Identification of novel fish sialidase genes responsible for KDN-cleaving activity.

2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN) is a minor component of sialic acids detected in vertebrates, such as human cancer cells, rat liver, and fish tissues. Although the enzyme activity of KDN-cleaving sialidase (KDN-sialidase) has been detected in rainbow trout, the gene responsible for its expression has not been identified in vertebrates. We evaluated sialidases in human and various fish for their KDN-cleaving activity using an artificial substrate, methylumbelliferyl-KDN (MU-KDN). Four of the human sialidases tested (NEU1, NEU2, NEU3, and NEU4) did not hydrolyze MU-KDN. Although most fish Neu1s showed negligible KDN-sialidase activity, two Neu1b sialidases from Oreochromis niloticus and Astyanax mexicanus, a paralog of Neu1, exhibited a potent KDN-sialidase activity. Further, O. niloticus and Oryzias latipes Neu3a exhibited a drastically high KDN-sialidase activity, while Danio rerio Neu3.1 showed moderate activities and other Neu3 proteins exhibited little activity. All the Neu4 sialidases tested in fish cleaved KDN and Neu5Ac from MU-KDN and MU-Neu5Ac, respectively, with equivalent potential. To our knowledge, this is the first report to identify KDN-sialidase genes in vertebrates and we believe that KDN-sialidase activity could be conserved among fish Neu4s.

Synergistic Effects of a Smac Mimetic with Doxorubicin Against Human Osteosarcoma.

BACKGROUND/AIM: Second mitochondria-derived activator of caspase (Smac) is a proapoptogenic mitochondrial protein that antagonizes inhibitors of apoptosis proteins (IAPs), resulting in induction of apoptosis. In the present study we investigated the effects of a Smac mimetic in combination with doxorubicin against osteosarcoma. MATERIALS AND METHODS: In vitro effects of the combination of a Smac mimetic AT-406 and doxorubicin on cell proliferation and apoptosis in osteosarcoma cell lines were examined using cell proliferation assays, flow cytometry, and immunoblot analyses. For in vivo experiments, human osteosarcoma xenografts were treated with combination of the two substances, and tumor volume and apoptotic activity in treated tumors were assessed. RESULTS: In vitro studies revealed that combination of the two substances significantly inhibited osteosarcoma proliferation with decreased cIAP1 expression and induced apoptosis in osteosarcoma cells. Combination of the two substances significantly suppressed osteosarcoma growth in vivo. Moreover, decreased cIAP1 expression and increased apoptotic activity were observed in tumors treated by their combination of the substances. CONCLUSION: The Smac mimetic AT-406 showed an apoptotic effect and a synergistic antitumor effect with doxorubicin on osteosarcoma. The combination of AT-406 and doxorubicin may serve as a novel therapeutic strategy for osteosarcoma treatment.

Optimization of antitumor treatment conditions for transcutaneous CO2 application: An in vivo study.

Carbon dioxide (CO2) therapy can be applied to treat a variety of disorders. We previously found that transcutaneous application of CO2 with a hydrogel decreased the tumor volume of several types of tumors and induced apoptosis via the mitochondrial pathway. However, only one condition of treatment intensity has been tested. For widespread application in clinical antitumor therapy, the conditions must be optimized. In the present study, we investigated the relationship between the duration, frequency, and treatment interval of transcutaneous CO2 application and antitumor effects in murine xenograft models. Murine xenograft models of three types of human tumors (breast cancer, osteosarcoma, and malignant fibrous histiocytoma/undifferentiated pleomorphic sarcoma) were used to assess the antitumor effects of transcutaneous CO2 application of varying durations, frequencies, and treatment intervals. In all human tumor xenografts, apoptosis was significantly induced by CO2 treatment for ≥10 min, and a significant decrease in tumor volume was observed with CO2 treatments of >5 min. The effect on tumor volume was not dependent on the frequency of CO2 application, i.e., twice or five times per week. However, treatment using 3- and 4-day intervals was more effective at decreasing tumor volume than treatment using 2- and 5-day intervals. The optimal conditions of transcutaneous CO2 application to obtain the best antitumor effect in various tumors were as follows: greater than 10 min per application, twice per week, with 3- and 4-day intervals, and application to the site of the tumor. The results suggest that this novel transcutaneous CO2 application might be useful to treat primary tumors, while mitigating some side effects, and therefore could be safe for clinical trials.

AICAR induces mitochondrial apoptosis in human osteosarcoma cells through an AMPK-dependent pathway.

The AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) modulates cellular energy metabolism, and promotes mitochondrial proliferation and apoptosis. Previous studies have shown that AICAR has anticancer effects in various cancers, however the roles of AMPK and/or the effects of AICAR on osteosarcoma have not been reported. In the present study, we evaluated the effects of AICAR on tumor growth and mitochondrial apoptosis in human osteosarcoma both in vitro and in vivo. For in vitro experiments, two human osteosarcoma cell lines, MG63 and KHOS, were treated with AICAR, and the effects of AICAR on cell growth and mitochondrial apoptosis were assessed by WST assays, TUNEL staining, and immunoblot analyses. In vivo, human osteosarcoma-bearing mice were treated with AICAR, and the mitochondrial proliferation and apoptotic activity in treated tumors were assessed. In vitro experiments revealed that AICAR activated AMPK, inhibited cell growth, and induced mitochondrial apoptosis in both osteosarcoma cell lines. In vivo, AICAR significantly reduced osteosarcoma growth without apparent body weight loss and AICAR increased both mitochondrial proliferation and apoptotic activity in treated tumor tissues. AICAR showed anticancer effects in osteosarcoma cells through an AMPK-dependent peroxisome proliferator­activated receptor-γ coactivator-1α (PGC-1α)/mitochondrial transcription factor A (TFAM)/mitochondrial pathway. The findings in this study strongly suggest that AICAR could be considered as a potent therapeutic agent for the treatment of human osteosarcoma.

Antitumor effect of YM155, a novel small-molecule survivin suppressant, via mitochondrial apoptosis in human MFH/UPS.

Survivin is a member of the inhibitor of apoptosis family, which is known to inhibit mitochondrial apoptosis. Survivin is highly expressed in cancers and plays an important role in cancer cell survival, and increased survivin expression is an unfavorable prognostic marker in cancer patients. YM155, a novel small-molecule survivin suppressant, selectively suppresses survivin expression, resulting in the induction of apoptosis in various malignancies. However, the roles of survivin in human malignant fibrous histiocytoma/undifferentiated pleomorphic sarcoma (MFH/UPS) have not been studied. In the present study, we examined survivin expression in human musculoskeletal tumor tissues, and the effect of survivin inhibition by siRNA or YM155 on apoptotic activity in human MFH/UPS cell lines. In tumor tissues, mRNA expression of survivin was significantly higher in MFH/UPS samples than in benign schwannomas. Moreover, in vitro studies revealed that both survivin siRNA and YM155 suppressed survivin expression and inhibited MFH/UPS cell proliferation in a dose- and a time-dependent manner. Further, the numbers of apoptotic cells significantly increased with YM155 treatment. in vivo, tumor volume in YM155-treated groups was significantly reduced without significant bodyweight loss. Increased apoptotic activity along with decreased survivin expression was also observed in YM155-treated tumors. The findings in this study strongly suggest that survivin suppressants, including YM155, contribute to the suppression of human MFH/UPS cell growth via promoting mitochondrial apoptosis, and that survivin may be a potent therapeutic target for the novel treatment of human MFH/UPS.

Transcutaneous carbon dioxide induces mitochondrial apoptosis and suppresses metastasis of oral squamous cell carcinoma in vivo.

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 in vivo. Our findings indicate that transcutaneous CO2 could be a novel therapeutic tool for treating human SCC.

Transcutaneous application of CO2 enhances the antitumor effect of radiation therapy in human malignant fibrous histiocytoma.

Sarcomas are relatively resistant because of hypoxia. We previously demonstrated that the transcutaneous CO(2) therapy reduced hypoxic conditions in human malignant fibrous histiocytoma (MFH). Therefore, we hypothesized that transcutaneous CO(2) therapy could enhance the antitumor effect of radiation therapy in human MFH. Our purpose was to evaluate the effects of transcutaneous CO(2) therapy on the antitumor efficacy of X-ray irradiation using MFH. First, in an in vitro study, we assessed apoptotic activity and reactive oxygen species (ROS) production using flow cytometric and immunoblot analysis at 24 h after X-ray irradiation under three different oxygen conditions (normoxic, reoxygenated and hypoxic). In addition, in the in vivo study, 24 male athymic BALB/c nude mice with MFH tumors that were inoculated in the dorsal subcutaneous area were randomized into four groups: control, CO(2), X-ray irradiation and combination (CO(2) and X-ray irradiation). Treatments were performed twice weekly for 2 weeks, four times in total. Tumor volume was calculated. All tumors were excised and apoptotic activity, ROS production, related proteins and HIF-1α expression were assessed using flow cytometric and immunoblot analysis. The in vitro study revealed that X-ray irradiation induced increased apoptosis and ROS production in MFH cells under normoxic and reoxygenated conditions relative to hypoxic conditions (P<0.01). In the in vivo study, tumor volume in the combination group was reduced to 28, 42 and 47% of that in the control, CO(2), and X-ray groups, respectively (P<0.05). Apoptotic activity and ROS production in the combination group were strongly increased with decreasing HIF-1α expression relative to the control, CO(2) and X-ray groups. The transcutaneous CO(2) system enhanced the antitumor action of X-ray irradiation and could be a novel therapeutic tool for overcoming radio-resistance in human malignancies.

Regulation of mitochondrial proliferation by PGC-1α induces cellular apoptosis in musculoskeletal malignancies.

A number of studies have reported that decreased mitochondrial numbers are linked with neoplastic transformation and/or tumor progression, including resistance to apoptosis. Peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) is a multi-functional transcriptional coactivator that regulates the activities of multiple nuclear receptors and transcriptional factors involved in mitochondrial biogenesis. In this study, we observed that the number of mitochondria in sarcoma tissues, such as osteosarcoma and malignant fibrous histiocytoma, is significantly lower than that in normal muscle tissue or benign tumors, and that increasing the number of mitochondria by PGC-1α overexpression induces mitochondrial apoptosis in human sarcoma cell lines. The findings suggest that decreased mitochondrial numbers may contribute to musculoskeletal tumor progression, and that regulation of mitochondrial numbers by PGC-1α could be a potent therapeutic tool for human malignancies.

'Decoy' and 'non-decoy' functions of DcR3 promote malignant potential in human malignant fibrous histiocytoma cells.

Decoy receptor 3 (DcR3) is a soluble secreted protein that belongs to the tumor necrosis factor receptor (TNFR) superfamily. DcR3 inhibits the Fas ligand (FasL)/Fas apoptotic pathway by binding to FasL, competitively with Fas receptor. Previous studies have reported that overexpression of DcR3 has been detected in various human malignancies and that DcR3 functions as a 'decoy' for FasL to inhibit FasL-induced apoptosis. In addition, recent studies have revealed that DcR3 has 'non-decoy' functions to promote tumor cell migration and invasion, suggesting that DcR3 may play important roles in tumor progression by decoy and non-decoy functions. We have previously reported that overexpression of DcR3 was observed in human malignant fibrous histiocytoma (MFH), however, the roles of DcR3 in MFH have not been studied. In the present study, to elucidate the roles of DcR3 in tumor progression of MFH, we examined the effects of DcR3 inhibition on cell apoptosis, migration and invasion in human MFH cells. siRNA knockdown of DcR3 enhanced the FasL-induced apoptotic activity and significantly decreased cell migration and invasion with a decrease in the activation of phosphatidylinositol 3 kinase (PI3K)/Akt and matrix metalloproteinase (MMP)-2. The findings in this study strongly suggest that DcR3 plays important roles in tumor progression of human MFH by decoy as well as non-decoy functions and that DcR3 may serve as a potent therapeutic target for human MFH.

Reoxygenation using a novel CO2 therapy decreases the metastatic potential of osteosarcoma cells.

Osteosarcoma is the most common primary solid malignant bone tumor. Despite substantial improvements in surgery and chemotherapy, metastasis remains a major cause of fatal outcomes, and the molecular mechanisms of metastasis are still poorly understood. Hypoxia, which is common in malignant tumors including osteosarcoma, increases expressions of hypoxia inducible factor (HIF)-1α, matrix metalloproteinase (MMP)-2 and MMP-9, and can induce invasiveness. As we previously showed a novel transcutaneous CO2 application to decrease HIF-1α expression and induce apoptosis in malignant fibrous histiocytoma, we hypothesize that transcutaneous CO2 application could suppress metastatic potential of osteosarcoma by improving hypoxic conditions. Here, we examined the effects of transcutaneous CO2 application on apoptosis, and development of pulmonary metastasis using a highly metastatic osteosarcoma cell line, LM8. Transcutaneous CO2 application significantly decreased tumor growth and pulmonary metastasis in LM8 cells. Apoptotic activity increased, and intratumoral hypoxia was improved with decreased expressions of HIF-1α, MMP-2 and MMP-9, significantly, in the CO2-treated tumors. In conclusion, we found that transcutaneous CO2 application can induce tumor cell apoptosis and might suppress pulmonary metastasis by improvement of hypoxic conditions with decreased expressions of HIF-1α and MMPs in highly metastatic osteosarcoma cell. These findings strongly indicate that this novel transcutaneous CO2 therapy could be a therapeutic breakthrough for osteosarcoma patients.

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 (CO(2)) 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 CO(2) 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 CO(2) 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 CO(2) 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 CO(2) treated tumors, highlighting the involvement of mitochondria in apoptosis. These data indicate that transcutaneous application of CO(2) may represent a novel therapeutic tool in the treatment of human MFH.