Publications by authors named "Masaru Hori"

68 Publications

Non-thermal plasma-induced DMPO-OH yields hydrogen peroxide.

Arch Biochem Biophys 2021 Jul 6;705:108901. Epub 2021 May 6.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, Showa-Ku, Nagoya, 466-8550, Japan; Center for Low-temperature Plasma Sciences, Nagoya University, Chikusa-ku, Nagoya, 464-8603, Japan. Electronic address:

Recent developments in electronics have enabled the medical applications of non-thermal plasma (NTP), which elicits reactive oxygen species (ROS) and reactive nitrogen species (RNS), such as hydroxyl radical (OH), hydrogen peroxide (HO), singlet oxygen (O), superoxide (O), ozone, and nitric oxide at near-physiological temperatures. In preclinical studies or human clinical trials, NTP promotes blood coagulation, eradication of bacterial, viral and biofilm-related infections, wound healing, and cancer cell death. To elucidate the solution-phase biological effects of NTP in the presence of biocompatible reducing agents, we employed electron paramagnetic resonance (EPR) spectroscopy to quantify OH using a spin-trapping probe, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO); O using a fluorescent probe; and O and HO using luminescent probes in the presence of thiols or tempol. NTP-induced OH was significantly scavenged by dithiothreitol (DTT), reduced glutathione (GSH), and oxidized glutathione (GSSG) in 2 or 5 mM DMPO. NTP-induced O was significantly scavenged by 10 μM DTT and GSH, while O was not efficiently scavenged by these compounds. GSSG degraded HO more effectively than GSH and DTT, suggesting that the disulfide bonds reacted with HO. In the presence of 1-50 mM DMPO, NTP-induced HO quantities were unchanged. The inhibitory effect of tempol concentration (50 and 100 μM) on HO production was observed in 1 and 10 mM DMPO, whereas it became ineffective in 50 mM DMPO. Furthermore, DMPO-OH did not interact with tempol. These results suggest that DMPO and tempol react competitively with O. Further studies are warranted to elucidate the interaction between NTP-induced ROS and biomolecules.
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http://dx.doi.org/10.1016/j.abb.2021.108901DOI Listing
July 2021

Lysosomal nitric oxide determines transition from autophagy to ferroptosis after exposure to plasma-activated Ringer's lactate.

Redox Biol 2021 Jul 23;43:101989. Epub 2021 Apr 23.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan; Center for Low Temperature Plasma Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8603, Japan; Sydney Medical School, The University of Sydney, Sydney, NSW, 2006, Australia. Electronic address:

Non-thermal plasma (NTP), an engineered technology to generate reactive species, induces ferroptosis and/or apoptosis specifically in various-type cancer cells. NTP-activated Ringer's lactate (PAL) is another modality for cancer therapy at preclinical stage. Here we found that PAL induces selective ferroptosis of malignant mesothelioma (MM) cells, where non-targeted metabolome screening identified upregulated citrulline-nitric oxide (NO) cycle as a PAL target. NO probe detected biphasic peaks transiently at PAL exposure with time-dependent increase, which was responsible for inducible NO synthase (iNOS) overexpression through NF-κB activation. NO and lipid peroxidation occupied lysosomes as a major compartment with increased TFEB expression. Not only ferrostatin-1 but inhibitors for NO and/or iNOS could suppress this ferroptosis. PAL-induced ferroptosis accompanied autophagic process in the early phase, as demonstrated by an increase in essential amino acids, LC3B-II, p62 and LAMP1, transforming into the later phase with boosted lipid peroxidation. Therefore, NO-mediated lysosomal impairment is central in PAL-induced ferroptosis.
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http://dx.doi.org/10.1016/j.redox.2021.101989DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8105670PMC
July 2021

Plasma-Treated Solutions (PTS) in Cancer Therapy.

Cancers (Basel) 2021 Apr 6;13(7). Epub 2021 Apr 6.

Plasma Engineering and Medicine Institute, Old Dominion University, Norfolk, VA 23508, USA.

Cold physical plasma is a partially ionized gas generating various reactive oxygen and nitrogen species (ROS/RNS) simultaneously. ROS/RNS have therapeutic effects when applied to cells and tissues either directly from the plasma or via exposure to solutions that have been treated beforehand using plasma processes. This review addresses the challenges and opportunities of plasma-treated solutions (PTSs) for cancer treatment. These PTSs include plasma-treated cell culture media in experimental research as well as clinically approved solutions such as saline and Ringer's lactate, which, in principle, already qualify for testing in therapeutic settings. Several types of cancers were found to succumb to the toxic action of PTSs, suggesting a broad mechanism of action based on the tumor-toxic activity of ROS/RNS stored in these solutions. Moreover, it is indicated that the PTS has immuno-stimulatory properties. Two different routes of application are currently envisaged in the clinical setting. One is direct injection into the bulk tumor, and the other is lavage in patients suffering from peritoneal carcinomatosis adjuvant to standard chemotherapy. While many promising results have been achieved so far, several obstacles, such as the standardized generation of large volumes of sterile PTS, remain to be addressed.
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http://dx.doi.org/10.3390/cancers13071737DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8038720PMC
April 2021

Differential data on the responsiveness of multiple cell types to cell death induced by non-thermal atmospheric pressure plasma-activated solutions.

Data Brief 2021 Jun 26;36:106995. Epub 2021 Mar 26.

Institute of Innovation for Future Society, Nagoya University, Japan.

A discovery that cells die of a novel and distinctive process, along with some characteristic events, such as cellular shrinkage and Programmed cell death 4 disappearance, has been done by using non-thermal atmospheric pressure plasma-activated solutions [1]. Data on the responsiveness of multiple cell types to the induction of cellular shrinkage and cell death and the loss of Programmed cell death 4 by exposure to the non-thermal atmospheric pressure plasma-activated solutions were collected. Human neuroblastoma SH-SY5Y cells, murine myoblast C2C12 cells, and murine embryonic fibroblasts were cultured for various periods in each of the non-thermal atmospheric pressure plasma-activated solutions and then examined by light field microscopic observation for their effects on cell morphology, by Trypan blue dye exclusion assay for those on cell death, and by Western blotting for those on Programmed cell death 4 disappearance. The data clarified some differences in the responsiveness to the induction of cellular shrinkage, cell death, and Pdcd4 disappearance by all the non-thermal atmospheric pressure plasma-activated solutions among the cells.
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http://dx.doi.org/10.1016/j.dib.2021.106995DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8056420PMC
June 2021

Preclinical Verification of the Efficacy and Safety of Aqueous Plasma for Ovarian Cancer Therapy.

Cancers (Basel) 2021 Mar 7;13(5). Epub 2021 Mar 7.

Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, Tsurumai-cho 65, Showa-ku, Nagoya 466-8550, Japan.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. The major cause of EOC's lethality is that intraperitoneal recurrence occurs with high frequency due to occult metastasis. We had demonstrated that plasma-activated medium (PAM) exerts a metastasis-inhibitory effect on ovarian cancer in vitro and in vivo. Here we investigated how PAM inhibits intraperitoneal metastasis. We studied PAM's inhibition of micro-dissemination onto the omentum by performing in vivo imaging in combination with a sequential histological analysis. The results revealed that PAM induced macrophage infiltration into the disseminated lesion. The iNOS-positive signal was co-localized at the macrophages in the existing lesion, indicating that PAM might induce M1-type macrophages. This may be another mechanism of the antitumor effect through a PAM-evoked immune response. Intraperitoneal lavage with plasma-activated lactate Ringer's solution (PAL) significantly improved the overall survival rate in an ovarian cancer mouse model. Our results demonstrated the efficiency and practicality of aqueous plasma for clinical applications.
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http://dx.doi.org/10.3390/cancers13051141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7962102PMC
March 2021

A novel and distinctive mode of cell death revealed by using non-thermal atmospheric pressure plasma: The involvements of reactive oxygen species and the translation inhibitor Pdcd4.

Chem Biol Interact 2021 Apr 11;338:109403. Epub 2021 Feb 11.

Institute of Innovation for Future Society, Nagoya University, Japan.

Cells death is indispensable for embryonic development, tissue homeostasis, and the elimination of cancer, virally infected, or degenerated cells in multicellular organisms. It occurs not only via existing modes but also via unidentified modes, whose elucidation requires. Exposure to non-thermal atmospheric pressure plasma (NTAPP) has been demonstrated to induce cell death, probably because of its ability to generate reactive oxygen species (ROS). However, the mode of this cell death and its underlying mechanism remained elusive. Here we show cell death occurring in a novel and distinctive mode different from apoptosis and necrosis/necroptosis through a mechanism that ROS mediate the loss of the translation inhibitor Programmed cell death 4 (Pdcd4) when cells are cultured in solutions activated by NTAPP irradiation. Thus, our study performed with NTAPP-activated solutions may provide insight into the existence of the atypical cell death in cells and some features of its distinguishing mode and underlying mechanism.
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http://dx.doi.org/10.1016/j.cbi.2021.109403DOI Listing
April 2021

Effects of Carbon Nanowalls (CNWs) Substrates on Soft Ionization of Low-Molecular-Weight Organic Compoundsin Surface-Assisted Laser Desorption/Ionization Mass Spectrometry (SALDI-MS).

Nanomaterials (Basel) 2021 Jan 20;11(2). Epub 2021 Jan 20.

Center for Low-temperature Plasma Sciences, Nagoya University, Furo, Chikusa, Nagoya 464-8603, Japan.

Carbon nanowalls (CNWs), which are vertically oriented multi-layer graphene sheets, were employed in surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) measurements to detect low-molecular-weight organic compounds. CNWs substrates with widely different wall-to-wall distances from 142 to 467 nm were synthesized using a radical-injection plasma-enhanced chemical vapor deposition (RI-PECVD) system with nanosecond pulse biasing to a sample stage. When survival yield (SY) values of -benzylpyridinium chloride (N-BP-Cl) were examined, which is commonly used to evaluate desorption/ionization efficiency, a narrower wall-to-wall distance presented a higher SY value. The highest SY value of 0.97 was realized at 4 mJ/cm for the highest-density CNWs with a wall-to-wall distance of 142 nm. The laser desorption/ionization effect of arginine, an amino acid, was also investigated. When CNWs with a narrower wall-to-wall distance were used, the signal-to-noise (SN) ratios of the arginine signals were increased, while the intensity ratios of fragment ions to arginine signals were suppressed. Therefore, the CNWs nanostructures are a powerful tool when used as a SALDI substrate for the highly efficient desorption/ionization of low-molecular-weight biomolecules.
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http://dx.doi.org/10.3390/nano11020262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7909522PMC
January 2021

L-Dehydroascorbate efficiently degrades non-thermal plasma-induced hydrogen peroxide.

Arch Biochem Biophys 2021 03 20;700:108762. Epub 2021 Jan 20.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, Showa-Ku, Nagoya, 466-8550, Japan; Center for Low-temperature Plasma Sciences, Nagoya University, Chikusa-ku, Nagoya, 464-8603, Japan. Electronic address:

Non-thermal plasma (NTP) devices generate reactive oxygen species (ROS) and reactive nitrogen species, such as singlet oxygen (O), superoxide (O), hydroxyl radical (OH), hydrogen peroxide (HO), ozone, and nitric oxide at near-physiological temperature. In preclinical studies, NTP promotes blood coagulation, wound healing with disinfection, and selective killing of cancer cells. Although these biological effects of NTP have been widely explored, the stoichiometric quantitation of ROS in the liquid phase has not been performed in the presence of biocompatible reducing agents, which may modify the final biological effects of NTP. Here, we utilized electron paramagnetic resonance spectroscopy to quantitate OH, using a spin-trapping probe 5,5-dimethyl-1-pyrroline-N-oxide; O, using a fluorescent probe; and O and HO, using luminescent probes, after NTP exposure in the presence of antioxidants. l-ascorbate (Asc) at 50 μM concentration (physiological concentration in serum) significantly scavenged OH, whereas (-)-epigallocatechin gallate (EGCG) and α-tocopherol were also effective at performing scavenging activities at 250 μM concentrations. Asc significantly scavenged O and HO at 100 μM. l-Dehydroascorbate (DHA), an oxidized form of Asc, degraded HO, whereas it did not quench OH or O, which are sources of HO. Furthermore, EGCG efficiently scavenged NTP-induced O, O, and HO in Chelex-treated water. These results indicate that the redox cycling of Asc/DHA and metabolites of DHA are important to be considered when applying NTP to cells and tissues. Additionally, ROS-reducing compounds, such as EGCG, affect the outcome. Further studies are warranted to elucidate the interaction between ROS and biomolecules to promote the medical applications of NTP.
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http://dx.doi.org/10.1016/j.abb.2021.108762DOI Listing
March 2021

Roles of Atomic Nitrogen/Hydrogen in GaN Film Growth by Chemically Assisted Sputtering with Dual Plasma Sources.

ACS Omega 2020 Oct 8;5(41):26776-26785. Epub 2020 Oct 8.

Center for Low-Temperature Plasma Sciences, Nagoya University, Furo-cho, Chikusa, Nagoya, Aichi 464-8601, Japan.

The growth of sputtered GaN at low temperature is strongly desired to realize the dissemination of low-cost GaN high electron mobility transistor devices for next-generation communication technology. In this work, the roles of atomic nitrogen (N)/hydrogen (H) in GaN film growth on AlN/sapphire substrates by chemically assisted dual source sputtering are studied at a low growth temperature of 600 °C under a pressure of 2 Pa using vacuum ultraviolet absorption spectroscopy. The lateral growth was strongly enhanced with an appropriate H/N flux ratio of 1.9 at a GaN growth rate of ∼1 μm h. X-ray photoelectron spectroscopy measurements indicated that N removal from the grown GaN surface by atomic hydrogen promoted the migration of Ga. A smooth GaN surface was achieved at a suitable N/Ga supply ratio of 53 and a H/N ratio of 1.9 with the addition of 0.5% chlorine to the Ar sputtering gas.
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http://dx.doi.org/10.1021/acsomega.0c03865DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7581250PMC
October 2020

Formation of spherical Sn particles by reducing SnO film in floating wire-assisted H/Ar plasma at atmospheric pressure.

Sci Rep 2020 Oct 20;10(1):17770. Epub 2020 Oct 20.

Nagoya University, Nagoya, 464-8601, Japan.

A green method to synthesize spherical Sn particles by reducing SnO film in atmospheric-pressure H/Ar plasma at low temperatures for various applications is presented. The floating wire-assisted remotely-generated plasma with a mixture of 0.05% H/Ar gas formed spherical metallic Sn particles by reducing a SnO layer on glass substrate. During the reduction process, H radical density was measured by using vacuum ultraviolet absorption spectroscopy, and plasma properties including electron density and gas temperature were diagnosed by optical emission spectroscopy. The inductively coupled generated plasma with a high electron density of 10 cm, a hydrogen atom density of 10 cm, and a gas temperature of 940 K was obtained at a remote region distance of 150 mm where the SnO/glass substrate was placed for plasma treatment. The process has been modeled on the spherical Sn formation based on the reduction of SnO films using H radicals. Depending on the treatment condition, the total reduction area, where spherical Sn particles formed, was enlarged and could reach 300 mm after 2 min. The substrate temperature affected the expansion rate of the total reduction area and the growth of the Sn spheres.
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http://dx.doi.org/10.1038/s41598-020-74663-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7576790PMC
October 2020

Small size gold nanoparticles enhance apoptosis-induced by cold atmospheric plasma via depletion of intracellular GSH and modification of oxidative stress.

Cell Death Discov 2020 10;6:83. Epub 2020 Sep 10.

Department of Radiology, Graduate School of Medicine and Pharmaceutical Sciences University of Toyama, Toyama, Japan.

Gold nanoparticles (Au-NPs) have attracted attention as a promising sensitizer owing to their high atomic number (Z), and because they are considered fully multifunctional, they are preferred over other metal nanoparticles. Cold atmospheric plasma (CAP) has also recently gained attention, especially for cancer treatment, by inducing apoptosis through the formation of reactive oxygen species (ROS). In this study, the activity of different sized Au-NPs with helium-based CAP (He-CAP) was analyzed, and the underlying mechanism was investigated. Treating cells with only small Au-NPs (2 nm) significantly enhanced He-CAP-induced apoptosis. In comparison, 40 nm and 100 nm Au-NPs failed to enhance cell death. Mechanistically, the synergistic enhancement was due to 2 nm Au-NPs-induced decrease in intracellular glutathione, which led to the generation of intracellular ROS. He-CAP markedly induced ROS generation in an aqueous medium; however, treatment with He-CAP alone did not induce intracellular ROS formation. In contrast, the combined treatment significantly enhanced the intracellular formation of superoxide (O) and hydroxyl radical (OH). These findings indicate the potential therapeutic use of Au-NPs in combination with CAP and further clarify the role of Au-NPs in He-CAP-aided therapies.
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http://dx.doi.org/10.1038/s41420-020-00314-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7483448PMC
September 2020

Non-thermal plasma-activated lactate solution kills U251SP glioblastoma cells in an innate reductive manner with altered metabolism.

Arch Biochem Biophys 2020 07 25;688:108414. Epub 2020 May 25.

Center for Low-temperature Plasma Sciences, Nagoya University, Furo-cho, Chikusa, Nagoya, 484-8601, Japan.

Ringer's lactate solution irradiated by non-thermal plasma, comprised of radicals, electrons, and ions, is defined as plasma-activated lactate (PAL). PAL exhibited antitumor effects in glioblastoma U251SP cells, which we termed PAL-specific regulated cell death. In contrast to the oxidative stress condition typical of cells incubated in plasma-activated medium (PAM), U251SP cells treated with Ringer's lactate solution or PAL exhibited changes in intracellular metabolites that were reductive in the redox state, as measured by the ratio of oxidative/reductive glutathione concentrations. In the metabolomic profiles of PAL-treated cells, the generation of acetyl-CoA increased for lipid metabolism from alanine and asparagine. PAL thus induces regulated death of U251SP glioblastoma cells in more innate microenvironments than PAM.
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http://dx.doi.org/10.1016/j.abb.2020.108414DOI Listing
July 2020

Growth of single crystalline films on lattice-mismatched substrates through 3D to 2D mode transition.

Sci Rep 2020 Mar 13;10(1):4669. Epub 2020 Mar 13.

Graduate School of Information Science and Electrical Engineering, Kyushu University, Fukuoka, 819-0395, Japan.

Regarding crystalline film growth on large lattice-mismatched substrates, there are two primary modes by which thin films grow on a crystal surface or interface. They are Volmer-Weber (VW: island formation) mode and Stranski-Krastanov (SK: layer-plus-island) mode. Since both growth modes end up in the formation of three-dimensional (3D) islands, fabrication of single crystalline films on lattice-mismatched substrates has been challenging. Here, we demonstrate another growth mode, where a buffer layer consisting of 3D islands initially forms and a relaxed two-dimensional (2D) layer subsequently grows on the buffer layer. This 3D-2D mode transition has been realized using impurities. We observed the 3D-2D mode transition for the case of ZnO film growth on 18%-lattice-mismatched sapphire substrates. First, nano-sized 3D islands grow with the help of nitrogen impurities. Then, the islands coalesce to form a 2D layer after cessation of the nitrogen supply, whereupon an increase in the surface energy may provide a driving force for the coalescence. Finally, the films grow in 2D mode, forming atomically flat terraces. We believe that our findings will offer new opportunities for high-quality film growth of a wide variety of materials that have no lattice-matched substrates.
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http://dx.doi.org/10.1038/s41598-020-61596-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7070095PMC
March 2020

Oxygen radical based on non-thermal atmospheric pressure plasma alleviates lignin-derived phenolic toxicity in yeast.

Biotechnol Biofuels 2020 28;13:18. Epub 2020 Jan 28.

1Faculty of Agriculture, Meijo University, Nagoya, Aichi 468-8502 Japan.

Background: Vanillin is the main byproduct of alkaline-pretreated lignocellulosic biomass during the process of fermentable-sugar production and a potent inhibitor of ethanol production by yeast. Yeast cells are usually exposed to vanillin during the industrial production of bioethanol from lignocellulosic biomass. Therefore, vanillin toxicity represents a major barrier to reducing the cost of bioethanol production.

Results: In this study, we analysed the effects of oxygen-radical treatment on vanillin molecules. Our results showed that vanillin was converted to vanillic acid, protocatechuic aldehyde, protocatechuic acid, methoxyhydroquinone, 3,4-dihydroxy-5-methoxybenzaldehyde, trihydroxy-5-methoxybenzene, and their respective ring-cleaved products, which displayed decreased toxicity relative to vanillin and resulted in reduced vanillin-specific toxicity to yeast during ethanol fermentation. Additionally, after a 16-h incubation, the ethanol concentration in oxygen-radical-treated vanillin solution was 7.0-fold greater than that from non-treated solution, with similar results observed using alkaline-pretreated rice straw slurry with oxygen-radical treatment.

Conclusions: This study analysed the effects of oxygen-radical treatment on vanillin molecules in the alkaline-pretreated rice straw slurry, thereby finding that this treatment converted vanillin to its derivatives, resulting in reduced vanillin toxicity to yeast during ethanol fermentation. These findings suggest that a combination of chemical and oxygen-radical treatment improved ethanol production using yeast cells, and that oxygen-radical treatment of plant biomass offers great promise for further improvements in bioethanol-production processes.
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http://dx.doi.org/10.1186/s13068-020-1655-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6988259PMC
January 2020

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

Sci Rep 2020 01 31;10(1):1614. Epub 2020 Jan 31.

Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, Nagoya, Japan.

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.
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http://dx.doi.org/10.1038/s41598-020-58667-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994502PMC
January 2020

Oxidative stress-dependent and -independent death of glioblastoma cells induced by non-thermal plasma-exposed solutions.

Sci Rep 2019 09 20;9(1):13657. Epub 2019 Sep 20.

Center for Low-temperature Plasma Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8603, Japan.

Non-thermal atmospheric pressure plasma has been widely used for preclinical studies in areas such as wound healing, blood coagulation, and cancer therapy. We previously developed plasma-activated medium (PAM) and plasma-activated Ringer's lactate solutions (PAL) for cancer treatments. Many in vitro and in vivo experiments demonstrated that both PAM and PAL exhibit anti-tumor effects in several types of cancer cells such as ovarian, gastric, and pancreatic cancer cells as well as glioblastoma cells. However, interestingly, PAM induces more intracellular reactive oxygen species in glioblastoma cells than PAL. To investigate the differences in intracellular molecular mechanisms of the effects of PAM and PAL in glioblastoma cells, we measured gene expression levels of antioxidant genes such as CAT, SOD2, and GPX1. Microarray and quantitative real-time PCR analyses revealed that PAM elevated stress-inducible genes that induce apoptosis such as GADD45α signaling molecules. PAL suppressed genes downstream of the survival and proliferation signaling network such as YAP/TEAD signaling molecules. These data reveal that PAM and PAL induce apoptosis in glioblastoma cells by different intracellular molecular mechanisms.
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http://dx.doi.org/10.1038/s41598-019-50136-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6754505PMC
September 2019

Monitoring of Surface Reactions during Atomic Layer Etching of Silicon Nitride Using Hydrogen Plasma and Fluorine Radicals.

ACS Appl Mater Interfaces 2019 Oct 25;11(40):37263-37269. Epub 2019 Sep 25.

ASM Japan K.K. , 23-1, 6-chome Nagayama, Tama, Tokyo 206-0025 , Japan.

The atomic layer etching (ALE) of silicon nitride (SiN) via a hydrogen plasma followed by exposure to fluorine radicals was investigated by using spectroscopic ellipsometry and attenuated total reflectance Fourier transform infrared (FTIR) spectroscopy to examine the surface reactions and etching mechanism. FTIR spectra of the surface following exposure to the hydrogen plasma showed an increase in the concentration of Si-H and N-H bonds, although the N-H bond concentration plateaued more quickly. In contrast, during fluorine radical exposure, the Si-H bond concentration decreased more rapidly. Secondary ion mass spectrometry demonstrated that the nitrogen atom concentration was decreased to a depth of 4 nm from the surface after the hydrogen plasma treatment and indicated a structure consisting of N-H rich, Si-H rich, and mixed layers. This indicated that Si-H bonds were primarily present near the surface, while N-H bonds were mainly located deeper into the film. The formations of the N-H and Si-H rich layers are important phenomena associated with modification by hydrogen plasma and fluorine radical etching, respectively.
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http://dx.doi.org/10.1021/acsami.9b11489DOI Listing
October 2019

Non-thermal plasma specifically kills oral squamous cell carcinoma cells in a catalytic Fe(II)-dependent manner.

J Clin Biochem Nutr 2019 Jul 1;65(1):8-15. Epub 2019 Jun 1.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, 65 Turumai-cho, Showa-ku, Nagoya 466-8550, Japan.

Oral cancer accounts for ~2% of all cancers worldwide, and therapeutic intervention is closely associated with quality of life. Here, we evaluated the effects of non-thermal plasma on oral squamous cell carcinoma cells with special reference to catalytic Fe(II). Non-thermal plasma exerted a specific killing effect on oral squamous cell carcinoma cells in comparison to fibroblasts. Furthermore, the effect was dependent on the amounts of catalytic Fe(II), present especially in lysosomes. After non-thermal plasma application, lipid peroxidation occurred and peroxides and mitochondrial superoxide were generated. Cancer cell death by non-thermal plasma was promoted dose-dependently by prior application of ferric ammonium citrate and prevented by desferrioxamine, suggesting the association of ferroptosis. Potential involvement of apoptosis was also observed with positive terminal deoxynucleaotidyl transferase-mediated dUTP nick end labeling and annexin V results. Non-thermal plasma exposure significantly suppressed the migratory, invasive and colony-forming abilities of squamous cell carcinoma cells. The oral cavity is easily observable; therefore, non-thermal plasma can be directly applied to the oral cavity to kill oral squamous cell carcinoma without damaging fibroblasts. In conclusion, non-thermal plasma treatment is a potential therapeutic option for oral cancer.
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http://dx.doi.org/10.3164/jcbn.18-91DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667380PMC
July 2019

l-Dehydroascorbic acid recycled by thiols efficiently scavenges non-thermal plasma-induced hydroxyl radicals.

Arch Biochem Biophys 2019 07 31;669:87-95. Epub 2019 May 31.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, Showa-Ku, Nagoya, 466-8550, Japan. Electronic address:

Recent development in electronics has enabled the use of non-thermal plasma (NTP) to strictly direct oxidative stress in a defined location at near-physiological temperature. In preclinical studies or human clinical trials, NTP promotes blood coagulation, wound healing with disinfection, and selective killing of cancer cells. Although these biological effects of NTP have been widely explored, the stoichiometric quantitation of free radicals in liquid phase has not been performed in the presence of biocompatible reducing agents, which may modify the final biological effects of NTP. Here we quantitated hydroxyl radicals, a major reactive oxygen species generated after NTP exposure, by electron paramagnetic resonance (EPR) spectroscopy using two distinct spin-trapping probes, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M4PO), in the presence of thiols or antioxidants. l-Ascorbic acid (AsA) at 25-50 μM concentrations (physiological concentration in the serum) significantly scavenged these hydroxyl radicals, whereas dithiothreitol (DTT), reduced glutathione (GSH), and N-acetyl-cysteine (NAC) as thiols were required in millimolar concentrations to perform scavenging activities. l-Dehydroascorbic acid (DHA), an oxidized form of AsA, necessitated the presence of 25-50 μM DTT or sub-millimolar concentrations of GSH and NAC for the scavenging of hydroxyl radicals and failed to scavenge hydroxyl radicals by itself. These results suggest that the redox cycling of AsA/DHA via thiols and cellular AsA metabolism are important processes to be considered while applying NTP to cells and tissues. Further studies are warranted to elucidate the interaction between other reactive species generated by NTP and biomolecules to promote biological and medical applications of NTP.
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http://dx.doi.org/10.1016/j.abb.2019.05.019DOI Listing
July 2019

Oriented Carbon Nanostructures by Plasma Processing: Recent Advances and Future Challenges.

Micromachines (Basel) 2018 Nov 1;9(11). Epub 2018 Nov 1.

Jožef Stefan Institute, Jamova cesta 39, SI-1000 Ljubljana, Slovenia.

Carbon, one of the most abundant materials, is very attractive for many applications because it exists in a variety of forms based on dimensions, such as zero-dimensional (0D), one-dimensional (1D), two-dimensional (2D), and-three dimensional (3D). Carbon nanowall (CNW) is a vertically-oriented 2D form of a graphene-like structure with open boundaries, sharp edges, nonstacking morphology, large interlayer spacing, and a huge surface area. Plasma-enhanced chemical vapor deposition (PECVD) is widely used for the large-scale synthesis and functionalization of carbon nanowalls (CNWs) with different types of plasma activation. Plasma-enhanced techniques open up possibilities to improve the structure and morphology of CNWs by controlling the plasma discharge parameters. Plasma-assisted surface treatment on CNWs improves their stability against structural degradation and surface chemistry with enhanced electrical and chemical properties. These advantages broaden the applications of CNWs in electrochemical energy storage devices, catalysis, and electronic devices and sensing devices to extremely thin black body coatings. However, the controlled growth of CNWs for specific applications remains a challenge. In these aspects, this review discusses the growth of CNWs using different plasma activation, the influence of various plasma-discharge parameters, and plasma-assisted surface treatment techniques for tailoring the properties of CNWs. The challenges and possibilities of CNW-related research are also discussed.
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http://dx.doi.org/10.3390/mi9110565DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6265782PMC
November 2018

Non-thermal plasma-activated medium modified metabolomic profiles in the glycolysis of U251SP glioblastoma.

Arch Biochem Biophys 2019 02 5;662:83-92. Epub 2018 Dec 5.

Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa, Nagoya, 484-8603, Japan; Plasma Medical Science Global Innovation Center, Nagoya University, Furo-cho, Chikusa, Nagoya, 484-8601, Japan; Institute of Innovation for Future Society, Nagoya University, Furo-cho, Chikusa, Nagoya, 484-8601, Japan.

Non-equilibrium atmospheric pressure plasma (NEAPP) is a mixture of radicals, electrons, anions, cations and light at near body temperature. Plasma-activated medium (PAM) is realized using NEAPP provided by engineered devices and irradiated to a cell culture medium for a period of 600 s. Glioblastoma cells U251SP cultivated in PAM previously indicated that antitumor effects induced PAM-specific apoptotic cell-death. Metabolomic profiles of a hundred intracellular metabolites were analyzed using capillary electrophoresis mass spectrometry. The metabolomic profiles of the PAM-treated U251SP cells were changed significantly with inhibition of the glycolysis pathway and with enhancement of the pentose phosphate pathway.
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http://dx.doi.org/10.1016/j.abb.2018.12.001DOI Listing
February 2019

A 65-nm CMOS Fully Integrated Analysis Platform Using an On-Chip Vector Network Analyzer and a Transmission-Line-Based Detection Window for Analyzing Circulating Tumor Cell and Exosome.

IEEE Trans Biomed Circuits Syst 2019 04 20;13(2):470-479. Epub 2018 Nov 20.

A fully integrated CMOS circuit based on a vector network analyzer and a transmission-line-based detection window for circulating tumor cell (CTC) and exosome analysis is presented for the first time. We have introduced a fully integrated architecture, which eliminates the undesired parasitic components and enables high-sensitivity, to analyze extremely low-concentration CTC in blood. The detection window was designed on the high-sensitive coplanar waveguide line. To validate the operation of the proposed system, a test chip was fabricated using 65-nm CMOS technology. Measurements were performed after adding a tiny lump of silicone or a droplet of water on its detection window. The measured results show |S_21| degradation of -1.96 dB and -6.04 dB for the silicone and the droplet, respectively, at 1.4 GHz. In addition, in another measurement using magnetic beads, it is confirmed that the proposed circuit can analyze even low concentrations of 20 beads/μL. As well as microbeads, measurement with CTCs was successfully demonstrated.
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http://dx.doi.org/10.1109/TBCAS.2018.2882472DOI Listing
April 2019

Molecular mechanisms of non-thermal plasma-induced effects in cancer cells.

Biol Chem 2018 12;400(1):87-91

Institute of Innovation for Future Society, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan.

Plasma is the fourth state of matter with higher energy than gas; non-thermal plasma (NTP) is currently available. As NTP is useful in sterilization, promoting wound healing and cancer treatments, the molecular mechanisms of plasma-induced effects in living cells and microorganisms are of significant interest in plasma medicine with medical-engineering collaboration. Molecular mechanisms of plasma-induced effects in cancer cells will be described in this minireview. Both direct and indirect methods to treat cancer cells with NTP have been developed. NTP interacts directly with not only cancer cells but also the liquids surrounding cancer cells and the immune cells that target them. Reactive oxygen and nitrogen species play key roles in NTP-induced effects; however, other mechanisms have been suggested. The complex interactions between NTP, cells and liquids have been extensively studied. In the future, details regarding NTP-induced effects on gene regulatory networks, signaling networks, and metabolic networks will be elucidated.
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http://dx.doi.org/10.1515/hsz-2018-0199DOI Listing
December 2018

Cell Deposition Microchip with Micropipette Control over Liquid Interface Motion.

Cell Med 2018 29;10:2155179017733152. Epub 2018 May 29.

Institute of Innovation for Future Society, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Japan.

Positioning single cells on a solid surface is a crucial technique for understanding the cellular functions and cell-cell interactions in cell culture assays. We developed a microfluidic chip for depositing single cells in microwells using a simple micropipette operation. Cells were delivered to microwells by the meniscus motion of liquid interface. The residue deposits of cells were redistributed with air injection, and the isolated single cells were stored in microwells. Different microwell sizes and depths were studied to evaluate the trapping possibility of cells. Medium replacement and cell viability staining with the isolated single cells were achieved in microwells. The chip will serve as a tool for single-cell patterning in an easy-to-use manner.
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http://dx.doi.org/10.1177/2155179017733152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6172985PMC
May 2018

Plasma-activated medium (PAM) kills human cancer-initiating cells.

Pathol Int 2018 Jan 13;68(1):23-30. Epub 2017 Dec 13.

Institute of Innovation for Future Society, Nagoya University, Furo-cho, Chikusa-ku Nagoya 464-8603, Japan.

Medical non-thermal plasma (NTP) treatments for various types of cancers have been reported. Cells with tumorigenic potential (cancer-initiating cells; CICs) are few in number in many types of tumors. CICs efficiently eliminate anti-cancer chemicals and exhibit high-level aldehyde dehydrogenase (ALDH) activity. We previously examined the effects of direct irradiation via NTP on cancer cells; even though we targeted CICs expressing high levels of ALDH, such treatment affected both non-CICs and CICs. Recent studies have shown that plasma-activated medium (PAM) (culture medium irradiated by NTP) selectively induces apoptotic death of cancer but not normal cells. Therefore, we explored the anti-cancer effects of PAM on CICs among endometrioid carcinoma and gastric cancer cells. PAM reduced the viability of cells expressing both low and high levels of ALDH. Combined PAM/cisplatin appeared to kill cancer cells more efficiently than did PAM or cisplatin alone. In a mouse tumor xenograft model, PAM exerted an anti-cancer effect on CICs. Thus, our results suggest that PAM effectively kills both non-CICs and CICs, as does NTP. Therefore, PAM may be a useful new anti-cancer therapy, targeting various cancer cells including CICs.
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http://dx.doi.org/10.1111/pin.12617DOI Listing
January 2018

Oxygen-radical pretreatment promotes cellulose degradation by cellulolytic enzymes.

Biotechnol Biofuels 2017 4;10:290. Epub 2017 Dec 4.

Faculty of Agriculture, Meijo University, Nagoya, Aichi 468-8502 Japan.

Background: The efficiency of cellulolytic enzymes is important in industrial biorefinery processes, including biofuel production. Chemical methods, such as alkali pretreatment, have been extensively studied and demonstrated as effective for breaking recalcitrant lignocellulose structures. However, these methods have a detrimental effect on the environment. In addition, utilization of these chemicals requires alkali- or acid-resistant equipment and a neutralization step.

Results: Here, a radical generator based on non-thermal atmospheric pressure plasma technology was developed and tested to determine whether oxygen-radical pretreatment enhances cellulolytic activity. Our results showed that the viscosity of carboxymethyl cellulose (CMC) solutions was reduced in a time-dependent manner by oxygen-radical pretreatment using the radical generator. Compared with non-pretreated CMC, oxygen-radical pretreatment of CMC significantly increased the production of reducing sugars in culture supernatant containing various cellulases from . The production of reducing sugar from oxygen-radical-pretreated CMC by commercially available cellobiohydrolases I and II was 1.7- and 1.6-fold higher, respectively, than those from non-pretreated and oxygen-gas-pretreated CMC. Moreover, the amount of reducing sugar from oxygen-radical-pretreated wheat straw was 1.8-fold larger than those from non-pretreated and oxygen-gas-pretreated wheat straw.

Conclusions: Oxygen-radical pretreatment of CMC and wheat straw enhanced the degradation of cellulose by reducing- and non-reducing-end cellulases in the supernatant of a culture of the white-rot fungus . These findings indicated that oxygen-radical pretreatment of plant biomass offers great promise for improvements in lignocellulose-deconstruction processes.
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http://dx.doi.org/10.1186/s13068-017-0979-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5713004PMC
December 2017

Effect of Plasma-Activated Lactated Ringer's Solution on Pancreatic Cancer Cells In Vitro and In Vivo.

Ann Surg Oncol 2018 Jan 14;25(1):299-307. Epub 2017 Nov 14.

Department of Gastroenterological Surgery (Surgery II), Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan.

Background: The medical applications of nonequilibrium atmospheric pressure plasma in cancer therapy have attracted attention. We previously reported on the antitumor effect of plasma-activated medium. However, this approach requires plasma-activated liquids that are administrable to the human body. In this study, we produced plasma-activated lactated Ringer's solution (PAL) and evaluated its antitumor effect and mechanism. Furthermore, we evaluated the effect of the intraperitoneal administration of PAL using a peritoneal dissemination mouse tumor model.

Methods: The antitumor effect of PAL on pancreatic cancer cell lines was evaluated using proliferation and apoptosis assays. In addition, cellular reactive oxygen species (ROS) generation was examined. The role of ROS was assessed using a proliferation assay with N-acetyl cysteine (NAC). An adhesion assay was performed to evaluate the effect of PAL on cell adhesion. Finally, pancreatic cancer cells stably expressing luciferase (AsPC-1/CMV-Luc) were injected intraperitoneally into mice, followed by intraperitoneal injection of PAL. Peritoneal dissemination was monitored using in vivo bioluminescent imaging.

Results: The antitumor effect of PAL was shown in all cell lines in vitro. The TUNEL assay showed that PAL induced apoptosis. ROS uptake was observed in PAL-treated cells, and the antitumor effect was inhibited by NAC. Cell adhesion also was suppressed by PAL. The intraperitoneal administration of PAL suppressed the formation of peritoneal nodules in vivo.

Conclusions: Our study demonstrated the antitumor effects of PAL in vitro and in vivo. Intraperitoneal administration of PAL may be a novel therapeutic option for peritoneal metastases.
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http://dx.doi.org/10.1245/s10434-017-6239-yDOI Listing
January 2018

Cold atmospheric helium plasma causes synergistic enhancement in cell death with hyperthermia and an additive enhancement with radiation.

Sci Rep 2017 09 15;7(1):11659. Epub 2017 Sep 15.

Department of Oral and Maxillofacial Surgery, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama Sugitani 2630, Toyama, 930-0194, Japan.

Cold atmospheric plasmas (CAPs) have been proposed as a novel therapeutic method for its anti-cancer potential. However, its biological effects in combination with other physical modalities remain elusive. Therefore, this study examined the effects of cold atmospheric helium plasma (He-CAP) in combination with hyperthermia (HT) 42 °C or radiation 5 Gy. Synergistic enhancement in the cell death with HT and an additive enhancement with radiation were observed following He-CAP treatment. The synergistic effects were accompanied by increased intracellular reactive oxygen species (ROS) production. Hydrogen peroxide (HO) and superoxide (O) generation was increased immediately after He-CAP treatment, but fails to initiate cell death process. Interestingly, at late hour's He-CAP-induced O generation subsides, however the combined treatment showed sustained increased intracellular O level, and enhanced cell death than either treatment alone. He-CAP caused marked induction of ROS in the aqueous medium, but He-CAP-induced ROS seems insufficient or not completely incorporated intra-cellularly to activate cell death machinery. The observed synergistic effects were due to the HT effects on membrane fluidity which facilitate the incorporation of He-CAP-induced ROS into the cells, thus results in the enhanced cancer cell death following combined treatment. These findings would be helpful when establishing a therapeutic strategy for CAP in combination with HT or radiation.
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http://dx.doi.org/10.1038/s41598-017-11877-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5600975PMC
September 2017

Lipid droplets exhaustion with caspases activation in HeLa cells cultured in plasma-activated medium observed by multiplex coherent anti-Stokes Raman scattering microscopy.

Biointerphases 2017 Aug 2;12(3):031006. Epub 2017 Aug 2.

Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8603, Japan.

The multiplex coherent anti-Stokes Raman scattering microscopy allowed label-free visualization of cytoplasmic lipid droplets (LDs). The LDs, which act to conserve energy storage, are usually accumulated during the normal apoptosis of HeLa cells with activation of caspase-3/7 leading to downregulation of the fatty acid catabolism pathways. During cultivating in nonthermal plasma-activated medium (PAM), while the activation of caspase-3/7 was induced, the authors found that a dynamic exhaustion of the intracellular LDs, underlying the metabolic mechanism of the PAM-induced apoptotic cell death of HeLa cells.
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http://dx.doi.org/10.1116/1.4997170DOI Listing
August 2017

Novel Intraperitoneal Treatment With Non-Thermal Plasma-Activated Medium Inhibits Metastatic Potential of Ovarian Cancer Cells.

Sci Rep 2017 07 20;7(1):6085. Epub 2017 Jul 20.

Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, Tsurumai-cho 65, Showa-ku, Nagoya, 466-8550, Japan.

Non-thermal atmospheric pressure plasma has been proposed as a new therapeutic tool for cancer treatment. Recently, plasma-activated medium (PAM) has been widely studied in various cancer types. However, there are only few reports demonstrating the anti-tumour effects of PAM in an animal model reflecting pathological conditions and the accompanying mechanism. Here we investigated the inhibitory effect of PAM on the metastasis of ovarian cancer ES2 cells in vitro and in vivo. We demonstrated that ES2 cell migration, invasion and adhesion were suppressed by PAM at a certain PAM dilution ratio, whereas cell viability remained unaffected. In an in vivo mouse model of intraperitoneal metastasis, PAM inhibited peritoneal dissemination of ES2 cells, resulting in prolonged survival. Moreover, we assessed the molecular mechanism and found that MMP-9 was decreased by PAM. On further investigation, we also found that PAM prevented the activation of the MAPK pathway by inhibiting the phosphorylation of JNK1/2 and p38 MAPK. These findings indicate that PAM inhibits the metastasis of ovarian cancer cells through reduction of MMP-9 secretion, which is critical for cancer cell motility. Our findings suggest that PAM intraperitoneal therapy may be a promising treatment option for ovarian cancer.
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http://dx.doi.org/10.1038/s41598-017-05620-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5519696PMC
July 2017