Elsevier

Archives of Biochemistry and Biophysics

Volume 605, 1 September 2016, Pages 109-116
Archives of Biochemistry and Biophysics

Biphasic effects of l-ascorbate on the tumoricidal activity of non-thermal plasma against malignant mesothelioma cells

https://doi.org/10.1016/j.abb.2016.05.016Get rights and content

Abstract

Non-thermal plasma (NTP) is a recently developed technology that elicits a variety of biological effects. This includes cancer cell-specific cytotoxicity, which is mainly attributed to the regional generation of reactive oxygen species (ROS). We studied the effects of NTP on malignant mesothelioma (MM) and its modulation by l-ascorbate. l-ascorbate is a major water-soluble anti-oxidant in vivo, but its pro-oxidant activity in vitro has been well recognized. Thus, the effects of ascorbate on the efficacy of NTP is important to examine. NTP exposure dose-dependently killed MM cells, whereas MM cells tolerated 1 mM l-ascorbate. However, brief pre-treatment with a pharmacological dose (250–750 μM) of l-ascorbate immediately prior to NTP exposure significantly increased its cytotoxicity in a dose-dependent manner, which was inhibited by the iron chelator, deferoxamine. However, paradoxically, this potentiating effect of l-ascorbate was completely abolished by a prolonged 4 h pre-incubation with l-ascorbate (500 μM). MM cytotoxicity induced by NTP was associated with immediate oxidative stress evaluated by 2′,7′-dichlorodihydrofluorecein diacetate, which was followed by an increase in the expression of the autophagosome marker, LC3B-II. In conclusion, MM can be a target for NTP treatment and l-ascorbate can increase or decrease its efficacy depending on the length of the pre-incubation period.

Introduction

Malignant mesothelioma (MM) is a rare, but aggressive neoplasm that is mostly induced by asbestos exposure [1], [2]. Although the International Agency for Research on Cancer (IARC) designated all asbestos types as Group 1 carcinogens in 1987, its importance as a major health concern has yet to be fully acknowledged in some countries [3], [4], [5]. Due to its long incubation period, MM is expected to increase in the near future [6], [7]. The treatments for advanced MM are limited and include: palliative surgery, chemotherapy and radiotherapy, but it has a very poor prognosis [8]. Therefore, new therapies based on innovative mechanisms of action with less side effects are desired [9].

Non-thermal plasma (NTP; non-equilibrium atmospheric pressure plasma) is a recently developed technology, which can generate reactive oxygen species (ROS) together with cations, electrons, UV and reactive nitrogen species at human body temperature at any desired location and intensity [10]. We previously identified the major biologically-active species in NTP as hydroxyl radicals and UV light, using a variety of chemical, biochemical and pathological analyses [11].

The use of NTP has already been applied to the control of air pollution [12], [13], sterilization [14], [15] and promotion of wound healing [16]. Furthermore, NTP has been proposed as a novel anti-cancer therapeutic strategy [17]. This is based on the fact that NTP induces its cytotoxic effects through generating oxidative stress [18], [19] specifically on cancer cells, including ovarian carcinoma [20], [21], head and neck carcinoma [22], colorectal cancer and glioblastoma [23]. Interestingly, a recent study examining pancreatic carcinoma cells (MIAPaCa2) revealed the merit of the combined use of NTP and the anti-metabolite, gemcitabine [24]. These latter studies indicated the potential of using a combinatorial approach of NTP with conventional chemotherapeutics. However, the effects of NTP on MM have not been reported and the precise molecular mechanisms of NTP-induced cancer-specific cytotoxicity remain to be elucidated.

Considering the implementation of agents that may be used in combination with NTP to enhance its efficacy, it is notable that l-ascorbate is a major water-soluble vitamin that is very well tolerated, and at high-doses, has shown anti-tumor activity against some cancers, including MM [25], [26]. Significantly, l-ascorbate has pro-oxidant properties under certain conditions [27], [28]. For example, ascorbate acts as a pro-oxidant when combined with Mn porphyrin and this effect mediates caspase-independent cancer cell death [29]. Such pro-oxidative effects of the vitamin may promote ROS generation by NTP and increase the efficacy of the treatment. Herein, we have investigated the combinational use of NTP and l-ascorbate on MM cells to assess its therapeutic potential and to understand the mechanism of the interaction.

Section snippets

NTP device and exposure

We used an NTP device that generates an ultra-high electron density which is in the order of 1.6 × 1016 cm−3 [30], as previously described [11]. Argon was used as the gas for generating NTP and implemented at a flow rate of 2 L/min. The distance between the plasma source and samples was fixed at L = 10 mm. Plasma was generated by applying 10 kV from a 60-Hz commercial power supply using 2 electrodes that were 20 mm apart. A quartz cover with a round window (diameter = 7 mm) was used to adjust

Cytotoxic effect of NTP exposure on MM cells

To evaluate the cytotoxic effect of NTP exposure on MM cells, we initially performed a viability assay using SM2 and EM2 cells. The results showed that NTP exposure of 30–120 s was dose-dependently cytotoxic, which was evaluated 24 h after NTP exposure (Fig. 1Ai). In contrast, using human IMR-90SV lung fibroblasts, NTP exposure at the same dose resulted in much less cytotoxicity (70–90% survival, Fig. 1Aii). These results demonstrate that NTP induces selective cytotoxicity against MM cells.

Addition of l-ascorbate promoted the cytotoxicity of NTP exposure

l

Discussion

In the present study, we showed for the first time that NTP exposure is effective and selective in terms of inducing MM cell cytotoxicity. In fact, experiments using the IMR 90SV fibroblast cell line revealed less susceptibility to NTP, suggesting a potential therapeutic window. Unfortunately, appropriate non-tumorous mesothelial cells could not be compared under the current experimental protocols, due to significant differences in culture media used and doubling times relative to MM cells.

Competing interests

The authors declare that they have no competing interests.

Acknowledgments

This work was supported, in part, by a Grant-in-aid for research from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan (24390094; 221S0001-04; 24108008), the National Cancer Center Research and Development Fund (25-A-5) and the Yasuda Medical Foundation. D.R.R. thanks the National Health and Medical Research Council of Australia for a Senior Principal Research Fellowship and Project Grants and also the Japan Society for the Promotion of Science (JSPS) for an

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    This article is part of a Special Issue entitled Low-temperature Plasma in biology and medicine, edited by Hori Masaru, Eun Ha Choi, and Shinya Toyokuni.

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