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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.

ABSTRACT: 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.

SUBMITTER: Li Y

PROVIDER: S-EPMC8197405 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Treatment of tumor cells with H2O2 and nitrite, two long-lived species derived from cold atmospheric plasma, induces a complex autoamplificatory, singlet oxygen-mediated process, which leads to catalase inactivation and reactivation of intercellular apoptosis-inducing signaling. Experimental dissection and quantification of this process is described in this study. When tumor cells were pretreated with H2O2 and nitrite, and then were added to untreated tumor cells, they propaged singlet oxygen mediated catalase inactivation and generation of singlet oxygen to the untreated cell population. This bystander effect allowed to analyze the biochemical requirements of a) induction of the bystander effect-inducing potential, b) transmission of the bystander effect to untreated neighbouring cells, and c) the biochemical consequences of these signaling events. The induction of bystander effect-inducing potential requires the generation of "primary singlet oxygen" through the reactions following the interaction between nitrite and H2O2, followed by local inactivation of a few catalase molecules. This primary effect seems to be very rare, but is efficiently enhanced by the generation of "secondary singlet oxygen" through the interaction between H2O2 and peroxynitrite at the site of inactivated catalase. Transmission of bystander signaling between pretreated and untreated tumor cells depends on the generation of secondary singlet oxygen by the pretreated cells and singlet oxygen-mediated catalase inactivation of the untreated recipient cells. This induces autoamplificatory propagation of secondary singlet oxygen generation in the population. This experimental approach allowed to quantify the efficiencies of primary and secondary singlet oxgen generation after CAP and PAM action, to dissect the system and to study the underlying chemical biology in detail. Our data confirm that CAP and PAM-derived components are merely the trigger for the activation of autoamplificatory mechanisms of tumor cells, whereas the tumor cells efficiently propagate their cell death through their own ROS/RNS signaling potential.

Project description:Nitrate reductases (NRs) are molybdoenzymes that reduce nitrate (NO3 -) to nitrite (NO2 -) in both mammals and plants. In mammals, the salival microbes take part in the generation of the NO2 - from NO3 -, which further produces nitric oxide (NO) either in acid-induced NO2 - reduction or in the presence of nitrite reductases (NiRs). Here, we report a new approach of VCl3 (V3+ ion source) induced step-wise reduction of NO3 - in a CoII-nitrato complex, [(12-TMC)CoII(NO3 -)]+ (2,{CoII-NO3 -}), to a CoIII-nitrosyl complex, [(12-TMC)CoIII(NO)]2+ (4,{CoNO}8), bearing an N-tetramethylated cyclam (TMC) ligand. The VCl3 inspired reduction of NO3 - to NO is believed to occur in two consecutive oxygen atom transfer (OAT) reactions, i.e., OAT-1 = NO3 - → NO2 - (r1) and OAT-2 = NO2 - → NO (r2). In these OAT reactions, VCl3 functions as an O-atom abstracting species, and the reaction of 2 with VCl3 produces a CoIII-nitrosyl ({CoNO}8) with VV-Oxo ({VV[double bond, length as m-dash]O}3+) species, via a proposed CoII-nitrito (3, {CoII-NO2 -}) intermediate species. Further, in a separate experiment, we explored the reaction of isolated complex 3 with VCl3, which showed the generation of 4 with VV-Oxo, validating our proposed reaction sequences of OAT reactions. We ensured and characterized 3 using VCl3 as a limiting reagent, as the second-order rate constant of OAT-2 (k 2 /) is found to be ∼1420 times faster than that of the OAT-1 (k 2) reaction. Binding constant (K b) calculations also support our proposition of NO3 - to NO transformation in two successive OAT reactions, as K b(CoII-NO2 -) is higher than K b(CoII-NO3 -), hence the reaction moves in the forward direction (OAT-1). However, K b(CoII-NO2 -) is comparable to K b{CoNO}8 , and therefore sequenced the second OAT reaction (OAT-2). Mechanistic investigations of these reactions using 15N-labeled-15NO3 - and 15NO2 - revealed that the N-atom in the {CoNO}8 is derived from NO3 - ligand. This work highlights the first-ever report of VCl3 induced step-wise NO3 - reduction (NRs activity) followed by the OAT induced NO2 - reduction and then the generation of Co-nitrosyl species {CoNO}8.

Project description:Cold Atmospheric Plasma (CAP) is a promising novel method for biofilm inactivation as log-reduction values up to 4.0 log10 (CFU/cm2) have been reported. Nevertheless, as the efficacy of CAP itself is not sufficient for complete inactivation of mature biofilms, the hurdle technology could be applied in order to obtain higher combined efficacies. In this study, CAP treatment was combined with a mild hydrogen peroxide (H2O2) treatment for disinfection of 1 and 7 day(s) old Listeria monocytogenes and Salmonella Typhimurium biofilms. Three different treatment sequences were investigated in order to determine the most effective treatment sequence, i.e., (i) first CAP, then H2O2, (ii) first H2O2, then CAP, and (iii) a simultaneous treatment of CAP and H2O2. Removal of the biofilm, induction of sub-lethal injury, and H2O2 breakdown due to the presence of catalase within the biofilms were investigated in order to comment on their possible contribution to the combined inactivation efficacy. Results indicated that the preferred treatment sequence was dependent on the biofilm forming species, biofilm age, and applied H2O2 concentration [0.05 or 0.20% (v/v)]. At the lowest H2O2 concentration, the highest log-reductions were generally observed if the CAP treatment was preceded by the H2O2 treatment, while at the highest H2O2 concentration, the opposite sequence (first CAP, then H2O2) proved to be more effective. Induction of sub-lethal injury contributed to the combined bactericidal effect, while the presence of catalase within the biofilms resulted in an increased resistance. In addition, high log-reductions were partially the result of biofilm removal. The highest overall log-reductions [i.e., up to 5.42 ± 0.33 log10 (CFU/cm2)] were obtained at the highest H2O2 concentration if CAP treatment was followed by H2O2 treatment. As this resulted in almost complete inactivation of the L. monocytogenes and S. Typhimurium biofilms, the combined treatment of CAP and H2O2 proved to be a promising method for disinfection of abiotic surfaces.

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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.

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