Plasma-activated Medium Treatment Research Articles

Cytotoxic Effect of Plasma Activated Medium on the Treated Breast Cancer

This study was aimed to evaluate the effect of plasma activated medium (PAM) on breast cancer cell lines (MCF7 and AMJ13) and compared it to non malignant one (ASCs). The cytotoxic effect of 10,15,20, and 25 min prepared PAM was measured and the results indicated that all cells were influenced in the exposure times (20 and 25)min, especially after 48 hours of incubation with lesser influence on normal cells. Based on the results 20 min was determined as the effective dose on 50% of cancer cells with minimal influence on normal one. Further, tumor necrosis factor-alpha (TNF-α) was assessed and the results indicated reduction in the level of this cytokine after PAM treatment, MCF7 showed significant 2reduction in both incubation intervals while AMJ13 and ASCs significantly varied only after 48hrs.Finally, caspase-9was analyzed to identify the apoptosis route and the obtained data revealed that both MCF7 and AMJ13raisedsignificantlyafter 24 and 48hrs.In contrast, ASC cells revealed non-significant changes in both intervals.

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.

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.

Plasma-activated medium induces ferroptosis by depleting FSP1 in human lung cancer cells

Cold atmospheric plasma (CAP) that generates reactive oxygen species (ROS) has received considerable scientific attentions as a new type of anticancer. In particular, an indirect treatment method of inducing cancer cell death through plasma-activated medium (PAM), rather than direct plasma treatment has been well established. Although various cell death pathways such as apoptosis, necroptosis, and autophagy have been suggested to be involved in PAM-induced cell death, the involvement of ferroptosis, another type of cell death regulated by lipid ROS is largely unknown. This study reports, that PAM promotes cell death via ferroptosis in human lung cancer cells, and PAM increases intracellular and lipid ROS, thereby resulting in mitochondrial dysfunction. The treatment of cells with N-acetylcysteine, an ROS scavenging agent, or ferrostatin-1, a ferroptosis inhibitor, protects cells against PAM-induced cell death. Interestingly, ferroptosis suppressor protein 1 (FSP1) is downregulated upon PAM treatment. Furthermore, the treatment of cells with iFSP1, an inhibitor of FSP1, further enhances PAM-induced ferroptosis. Finally, this study demonstrates that PAM inhibits tumor growth in a xenograft model with an increase in 4-hydroxynoneal and PTGS2, a byproduct of lipid peroxidation, and a decrease in FSP1 expression. This study will provide new insights into the underlying mechanism and therapeutic strategies of PAM-mediated cancer treatment.

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.

Effects and Mechanism of Plasma-Activated Medium on Angiogenesis of Vascular Endothelial Cells

After cell culture medium is treated with low temperature plasma (LTP), the liquid is rich in reactive oxygen and nitrogen species (RONS), and becomes plasma-activated medium (PAM). PAM, as the supplier of RONS, can affect the angiogenesis of cells. The purpose of this study is to investigate the effects and related mechanism of PAM on human umbilical vein endothelial cells (HUVECs). Cell viability and cell cycle were evaluated after HUVECs were treated with PAM for 24 h. Changes in cell angiogenesis, migration and adhesion, secretion of cytokines such as VEGF and bFGF, expression of VEGFR-2 and phosphorylation of the key proteins in the MEK/ERK signaling pathway, concentrations of H2O2 and NO2− in PAM and in cells were also investigated. The results showed that PAM obtained by LTP treatment had dual effects on the angiogenesis of HUVECs: PAM obtained by short-term LTP treatment promoted the angiogenesis of HUVECs, while PAM obtained by long-term LTP treatment inhibited the angiogenesis of HUVECs. The mechanism may be that PAM treatment changes the content of RONS, affects the VEGF-VEGFR-2 signaling pathway, and ultimately affects the angiogenesis of HUVECs.

Epithelial-to-Mesenchymal Transition Enhances Cancer Cell Sensitivity to Cytotoxic Effects of Cold Atmospheric Plasmas in Breast and Bladder Cancer Systems.

Simple SummaryCold atmospheric plasma (CAP) and plasma-activated medium (PAM) are known to selectively kill cancer cells, however the efficacy of CAP in cancer cells following epithelial-mesenchymal transition (EMT), a process which endows cancer cells with increased stemness, metastatic potential, and resistance to conventional therapies, has not been previously examined. We have used several established models of EMT to show that PAM is significantly more active in cancer cells exhibiting EMT than their epithelial counterparts. We further show that this enhancement correlated with increased levels of reactive oxygen species (ROS) in the mesenchymally-shifted cell lines. Cold atmospheric plasma (CAP) has emerged as a highly selective anticancer agent, most recently in the form of plasma-activated medium (PAM). Since epithelial–mesenchymal transition (EMT) has been implicated in resistance to various cancer therapies, we assessed whether EMT status is associated with PAM response. Mesenchymal breast cancer cell lines, as well as the mesenchymal variant in an isogenic EMT/MET human breast cancer cell system (PMC42-ET/LA), were more sensitive to PAM treatment than their epithelial counterparts, contrary to their responses to other therapies. The same trend was seen in luminal muscle-invasive bladder cancer model (TSU-Pr1/B1/B2) and the non-muscle-invasive basal 5637 bladder cancer cell line. Three-dimensional spheroid cultures of the bladder cancer cell lines were less sensitive to the PAM treatment compared to their two-dimensional counterparts; however, incrementally better responses were again seen in more mesenchymally-shifted cell lines. This study provides evidence that PAM preferentially inhibits mesenchymally-shifted carcinoma cells, which have been associated with resistance to other therapies. Thus, PAM may represent a novel treatment that can selectively inhibit triple-negative breast cancers and a subset of aggressive bladder cancers, which tend to be more mesenchymal. Our approach may potentially be utilized for other aggressive cancers exhibiting EMT and opens new opportunities for CAP and PAM as a promising new onco-therapy.

Plasma-Activated Medium Potentiates the Immunogenicity of Tumor Cell Lysates for Dendritic Cell-Based Cancer Vaccines

Simple SummaryDendritic cells (DCs)-based anti-cancer vaccines displayed limited efficacy in clinical trials, mostly due to a lack of protocols for preparing immunogenic tumor antigens used in the vaccine. Here, a unique atmospheric pressure plasma jet was used to prepare a plasma-activated medium (PAM) which induced immunogenic cell death in tumor cells. This procedure increased the efficacy of tumor lysates in enhancing the immunogenicity of DCs according to their increased maturation, production of IL-12, and the capacity to induce cytotoxic CD8 T cells able to kill tumor cells. In contrast to the tumor lysates commonly used in DC vaccines, PAM-tumor lysates lacked the capacity to increase IL-10 production by DCs, and their potential to induce protumorogenic Th2 and regulatory T cells. Cumulatively, these results suggest that the novel method for preparing immunogenic tumor lysates with PAM could be suitable for improved DC-based immunotherapy of cancer patients.Autologous dendritic cells (DCs)-based vaccines are considered quite promising for cancer immunotherapy due to their exquisite potential to induce tumor antigen-specific cytotoxic T cells. However, a lack of efficient protocols for inducing immunogenic tumor antigens limits the efficacy of DC-based cancer vaccines. Here, we found that a plasma-activated medium (PAM) induces immunogenic cell death (ICD) in tumor cells but not in an immortalized L929 cell line or human peripheral blood mononuclear cells. PAM induced an accumulation of reactive oxygen species (ROS), autophagy, apoptosis, and necrosis in a concentration-dependent manner. The tumor lysates prepared after PAM treatment displayed increased immunogenicity in a model of human monocyte-derived DCs, compared to the lysates prepared by a standard freezing/thawing method. Mature DCs loaded with PAM lysates showed an increased maturation potential, as estimated by their increased expression of CD83, CD86, CD40, IL-12/IL-10 production, and attenuated PDL1 and ILT-4 expression, compared to the DCs treated with control tumor lysates. Moreover, in co-culture with allogeneic T cells, DCs loaded with PAM-lysates increased the proportion of cytotoxic IFN-γ+ granzyme A+ CD8+ T cells and IL-17A-producing T cells and preserved the Th1 response. In contrast, control tumor lysates-treated DCs increased the frequency of Th2 (CD4+IL-4+), CD4, and CD8 regulatory T cell subtypes, none of which was observed with DCs loaded with PAM-lysates. Cumulatively, these results suggest that the novel method for preparing immunogenic tumor lysates with PAM could be suitable for improved DC-based immunotherapy of cancer patients.

Plasma-activated medium as adjuvant therapy for lung cancer with malignant pleural effusion

This study compared effects of plasma-activated medium (PAM) with effects of conventional clinical thermal therapy on both lung cancer cells and benign cells for management of malignant pleural effusion (MPE). For MPE treatment, chemotherapy, photodynamic therapy, and thermal therapy are used but caused systemic side effects, patient photosensitivity, and edema, respectively. Recent studies show that plasma induces apoptosis in cancer cells with minor effects on normal cells and is cost-effective. However, the effects of plasma on MPE have not been investigated previously. This study applied a nonthermal atmospheric-pressure plasma jet to treat RPMI medium to produce PAM, carefully controlled the long-life reactive oxygen and nitrogen species concentration in PAM, and treated the cells. The influence of PAM treatment on the microenvironment of cells was also checked. The results indicated that PAM selectively inhibited CL1–5 and A549 cells, exerting minor effects on benign mesothelial and fibroblast cells. In contrast to selective lethal effects of PAM, thermal therapy inhibited both CL1–5 and benign mesothelial cells. This study also found that fibroblast growth factor 1 is not the factor explaining why PAM can selectively inhibit CL1–5 cells. These results indicate that PAM is potentially a less-harmful and cost-effective adjuvant therapy for MPE.

Plasma-activated medium promotes autophagic cell death along with alteration of the mTOR pathway

The biological function of non-thermal atmospheric pressure plasma has been widely accepted in several types of cancer. We previously developed plasma-activated medium (PAM) for clinical use, and demonstrated that PAM exhibits a metastasis-inhibitory effect on ovarian cancer through reduced MMP-9 secretion. However, the anti-tumor effects of PAM on endometrial cancer remain unknown. In this study, we investigated the inhibitory effect of PAM on endometrial cancer cell viability in vitro. Our results demonstrated that AMEC and HEC50 cell viabilities were reduced by PAM at a certain PAM ratio, and PAM treatment effectively increased autophagic cell death in a concentration dependent manner. In addition, we evaluated the molecular mechanism of PAM activity and found that the mTOR pathway was inactivated by PAM. Moreover, our results demonstrated that the autophagy inhibitor MHY1485 partially inhibited the autophagic cell death induced by PAM treatment. These findings indicate that PAM decreases the viability of endometrial cancer cells along with alteration of the mTOR pathway, which is critical for cancer cell viability. Collectively, our data suggest that PAM inhibits cell viability while inducing autophagic cell death in endometrial cancer cells, representing a potential novel treatment for endometrial cancer.

Non-thermal Plasma-activated Medium Induces Apoptosis of Aspc1 Cells Through the ROS-dependent Autophagy Pathway.

Numerous studies on various cancer cell lines have reported that direct exposure to non-thermal plasma treatment using plasma-activated medium (PAM) can be applied as a novel technology for cancer therapy. In this study, we investigated the inhibitory effects of PAM on Aspc1 pancreatic cancer cells and the mechanisms responsible for the cell death observed. A colony-formation, sphere-formation, wound-healing and transwell assays, immunocytochemistry and western blot analysis were used monitor effects of PAM. PAM induced a greater cytotoxic effect in pancreatic cancer cells compared to that induced in NIH3T3 cells and 293T cells, and significantly inhibited colony and sphere formation, and cell migration of Aspc1 cells. Furthermore, PAM treatment increased the accumulation of reactive oxygen species (ROS) and reduced the mitochondrial membrane potential in Aspc1 cells. In addition, PAM treatment down-regulated the AKT serine/threonine kinase 1/signal transducer and activator of transcription 3 signaling pathway and induced ROS-dependent cellular autophagy. Our findings suggest that PAM can induce apoptosis of Aspc1 cells through ROS-dependent autophagy and may be a candidate for use in pancreatic cancer therapeutics.

Sublethal treatment with plasma-activated medium induces senescence-like growth arrest of A549 cells: involvement of intracellular mobile zinc.

Plasma-activated medium (PAM) is a solution produced by exposing a liquid medium to non-thermal atmospheric pressure plasma (NTAPP). A number of reactive molecules, such as reactive oxygen species and reactive nitrogen species, are contained in PAM. Therefore, exposure to high doses of PAM results in cell death. We previously demonstrated that intracellular zinc (Zn2+) serves as an important mediator in PAM-induced cell death; however, the effects of sublethal treatment with PAM on cell functions are not fully understood. In the present study, we found that sublethal PAM treatment suppressed cell proliferation and induced senescence-like changes in lung adenocarcinoma A549 cells. Cell cycle analysis revealed that PAM induced cell cycle arrest at the G2/M phase. PAM increased the level of intracellular free Zn2+ and the Zn2+ chelator TPEN counteracted PAM-induced growth suppression, suggesting that Zn2+ functions in PAM-induced growth suppression. In addition, sublethal treatment with PAM induced phosphorylation of ATM kinase, accumulation of p53 protein, and expression of p21 and GADD45A, which are known p53 target genes, in a Zn2+-dependent manner. These results suggest that the induction of growth arrest and cellular senescence by sublethal PAM treatment is mediated by Zn2+-dependent activation of the ATM/p53 pathway.

Introducing Asia-Pacific Plasma Oncology Network: Progress On Triple Negative Breast Cancer

Here we introduce the Asia-Pacific Plasma Oncology network representing researchers based in Australia, China, USA, Korea, Malaysia and Singapore and welcome other members to join. Triple negative breast cancers (TNBC), among the many subtypes of such cancers, have the worst prognosis due to lack of surficial marker expression and effective targeted therapy as well as highest aggressiveness and cancer stemness. Plasma activated medium (PAM) was found, from our work, to be more effective in selectively killing TNBCs and was applied in the following three lines of studies. Part 1: Establishment of computational equation predicting cells’ response to PAM We designed orthogonal experiments to analyze the effects of various parameters of PAM in treating TNBC cells. Among 7 independent parameters, i.e., cell number (C), treatment time (T), output voltage (U), helium flow rate (F), well size (A), distance from the tail of plasma jet to medium surface (D1), and medium thickness (D2), we identified 4 primary influential factors that collectively determine the efficacy of PAM in treating TNBC. Part 2: Internal plasma treatment device development and efficacy comparison Novel plasma needle-like punctuation approach and device were developed for internal animal treatment, using which our in vivo experiments demonstrate the advantages of internal plasma treatment over PAM and surficial treatment. Part 3: Exploration on mechanisms leading to PAM selectivity in TNBC treatment PAM was found capable of selectively killing and inhibiting the migration ability of TNBC cells as compared with other BC subtypes. The cell cohort with ALDH over-expression, representing cancer stem cells, was found to be more sensitive to PAM treatment. High throughput sequencing on TNBC and non-TNBC cell lines treated with PAM under different time points revealed a dynamic network integrating data at mRNA, miRNA, lncRNA and circRNA levels. Pathway analyses consolidate our hypothetical network where ALDH1 mediated cancer stem cell signaling is more sensitive to PAM treatment that leads to higher fraction of cell death among cancer stem cells than bulk tumor cells. Our current network involves more than 20 active faculty members from Australia, China, Korea, USA, Singapore and Malaysia with their researchers and students, and we sincerely thank all of them.

Effect of plasma-activated medium on the decrease of tumorigenic population in lymphoma

Nonequilibrium atmospheric pressure plasma (NEAPP) is a novel approach for blood coagulation, wound healing, and tumor elimination. NEAPP not only directly but also indirectly affects living cells via the medium exposed to NEAPP-yielding devises, called plasma-activated medium (PAM). The conservable and portable PAM serves as an alternative and advantageous approach over direct NEAPP. Here we examined the effect of PAM on lymphoplasmacytic lymphoma (LPL) cell lines. We found that PAM induced plasma cell differentiation and reduced tumorigenic population. PAM increased the expression level of PRDM1α, which is a transcription factor promoting plasma cell differentiation, suggesting that plasma cell differentiation of LPL might be mediated by PRDM1α. We previously reported that plasma cell component of LPL is vulnerable to apoptosis and less tumorigenic. These findings suggested that PAM treatment might become a novel therapy against LPL by inducing the transition from tumorigenic to non-tumorigenic population.

Intraperitoneal Administration of Plasma-Activated Medium: Proposal of a Novel Treatment Option for Peritoneal Metastasis From Gastric Cancer.

The administration of fluid irradiated with non-equilibrium atmospheric pressure plasma (NEAPP) has attracted much interest as a novel therapeutic method for cancer. The authors previously reported on the efficacy of plasma-activated medium (PAM) for treating cancer cell lines through the induction of apoptosis. In this study, the therapeutic effect of PAM was evaluated in vivo using a peritoneal metastasis mouse model. Two gastric cancer cell lines were used in proliferation assays performed to optimize the production of PAM by changing the distance between the plasma source and the medium surface and by altering the volume of irradiated medium. Wound-healing and adhesion assays were conducted to determine the effect of PAM therapy on cell migration and adhesion capacity in vitro. Finally, a mouse model established by the intraperitoneal injection of enhanced green fluorescent protein-tagged gastric cancer cells was used to explore the efficacy of PAM administered intraperitoneally in inhibiting peritoneal metastasis formation. Shorter distances between the plasma source and the medium surface and smaller volumes of treated medium increased the anti-tumor effect of PAM. The PAM treatment attenuated gastric cancer cell migration and adhesion in vitro. The intraperitoneal administration of PAM decreased the formation of peritoneal metastatic nodules by 60% in the mouse model, and no adverse events were observed. Plasma-activated liquids may represent a novel therapeutic method for the treatment of peritoneal metastases in gastric cancer.

Effectiveness of plasma treatment on pancreatic cancer cells.

Non-equilibrium atmospheric pressure plasma (NEAPP) has attracted attention in cancer therapy. We explored the indirect effect of NEAPP through plasma-activated medium (PAM) on pancreatic cancer cells in vitro and in vivo. In this study, four pancreatic cancer cell lines were used and the antitumor effects of PAM treatment were evaluated using a cell proliferation assay. To explore functional mechanisms, morphological change and caspase-3/7 activation in cells were also assessed. Furthermore, reactive oxygen species (ROS) generation in cells was examined and N-acetyl cysteine (NAC), an intracellular ROS scavenger, was tested. Finally, the antitumor effect of local injection of PAM was investigated in a mouse xenograft model. We found that PAM treatment had lethal effect on pancreatic cancer cells. Typical morphological findings suggestive of apoptosis such as vacuolization of cell membranes, small and round cells and aggregation of cell nuclei, were observed in the PAM treated cells. Caspase-3/7 activation was detected in accordance with the observed morphological changes. Additionally, ROS uptake was observed in all cell lines tested, while the antitumor effects of PAM were completely inhibited with NAC. In the mouse xenograft model, the calculated tumor volume on day 28 in the PAM treatment group was significantly smaller compared with the control group [28±22 vs. 89±38 (mm3 ± SD), p=0.0031]. These results show that PAM treatment of pancreatic cancer might be a promising therapeutic strategy.