Synergistic effects of plasma-activated medium in combination with Baicalin against neuronal damage.

Neurodegenerative disorders are chronic conditions that progressively damage and destroy parts of the nervous system, and are currently considered permanent and incurable. Alternative strategies capable of effectively healing neuronal damage have been actively pursued. Here, we report the neuroprotective effects of baicalin (BA) combined with plasma-activated medium (PAM) against glutamate-induced excitotoxicity in SH-SY5Y cells. Through in vitro assays, the cell viability, inflammation, apoptosis, and oxidative stress were evaluated. The co-application of BA and PAM significantly enhanced cell viability, reduced pro-inflammatory markers (TNF-α and NF-κB), decreased apoptotic proteins (Bax and Caspase-3) and boosted antioxidative defenses (increased SOD activity and lowered ROS levels). This study confirms the potential of combining BA with PAM as an effective therapeutic strategy for mitigating the effects of excitotoxicity. PAM is a promising adjunct and potential drug delivery method in neuroprotective therapy, providing a new avenue for developing treatments for diseases characterized by neuronal damage.

Plasma-activated medium exerts tumor-specific inhibitory effect on hepatocellular carcinoma via disruption of the salvage pathway.

Hepatocellular carcinoma has high fatality and poor prognosis. For curing hepatocellular carcinoma, the demand for effective therapeutic reagents with low toxicity is urgent. Herein, we investigated plasma-activated medium, an emerging reagent obtained via irradiation of cell-free medium with cold atmospheric plasma. Plasma-activated medium exerts inhibitory effect on many types of tumor cells with little toxicity to non-cancerous cells. In present study, we verified the tumor-specific inhibition of plasma-activated medium on hepatocellular carcinoma cell lines. Under the effect of plasma-activated medium, oxidative stress, mitochondrial dysfunction, and loss of intracellular NAD+ and ATP were detected inside cells, suggesting an energy depletion. Through investigating the salvage pathway which synthesizes NAD+ and maintains the respiratory chain in hepatocellular carcinoma, we found that the energy failure was resulted by the blockage of the salvage pathway. Moreover, nicotinamide phosphoribosyltransferase, the rate-limiting enzyme in the salvage pathway, was determined as an important target to be inactivated by the effect of plasma-activated medium. Additionally, the blockage of the salvage pathway activates AMPKα and suppresses mTOR pathway, which reinforces the cell growth inhibition. Overall, our findings demonstrated that the disruption of functions of nicotinamide phosphoribosyltransferase and the salvage pathway contribute to the tumor-specific cytotoxicity of plasma-activated medium.

Cold Atmospheric Pressure Plasma-Activated Medium Modulates Cellular Functions of Human Mesenchymal Stem/Stromal Cells In Vitro.

Cold atmospheric pressure plasma (CAP) offers a variety of therapeutic possibilities and induces the formation of reactive chemical species associated with oxidative stress. Mesenchymal stem/stromal cells (MSCs) play a central role in tissue regeneration, partly because of their antioxidant properties and ability to migrate into regenerating areas. During the therapeutic application, MSCs are directly exposed to the reactive species of CAP. Therefore, the investigation of CAP-induced effects on MSCs is essential. In this study, we quantified the amount of ROS due to the CAP activation of the culture medium. In addition, cell number, metabolic activity, stress signals, and migration were analyzed after the treatment of MSCs with a CAP-activated medium. CAP-activated media induced a significant increase in ROS but did not cause cytotoxic effects on MSCs when the treatment was singular and short-term (one day). This single treatment led to increased cell migration, an essential process in wound healing. In parallel, there was an increase in various cell stress proteins, indicating an adaptation to oxidative stress. Repeated treatments with the CAP-activated medium impaired the viability of the MSCs. The results shown here provide information on the influence of treatment frequency and intensity, which could be necessary for the therapeutic application of CAP.

No-ozone cold plasma induces apoptosis in human neuroblastoma cell line via increased intracellular reactive oxygen species (ROS).

BACKGROUND: This study aimed to evaluate the effect of argon-based No-ozone Cold Plasma (NCP) on neuroblastoma cancer cell apoptosis. METHODS: Experiments were performed with SK-N-SH and HS 68. Cell cultures were treated with NCP for 1, 3, and 5 min. NCP was applied using three different strategies: direct NCP application to cell cultures, to only media, and to only cells. Evaluation of cell viability and the level of the reactive oxygen species (ROS) was performed. N-acetyl-L-cysteine (NAC) was also used to antagonize intracellular ROS. Cleaved caspase 3, PARP, aquaporin (AQP) 3 and 8 were detected. RESULTS: NCP induced a gradual decrease in the SK-N-SH cell viability. In contrast, the viability of HS 68 cells did not change. SK-N-SH cells viability was reduced the most when the only media-NCP application strategy was employed. Intracellular ROS levels were significantly increased with time. Cleaved caspase 3 and PARP were increased at 6 h after NCP application. SK-N-SH cells remained viable with NAC after NCP application. AQP 3 and 8 were over-expressed in SK-N-SH cells. CONCLUSION: These findings demonstrate the anti-cancer effect of NCP on neuroblastoma cells. NCP enhanced the selective apoptosis of neuroblastoma cells due to the increased intracellular ROS.

Plasma-Activated Media Produced by a Microwave-Excited Atmospheric Pressure Plasma Jet Is Effective against Cisplatin-Resistant Human Bladder Cancer Cells In Vitro.

Media exposed to atmospheric pressure plasma (APP) produce reactive oxygen and nitrogen species (RONS), with hydrogen peroxide (H2O2), nitrite (NO2-), and nitrate (NO3-) being among the most detected species due to their relatively long lifetime. In this study, a standardized microwave-excited (ME) APP jet (APPJ) source was employed to produce gaseous RONS to treat liquid samples. The source was a commercially available plasma jet, which generated argon plasma utilizing a coaxial transmission line resonator at the operating frequency of 2.45 GHz. An ultraviolet-visible spectrophotometer was used to measure the concentrations of H2O2 and NO3- in plasma-activated media (PAM). Three different types of media (deionized water, Hank's balanced salt solution, and cell culture solution Dulbecco's modified eagles medium [DMEM]) were utilized as liquid samples. Among these media, the plasma-treated DMEM was observed to have the highest levels of H2O2 and NO3-. Subsequently, the feasibility of using argon ME-APPJ-activated DMEM (PAM) as an adjuvant to enhance the therapeutic effects of cisplatin on human bladder cancer cells (T-24) was investigated. Various cancer cell lines, including T-24 cells, treated with PAM were observed in vitro for changes in cell viability using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. A viability reduction was detected in the various cancer cells after incubation in PAM. Furthermore, the study's results revealed that PAM was effective against cisplatin-resistant T-24 cells in vitro. In addition, a possible connection between HER expression and cell viability was sketched.

Effects of cold atmospheric-pressure plasma in combination with doxorubicin drug against breast cancer cells in vitro and invivo.

Cold atmospheric plasma (CAP) has been suggested for medical applications that can be applied indirectly through plasma-activated medium (PAM) and recently it has been introduced as an innovative therapeutic approach for all cancer types. Studies have exhibited that ROS/RNS are key factors in CAP-dependent apoptosis; nevertheless, ROS/RNS stability are weak. Combination therapy is considered an effective strategy to overcome these problems. In the present research, we revealed that the combination of CAP and doxorubicin (DOX) significantly induces the apoptosis of breast cancer cells both in vitro and in vivo. Our results indicated that both Ar and He/O2 CAP treatment as well as DOX drug alone reduced cell growth. CAP/PAM treatment in combination with DOX induced apoptosis in MCF-7 breast cancer cells and 4T1-implanted BALB/c mice, resulting in a significant increase in antitumor activity. The apoptotic effects of CAP-DOX on MCF-7 cells were inferred from altered expression of BAX and cleaved-caspase-3 which mechanistically take place through the mitochondrial pathway mediated by Bcl-2 family members. Besides, the BAX/BCL-2 ratio is significantly higher in the simultaneous treatment of CAP and DOX. This ratio was equal to 2.82 ± 0.24, 2.54 ± 0.30, and 11.27 ± 0.31 for treatment with DOX, He/O2 plasma, and combination treatment, respectively. Additionally, the tumor growth rate of He/O2-PAM + DOX and Ar-PAM + DOX treatments was significantly inhibited by PAM-injection, and the tumor growth rate of PAM alone or DOX alone was slightly reduced. It can be concluded that the effect of PAM + DOX may increase the anticancer activity and decrease the dose required for the chemotherapeutic treatment.

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.

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.

Low-Temperature Plasma-Activated Medium Inhibits the Migration of Non-Small Cell Lung Cancer Cells via the Wnt/ß-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/ß-catenin signaling pathway. These results suggested that the high ROS levels generated by the PAM treatment reversed the EMT process by inhibiting the WNT/ß-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.

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 for preventing infection of viruses that use ACE2 for entry.

Rational: The mutating SARS-CoV-2 potentially impairs the efficacy of current vaccines or antibody-based treatments. Broad-spectrum and rapid anti-virus methods feasible for regular epidemic prevention against COVID-19 or alike are urgently called for. Methods: Using SARS-CoV-2 virus and bioengineered pseudoviruses carrying ACE2-binding spike protein domains, we examined the efficacy of cold atmospheric plasma (CAP) on virus entry prevention. Results: We found that CAP could effectively inhibit the entry of virus into cells. Direct CAP or CAP-activated medium (PAM) triggered rapid internalization and nuclear translocation of the virus receptor, ACE2, which began to return after 5 hours and was fully recovered by 12 hours. This was seen in vitro with both VERO-E6 cells and human mammary epithelial MCF10A cells, and in vivo. Hydroxyl radical (·OH) and species derived from its interactions with other species were found to be the most effective CAP components for triggering ACE2 nucleus translocation. The ERα/STAT3(Tyr705) and EGFR(Tyr1068/1086)/STAT3(Tyr705) axes were found to interact and collectively mediate the effects on ACE2 localization and expression. Conclusions: Our data support the use of PAM in helping control SARS-CoV-2 if developed into products for nose/mouth spray; an approach extendable to other viruses utilizing ACE2 for host entry.

The Impacts of Prepared Plasma-Activated Medium (PAM) Combined with Doxorubicin on the Viability of MCF-7 Breast Cancer Cells: A New Cancer Treatment Strategy.

Background: For many years, the chemotherapeutic agent doxorubicin (DOX) has been used to treat various cancers; however, DOX initiates several critical adverse effects. Many studies have reported that non-thermal atmospheric pressure plasma can provide novel, but challenging, treatment strategies for cancer patients. To date, tissues and cells have been treated with plasma-activated medium (PAM) as a practical therapy. Consequently, due to the harmful adverse effects of DOX, we were motivated to elucidate the impact of PAM in the presence of DOX on MCF-7 cell proliferation. Methods: MTT assay, N-acetyl-L-cysteine (NAC) assay, and flow cytometry analysis were utilized in this research. Results: The results demonstrated that 0.45 µM DOX combined with 3-min PAM significantly induced apoptosis (p< 0.01) through intracellular ROS generation in MCF-7 when compared with 0.45 µM DOX alone or 3-min PAM alone. In contrast, after treatment with 0.45 µM DOX plus 4-min PAM, cell necrosis was increased. Hence, DOX combined with 4-min PAM has cytotoxic effects with different mechanisms than 4-min PAM alone, in which the number of apoptotic cells increases. Conclusion: Although further investigations are crucial, low doses of DOX plus 3-min PAM could be a promising strategy for cancer therapy. The findings from this research may offer advantageous and innovative clinical strategies for cancer therapy using PAM.

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.

Air Plasma-Activated Medium Evokes a Death-Associated Perinuclear Mitochondrial Clustering.

Intractable cancers such as osteosarcoma (OS) and oral cancer (OC) are highly refractory, recurrent, and metastatic once developed, and their prognosis is still disappointing. Tumor-targeted therapy, which eliminates cancers effectively and safely, is the current clinical choice. Since aggressive tumors are substantially resistant to multidisciplinary therapies that target apoptosis, tumor-specific activation of another cell death modality is a promising avenue for meeting this goal. Here, we report that a cold atmospheric air plasma-activated medium (APAM) can kill OS and OC by causing a unique mitochondrial clustering. This event was named monopolar perinuclear mitochondrial clustering (MPMC) based on its characteristic unipolar mitochondrial perinuclear accumulation. The APAM caused apoptotic and nonapoptotic cell death. The APAM increased mitochondrial ROS (mROS) and cell death, and the antioxidants such as N-acetylcysteine (NAC) prevented them. MPMC occurred following mitochondrial fragmentation, which coincided with nuclear damages. MPMC was accompanied by mitochondrial lipid peroxide (mLPO) accumulation and prevented by NAC, Ferrostatin-1, and Nocodazole. In contrast, the APAM induced minimal cell death, mROS generation, mLPO accumulation, and MPMC in fibroblasts. These results suggest that MPMC occurs in a tumor-specific manner via mitochondrial oxidative stress and microtubule-driven mitochondrial motility. MPMC induction might serve as a promising target for exerting tumor-specific cytotoxicity.

Cold helium plasma jet does not stimulate collagen remodeling in a 3D human dermal substitute.

Cold Atmospheric Plasma (CAP) is an emerging physical approach displaying encouraging antitumor and wound healing effects both in vitro and in vivo. In this study, we assessed the potential of direct CAP to remodel skin collagens using an original tissue-engineered human dermal substitute model rich in endogenous extracellular matrix (ECM) covered with 600 µl of culture medium and treated with CAP for 30 and 120 s. Our results indicated that Reactive Oxygen and Nitrogen Species (RONS) such as H2O2, NO3- and NO2- were produced in the medium during treatment. It appeared that in the CAP-treated dermal substitutes 1) cell viability was not altered, 2) pro-collagen I secretion was not modified over 48 h of culture after treatment, 3) global activity of matrix metalloproteinases MMPs was not modulated over 48 h after treatment, and 4) no change in hydroxyproline content was observed over 5 days after treatment. In order to confirm the efficiency of our device, we showed that the plasma-activated culture medium induced cell apoptosis and growth delay using a 3D human tumor spheroid model. In conclusion, no effect of direct CAP treatment was monitored on dermal ECM production and degradation, indicating that CAP does not stimulate collagen remodeling at the tissue scale.

[Molecular Mechanisms Underlying Cellular Responses to the Loading of Non-thermal Atmospheric Pressure Plasma-activated Solutions].

Plasma medicine is a rapidly expanding new field of interdisciplinary research that combines physics, chemistry, biology, and medicine. Non-thermal atmospheric pressure plasma (NTAPP) has recently been applied to living cells and tissues, and has emerged as a novel technology for medical applications, such as wound healing, blood coagulation, and cancer treatment. NTAPP was found to affect cells indirectly through the treatment of cells with previously prepared medium irradiated by NTAPP, termed plasma-activated medium (PAM). The treatment of culture media with NTAPP results in the generation of a large amount of reactive oxygen species and reactive nitrogen species, and their derived species. We found that PAM triggered a spiral apoptotic cascade in the mitochondrial-nuclear network in A549 cancer cells. This process induced the depletion of total cellular NAD+ and elevations in intracellular calcium ion, ultimately leading to cell death. We also detected the production of hydroxyl radical and elevations in intracellular ferrous ions in PAM-treated cells. The elevations observed in ferrous ions may have been due to their release from the intracellular iron store, ferritin. However, difficulties are associated with applying PAM to the clinical phase because culture media cannot be used for medical treatments. The anti-tumor activity of plasma-activated Ringer's solution was significantly stronger than that of PAM. At the end, we herein demonstrated the advantages of the combined application of plasma-activated acetate Ringer's solution and hyperthermia, a heat treatment at 42℃, for A549 cancer cell death and elucidated the underlying mechanisms.

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

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.

Cold Atmospheric Pressure Plasma-Activated Medium Induces Selective Cell Death in Human Hepatocellular Carcinoma Cells Independently of Singlet Oxygen, Hydrogen Peroxide, Nitric Oxide and Nitrite/Nitrate.

Cold atmospheric pressure plasma (CAP) and plasma-activated medium (PAM) induce cell death in diverse cancer cells and may function as powerful anti-cancer agents. The main components responsible for the selective anti-cancer effects of CAP and PAM remain elusive. CAP or PAM induces selective cell death in hepatocellular carcinoma cell lines Hep3B and Huh7 containing populations with cancer stem cell markers. Here, we investigated the major component(s) of CAP and PAM for mediating the selective anti-proliferative effect on Hep3B and Huh7 cells. The anti-proliferative effect of CAP was mediated through the medium; however, the reactive oxygen species scavenger N-acetyl cysteine did not suppress PAM-induced cell death. Neither high concentrations of nitrite or nitrite/nitrate nor a low concentration of H2O2 present in the PAM containing sodium pyruvate affected the viability of Hep3B and Huh7 cells. Inhibitors of singlet oxygen, superoxide anions, and nitric oxide retained the capacity of PAM to induce anti-cancer effects. The anti-cancer effect was largely blocked in the PAM prepared by placing an aluminum metal mesh, but not a dielectric PVC mesh, between the plasma source and the medium. Hence, singlet oxygen, hydrogen peroxide, nitric oxide, and nitrite/nitrate are not the main factors responsible for PAM-mediated selective death in Hep3B and Huh7 cells. Other factors, such as charged particles including various ions in CAP and PAM, may induce selective anti-cancer effects in certain cancer cells.

Cancer Treatments Using Low-Temperature Plasma.

Low-temperature plasma (LTP) is a partially ionized gas that contains electrons, ions, radicals, light, etc. Recently, the bio-medical application of LTP has become a hot topic in plasma science and biological science. Cancer treatment with plasma is the most challenging topic in plasma bio-medical applications. Many in vitro and in vivo experiments have been conducted to investigate the anti-tumor effects of LTP. Extracellular reactive oxygen and nitrogen species (RONS) in plasma-activated solutions are key factors for the anti-tumor effects, and amino acid modifications by LTP may affect cellular responses. Intracellular RONS are also key factors for the anti-tumor effects. Various signaling pathways, such as p53 signaling pathways, survival and proliferation signaling pathways, and oxidative stress-dependent signaling pathways are activated by LTP.

Selective Anti-Cancer Effects of Plasma-Activated Medium and Its High Efficacy with Cisplatin on Hepatocellular Carcinoma with Cancer Stem Cell Characteristics.

Hepatocellular carcinoma (HCC) is a major histological subtype of primary liver cancer. Ample evidence suggests that the pathological properties of HCC originate from hepatic cancer stem cells (CSCs), which are responsible for carcinogenesis, recurrence, and drug resistance. Cold atmospheric-pressure plasma (CAP) and plasma-activated medium (PAM) induce apoptosis in cancer cells and represent novel and powerful anti-cancer agents. This study aimed to determine the anti-cancer effect of CAP and PAM in HCC cell lines with CSC characteristics. We showed that the air-based CAP and PAM selectively induced cell death in Hep3B and Huh7 cells with CSC characteristics, but not in the normal liver cell line, MIHA. We observed both caspase-dependent and -independent cell death in the PAM-treated HCC cell lines. Moreover, we determined whether combinatorial PAM therapy with various anti-cancer agents have an additive effect on cell death in Huh7. We found that PAM highly increased the efficacy of the chemotherapeutic agent, cisplatin, while enhanced the anti-cancer effect of doxorubicin and the targeted-therapy drugs, trametinib and sorafenib to a lesser extent. These findings support the application of CAP and PAM as anti-cancer agents to induce selective cell death in cancers containing CSCs, suggesting that the combinatorial use of PAM and some specific anti-cancer agents is complemented mechanistically.