[Pharmacological actions of anifrolumab (Saphnelo®) and clinical trial results as a treatment for systemic lupus erythematosus].

Systemic lupus erythematosus (SLE) is an autoimmune disease which causes damaging inflammation in multiple organs via the accumulation of immune complexes. SLE pathogenesis is associated with type I interferons (IFNs), which are central and reflective of disease activity in SLE. Even before clinical development of disease, genetic and environmental contributions to IFN production lead to abnormal innate and adaptive immune activation. Through the Janus kinase-signal transducer and activator of transcription signaling pathway, IFN play a central role in the immunopathogenicity of SLE. Thus, IFN-blocking therapy may be used to regulate inflammation in individuals with SLE. Food and Drug Administration (FDA)-approved anifrolumab (Saphnelo®), which is a human IgG1κ monoclonal antibody that binds to subunit 1 of the type I interferon receptor with high specificity and affinity, was also approved for the treatment of adult patients with moderate to severe SLE who are receiving standard therapy by Pharmaceuticals and Medical Device Agency (PMDA), in Japan in September 2021; anifrolumab is administered as an intravenous infusion, 300 mg over a 30-minute period, every 4 weeks. In this article, we reviewed the actions of type I IFN and anifrolumab as a treatment for SLE.

In vivo evaluation of percutaneous carbon dioxide treatment for improving intratumoral hypoxia using 18F-fluoromisonidazole PET-CT.

Carbon dioxide (CO2) treatment is reported to have an antitumor effect owing to the improvement in intratumoral hypoxia. Previous studies were based on histological analysis alone. In the present study, the improvement in intratumoral hypoxia by percutaneous CO2 treatment in vivo was determined using 18F-fluoromisonidazole positron emission tomography-computed tomography (18F-FMISO PET-CT) images. Twelve Japanese nude mice underwent implantation of LM8 tumor cells in the dorsal subcutaneous area 2 weeks before percutaneous CO2 treatment and 18F-FMISO PET-CT scans. Immediately after intravenous injection of 18F-FMISO, CO2 and room air were administered transcutaneously in the CO2-treated group (n=6) and a control group (n=6), respectively; each treatment was performed for 10 minutes. PET-CT was performed 2 h after administration of 18F-FMISO. 18F-FMISO tumor uptake was quantitatively evaluated using the maximum standardized uptake value (SUVmax), tumor-to-liver ratio (TLR), tumor-to-muscle ratio (TMR), metabolic tumor volume (MTV) and total lesion glycolysis (TLG). Mean ± standard error of the mean (SEM) of the tumor volume was not significantly different between the two groups (CO2-treated group, 1.178±0.450 cm3; control group, 1.368±0.295 cm3; P=0.485). Mean ± SEM of SUVmax, TLR, MTV (cm3) and TLG were significantly lower in the CO2-treated group compared with the control group (0.880±0.095 vs. 1.253±0.071, P=0.015; 1.063±0.147361 vs. 1.455±0.078, P=0.041; 0.353±0.139 vs. 1.569±0.438, P=0.015; 0.182±0.070 vs. 1.028±0.338, P=0.015), respectively. TMR was not significantly different between the two groups (4.520±0.503 vs. 5.504±0.310; P=0.240). In conclusion, 18F-FMISO PET revealed that percutaneous CO2 treatment improved intratumoral hypoxia in vivo. This technique enables assessment of the therapeutic effect in CO2 treatment by imaging, and may contribute to its clinical application.

Transcutaneous carbon dioxide application inhibits muscle atrophy after fracture in rats.

BACKGROUND: Muscle atrophy causes difficulty in resuming daily activities after a fracture. Because transcutaneous carbon dioxide (CO2) application has previously upregulated oxygen pressure in the local tissue, thereby demonstrating its potential in preventing muscle atrophy, here we investigated effects of CO2 application on muscle atrophy after femoral shaft fracture. METHODS: Thirty fracture model rats were produced and randomly divided into a no treatment (control group) and treatment (CO2 group) groups. After treatment, the soleus muscle was dissected at post-fracture days 0, 14, and 21. Evaluations were performed by measuring muscle weight and performing histological examination and gene expression analysis. RESULTS: Muscle weight was significantly higher in the CO2 group than in the control group. Histological analysis revealed that the muscle fiber cross-sectional area was reduced in both groups. Nevertheless, the extent of atrophy was lesser in the CO2 group. Muscle fibers in the control group tended to change into fast muscle fibers. Vascular staining revealed that more capillary vessels surrounded the muscle fibers in the CO2 group than in the control group. Messenger RNA (mRNA) analysis revealed that the CO2 group had a significantly enhanced expression of genes that were related to muscle synthesis. CONCLUSION: Transcutaneous CO2 application may be a novel therapeutic strategy for preventing skeletal muscle atrophy after fracture.

Induced Pluripotent Stem Cell-related Genes Correlate With Poor Prognoses of Oral Squamous Cell Carcinoma.

BACKGROUND/AIM: We recently investigated the contribution of the iPS-related genes SOX2, OCT4, and Nanog to de-differentiation by assaying for their mRNA levels. Given that mRNA expression does not always correlate with the protein levels, the aim of this study was to retrospectively determine the expression of these four iPS-related factors in human OSCC specimens by immunohistochemistry and examine their association with patient prognosis. MATERIALS AND METHODS: iPS cell-related gene expression in 89 OSCC patients by tissue microarray, and its correlation with clinicopathological factors, differentiation, metastasis, and poor prognoses were investigated. RESULTS: No evidence of statistically significant relationships was found between the expression of iPS cell-related genes and clinicopathological parameters. However, our data indicated that KLF4 expression was associated with survival, and poor tumor differentiation. In addition, high expression of KLF4 was an independent poor prognostic factor (p=0.004) for OSCC patients. CONCLUSION: In preoperative biopsies, higher KLF4 and poor differentiation may be clinically effective predictors for the prognosis of oral cancer.

Transcutaneous carbon dioxide application suppresses bone destruction caused by breast cancer metastasis.

Hypoxia plays a significant role in cancer progression, including metastatic bone tumors. We previously reported that transcutaneous carbon dioxide (CO2) application could decrease tumor progression through the improvement of intratumor hypoxia. Therefore, we hypothesized that decreased hypoxia using transcutaneous CO2 could suppress progressive bone destruction in cancer metastasis. In the present study, we examined the effects of transcutaneous CO2 application on metastatic bone destruction using an animal model. The human breast cancer cell line MDA-MB-231 was cultured in vitro under three different oxygen conditions, and the effect of altered oxygen conditions on the expression of osteoclast-differentiation and osteolytic factors was assessed. An in vivo bone metastatic model of human breast cancer was created by intramedullary implantation of MDA-MB-231 cells into the tibia of nude mice, and treatment with 100% CO2 or a control was performed twice weekly for two weeks. Bone volume of the treated tibia was evaluated by micro-computed tomography (µCT), and following treatment, histological evaluation was performed by hematoxylin and eosin staining and immunohistochemical staining for hypoxia-inducible factor (HIF)-1α, osteoclast-differentiation and osteolytic factors, and tartrate-resistant acid phosphatase (TRAP) staining for osteoclast activity. In vitro experiments revealed that the mRNA expression of RANKL, PTHrP and IL-8 was significantly increased under hypoxic conditions and was subsequently reduced by reoxygenation. In vivo results by µCT revealed that bone destruction was suppressed by transcutaneous CO2, and that the expression of osteoclast-differentiation and osteolytic factors, as well as HIF-1α, was decreased in CO2-treated tumor tissues. In addition, multinucleated TRAP-positive osteoclasts were significantly decreased in CO2-treated tumor tissues. Hypoxic conditions promoted bone destruction in breast cancer metastasis, and reversal of hypoxia by transcutaneous CO2 application significantly inhibited metastatic bone destruction along with decreased osteoclast activity. The findings in this study strongly indicated that transcutaneous CO2 application could be a novel therapeutic strategy for treating metastatic bone destruction.

Effect of local application of transcutaneous carbon dioxide on survival of random-pattern skin flaps.

INTRODUCTION: Skin flap procedures are widely used to reconstruct skin and soft tissue defects. Skin flap necrosis is a serious postoperative complication. Many researchers have introduced pharmacological agents to improve flap ischemia in experimental studies. However, outcomes of these studies remain controversial. We previously demonstrated that transcutaneous CO2 application improves hypoxia in fracture repair. In this study, we hypothesized that improving hypoxia by transcutaneous CO2 application can improve the blood flow in skin flaps and increase angiogenesis. We investigated whether transcutaneous CO2 application can increase the survival of random-pattern skin flaps. MATERIALS AND METHODS: Six-week-old male Sprague-Dawley rats were divided into two equal groups: the control group (n = 6) and CO2 group (n = 6). A random-pattern skin flap was constructed in these rats. Topical CO2 was applied using a hydrogel every day for 5 days in the CO2 group. The flap survival area was measured on postoperative days 1, 3, and 5. The vessel density and expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and hypoxia-inducible factor-1α (HIF-1α) were evaluated on postoperative day 5. RESULTS: A statistically significant difference was found in the percentage of the flap survival area between the two groups on postoperative days 3 and 5 (p < 0.05). Furthermore, the expression of VEGF and bFGF was significantly higher and that of HIF-1α was significantly lower in the CO2 than in the control group (p < 0.05). CONCLUSIONS: Transcutaneous CO2 application can improve the blood flow in skin flaps and increase angiogenesis, thus increasing the survival of random-pattern skin flaps.

Transcutaneous carbon dioxide enhances the antitumor effect of radiotherapy on oral squamous cell carcinoma.

Radiotherapy (RT) is one of the main treatment modalities for oral squamous cell carcinoma (OSCC), however, radioresistance is a major impediment to its clinical success and poses as a concern that needs to be addressed. Tumor hypoxia is known to be significantly associated with radioresistance in various malignancies, hence, resolving the hypoxic state of a tumor may improve the antitumor effect of RT on OSCC. We have previously revealed that transcutaneous CO2 induced mitochondrial apoptosis and suppressed tumor growth in OSCC by resolving hypoxia. Considering the previous study, we hypothesized that transcutaneous CO2 may enhance the antitumor effect of RT on OSCC by improving intratumoral hypoxia, thereby overcoming radioresistance. In the present study, the combination of transcutaneous CO2 and RT significantly inhibited tumor growth compared with other treatments. This combination therapy also led to decreased expression of HIF-1α in parallel with increased expression of the cleaved forms of caspase-3-8-9 and PARP, which play essential roles in mitochondrial apoptosis. Additionally, the combination therapy increased the expression of ROS modulator 1 and subsequent mitochondrial ROS production, compared to RT alone. These results indicated that transcutaneous CO2 could potentially improve the antitumor effect of RT by decreasing the intratumoral hypoxia and increasing the mitochondrial apoptosis. Our findings indicated that CO2 therapy may be a novel adjuvant therapy in combination with RT for OSCC.

The Influence of Ruptured Scar Pattern on the Healing Potential of Anterior Cruciate Ligament Remnant Cells.

BACKGROUND: Vascular CD34+ cells in anterior cruciate ligament (ACL) tissues have a potential for high proliferation and multilineage differentiation, which can accelerate tendon-bone healing after ACL reconstruction. To predict outcomes of ACL reconstruction with remnant preservation or ruptured tissue incorporation, patient characteristics should be considered. However, the influence of ACL remnant morphologic pattern on healing potential remains unknown. HYPOTHESIS: The healing potential of ACL remnants could differ among their morphologic patterns. STUDY DESIGN: Descriptive laboratory study. METHODS: ACL remnant tissues were harvested from patients aged <35 years who received primary ACL reconstruction within 3 months after injury. The tissues were evaluated according to the Crain classification (4 patterns). The patterns were divided into 2 groups: the reattachment group (Crain I-III) and the nonreattachment group (Crain IV). ACL remnant cells were characterized via fluorescence-activated cell sorting. The potential for proliferation and multilineage differentiation was assessed and compared between the groups. RESULTS: The ratio of CD34+ cells was significantly higher in the nonreattachment group than in the reattachment group. In early passages, the nonreattachment group had a significantly higher expansion potential than the reattachment group. In the evaluation of osteogenic and endothelial differentiation potential, the nonreattachment group showed a higher potential in immunohistochemical/histochemical staining and quantitative real-time polymerase chain reaction analysis as compared with the reattachment group. CONCLUSION: In the subacute phase, ACL remnant tissue of the nonreattachment group possibly has a higher healing potential than that of the reattachment group. CLINICAL RELEVANCE: If healing potential differs among the morphologic patterns of ACL remnants, surgeons may expect the healing potential when preserving remnants.

Methotrexate upregulates circadian transcriptional factors PAR bZIP to induce apoptosis on rheumatoid arthritis synovial fibroblasts.

BACKGROUND: Effects of methotrexate (MTX) on the proliferation of rheumatoid arthritis (RA) synovial fibroblasts are incompletely understood. We explored actions of MTX in view of circadian transcriptions of synovial fibroblasts. METHODS: Under treatment with MTX, expression of core circadian clock genes, circadian transcriptional factor proline and acidic amino acid-rich basic leucine zipper (PAR bZIP), and proapoptotic molecule Bcl-2 interacting killer (Bik) was examined by real-time polymerase chain reaction. Protein expression of circadian clock gene PERIOD2 (PER2) and CYTOCHROME C was also examined by western blotting and ELISA. Promoter activities of Per2 and Bik were measured by Luciferase assay. Expression of PER2, BIK, and CYTOCHROME C and morphological changes of the nucleus were observed by fluorescent immunostaining. Synovial fibroblasts were transfected with Per2/Bik small interfering RNA, and successively treated with MTX to determine cell viabilities. Finally, synovial fibroblasts were treated with MTX according to the oscillation of Per2/Bik expression. RESULTS: MTX (10 nM) significantly decreased cell viabilities, but increased messenger RNA expression of Per2, Bik, and PAR ZIP including D site of the albumin promoter binding protein (Dbp), hepatic leukemia factor (Hlf), and thyrotroph embryonic factor (Tef). MTX also increased protein expression of PER2 and CYTOCHROME C, and promoter activities of Per2 and Bik via D-box. Under fluorescent observations, expression of PER2, BIK, and CYTOCHROME C was increased in apoptotic cells. Cytotoxicity of MTX was attenuated by silencing of Per2 and/or Bik, and revealed that MTX was significantly effective in situations where Per2/Bik expression was high. CONCLUSIONS: We present here novel unique action of MTX on synovial fibroblasts that upregulates PAR bZIP to transcribe Per2 and Bik, resulting in apoptosis induction. MTX is important in modulating circadian environments to understand a new aspect of pathogenesis of RA.

Increase in carbon dioxide accelerates the performance of endurance exercise in rats.

Endurance exercise generates CO2 via aerobic metabolism; however, its role remains unclear. Exogenous CO2 by transcutaneous delivery promotes muscle fibre-type switching to increase endurance power in skeletal muscles. Here we determined the performance of rats running in activity wheels with/without transcutaneous CO2 exposure to clarify its effect on endurance exercise and recovery from muscle fatigue. Rats were randomised to control, training and CO2 groups. Endurance exercise included activity-wheel running with/without transcutaneous CO2 delivery. Running performance was measured after exercise initiation. We also analysed changes in muscle weight and muscle fibres in the tibialis anterior muscle. Running performance improved over the treatment period in the CO2 group, with a concomitant switch in muscle fibres to slow-type. The mitochondrial DNA content and capillary density in the CO2 group increased. CO2 was beneficial for performance and muscle development during endurance exercise: it may enhance recovery from fatigue and support anabolic metabolism in skeletal muscles.

Transcutaneous carbon dioxide application with hydrogel prevents muscle atrophy in a rat sciatic nerve crush model.

The acceleration of nerve regeneration remains a clinical challenge. We previously demonstrated that transcutaneous CO2 application using a novel hydrogel increases the oxygen concentration in local tissue via an "artificial Bohr effect" with the potential to prevent muscle atrophy. In this study, we investigated the effect of transcutaneous CO2 administration on limb function after peripheral nerve injury in a rat sciatic nerve injury model. In total, 73 Sprague-Dawley rats were divided into a sham group, a control group (crush injury to sciatic nerve and no treatment) or a CO2 group (crush injury with transcutaneous CO2 application). CO2 was administered percutaneously for 20 min five times per week. Scores for the sciatic function index and pinprick test were significantly higher in the CO2 group than control group. The muscle wet weight ratios of the tibialis anterior and soleus muscles were higher in the CO2 group than control group. Electrophysiological examination showed that the CO2 group had higher compound motor action potential amplitudes and shorter distal motor latency than the control group. Histological examination of the soleus muscle sections at postoperative week 2 showed shorter fiber diameter in the control group than in the CO2 group. The mRNA expression of Atrogin-1 and MuRF-1 was lower, mRNA expression of VEGF and myogenin and MyoD was higher in CO2 group at postoperative week 2 compared to the control group. CLINICAL SIGNIFICANCE: Transcutaneous CO2 application has the therapeutic potential to accelerate the recovery of muscle atrophy in peripheral nerve injury. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1653-1658, 2018.

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.

Intra-arterially infused carbon dioxide-saturated solution for sensitizing the anticancer effect of cisplatin in a rabbit VX2 liver tumor model.

The present study aimed to evaluate the efficacy of an intra-arterially infused carbon dioxide (CO2)-saturated solution in sensitizing the anticancer effect of cisplatin in a rabbit VX2 liver tumor model. Forty VX2 liver tumor-bearing Japanese white rabbits were randomly divided into four groups and infused via the proper hepatic artery with a saline solution (control group), CO2-saturated solution (CO2 group), cisplatin solution (cisplatin group), or CO2-saturated solution and cisplatin solution (combined group). The tumor volume (TV) and the relative tumor volume (RTV), RTV = (TV on day 3 or 7)/(TV on day 0) x 100, were calculated using contrast-enhanced computed tomography. Hypoxia-inducible factor-1α (HIF­1α) and carbonic anhydrase IX (CA IX) staining were used to evaluate cellular hypoxia. Cleaved caspase-3 and cleaved caspase-9 were analyzed to assess tumor apoptosis. The mean RTV on days 3 and 7 were 202.6±23.7 and 429.2±94.8%, respectively, in the control group; 172.2±38.1 and 376.5±61.1% in the CO2 group; 156.1±15.1 and 269.6±45.2% in the cisplatin group; and 118.3±28.1 and 210.3±55.1% in the combined group. RTV was significantly lower in the CO2 group than in the control group (day 3; P<0.05), and in the combined group than in the cisplatin group (days 3 and 7; P<0.05). HIF-1α and CA IX suppression, and increased cleaved caspase-3 and cleaved caspase-9 expression, were detected in the CO2 and combined groups, compared with the other two groups. An intra-arterially infused CO2-saturated solution inhibits liver VX2 tumor growth and sensitizes the anticancer effect of cisplatin.

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.

Induced Pluripotent-stem-cell Related Genes Contribute to De-differentiation in Oral Squamous Cell Carcinoma.

BACKGROUND/AIM: Cancer stem cells are suspected to contribute to malignancy in tumors. Hypoxia affects cell differentiation and induces stem-cell-like characteristics in malignancies. Induced pluripotency was demonstrated in mouse fibroblasts by reprogramming with four transcriptional factors: Oct3/4, Sox2, c-Myc, and Klf4. Conversely, oncogenic transformations frequently express transcriptional factors and Nanog. Therefore, cancer cells present some similarities with induced pluripotent stem (iPS) cells. MATERIALS AND METHODS: We investigated the expression of iPS-related genes in vitro and in clinical samples to identify their relationships with hypoxia and tumorigenesis. RESULTS: Oral squamous cell carcinoma (SCC) cells were used to show that expression levels of Oct3/4, Sox2, and Nanog were significantly increased in hypoxic condition in vitro and in moderately- and poorly-differentiated samples. CONCLUSION: We propose that Oct3/4, Sox2 and Nanog are associated with tumor hypoxia characterized in oral SCC and that these factors may also contribute to the undifferentiated potency observed in oral SCC clinically.

Transcutaneous carbon dioxide application accelerates muscle injury repair in rat models.

PURPOSE: Skeletal muscle injuries are commonly observed in sports and traumatology medicine. Previously, we demonstrated that transcutaneous application of carbon dioxide (CO2) to lower limbs increased the number of muscle mitochondria and promoted muscle endurance. Therefore, we aimed to investigate whether transcutaneous CO2 application could enhance recovery from muscle injury. METHODS: Tibialis anterior muscle damage was induced in 27 Sprague Dawley rats via intramuscular injection of bupivacaine. After muscle injury, rats were randomly assigned to transcutaneous CO2-treated or -untreated groups. From each group, three rats were sacrificed at weeks one, two, four and six. At each time point, histology and immunofluorescence analyses were performed, and changes in muscle weight, muscle weight/body weight ratio, muscle fibre circumference, gene expression levels and capillary density were measured. RESULTS: Injured muscle fibres were completely repaired at week six in the CO2-treated group but only partially repaired in the untreated group. The repair of basement and plasma membranes did not differ significantly between groups. However, expression levels of genes and proteins related to muscle protein synthesis were significantly higher in the CO2-treated group and significantly more capillaries four weeks after injury. CONCLUSION: Transcutaneous CO2 application can accelerate recovery after muscle injury in rats.

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.

Decreased mitochondrial copy numbers in oral squamous cell carcinoma.

BACKGROUND: Mitochondrial dysfunction and altered respiration have long been suspected to affect the development and progression of cancer. Although quantitative changes in mitochondrial DNA (mtDNA) have been reported in head and neck squamous cell carcinoma (SCC), differences in mtDNA copy numbers between normal and cancerous tissues from same patients have not been assessed. METHODS: We compared mtDNA copy numbers and expressions of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) and mitochondrial transcription factor A (TFAM) between normal mucous membrane and cancerous tissues resected from 35 patients with oral SCC, using TaqMan quantitative real-time polymerase chain reaction (PCR) and immunohistochemical staining. RESULTS: We found mtDNA copy numbers and expressions of PGC-1α and TFAM were decreased in cancerous tissues compared with normal tissues from the same patients. CONCLUSION: The PGC-1α-TFAM mitochondrial pathway may be associated with malignant potential in human oral SCC, and could be an attractive therapeutic target. © 2016 Wiley Periodicals, Inc. Head Neck 38:1170-1175, 2016.

Transcutaneous carbon dioxide suppresses epithelial-mesenchymal transition in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is the most common form of oral cancers. Recent studies have shown that the malignant transformation of various carcinomas, including OSCC, is associated with epithelial-mesenchymal transition (EMT), and that expression of the EMT factors are significantly associated with tumor invasion, tumor metastasis, and survival rates in OSCC patients. Hence, there is a possibility that EMT suppression may improve the prognosis of OSCC patients. Hypoxia inducible factor-1α (HIF-1α) is a crucial microenvironmental factor in tumor progression, which induces the expression of EMT factors. We previously reported that transcutaneous CO2 suppresses both human OSCC tumor growth and metastasis to the regional lymph nodes by improving hypoxia in treated tissue. According to this background, we hypothesized that increased EMT with HIF-1α expression may increase the progression and the metastatic potential of OSCC, and that decreased hypoxia by transcutaneous CO2 could suppress EMT. In the present study, in vitro studies showed that hypoxic conditions increased the expression of HIF-1α and EMT factors in OSCC cells. In addition, in vivo studies revealed that transcutaneous CO2 increased E-cadherin expression with the decreased expression of HIF-1α, Snail, Slug, N-cadherin, and Vimentin in tumor treatment. These results suggest that transcutaneous CO2 could suppress EMT by improving hypoxia, resulting in the reduction of metastatic potential of OSCC. The findings indicate that transcutaneous CO2 may be able to improve the prognosis of OSCC patients through the suppression of EMT.

Recombinant human SIRT1 protects against nutrient deprivation-induced mitochondrial apoptosis through autophagy induction in human intervertebral disc nucleus pulposus cells.

INTRODUCTION: Nutrient deprivation is a likely contributor to intervertebral disc (IVD) degeneration. Silent mating type information regulator 2 homolog 1 (SIRT1) protects cells against limited nutrition by modulation of apoptosis and autophagy. However, little evidence exists regarding the extent to which SIRT1 affects IVD cells. Therefore, we conducted an in vitro study using human IVD nucleus pulposus (NP) cells. METHODS: Thirty-two IVD specimens were obtained from patients who underwent surgical intervention and were categorized based on Pfirrmann IVD degeneration grades. Cells were isolated from the NP and cultured in the presence of recombinant human SIRT1 (rhSIRT1) under different serum conditions, including 10 % (v/v) fetal bovine serum (FBS) as normal nutrition (N) and 1 % (v/v) FBS as low nutrition (LN). 3-Methyladenine (3-MA) was used to inhibit autophagy. Autophagic activity was assessed by measuring the absorbance of monodansylcadaverine and immunostaining and Western blotting for light chain 3 and p62/SQSTM1. Apoptosis and pathway analyses were performed by flow cytometry and Western blotting. RESULTS: Cells cultured under LN conditions decreased in number and exhibited enhanced autophagy compared with the N condition. Medium supplementation with rhSIRT1 inhibited this decrease in cell number and induced an additional increase in autophagic activity (P < 0.05), whereas the combined use of rhSIRT1 and 3-MA resulted in drastic decreases in cell number and autophagy (P < 0.05). The incidence of apoptotic cell death increased under the LN condition, which was decreased by rhSIRT1 (P < 0.05) but increased further by a combination of rhSIRT1 and 3-MA (P < 0.05). Under LN conditions, NP cells showed a decrease in antiapoptotic Bcl-2 and an increase in proapoptotic Bax, cleaved caspase 3, and cleaved caspase 9, indicating apoptosis induction via the mitochondrial pathway. These changes were suppressed by rhSIRT1 but elevated further by rhSIRT1 with 3-MA, suggesting an effect of rhSIRT1-induced autophagy on apoptosis inhibition. Furthermore, the observed autophagy and apoptosis were more remarkable in cells from IVDs of Pfirrmann grade IV than in those from IVDs of Pfirrmann grade II. CONCLUSIONS: SIRT1 protects against nutrient deprivation-induced mitochondrial apoptosis through autophagy induction in human IVD NP cells, suggesting that rhSIRT1 may be a potent treatment agent for human degenerative IVD disease.