma medicine: Plasma-activated medium (PAM) and its effect on cell viability’” [Phys. Plasmas 29, 094701 (2022)

Response to “Comment on ‘On the dose of plasma medicine: Plasma-activated medium (PAM) and its effect on cell viability’” [Phys. Plasmas <b>29</b>, 094701 (2022)

Plasma activated medium prepared by a bipolar microsecond-pulsed atmospheric pressure plasma jet array induces mitochondria-mediated apoptosis in human cervical cancer cells.

Plasma activated medium (PAM) was prepared by a bipolar microsecond-pulsed atmospheric pressure plasma jet (APPJ) array source and was utilized for cancer cell treatment. APPJ array-produced plasma were characterized. APPJ array treatment of three different solutions (deionized water (DW), HBSS (serum-free Hanks’ balanced salt solution), and DMEM (Dulbecco’s Modified Eagle Medium) + 10% FBS (fetal bovine serum)) were performed to induce the changes in the concentration of reactive oxygen and nitrogen species (RONS) as functions of the operating parameters. Human cervical cancer cells (HeLa) injected with plasma-treated media were investigated for changes in cell viability using MTT assay. It was observed that PAM-induced ROS can regulate the protein expression associated with mitochondria, and PAM causes apoptosis through Cyto C/JNK/p38 signaling on human cervical cancer cells.

Investigation of different solutions activated by air plasma jet and their anticancer effect

In the field of plasma biomedicine, research on a plasma-activated medium (PAM) has attracted increasing attention in recent years because of its excellent characteristics. In this study, we used an atmospheric pressure air plasma jet to treat four different solutions: de-ionized water, RPMI 1640 medium, phosphate buffered saline (PBS), and saline. In order to investigate the property differences of different PAM, we mainly analyzed the physical and chemical properties and liquid-phase active species of different PAM and evaluated the inactivation of A549 lung cancer cells. The results show that the concentrations of long-lived reactive species (H2O2, NO2−, and NO3−) in different PAM increased with increasing treatment time. Biological experiments showed that the antitumor effects were in the order of PBS &amp;gt; saline &amp;gt; RPMI 1640 medium, and the best inactivation effect of plasma-activated PBS for 12 min was 89%. Meanwhile, plasma-activated PBS effectively promoted apoptosis in A549 cells, and the highest apoptosis rate was 91.3%. Therefore, this study demonstrates the medical application of different PAM in killing cancer cells and promotes the understanding of plasma–liquid interaction.

On the dose of plasma medicine: Plasma-activated medium (PAM) and its effect on cell viability

Plasma-activated medium (PAM) has a broad prospect in the medical field. However, how to define the plasma dose of PAM and what is the dose–response relationship of PAM on cells are still open questions of plasma medicine. In this paper, the plasma dose of PAM based on equivalent total oxidation potential (ETOP) is introduced, and the S-logistic model is used to describe the relationship between PAM dose and the corresponding lethal effects of cells. Experiments of PAM on A875/HaCaT cell viabilities indicate that ETOP as a plasma dose is suitable for PAM. Evidence of dose discrepancies in 50% response intensity suggests that ETOP can be used to maximize the lethality difference between normal/cancer cells. Further validation by the published literature again indicates that ETOP may provide a well-defined strategy in evaluating the selectivity of PAM treatment on different cell types.

Knockdown of Peroxiredoxin V increased the cytotoxicity of non-thermal plasma-treated culture medium to A549 cells.

Administration of non-thermal plasma therapy via the use of plasma-activated medium (PAM) might be a novel strategy for cancer treatment, as it induces apoptosis by increasing reactive oxygen species (ROS) levels. Peroxiredoxin V (PRDX5) scavenges ROS and reactive nitrogen species and is known to regulate several physiological and pathological reactions. However, its role in lung cancer cells exposed to PAM is unknown. Here, we investigated the effect of PRDX5 in PAM-treated A549 lung cancer cells and determined the mechanism underlying its cytotoxicity. Cell culture medium was treated with low temperature plasma at 16.4 kV for 0, 60, 120, or 180 s to develop PAM. PRDX5 was knocked down in A549 cells via transfection with short hairpin RNA targeting PRDX5. Colony formation and wound healing assays, flow cytometry, fluorescence microscopy, and western blotting were performed to detect the effect of PRDX5 knockdown on PAM-treated A549 cells. PAM showed higher cytotoxicity in lung cancer cells than in control cells, downregulated the mitogen-activated protein kinase signaling pathway, and induced apoptosis. PRDX5 knockdown significantly inhibited cell colony formation and migration, increased ROS accumulation, and reduced mitochondrial membrane potential in lung cancer cells. Hence, PRDX5 knockdown combined with PAM treatment represents an effective option for lung cancer treatment.

Cold Atmospheric Plasma-Activated Media Improve Paclitaxel Efficacy on Breast Cancer Cells in a Combined Treatment Model.

Simple SummaryBreast cancer is the most commonly diagnosed cancer and the leading cause of cancer mortality in females, with chemotherapy currently the only adjunctive therapy. However, the disadvantages of chemotherapy are represented by toxicity/side effects, and for many women the benefit is uncertain. Cold atmospheric plasma in in vitro and in vivo experiments proved that it is able to selectively inhibit the growth of cancer cells in various cancer types, with less deleterious side effects. Current guidelines include paclitaxel as a chemotherapy of choice for various types of breast cancers. The aim of the study was to evaluate in vitro, on human breast cancer cell lines, a plasma-activated media as a cytotoxic agent and as an associative agent in a combined treatment for breast cancer with paclitaxel. The collected data indicated that the plasma-activated media amplified the cytotoxic effect of paclitaxel.The use of plasma-activated media (PAM), an alternative to direct delivery of cold atmospheric plasma to cancer cells, has recently gained interest in the plasma medicine field. Paclitaxel (PTX) is used as a chemotherapy of choice for various types of breast cancers, which is the leading cause of mortality in females due to cancer. In this study, we evaluated an alternative way to improve anti-cancerous efficiency of PTX by association with PAM, the ultimate achievement being a better outcome in killing tumoral cells at smaller doses of PTX. MCF-7 and MDA-MB-231 cell lines were used, and the outcome was measured by cell viability (MTT assay), the survival rate (clonogenic assay), apoptosis occurrence, and genotoxicity (COMET assay). Treatment consisted of the use of PAM in combination with under IC50 doses of PTX in short- and long-term models. The experimental data showed that PAM had the capacity to improve PTX’s cytotoxicity, as viability of the breast cancer cells dropped, an effect maintained in long-term experiments. A higher frequency of apoptotic, dead cells, and DNA fragmentation was registered in cells treated with the combined treatment as compared with those treated only with PT. Overall, PAM had the capacity to amplify the anti-cancerous effect of PTX.

Oxidative Stress Pathways Linked to Apoptosis Induction by Low-Temperature Plasma Jet Activated Media in Bladder Cancer Cells: An In Vitro and In Vivo Study

Current methods used to treat non-muscle invasive bladder cancer are inadequate due to a high recurrence rate after surgery and the occurrence of adverse events such as interstitial pneumonia following intravesical instillation therapy. Low-temperature plasma is a new form of physical therapy that provides a rich source of reactive oxygen species (ROS). Oxidative solutions, created by pre-treatment of aqueous media with plasma before application to target cells, lead to the destruction of cancer cells through oxidative stress pathways. This study focuses on the effects of plasma-activated media (PAM) in bladder cancer cells. PAM treatment increases oxidative stress that leads to cell cycle arrest and concomitantly depolarises the mitochondrial membrane leading to increased mitochondrial ROS production. Cell cycle arrest and increased mitochondrial ROS production led to an increase in caspase 3/cytochrome c activity, which might explain the induction of apoptosis in bladder cancer cells in vitro and in a bladder cancer tumour in vivo. These observations highlight the potential of plasma activated solutions as a new adjuvant therapy in the clinical treatment of bladder cancer.

Cell Type-Specific Anti-Adhesion Properties of Peritoneal Cell Treatment with Plasma-Activated Media (PAM)

Postoperative abdominal adhesions are responsible for serious clinical disorders. Administration of plasma-activated media (PAM) to cell type-specific modulated proliferation and protein biosynthesis is a promising therapeutic strategy to prevent pathological cell responses in the context of wound healing disorders. We analyzed PAM as a therapeutic option based on cell type-specific anti-adhesive responses. Primary human peritoneal fibroblasts and mesothelial cells were isolated, characterized and exposed to different PAM dosages. Cell type-specific PAM effects on different cell components were identified by contact- and marker-independent Raman imaging, followed by thorough validation by specific molecular biological methods. The investigation revealed cell type-specific molecular responses after PAM treatment, including significant cell growth retardation in peritoneal fibroblasts due to transient DNA damage, cell cycle arrest and apoptosis. We identified a therapeutic dose window wherein specifically pro-adhesive peritoneal fibroblasts were targeted, whereas peritoneal mesothelial cells retained their anti-adhesive potential of epithelial wound closure. Finally, we demonstrate that PAM treatment of peritoneal fibroblasts reduced the expression and secretion of pro-adhesive cytokines and extracellular matrix proteins. Altogether, we provide insights into biochemical PAM mechanisms which lead to cell type-specific pro-therapeutic cell responses. This may open the door for the prevention of pro-adhesive clinical disorders.

Low-Temperature Plasma-Activated Medium Inhibited Proliferation and Progression of Lung Cancer by Targeting the PI3K/Akt and MAPK Pathways.

Low-temperature plasma, an engineered technology to generate various reactive species, is actively studied in cancer treatment in recent years, yet mainly by using a traditional 2D cell culture system. In this study, we explored the effect of the plasma-activated medium (PAM) on lung cancer cells in vitro and in vivo by using a 3D cell culture model. The results showed that PAM markedly inhibited 3D spheroid formation and downregulated stemness-related gene expression. We found that reactive oxygen species (ROS) penetrated throughout the whole spheroids and induced cell death surrounding and in the core of the tumor spheroid. Besides, PAM treatment suppressed migration and invasion of lung cancer cells and downregulated epithelial-mesenchymal transition- (EMT-) related gene expression. In the mouse xenograft model, the tumor volume was significantly smaller in the PAM-treated group compared with the control group. By using transcriptome sequencing, we found that PI3K/Akt and MAPK pathways were involved in the inhibition effects of PAM on lung cancer cells. Therefore, our results indicated that PAM exhibits potential anticancer effects on lung cancer and provides insight into further exploration of PAM-induced cell death and translational preclinical use.

Periodic Exposure of Plasma-Activated Medium Alters Fibroblast Cellular Homoeostasis.

Excess amounts of redox stress and failure to regulate homeostatic levels of reactive species are associated with several skin pathophysiologic conditions. Nonmalignant cells are assumed to cope better with higher reactive oxygen and nitrogen species (RONS) levels. However, the effect of periodic stress on this balance has not been investigated in fibroblasts in the field of plasma medicine. In this study, we aimed to investigate intrinsic changes with respect to cellular proliferation, cell cycle, and ability to neutralize the redox stress inside fibroblast cells following periodic redox stress in vitro. Soft jet plasma with air as feeding gas was used to generate plasma-activated medium (PAM) for inducing redox stress conditions. We assessed cellular viability, energetics, and cell cycle machinery under oxidative stress conditions at weeks 3, 6, 9, and 12. Fibroblasts retained their usual physiological properties until 6 weeks. Fibroblasts failed to overcome the redox stress induced by periodic PAM exposure after 6 weeks, indicating its threshold potential. Periodic stress above the threshold level led to alterations in fibroblast cellular processes. These include consistent increases in apoptosis, while RONS accumulation and cell cycle arrest were observed at the final stages. Currently, the use of NTP in clinical settings is limited due to a lack of knowledge about fibroblasts’ behavior in wound healing, scar formation, and other fibrotic disorders. Understanding fibroblasts’ physiology could help to utilize nonthermal plasma in redox-related skin diseases. Furthermore, these results provide new information about the threshold capacity of fibroblasts and an insight into the adaptation mechanism against periodic oxidative stress conditions in fibroblasts.

Plasma-activated medium inhibits cancer stem cell-like properties and exhibits a synergistic effect in combination with cisplatin in ovarian cancer

Ovarian cancer stem-like cells (CSCs) have been implicated in tumor recurrence, metastasis, and drug resistance. Accumulating evidence has demonstrated the antitumor effect of plasma-activated medium (PAM) in various carcinomas, including ovarian cancer. Thus, PAM represents a novel onco-therapeutic strategy. However, its impact on ovarian CSCs is unclear. Here, we show that ovarian CSCs resistant to high-dose conventional chemotherapeutic agents used for ovarian cancer treatment exhibited dose-dependent sensitivity to PAM. In addition, PAM treatment reduced the expression of stem cell markers and sphere formation, along with the aldehyde dehydrogenase- or CD133-positive cell population. We further investigated the effect of PAM in combination with other chemotherapeutics on ovarian CSCs in vitro. PAM exhibited synergistic cytotoxicity with cisplatin (CDDP) but not with paclitaxel and doxorubicin. In a peritoneal metastasis xenograft model established via intraperitoneal spheroid injection, PAM intraperitoneal therapy significantly suppressed peritoneal carcinomatosis (tumor size and number), with a more significant decrease observed due to the combined effects of PAM and CDDP with no side effects. Taken together, our results indicate that PAM inhibits ovarian CSC traits and exhibits synergetic cytotoxicity with CDDP, demonstrating PAM as a promising intraparietal chemotherapy for enhancing antitumor efficacy and reducing side effects.