High dose of ascorbic acid induces cell death in mesothelioma cells

Biochemical and Biophysical Research Communications 394 (2010) 249–253

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High dose of ascorbic acid induces cell death in mesothelioma cells Yukitoshi Takemura a, Motohiko Satoh a, Kiyotoshi Satoh a, Hironobu Hamada b, Yoshitaka Sekido c,d, Shunichiro Kubota a,* a

Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan Department of Integrated Medicine and Informatics, Ehime University Graduate School of Medicine, Toon, Ehime 791-0295, Japan c Division of Molecular Oncology, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-ku, Nagoya, Aichi 464-8681, Japan d Department of Cancer Genetics, Nagoya University Graduate School of Medicine 65 Tsurumai, Showa-ku, Nagoya, Aichi 466-8550, Japan b

a r t i c l e

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Article history: Received 26 January 2010 Available online 19 February 2010 Keywords: Mesothelioma Ascorbic acid Reactive oxygen species Cell death

a b s t r a c t Malignant mesothelioma is an asbestos-related fatal disease with no effective cure. Recently, high dose of ascorbate in cancer treatment has been reexamined. We studied whether high dose of ascorbic acid induced cell death of four human mesothelioma cell lines. High dose of ascorbic acid induced cell death of all mesothelioma cell lines in a dose-dependent manner. We further clarified the cell killing mechanism that ascorbic acid induced reactive oxygen species and impaired mitochondrial membrane potential. In vivo experiment, intravenous administration of ascorbic acid significantly decreased the growth rate of mesothelioma tumor inoculated in mice. These data suggest that ascorbic acid may have benefits for patients with mesothelioma. Ó 2010 Elsevier Inc. All rights reserved.

Introduction Malignant mesothelioma is an aggressive tumor associated with asbestos exposure. Worldwide incidence of mesothelioma is expected to increase, particularly in Europe, Japan, and Australia [1,2]. Although many clinical trials including surgery, radiotherapy, and chemotherapy have been reported, the prognosis of patients remains poor [3].

The effect of ascorbic acid (vitamin C) as a potential chemotherapy agent in cancer treatment has a controversial history [4]. More than 30 years ago, several studies reported about high dose ascorbic acid treatment [5–7]. These studies concluded that ascorbic acid treatment brought enhanced quality and prolongation of life.

However, afterward these studies were considered to be no benefits for cancer patients [8,9]. Recently, emerging evidence indicates that ascorbic acid in cancer treatment deserves reevaluation anew [10,11]. Pharmacological ascorbic acid concentration (high dose) resulted in effective cell death in vitro and inhibition of tumor growth in vivo [10,12]. As an alternative and complementary therapy, ascorbic acid continues to be widely used in some patients by clinicians [13–16]. For superior efficacy of ascorbic acid treatment, administration methods should be considered as an important factor. Among several methods, intravenous injection produces plasma concentrations of ascorbic acid as much as 70-fold greater than those by oral dosing [11].

* Corresponding author. Fax: +81 3 5454 6869.

E-mail address: kubota@idaten.c.u-tokyo.ac.jp (S. Kubota).

0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.02.012

Although mechanism and physiologic relevance remains to be fully elucidated, ascorbic acid leads to production of H2O2 [10,17–19]. It is known that H2O2 is involved in the redox control of several physiological processes including cell proliferation and apoptosis [20,21]. For both ascorbic acid and H2O2, cell death-type changed from apoptosis to necrosis as the concentrations increased [10].

As little information is available concerning the effect of ascorbic acid on mesothelioma, we studied whether ascorbic acid induced mesothelioma cell death. We found that high dose of ascorbic acid induced cell death of four human mesothelioma cell lines.

Materials and methods Cells and reagents. Four mesothelioma cell lines, ACC-MESO 1 (ACC-MESO) [22], Y-MESO 8A (Y-MESO) [22], EHMES-10 [23,24], and EHMES-1 [23], were used. ACC-MESO and Y-MESO were cultured in DMEM (Dulbecco’s modified Eagle’s medium) (Sigma, St. Louis, MO) supplemented with 10% fetal calf serum and 1 penicillin–streptomycin antibiotics (Wako Pure Chemical Industries Ltd., Osaka, Japan). EHMES-10 and EHMES-1 were cultured in RPMI1640 (Sigma) supplemented with 10% fetal calf serum and 1 penicillin–streptomycin antibiotics. All cell lines were incubated at 37 °C in 5% CO2. Ascorbic acid and sodium hydroxide were purchased form Wako Pure Chemical Industries Ltd. Cell viability assay. Cells were seeded at a density of 2000 cells/ well in 96-well plate and treated with ascorbic acid (adjusted to pH 7.4 with sodium hydroxide) at various concentrations for 24 h. For

Y. Takemura et al. / Biochemical and Biophysical Research Communications 394 (2010) 249–253

caliper as length (width)2 0.5 (mm3). This study was approved by an Animal Committee, The University of Tokyo and all procedures involving Animal Care were in accord with Institutional Guidelines in compliance with National Laws.

Results Inhibition of mesothelioma cell growth by ascorbic acid We investigated whether ascorbic acid affected the survival of mesothelioma cell lines. Four cell lines were treated with ascorbic acid for 24 h at concentrations from 50 lM to 1000 lM. Ascorbic acid reduced all cell viability in a dose-dependent manner (Fig. 1A). EC50 values of each cell, the concentrations of ascorbic acid that decreased survival by 50%, were as follows: 120 lM (ACCMESO), 610 lM (Y-MESO), 80 lM (EHMES-1), and 640 lM (EHMES-10). For application of clinical use, cell lines were incubated for 1 h with high dose (1, 10, and 100 mM) of ascorbic acid. Although treatment with ascorbic acid for 1 h needed higher dose to decrease the cell number than that for 24 h, ascorbic acid also induced cell death in a dose-dependent manner (Fig. 1B). ACC-MESO and EHMES-1 cells were not viable at all with 10 mM for 1 h while YMESO and EHMES-10 cells were viable with 10 mM for 1 h.

To examine whether growth inhibition was due to apoptotic cell death, we performed TUNEL assay in EHMES-10 cells. TUNEL-positive cells were increased with time and nuclear shrinkage was also observed in 24 h (Supplementary Fig. 1). Using other three mesothelioma cell lines, TUNEL-positive cells were also

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assay of EHMES-10 using anti-oxidative enzyme, cells were treated with combination of ascorbic acid and catalase (Sigma) or ascorbic acid and tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl, Sigma) for 24 h. Each well was washed with medium three times to remove ascorbic acid. For assay of EHMES-10 using cisplatin (Wako Pure Chemical Industries Ltd.), cells were treated with cisplatin at various concentrations for 24 h. Cell viability was determined using Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan). The color intensity was measured in microplate reader (Thermo Electron Corporation, Vantaa, Finland) at 450 nm.

TUNEL assay. EHMES-10 cells were seeded on LabTek chamber slide (Nalge Nunc International, Rochester, NY) and incubated for 12 h at 37 °C in 5% CO2. After treatment with 800 lM ascorbic acid (pH 7.4), cells were washed twice with PBS (phosphate–buffered saline) and fixed with 3% formaldehyde in PBS for 30 min.

The fixed cells were stained with the terminal dUTP nick-end labeling (TUNEL) method, using DeadEnd Colorimetric TUNEL System (Roche Applied Science, Mannheim, Germany), according to the manufacturer’s instruction.

Western blotting. After ascorbic acid treatment, cells were lysed in Triton X-100 lysis buffer (1% Triton X-100, 10% glycerol, 150 mM NaCl, 2 mM EDTA, 0.02% NaN3, 10 lg/ml PMSF, and 1 mM Na3VO4).

Total cell lysates were separated on SDS–PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were reacted with rabbit anti-Bax Ab (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) followed by peroxidase-conjugated anti-rabbit IgG Ab (New England Biolabs, Ipswich, MA), mouse anti-Bcl-2 mAb (Santa Cruz Biotechnology, Inc.) followed by peroxidase-conjugated antimouse IgG Ab (New England Biolabs), or goat anti-actin Ab (Santa Cruz Biotechnology, Inc.) followed by peroxidase-conjugated antigoat IgG Ab (Vector Laboratories, Inc., Burlingame, CA). Proteins were then visualized using Immobilon Western (Millipore, Billerica, MA).

Mitochondrial membrane potential and superoxide. J-aggregateforming lipophilic cation (JC-1) (Wako Pure Chemical Industries Ltd.) was used to evaluate mitochondrial potential. EHMES-10 cells were seeded on 24-well plate. After ascorbic acid treatment for 24 h, cells were washed with PBS containing 10% fetal calf serum (10% FCS-PBS) and incubated with 2 lg/ml JC-1 (final concentration) in 10% FCS-PBS for 30 min at 37 °C in 5% CO2 incubator. Mitochondrial superoxide was detected by MitoSOX (Invitrogen, Eugene, OR). Before incubation with ascorbic acid, the cells were incubated with 1 lM MitoSOX (final concentration) for 30 min at 37 °C in 5% CO2.

After washing with medium, the cells were treated with ascorbic acid for 2 h. Phase contrast and fluorescence images were obtained using fluorescence microscope (Olympus Corporation, Tokyo, Japan). In case of JC-1, the red and green fluorescence images were merged using DP manager software (Olympus Corporation).

Flow cytometry. EHMES-10 cells were incubated for 24 h in the absence and presence of different concentrations of ascorbic acid.

The cells were incubated with JC-1 (final concentration 10 lg/ml) for 30 min at 37 °C. After washing with culture medium, cells were re-suspended in 500 ll of warm culture medium and were analyzed by FACS Calibur (BD, Franklin Lakes, NJ). For superoxide detection, EHMES-10 cells were incubated for 24 h as described above. Then, cells were incubated with MitoSOX Red (final concentration 5 lg/ml) for 15 min at 37 °C. After washing with warm culture medium, cells were re-suspended in 500 ll of warm culture medium and analyzed by FACS Calibur.

Xenograft of tumor cells. EHMES-10 cells (5 106 cells) suspended in 100 ll PBS were inoculated subcutaneously into the right flank of male SCID mice (C.B-17/lcr-scid/scidJcl aged 6– 7 weeks, CLEA Japan, Tokyo, Japan). When tumor size reached 50 mm3, ascorbic acid of 50 ll at each concentration was administrated intravenously into tail vein. Ascorbic acid was neutralized to pH 7.4 with sodium hydroxide. Tumor size was calculated using

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Fig. 1. Effect of ascorbic acid on human mesothelioma cell lines. Cells were treated with ascorbic acid of indicated concentration (AA concentration) for 24 h (A), and 1 h (B). Values are expressed as absorbance at 450 nm using cell counting kit. Error bars represent the SD for six samples.

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detected (data not shown). To determine whether ascorbic acid treatment influenced Bcl-2 family members which are involved in the execution of apoptosis, the protein levels of Bcl-2 and Bax were examined by Western blot analysis (Supplementary Fig. 2).

Treatment with ascorbic acid in tested concentration for 0, 3, 6, 9, and 12 h decreased the expression of Bcl-2 and the ratio of Bcl-2/Bax expression significantly.

These data indicate that ascorbic acid induced apoptosis of four cell lines.

Production of reactive oxygen species by ascorbic acid Previous reports show that ascorbic acid generates H2O2 in the extracellular milieu [10,19]. Because H2O2 is membrane permeable, we investigated ROS (reactive oxygen species) production inside the cells. To determine whether ascorbic acid lead to ROS production, preliminarily experiment was performed using APF or HPF which can detect ROS. We detected the fluorescence intensity in ascorbic acid-treated cells (data not shown). The results suggest that ascorbic acid induced ROS production inside the cells. Therefore, we focused on mitochondria where ROS was produced by energy metabolism.

We observed mitochondrial membrane potential using JC-1 and assessed by FACS Calibur (Fig. 2). A direct observation using JC-1 revealed that ascorbic acid impaired mitochondrial membrane potential in a dose-dependent manner (Fig. 2A). The observation

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was confirmed by FACS analysis (Fig. 2B). The number of cells with conventional mitochondrial membrane potential (JC-1 red fluorescence) was decreased while that of cells with low mitochondrial membrane potential (JC-1 green fluorescence) was increased in a dose-dependent manner. Then, to examine the production of ROS especially superoxide in mitochondria, we used MitoSOX for livecell imaging and FACS analysis (Supplementary Fig. 3). Though the fluorescence by low dose of ascorbic acid was not detected using fluorescent microscope, we detected the fluorescence of high dose ascorbic acid-treated cells, but not in non-treated cells (Supplementary Fig. 3A). Concerning relatively low dose of ascorbic acid treatment, the fluorescence was also detected by FACS analysis (Supplemental Fig. 3B). The mean fluorescence intensity of MitoSOX was increased in a dose-dependent manner. Moreover, to examine the production of ROS related to the cell death, we tested the effects of H2O2-scavenger catalase and superoxide-scavenger tempol. Catalase completely prevented ascorbic acid-induced cell death (Fig. 3A).

Tempol, membrane-permeable radical scavenger [25–27], was also completely protective against ascorbic acid-inducible cell death (Fig. 3B).

Ascorbic acid treatment decreases tumor growth The efficacy of ascorbic acid administration on growth of EHMES-10 tumor was examined in SCID mice. Among the several

Fig. 2. Effect of ascorbic acid on mitochondrial membrane potential in mesothelioma cells. EHMES-10 cells were treated with ascorbic acid of indicated concentrations for 24 h. JC-1 fluorescence images (A) were obtained using fluorescence microscope. Bars indicate 200 lm. (B) Representative FACS plots of cells treated with indicated concentrations of ascorbic acid. JC-1 fluorescence was detected by flow cytometry. Percentages indicate the cells in encircled area with conventional (JC-1 red fluorescence) and low (JC-1 green fluorescence) mitochondrial membrane potential.

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Fig. 3. Catalase and tempol restore the cell death induced by ascorbic acid. EHMES10 cells incubated with ascorbic acid at 0, 700, and 900 lM together with catalase (A) or tempol (B) at indicated concentrations for 24 h. Values are expressed as absorbance at 450 nm using cell counting kit. Error bars represent the SD for six samples.

administration ways, intravenous administration is a better method to achieve higher concentration of ascorbic acid in plasma [11,12]. Therefore, single infusion of ascorbic acid was performed in vivo to investigate the efficacy of intravenous administration.

Xenograft experiment showed that tumor growth of both ascorbic acid treatment groups was significantly decreased on day 20 compared with control group while on day 34 there were no significant differences in size between any groups (Fig. 4).

Discussion

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dose-dependent manner. To our knowledge, this is the first report of ascorbic acid that can induce mesothelioma cell death.

Ascorbic acid-mediated cell death was at least due to ROS, especially hydrogen peroxide, accompanied by the disruption of mitochondria structure. Intravenously administration of ascorbic acid was effective in tumor xenograft experiment.

Concerning the mode of cell death, ascorbic acid induces apoptosis and a gradual shift toward necrosis in dose-dependent manner [10]. Ascorbic acid induced apoptosis in mesothelioma cells in the present study. In addition, we observed mesothelioma cell death with necrosis induced by high dose ascorbic acid using FACS analysis (data not shown). The data are consistent with previous report [10]. The cell death induced by ascorbic acid was restored with catalase (Supplemental Fig. 3A). This result suggested that ascorbic acid produced hydrogen peroxide. Because the catalase was added extracellularly, this enzyme could decrease hydrogen peroxide induced by ascorbic acid extracellularly. In contrast, tempol, a membrane-permeable radical scavenger, also restored ascorbic acid-induced cell death. This reagent reduced the formation of hydroxyl radical by scavenging superoxide anions [28]. It suggested that superoxide anion intracellularly resulted from ascorbic acid treatment could lead to cell death either directly or indirectly.

Therefore, high dose of ascorbic acid treatment may induce the disruption of mitochondrial membrane potential inside cells.

In the present study, single injection of ascorbic acid significantly brought inhibition of tumor growth on the 20th day while no significant difference was observed on the 34th day. The data indicate that several time injection of ascorbic acid is needed for consecutive inhibition of tumor growth. Low concentrations of hydrogen peroxide have proliferative effect on cell growth [29– 32]. Moreover, our results indicated the different sensitivity to ascorbic acid among mesothelioma cells (Fig. 1). As discussed in the mini-review [33], the concentration of ascorbic acid for cell death in the tissues should be high as much as possible. As therapies in cancer are often combined, ascorbic acid in combination with other therapies is worthy of further examinations for treatment of mesothelioma that otherwise have poor prognosis.

It is considered that the anticancer effect of chemotherapeutic agents is mediated at least in part by production of hydrogen peroxide [33]. As is increased concentration of anticancer drug, cell death is induced dose-dependently. However, elevated dose of anticancer drugs would result in severe side effects which should be attenuated for patients. When EHMES-10 cells were treated with cisplatin, a number of cells could survive even at 250 or 500 lM (Supplementary Fig. 4). Two hundred and fifty or 500 lM was very high dose for clinical use [34]. In contrast, ascorbic acid for clinical use brings less side effects to the patients [13,14] and facilitates the QOL [16].

In recent years, chemotherapy using ascorbic acid has been reexaminated. Therefore, ascorbic acid would bring therapeutic benefits for patients with mesothelioma.

In conclusion, our results indicate that high dose of ascorbic acid induced cell death in all mesothelioma cells tested. For clinical application of mesothelioma, further investigations will be necessary.

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This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology, Japan and from Nichias Corporation.

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Fig. 4. Effect of high dose of ascorbic acid (V.C) on tumor growth by intravenously injected. (A) Data show growth curves of tumor volume with PBS or 0.1 M ascorbic acid or 1 M ascorbic acid treatment in mice bearing EHMES-10 tumor. Tumor volumes with ascorbic acid treatment were compared with control (PBS) on 20th day (B) and 34th day (C) from the day of injection. P values was calculated by Unpaired t-test: *P < 0.05; **P < 0.01.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2010.02.012.

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