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

<div><p>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 <em>in vitro</em>. 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.</p> </div

Transcutaneous Carbon Dioxide Induces Mitochondrial Apoptosis and Suppresses Metastasis of Oral Squamous Cell Carcinoma <i>In Vivo</i>

<div><p>Squamous cell carcinoma (SCC) is the main histological type of oral cancer. Its growth rate and incidence of metastasis to regional lymph nodes is influenced by various factors, including hypoxic conditions. We have previously reported that transcutaneous CO₂ induces mitochondrial apoptosis and decreases lung metastasis by reoxygenating sarcoma cells. However, previous studies have not determined the sequential mechanism by which transcutaneous CO₂ suppresses growth of epithelial tumors, including SCCs. Moreover, there is no report that transcutaneous CO₂ suppresses lymphogenous metastasis using human cell lines xenografts. In this study, we examined the effects of transcutaneous CO₂ on cancer apoptosis and lymphogenous metastasis using human SCC xenografts. Our results showed that transcutaneous CO₂ affects expressions of PGC-1α and TFAM and protein levels of cleavage products of caspase-3, caspase-9 and PARP, which relatives mitochondrial apoptosis. They also showed that transcutaneous CO₂ significantly inhibits SCC tumor growth and affects expressions of HIF-1α, VEGF, MMP-2 and MMP-9, which play essential roles in tumor angiogenesis, invasion and metastasis. In conclusion, transcutaneous CO₂ suppressed tumor growth, increased mitochondrial apoptosis and decreased the number of lymph node metastasis in human SCC by decreasing intra-tumoral hypoxia and suppressing metastatic potential with no observable effect <i>in vivo</i>. Our findings indicate that transcutaneous CO₂ could be a novel therapeutic tool for treating human SCC.</p></div

Effect of transcutaneous CO₂ application on intracellular Ca²⁺ concentration in a mouse model of human MFH.

<p>Implanted tumors were isolated from mice at 0 (n = 12), 6 (n = 6) and 24 hours (n = 12) after transcutaneous CO₂ exposure, and the intracellular Ca²⁺ concentration was assessed using the Calcium Assay Kit. Data represent the mean ± S.E. of at least three independent experiments (*<i>p</i><0.05, **<i>p</i><0.01).</p

Procedure for administering transcutaneous CO₂ in animal model of human SCC.

<p>The area of skin around the implanted tumor was covered with CO₂ hydrogel and sealed with a polyethylene bag, through which 100% CO₂ gas was administered. Treatment commenced 7 days after HSC-3 cell implantation, and was performed twice a week for 3 weeks.</p

Effect of transcutaneous application of CO₂ treatment on mitochondrial proliferation in tumors.

<p>qRT-PCR for <i>PGC-1α</i> (A) and <i>TFAM</i> (B) in CO₂ treated or control tumor specimens collected two weeks post-treatment. Expression was normalized to <i>β-actin</i> control. Data represent the mean ± S.E of at least three independent experiments (*<i>p</i><0.05). (C) mtDNA was measured in CO₂ treated or control tumor samples by PCR and the relative copy number was determined by normalizing to nDNA. Data represent the mean ± S.E. of at least three independent experiments (*<i>p</i><0.05). (D) Immunofluorescence staining of mitochondria in CO₂ treated or control tumors after two weeks (<i>Blue</i>, nuclear; <i>Red</i>, mitochondria).</p