Transcutaneous Application of Carbon Dioxide (CO2) Induces Mitochondrial Apoptosis in Human Malignant Fibrous Histiocytoma In Vivo

Mitochondria play an essential role in cellular energy metabolism and apoptosis. Previous studies have demonstrated that decreased mitochondrial biogenesis is associated with cancer progression. In mitochondrial biogenesis, peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) regulates the activities of multiple nuclear receptors and transcription factors involved in mitochondrial proliferation. Previously, we showed that overexpression of PGC-1α leads to mitochondrial proliferation and induces apoptosis in human malignant fibrous histiocytoma (MFH) cells in vitro. We also demonstrated that transcutaneous application of carbon dioxide (CO2) to rat skeletal muscle induces PGC-1α expression and causes an increase in mitochondrial proliferation. In this study, we utilized a murine model of human MFH to determine the effect of transcutaneous CO2 exposure on PGC-1α expression, mitochondrial proliferation and cellular apoptosis. PGC-1α expression was evaluated by quantitative real-time PCR, while mitochondrial proliferation was assessed by immunofluorescence staining and the relative copy number of mitochondrial DNA (mtDNA) was assessed by real-time PCR. Immunofluorescence staining and DNA fragmentation assays were used to examine mitochondrial apoptosis. We also evaluated the expression of mitochondrial apoptosis related proteins, such as caspases, cytochorome c and Bax, by immunoblot analysis. We show that transcutaneous application of CO2 induces PGC-1α expression, and increases mitochondrial proliferation and apoptosis of tumor cells, significantly reducing tumor volume. Proteins involved in the mitochondrial apoptotic cascade, including caspase 3 and caspase 9, were elevated in CO2 treated tumors compared to control. We also observed an enrichment of cytochrome c in the cytoplasmic fraction and Bax protein in the mitochondrial fraction of CO2 treated tumors, highlighting the involvement of mitochondria in apoptosis. These data indicate that transcutaneous application of CO2 may represent a novel therapeutic tool in the treatment of human MFH.


Introduction
Musculoskeletal malignancies, particularly high-grade sarcomas such as malignant fibrous histiocytoma (MFH), are clinically aggressive and demonstrate high metastatic behavior in various organs. Although many chemotherapeutic protocols are used to treat human sarcomas, current treatment strategies for high-grade sarcomas are ineffective and the prognosis of patients is poor due to local recurrence and metastases [1]. Therefore, new therapeutic strategies against high-grade sarcomas are required.
Mitochondria are cytoplasmic organelles that play an essential role in cellular energy metabolism and programmed cell death [2]. Previous studies have linked decreases in mitochondrial metabolism and/or mitochondrial number to cancer progression [3,4,5]. Mitochondrial proliferation has also been shown to play an important role in cellular apoptosis and may be an integral part of a cascade of apoptotic events [6]. Peroxisome proliferatoractivated receptor gamma coactivator-1 alpha (PGC-1a) is a multi-functional transcriptional coactivator that regulates the activities of multiple nuclear receptors and transcription factors involved in mitochondrial biogenesis [7]. Specifically, PGC-1a transcriptionally regulates the gene encoding mitochondrial transcription factor A (TFAM), which plays an important role in mitochondrial biogenesis [8]. TFAM expression mirrors the fluctuating levels of mitochondrial DNA (mtDNA) in the cell, and mitochondrial synthesis is stimulated by the PGC-1a/TFAM pathway [8]. We have previously shown that mitochondria abundance is significantly decreased in several human sarcomas compared to benign tumors (unpublished data). Furthermore, we demonstrated that PGC-1a overexpression increases mitochondrial proliferation and induces mitochondrial apoptosis in human MFH cells in vitro (unpublished data). These results suggest that regulation of mitochondrial proliferation via modulation of PGC-1a expression, may be utilized as a useful therapeutic tool for the treatment of human musculoskeletal malignancies.
Carbon dioxide (CO 2 ) therapy in the form of a carbonated spa has been historically used in Europe as an effective treatment for cardiac diseases and skin lesions [9,10]. The therapeutic effects of CO 2 are caused by an increase in blood flow and microcirculation, nitric oxide-dependent neocapillary formation, and a partial increase in O 2 pressure in the local tissue, known as the Bohr effect [9,10,11]. Previously, we demonstrated that our transcutaneous CO 2 therapy to rat skeletal muscle induced PGC-1a expression, and led to an increase in mitochondria [12]. These findings suggest that our transcutaneous CO 2 therapy can upregulate the mitochondrial biogenesis through an increase of PGC-1a expression in the treated tissue.
Based on our previous studies in skeletal muscle, we hypothesized that transcutaneous application of CO 2 may also induce PGC-1a expression and mitochondrial proliferation in tumor tissue, but in this context lead to tumor cell apoptosis. In this study, we use a murine model of human MFH to investigate the effects of transcutaneous application of CO 2 on mitochondrial biogenesis and tumor cell apoptosis.

Transcutaneous Application of CO 2 Significantly Reduced MFH Cell Growth in vivo
To determine the effect of our CO 2 treatment on MFH cell growth in vivo, we constructed a murine model of human MFH by transplanting the Nara-H cell line into the dorsal subcutaneous area of mice. Transcutaneous application of CO 2 reduced tumor volume by 48% in treated mice compared to controls (p,0.01) ( Figure 1A and B). No significant difference in body weight was observed between CO 2 treated and control groups ( Figure 1C). Thus, transcutaneous application of CO 2 had an inhibitory effect on MFH tumor growth in vivo, with no observable negative side effects.
Transcutaneous Application of CO 2 Up-regulated the PGC-1a-TFAM-mitochondria pathway To investigate the mechanisms underlying the decrease in tumor volume in CO 2 treated mice, we examined the expression of PGC-1a and TFAM in tumor tissue using quantitative real-time PCR (qRT-PCR). PGC-1a and TFAM expression was significantly increased in the CO 2 group compared to control animals (p,0.05) (Figure 2A and B). Previous studies have shown that mitochondrial synthesis is stimulated by the PGC-1a/TFAM pathway [8]. Thus, we measured the relative levels of mtDNA to nuclear DNA (nDNA) in both CO 2 treated and control groups. mtDNA copy number was significantly higher in the tumors from CO 2-treated animals compared to controls (p,0.05) ( Figure 2C). Consistent with these findings, immunofluorescence staining of mitochondria revealed that mitochondria levels were elevated in the CO 2 treated tumors relative to controls ( Figure 2D). Staining of normal muscle tissues as a positive control revealed strong staining ( Figure S1).

Mitochondrial Apoptosis was Induced by Transcutaneous Application of CO 2 Treatment in Human MFH Cells in vivo
We performed immunofluorescence staining for DNA breaks to evaluate the effect of CO 2 treatment on MFH cell apoptosis in vivo.
We observed an increase in cells with apoptotic nuclei in tumors from the CO 2 treated group compared to controls ( Figure 3A). Flow cytometry revealed that DNA fragmentation, a measure of apoptosis, was increased in CO 2 treated tumors compared to controls ( Figure 3B). Taken together, these results indicate that CO 2 treatment induced apoptosis in human MFH cells in vivo.
We also examined the cleavage of caspases and PARP, and evaluated the expression of cytochrome c and Bax in the mitochondrial and cytoplasmic fractions separately to determine the involvement of mitochondria in the observed apoptosis. Immunoblot analyses revealed increased cleavage products of caspase 3 and 9, and PARP in CO 2 treated tumors, but not in the control tumors ( Figure 3C). Furthermore, we observed decreased expression of cytochrome c in the mitochondrial fraction and increased expression in the cytoplasmic fraction in the CO 2 treated group compared to controls. Conversely, Bax protein was increased in the mitochondrial fraction and was decreased in the cytoplasmic fraction ( Figure 3D). Positive bands in immunoblot analyses were semiquantified using densitometrical analyses using the Image J program (NIH, USA, http://rsb.info.nih.gov/ij/). Taken together, these results indicated that the anti-tumoral effect of transcutaneous CO 2 treatment in a murine model of human MFH may be mediated via mitochondria induced apoptosis.

Transcutaneous Application of CO 2 Treatment Increased Intracellular Ca 2+ in MFH Cells
We finally investigated the mechanism of the induction of the PGC-1a-TFAM-mitochondria pathway by our system in MFH cells. It has been reported that raising intracellular calcium (Ca 2+ ) concentration induces the PGC-1a expression [13,14], and mitochondrial biogenesis [13,14]. Therefore, we examined the effect of our CO 2 treatment on the intracellular Ca 2+ concentration in human MFH cells in vivo. We isolated implanted tumors from mice at 0, 6 and 24 hours after our transcutaneous CO 2 treatment, and we evaluated the intracellular Ca 2+ in the tumors. At 0 and 6 hours after treatment, Ca 2+ concentration in CO 2 treated tumors was significantly higher than that in the control tumors ( Figure 4). The elevated relative Ca 2+ concentration of the CO 2 treated cells fell in a time-dependent manner after treatment and was equivalent to that of the cells in untreated control tumors within 24 hours ( Figure 4). The results indicated that transcutaneous CO 2 exposure increased the intracellular Ca 2+ concentration in human MFH in vivo.

Discussion
A number of studies have shown that decreased mtDNA levels are associated with neoplastic transformation and/or tumor progression [15,16], and that mtDNA plays an important role in apoptosis [17]. Higuchi et al. investigated the role of mitochondrial respiration in apoptosis signaling in the human myelogenous leukemia cell line, ML-1a. These studies revealed that respirationdeficient clones were resistant to the tumor necrosis factor-induced apoptosis, whereas the clones reconstituted with normal mtDNA were sensitive [18]. We previously demonstrated that both mtDNA levels and PGC-1a expression were significantly decreased in human sarcoma tissues (unpublished data). Our studies showed that apoptosis could be induced by PGC-1a overexpression, which led to an increase in mtDNA number in human MFH cells (unpublished data). These results indicated that loss of PGC-1a expression and the subsequent decrease in mtDNA may render cells resistant to a certain apoptotic pathway.
The relationship between cancer and CO 2 is controversial [19,20,21]. It is well established that CO 2 alters the functions of macrophages and polymorphonuclear cells in the peritoneal and thoracic cavity [22]. While studies exposing tumor cells to CO 2 are not conclusive [23], numerous studies confirm that CO 2 affects the behavior of tumor cells derived from colon carcinomas, adenocarcinomas and breast cancers [19,20,24]. Although several studies have identified a CO 2 -associated increase in tumor cell growth and invasiveness in various cancer cell lines [19,21], there are also reports that show CO 2 can inhibit tumor cells [23,24]. Gutt et al. demonstrated that CO 2 increased cell necrosis and decreased proliferation in colonic and pancreatic carcinoma cells [25]. Hao et al. reported that exposure of gastric cancer cells to CO 2 pneumoperitoneum significantly induced apoptosis [26]. In the current study, we demonstrate that transcutaneous application of CO 2 to human MFH cells in an engrafted tumor model, upregulated the expression of PGC-1a and TFAM, increased the number of mitochondria, and led to mitochondrial-induced apoptosis. Furthermore, we previously observed a similar effect on human breast cancer cells in vivo ( Figure S2). Therefore, our transcutaneous CO 2 therapy may have an antitumoral effect on various human malignancies. However, the mechanisms underlying this observation remain unknown. In muscle tissue, mitochondrial respiration is regulated by PGC-1a, which stimulates various genes associated with mtDNA replication and transcription [7]. Generally, PGC-1a is induced by exercise in muscles, and mediates known responses to exercise such as muscle fiber-type switching and mitochondrial biogenesis [27]. PGC-1a expression is also induced by other stimuli, such as thyroid hormone treatment or 5-aminoimidazole-4-carboxamide-1-b-dribofuranoside (AICAR)-induced AMPK activation [28], as well as contractile activity in skeletal muscle [28,29]. Several signaling kinases, after activation of calcium influx, such as p38 [30], AMPK [31] and CaMKIV [32], have also been implicated in mediating transcriptional activation of PGC-1a [33]. We recently demonstrated that transcutaneous application of CO 2 upregulates PGC-1a expression in rat skeletal muscle, establishing a potential link between CO 2 exposure and the induction of mitochondrial biogenesis [12]. It is reported that CO 2 increased the intracellular Ca 2+ concentration in various cells [34,35], and that the increase in intracellular Ca 2+ increases the expression of PGC-1a and the amount of mitochondria [13,14,36]. These reports indicated that CO 2 induced the PGC-1a expression and mitochondrial biogenesis through raising the intracellular Ca 2+ concentration. In the current study, we have demonstrated that our transcutaneous CO 2 treatment increased the intracellular Ca 2+ in human MFH cells in vivo. The results strongly support that the effect of our transcutaneous CO 2 system on the induction of mitochondrial apoptosis through PGC-1a expression was caused by raising intracellular Ca 2+ concentration.
We here show that localized, transcutaneous application of CO 2 to an in vivo model of human MFH led to mitochondria-mediated apoptosis and impaired tumor growth, with no observable effects on body weight, a side effect typically observed following chemotherapy. Although further studies are needed to elucidate the mechanisms of the effects of the treatment on tumor cell apoptosis, our data indicate that transcutaneous application of CO 2 may be a useful therapeutic tool for human MFH.

Animal Models
Male athymic BALB/c nude mice, aged 5-8 weeks were obtained from CLEA Japan, Inc (Tokyo, Japan). Animals were maintained under pathogen-free conditions, in accordance with institutional principles. All animal experiments were performed according to the Guide for the Care and Use of Laboratory Animals at the host institution and were approved by the institutional animal committee (P-101203). Nara-H cells (4.0610 6 cells in 500 ml PBS) were injected into dorsal, subcutaneous area of mice as previously described [38].

Transcutaneous CO 2 Treatment
Transcutaneous application of CO 2 was performed as previously described [12]. Briefly, the area of skin around the implanted tumor was treated with CO 2 hydrogel. This area was then sealed with a polyethylene bag and 100% CO 2 gas was administered into the bag ( Figure S3). Each treatment was performed for 10 minutes. Control animals were treated similarly, replacing CO 2 with an ambient air.

In vivo MFH Tumor Studies
Twenty-four mice were randomly divided into two groups: CO 2 group (n = 12) and control group (n = 12). Treatment commenced three days after MFH cell implantation, and was performed twice weekly for 2 weeks. Tumor volume and body weight in mice were monitored twice weekly until the end of the treatment. Tumor volume was calculated as previously described [38] according to the formula V = p/66a 2 6b, where a and b represent the shorter and the longer dimensions of the tumor, respectively. At the completion of treatment, all tumors were excised from mice and tissue was stored at 280uC.

Evaluation of Mitochondrial Proliferation
Mitochondrial proliferation was assessed by determining the relative amount of mtDNA to nuclear (nDNA) in tumor samples. Genomic DNA was isolated from tumor specimens using the GenElute Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich), and PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems) with primers designed to amplify a region corresponding to nucleotides 16-408 of a D-loop of human mtDNA. The primers used were 59-GCAGATTTGGG-TACCACCCAAGTATTGACTCACCC-39 (forward) and 59-GCATGGAGAGCTCCCGTGAGTGGTTAATAGGGTGA-TAG-39 (reverse).

Immunofluorescence Staining
To assess the mitochondrial proliferation and the apoptotic activity in treated tumors, we performed the immunofluorescence staining using the MitoTracker Deep Red FM (Invitrogen) and the APO-DIRECT Kit (BD Pharmingen, Franklin Lakes, NJ, USA) following the manufacturer's protocol, respectively. The nucleus was stained with DAPI. The images were obtained using a BZ-8000 confocal microscope (Keyence).

Measurement of Intracellular Ca 2+
To investigate the effect of transcutaneous CO 2 treatment on intracellular Ca 2+ in MFH tumor tissues, we isolated implanted tumors from mice at 0 (n = 12), 6 (n = 6) and 24 hours (n = 12) after our transcutaneous CO 2 treatment, and evaluated the intracellular Ca 2+ concentration using Calcium Assay Kit according to the manufacturer's protocol (Cayman Chemical Company, Ann Arbor, Michigan, USA). Briefly, implanted tumors were excised, minced and rinsed with PBS containing 0.16 mg/ml heparin to remove any extraneous red blood cells and clots, and the tissues were homogenized in PBS containing 0.16 mg/ml heparin. Suspensions were centrifuged at 100006g for 15 minutes at 4uC, and the supernatant was removed. Then, the detector was added, and the optical density was measured at a wavelength of 570 nm using a Model 680 Microplate Reader (Bio-Rad) after 5 minutes of incubation. The relative number of Ca 2+ concentration was calculated.

Statistical Analyses
Experiments were performed independently at least three times, and data are presented as the mean 6 standard error unless otherwise indicated. Significance of differences between groups was evaluated using a two-tailed Student's t-test, and by ANOVA with post hoc test to compare for continuous values. All tests were considered significant at p,0.05. Figure S1 Immunofluorescence staining were performed in normal muscle tissues of mice as the control images of staining using the MitoTracker Deep Red FM (Invitrogen). The nucleus was stained with DAPI. The images were obtained using a BZ-8000 confocal microscope (Keyence). (TIFF) Figure S2 Effect of our transcutaneous CO 2 treatment on the in vivo tumor growth of human breast cancer cell line, MDA-MB-231. Tumor model mice were created by subcutaneous implantation of the cells (1.5610 6 cells in 500 ml PBS). Mice were randomly divided into CO 2 group (n = 5) or control group (n = 5), and treatment was performed twice weekly for 15 days. Tumor volume (A) and body weight (B) in mice were monitored until the end of the treatment. (A) At the end of the treatment, we observed a significant decrease in tumor volume in CO 2 group compared with the control group (*p,0.05). (B) No significant difference in body weight was observed between CO 2 treated and control groups. (TIFF) Figure S3 Transcutaneous application of CO 2 for a model mouse of human MFH. (TIFF)