Adipocytes contribute to tumor progression and invasion of peritoneal metastasis by interacting with gastric cancer cells as cancer associated fibroblasts.

BACKGROUND: Peritoneal metastasis (PM) is one of the most common causes of noncurative surgery and the most frequent recurrence pattern in gastric cancer (GC). During the process of PM, GC cells detached from primary tumor interact with human peritoneal mesothelial cells (HPMC) overlapped with adipose tissues such as the omentum or mesentery. Although the interaction with HPMC promotes the malignancy of GC, the role of adipose tissues remains unclear. AIMS: We aimed to clarify how adipose tissue are affected by adjacent primary tumors during the expression of adipokines and to elucidate whether GC cells transform adipocytes into CAFs in vitro. In addition, we investigated whether GC cells are affected by adipocytes in their ability to infiltrate. METHODS: We investigated the phenotypic conversion of adipocytes during the malignant process of GC cells in vivo and in vitro. We evaluated the expression levels of adiponectin in the omental adipose tissue of gastric cancer patients by western blotting. Following adipocytes/gastric cancer cells coculture, adipocyte markers, adiponectin receptors, and inflammatory cytokine markers were detected by real-time PCR and/or western blotting in the single-cultured and co-cultured adipocytes; cancer-associated fibroblast (CAF) markers were detected by immunofluorescence and western blotting in the single-cultured and co-cultured adipocytes; invasion assays were performed in single cultured and co-cultured MKN45 and OCUM. RESULTS: In omental adipose tissues that are situated close to the primary tumors, the expression of adiponectin tended to decrease in patients with subserosal or serosal invasion. By co-culturing with GC cells, adipocytes were dedifferentiated and the expression levels of CAF marker FSP1 and inflammatory cytokines, PAI-1 and IL-6, significantly increased (p < 0.05). Furthermore, GC cells co-cultured with adipocytes showed enhanced invasion ability. CONCLUSION: Our findings suggest that the phenotypic conversion of adipocytes may promote the malignancy of GC in the construction of the cancer microenvironment of PM.

Hypoxia-Induced CD36 Expression in Gastric Cancer Cells Promotes Peritoneal Metastasis via Fatty Acid Uptake.

BACKGROUND: The lipid scavenger receptor cluster of differentiation 36 (CD36) has been shown to have a pro-metastatic function in several cancers. Adipose tissue, a favorable site for peritoneal metastasis (PM) from gastric cancer (GC), promotes this process by providing free fatty acids (FFAs); however, the role of CD36 in PM progression from GC remains to be elucidated. MATERIALS AND METHODS: We evaluated CD36 expression in the GC cells under various conditions. CD36 overexpressing (CD36OE) MKN45 cells were prepared and their migration and invasive properties were assessed. A PM mouse model was used to investigate the biological effects of palmitic acid (PA) and CD36. Furthermore, we examined the clinical role of CD36 expression in 82 human PM samples by immunohistochemical staining. RESULTS: Hypoxia markedly increased CD36 expression in GC cells. In normoxia, only CD36OE MKN45 cells treated with PA showed an increase in migration and invasion abilities. An increased expression of active Rac1 and Cdc42 was observed, which decreased following etomoxir treatment. Conversely, hypoxia increased those capacities of both vector and CD36OE MKN45 cells. In a mouse model transplanted with CD36OE MKN45 cells, more peritoneal tumors were observed in the high-fat diet group than those in the normal diet group. In clinical samples, 80% of PM lesions expressed CD36, consistent with hypoxic regions, indicating a significant association with prognosis. CONCLUSION: Our findings indicate that a hypoxia in the peritoneal cavity induces CD36 expression in GC cells, which contributes to PM through the uptake of FFAs.

Crosstalk between cancer-associated fibroblasts and immune cells in peritoneal metastasis: inhibition in the migration of M2 macrophages and mast cells by Tranilast.

BACKGROUND: The role of tumor-stroma interactions in tumor immune microenvironment (TME) is attracting attention. We have previously reported that cancer-associated fibroblasts (CAFs) contribute to the progression of peritoneal metastasis (PM) in gastric cancer (GC), and M2 macrophages and mast cells also contribute to TME of PM. To elucidate the role of CAFs in TME, we established an immunocompetent mouse PM model with fibrosis, which reflects clinical features of TME. However, the involvement of CAFs in the immunosuppressive microenvironment remains unclear. In this study, we investigated the efficacy of Tranilast at modifying this immune tolerance by suppressing CAFs. METHODS: The interaction between mouse myofibroblast cell line LmcMF and mouse GC cell line YTN16 on M2 macrophage migration was investigated, and the inhibitory effect of Tranilast was examined in vitro. Using C57BL/6J mouse PM model established using YTN16 with co-inoculation of LmcMF, TME of resected PM treated with or without Tranilast was analyzed by immunohistochemistry. RESULTS: The addition of YTN16 cell-conditioned medium to LmcMF cells enhanced CXCL12 expression and stimulated M2 macrophage migration, whereas Tranilast inhibited the migration ability of M2 macrophages by suppressing CXCL12 secretion from LmcMF. In PM model, Tranilast inhibited tumor growth and fibrosis, M2 macrophage, and mast cell infiltration and significantly promoted CD8 + lymphocyte infiltration into the tumor, leading to apoptosis of cancer cells by an immune response. CONCLUSION: Tranilast improved the immunosuppressive microenvironment by inhibiting CAF function in a mouse PM model. Tranilast is thus a promising candidate for the treatment of PM.

Interleukin-17A derived from mast cells contributes to fibrosis in gastric cancer with peritoneal dissemination.

OBJECTIVES: Interleukin-17A (IL-17A) is pro-inflammatory cytokine and acts as profibrotic factor in the fibrosis of various organs. Fibrosis tumor-like peritoneal dissemination of gastric cancer interferes with drug delivery and immune cell infiltration because of its high internal pressure. In this study, we examined the relationship between IL-17A and tissue fibrosis in peritoneal dissemination and elucidated the mechanism of fibrosis induced by IL-17A using human peritoneal mesothelial cells (HPMCs) and a mouse xenograft model. METHODS: Seventy gastric cancer patients with peritoneal dissemination were evaluated. The correlation between IL-17A and fibrosis was examined by immunofluorescence and immunohistochemistry. A fibrosis tumor model was developed based on subcutaneous transplantation of co-cultured cells (HPMCs and human gastric cancer cell line MKN-45) into the dorsal side of nude mice. Mice were subsequently treated with or without IL-17A. We also examined the effect of IL-17A on HPMCs in vitro. RESULTS: There was a significant correlation between IL-17A expression, the number of mast cell tryptase (MCT)-positive cells, and the degree of fibrosis (r = 0.417, P < 0.01). In the mouse model, IL-17A enhanced tumor progression and fibrosis. HPMCs treated with IL-17A revealed changes to a spindle-like morphology, decreased E-cadherin expression, and increased α-SMA expression through STAT3 phosphorylation. Moreover, HPMCs treated with IL-17A showed increased migration. CONCLUSIONS: IL-17A derived from mast cells contributes to tumor fibrosis in peritoneal dissemination of gastric cancer. Inhibiting degranulation of mast cells might be a promising treatment strategy to control organ fibrosis.

Effect of Oral Branched-Chain Amino Acids and Glutamine Supplementation on Skeletal Muscle Atrophy After Total Gastrectomy in Rat Model.

BACKGROUND: Sarcopenia is closely related to short-term outcomes of surgery and long-term prognosis. After gastrectomy, a decrease in muscle strength occurs because of insufficient nutrient intake and disturbed digestive function. Branched-chain amino acids (BCAAs) and glutamine (Gln) play vital roles in the signaling pathways regulating protein synthesis and protein degradation. In this study, we investigated the effects of BCAA and Gln supplementation alone or in combination on skeletal muscle atrophy after total gastrectomy in a rat model. METHODS: Male Wistar rats were divided into five groups: (1) sham operation (n = 8); (2) total gastrectomized rats (TG [control group], n = 16); (3) TG with BCAA (TG-B, n = 16); (4) TG with Gln (TG-G, n = 16); and (5) TG with BCAA and Gln (TG-BG, n = 16). In all groups, body weight, muscle weight, and marker for muscle metabolism were examined. RESULTS: Weight gain was significantly greater in the TG-BG group (130.5%) than in the TG group (108.1%) at 15 wk (P < 0.05). The gastrocnemius muscle weight was significantly higher for TG-BG (2.84 g) than for TG (2.44 g) at 15 wk (P < 0.05). Western blotting indicated that atrogin-1 and MuRF1 levels were lower in the TG-BG group than in the TG group but were not suppressed in the TG-B or TG-G group. CONCLUSIONS: In a rodent sarcopenia model induced by TG, the administration of BCAA in combination with Gln more effectively inhibited muscle atrophy than the administration of BCAA or Gln alone.

The effect of HIF-1α and PKM1 expression on acquisition of chemoresistance.

BACKGROUND: In patients with gastric cancer, one of the greatest obstacles to effective chemotherapy is the development of chemoresistance. It has been previously reported that hypoxia-inducible factor-1 alpha (HIF-1α) is associated with acquisition of chemoresistance, and more recent studies have also noted an association of pyruvate kinase muscle 1 (PKM1) and chemoresistance. The purpose of this study was to identify the effect of HIF-1α and PKM1 expression on the development of acquired chemoresistance using a paclitaxel (PTX)-resistant gastric cancer cell line. MATERIALS AND METHODS: A cancer cell line resistant to PTX was established from MKN45 cells by stepwise exposure to drug (rMKN45-PTX). The expressions of HIF-1α, apoptosis, vascular endothelial growth factor (VEGF), multidrug transporters and glycolytic enzyme were examined by Western blotting, enzyme-linked immunosorbent assay and immunohistochemistry. We also assessed the tumor proliferation by subcutaneous tumor and peritoneal dissemination of mouse xenograft model. RESULTS: The resistance index was 6.1 by determining as the ratio of the 50% growth inhibition (IC50) of rMKN45-PTX/IC50 of MKN45. Expression of nuclear factor kappa B and HIF-1α was increased in rMKN45-PTX cells compared with the parent cells. Expression of Bax and caspase-3 was significantly downregulated, whereas expression of Bcl-xL, P-glycoprotein, multidrug resistance-associated protein and VEGF was increased in rMKN45-PTX. The expression level of PKM1 was upregulated in rMKN45-PTX, leading to an increase in the PKM1/PKM2 ratio. Using xenograft models, we demonstrated that mouse subcutaneous tumors derived from rMKN45-PTX were significantly larger than those derived from MKN45 cells. CONCLUSION: Under the stress of chemotherapeutic agent exposure, high expression of HIF-1α affects various downstream genes. Although the underlying mechanism is unknown, our data suggest that PKM1 is also a molecular target for gastric cancer treatment.

Sodium-glucose transporter as a novel therapeutic target in disease.

Glucose is the primary energy fuel of life. A glucose transporter, the sodium-glucose transporter (SGLT), is receiving attention as a novel therapeutic target in disease. This review summarizes the physiological role of SGLT in cerebral ischemia, cancer, cardiac disease, and intestinal ischemia, which has encouraged analysis of SGLT function. In cerebral ischemia and cardiomyopathy, SGLT-1 is involved in worsening of the injury. In addition, SGLT-1 promotes the development of cancer. On the other hand, SGLT-1 has a protective effect against cardiac and intestinal ischemia. Interestingly, SGLT-1 expression levels are increased in some diseased tissue, such as in cerebral ischemia and cancer. This suggests that SGLT-1 may have an important role in many diseases. This review discusses the potential of SGLT as a target for novel therapeutic agents.

Phosphodiesterase III inhibitor attenuates rat sinusoidal obstruction syndrome through inhibition of platelet aggregation in Disse's space.

BACKGROUND AND AIM: Sinusoidal obstruction syndrome (SOS) is a serious drug-induced liver injury. However, the pathophysiology of the disease remains unclear. This study investigated the effects of cilostazol (CZ), a phosphodiesterase III inhibitor, in a monocrotaline (MCT)-induced rat model of SOS. METHODS: Male Wistar rats were administrated MCT to induce SOS. Rats were divided into control, MCT, and MCT + CZ groups. In the MCT + CZ group, CZ was administered at 48 h, 24 h, and 30 min prior to and 8 h and 24 h after MCT administration. The MCT group was treated with water instead of CZ. At 48 h after MCT administration, blood and liver samples were collected to assess biochemistry and liver histology. Expression of rat endothelial cell antigen, CD34, CD41, P-selectin, and caspase-3 in the liver were analyzed. Plasminogen activator inhibitor-1 (PAI-1) in hepatocytes was analyzed using western blotting and polymerase chain reaction. RESULTS: In the MCT group, macroscopic findings showed a dark-red liver surface. Histological findings showed sinusoidal dilatation, coagulative necrosis of hepatocytes, and endothelial damage of the central vein. These changes were attenuated in the MCT + CZ group. Elevated serum transaminase and decreased platelet counts were observed in the MCT + CZ group compared with those in the MCT group. Treatment with CZ reduced MCT-induced damage to the liver sinusoidal endothelial cells, inhibited extravasated platelet aggregation, and suppressed hepatocyte apoptosis around the central vein. CZ attenuated hepatic PAI-1 protein and mRNA levels. CONCLUSIONS: Cilostazol attenuated MCT-induced SOS by preventing damage to liver sinusoidal endothelial cells and extravasated platelet aggregation. Hepatic PAI-1 levels were suppressed with CZ treatment.

DHA supplementation prevent the progression of NASH via GPR120 signaling.

Nonalcoholic steatohepatitis (NASH) is one of the most common liver diseases involving chronic accumulation of fat and inflammation, often leading to advanced fibrosis, cirrhosis and carcinoma. However, the pathological mechanism for this is unknown. GPR120/FFAR4 has been recognized as a functional fatty acid receptor and an attractive therapeutic target for metabolic diseases. In this study, we investigated the involvement of GPR120/FFAR4 in the pathogenesis of NASH. Mice fed with a 0.1% methionine and choline deficient high-fat (CDAHF) diet showed a significant increase in plasma aspartate transaminase and alanine transaminase levels, fatty deposition, inflammatory cell infiltration, and mild fibrosis. Docosahexaenoic acid (DHA, GPR120/FFAR4 agonist) suppressed the inflammatory cytokines in the liver tissues and prevented fibrosis in the wild type (WT) mice fed CDAHF diet, but not GPR120/FFAR4 deficient (GPR120KO) mice. GPR120KO mice fed CDAHF diet showed increment of the number of crown like structures and the immunoreactivity for F4/80 positive cells, and increased TNF-α mRNA in the liver compared to WT mice fed CDAHF diet. GPR120 KO mice fed CDAHF diet showed more severe liver inflammation than that of WT mice fed CDAHF diet, but not fibrosis. Our findings suggest that DHA supplementation could be prevented the development of NASH via GPR120/FFAR4 signaling. Furthermore, decrease of GPR120/FFAR4 signaling could be facilitated an inflammatory response in the process of NASH progression.

Importance of human peritoneal mesothelial cells in the progression, fibrosis, and control of gastric cancer: inhibition of growth and fibrosis by tranilast.

BACKGROUND: Scirrhous gastric cancer is an intractable disease with a high incidence of peritoneal dissemination and obstructive symptoms (e.g., ileus, jaundice, and hydronephrosis) arising from accompanying marked fibrosis. Microenvironmental interactions between cancer cells and cancer-associated fibroblasts are the suggested cause of the disease. We elucidated the mechanisms of tumor growth and fibrosis using human peritoneal mesothelial cells (HPMCs) and investigated the effects of tranilast treatment on cells and a xenograft mouse model of fibrosis. METHODS: HPMCs were isolated from surgically excised omentum and their interaction with MKN-45 gastric cancer cells was investigated using co-culture. Furthermore, a fibrosis tumor model was developed based on subcutaneous transplantation of co-cultured cells into the dorsal side of nude mice to form large fibrotic tumors. Mice were subsequently treated with or without tranilast. RESULTS: The morphology of HPMCs treated with transforming growth factor (TGF)-ß1 changed from cobblestone to spindle-type. Moreover, E-cadherin was weakly expressed whereas high levels of α-smooth muscle actin expression were observed. TGF-ß-mediated epithelial-mesenchymal transition-like changes in HPMCs were inhibited in a dose-dependent manner following tranilast treatment through inhibition of Smad2 phosphorylation. In the mouse model, tumor size decreased significantly and fibrosis was inhibited in the tranilast treatment group compared with that in the control group. CONCLUSIONS: Tranilast acts on the TGF-ß/Smad pathway to inhibit interactions between cancer cells and cancer-associated fibroblasts, thereby inhibiting tumor growth and fibrosis. This study supports the hypothesis that tranilast represents a novel strategy to prevent fibrous tumor establishment represented by peritoneal dissemination.

Spontaneous Tumor Lysis Syndrome in a Patient with a Dedifferentiated Endometrial Adenocarcinoma.

Tumor lysis syndrome (TLS) is an oncological emergency caused by massive cytolysis of malignant cells. This syndrome eventually induces metabolic abnormalities. TLS is observed mainly among tumors with rapid cell proliferation or high sensitivity to antineoplastic treatment. In rare cases, TLS occurs without any cytotoxic treatment. Previous reports have shown that alternative stress including proceeding infection or an operation might play a role in TLS. However, exact mechanism of spontaneous TLS remains unknown. Here, we describe a case of a 59-year-old woman who presented with dedifferentiated endometrial adenocarcinoma and developed TLS without any cytotoxic chemotherapy. Although spontaneous TLS in solid malignancies are extremely rare, clinicians should consider the possibilities of TLS especially in aggressive solid tumors.

Low-dose paclitaxel suppresses the induction of M2 macrophages in gastric cancer.

Tumor-associated macrophages of the M2 phenotype promote tumor proliferation and are associated with a poor prognosis in patients with various malignancies, including gastric cancer with peritoneal dissemination. The present study assessed whether paclitaxel (PTX) suppresses M2 macrophages, by acting as a Toll-like receptor 4 (TLR4) agonist. Macrophages derived from the THP-1 monocytic cell line and peripheral blood mononuclear cell (PBMC)-derived macrophages were cultured with gastric cancer cells in medium containing PTX, at a concentration that did not affect cell proliferation. The effects of PTX on macrophage expression of CD204, a marker of M2 macrophages and NOS2, a marker of M1 macrophages, was evaluated by western blotting. The ability of PTX to stimulate intranuclear translocation of NF-κB was determined by evaluating the expression of the p65 subunit of NF-κB. In THP-1 macrophages, low-dose PTX (1 and 5 nM) inhibited the expression of CD204, enhanced the expression of NOS2, and significantly suppressed the phosphorylation of Stat3, which is essential for the M2 phenotype. Low-dose PTX also inhibited CD204 expression in primary macrophages derived from PBMCs. PTX treatment of THP-1 macrophages for 1 h induced marked intranuclear translocation of NF-κB p65. Low-dose PTX inhibited the M2 phenotype and induced the M1 phenotype via TLR4 signaling, suggesting that low-dose PTX can alter the macrophage phenotype, whereas clinical doses can kill cancer cells. These results suggest that the anticancer effects of PTX are due both to its cytotoxic and immunomodulatory activities.

Extravasated platelet aggregation in the livers of rats with drug­induced hepatic sinusoidal obstruction syndrome.

Oxaliplatin-based chemotherapy plays an important role in the treatment of colorectal liver metastases. Oxaliplatin, however, causes sinusoidal obstruction syndrome (SOS), which is characterized by portal hypertension, splenomegaly, thrombocytopenia, and liver dysfunction. SOS is diagnosed histopathologically by disruption of the sinusoidal endothelium, collagen deposition, fibrosis especially around zone 3, dilatation of the sinusoidal space and congestion. This study assessed the characteristics of a rat model of SOS. SOS was induced in rats by administration of monocrotaline (MCT). Blood chemistries and macroscopic and microscopic findings were compared in rats administered MCT and vehicle (control group). Levels of expression in the liver of CD41, P­selectin, rat endothelial cell antigen­1, CD34, and cleaved caspase­3 were analyzed immunohistochemically. Moreover, livers of these rats were analyzed by electron microscopy. Macroscopically, MCT­treated rats showed accumulation of bloody ascites and blue liver and were diagnosed with SOS histologically. Serum concentrations of aspartate aminotransferase (P=0.003), alanine aminotransferase (P=0.008), total­bilirubin (P=0.012), direct­bilirubin (P=0.007), indirect­bilirubin (P=0.003), lactate dehydrogenase (P<0.001) and hyaluronic acid (P=0.016) were significantly higher, and platelet counts significantly lower (P=0.004), in MCT­treated than in control rats. The livers of MCT­treated rats were immunohistochemically positive for CD41 and P­selectin, suggesting platelet aggregates; for rat endothelial cell antigen­1 and CD34, suggesting sinusoidal endothelial disorder; and for cleaved caspase­3, suggesting hepatocyte apoptosis. Electron microscopic findings revealed platelet aggregation in the space of Disse in the MCT group. Extravasated platelet aggregation in Disse's space may be involved in the development of SOS.

Influence of hyperglycemia on liver inflammatory conditions in the early phase of non-alcoholic fatty liver disease in mice.

OBJECTIVES: A non-alcoholic fatty liver disease (NAFLD) has high prevalence and now important issue of public health. In general, there exists strong interaction between NAFLD and diabetes, but the detailed mechanism is unclear. In this study, we determined the effects of hyperglycemia on progression in the early phase of NAFLD in mice. METHODS: Male ddY mice were fed a choline-deficient, l-amino acid-defined, high-fat diet (CDAHFD) consisting of 60% of kcal from fat and 0.1% methionine by weight. Hyperglycemic condition was induced by streptozotocin (STZ) treatment. The assessment of liver function used serum AST and ALT levels, and histological analysis. Hepatic tumour necrosis factor (TNF)-α mRNA levels was estimated by qRT-PCR. KEY FINDINGS: During the 3-42 days that the mice were fed CDAHFD, the livers gradually caused accumulation of fat, and infiltration of inflammation cells gradually increased. Serum AST and ALT levels and significantly increased after being fed CDAHFD for 3 days and were exacerbated by the STZ-induced hyperglycemic condition. In addition, hepatic TNF-α mRNA also significantly increased. These phenomena reversed by insulin administration. CONCLUSIONS: The results showed that progression in the early phase of NAFLD may be exacerbated by hyperglycemia-induced exacerbation of inflammation.

RETRACTED: Cerebral ischemia-induced elevation of hepatic inflammatory factors accompanied by glucose intolerance suppresses hypothalamic orexin-A-mediated vagus nerve activation.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the authors following an investigation into data manipulation (Fig.3A-D and Fig.4A-F) by an Investigation Committee at Kobe Gakuin University. Namely: Fig.3A-D and Fig.4A-F ­ numerical disagreement (numbers removed) was found in some parts between the raw data and the article data, hence the significant difference illustrated in the published article was not obtained.

Establishing a xenograft mouse model of peritoneal dissemination of gastric cancer with organ invasion and fibrosis.

BACKGROUND: The clinical prognosis of gastric cancer with peritoneal dissemination is poor because of its chemoresistance and rich fibrosis. While several gastric cancer cell lines have been used to establish models of peritoneal dissemination by intraperitoneal injection, most peritoneal tumors that form adopt a medullary pattern in microscopic appearance. This histological finding for the model differs from that in the clinical situation. This study was performed to demonstrate the contribution of human peritoneal mesothelial cells (HPMCs) to fibrotic tumor formation and to establish a new xenograft model with high potential for peritoneal dissemination with organ invasion and extensive fibrosis. METHODS: We established four types of xenograft model: i) intraperitoneal injection of MKN45-P cells alone (control group), ii) injection of MKN45-P cells co-cultured with HPMCs (co-cultured group), iii) scratching the parietal peritoneum (parietal group), and iv) scratching the visceral peritoneum (visceral group) with a cotton swab before injection of co-cultured cells. Fibrosis, α-smooth muscle actin expression, and organ invasion by tumor cells were all assessed by immunohistochemical examination. RESULTS: All mice developed abdominal swelling with peritoneal tumors and bloody ascites. Tumors of the control and co-cultured groups were not invasive or fibrotic. Contrastingly, tumors of the scratch groups exhibited rich stromal fibrosis and possessed increased α-smooth muscle actin (α-SMA) expression. In particular, the visceral group showed edematous and spreading tumors invading the intestinal wall. CONCLUSION: We established a model of peritoneal dissemination with organ invasion and stromal fibrosis. Formation of peritoneal dissemination required a favorable environment for cell adhesion, invasion, and growth. This model may be useful for analyzing the pathogenesis and treatment of peritoneal dissemination of gastric cancer.

Valproic acid inhibits irradiation-induced epithelial-mesenchymal transition and stem cell-like characteristics in esophageal squamous cell carcinoma.

Esophageal carcinoma is one of the most aggressive malignancies, and is characterized by poor response to current therapy and a dismal survival rate. In this study we investigated whether irradiation induces epithelial-mesenchymal transition (EMT) in esophageal squamous cell carcinoma (ESCC) TE9 cells and whether the classic histone deacetylase (HDAC) inhibitor valproic acid (VPA) suppresses these changes. First, we showed that 2 Gy irradiation induced spindle cell-like morphologic changes, decreased expression of membranous E-cadherin, upregulated vimentin expression, and altered the localization of ß-catenin from its usual membrane-bound location to cytoplasm in TE9 cells. Irradiation induced upregulation of transcription factors including Slug, Snail, and Twist, which regulate EMT. Stimulation by irradiation resulted in increased TGF-ß1 and HIF-1α expression and induced Smad2 and Smad3 phosphorylation. Furthermore, irradiation enhanced CD44 expression, indicating acquisition of cancer stem-like cell properties. In addition, irradiation enhanced invasion and migration ability with upregulation of matrix metalloproteinases. These findings indicate that single-dose irradiation can induce EMT in ESCC cells. Second, we found that treatment with 1 mM VPA induced reversal of EMT caused by irradiation in TE9 cells, resulting in attenuated cell invasion and migration abilities. These results suggest that VPA might have clinical value to suppress irradiation-induced EMT. The reversal of EMT by HDAC inhibitors may be a new therapeutic strategy to improve the effectiveness of radiotherapy in ESCC by inhibiting the enhancement of invasion and metastasis.

Metformin inhibits the radiation-induced invasive phenotype of esophageal squamous cell carcinoma.

Esophageal cancer is one of the most aggressive tumor types because of its invasiveness and metastatic potential. Several reports have described an association between increased invasiveness after ionizing radiation (IR) treatment and epithelial-to-mesenchymal transition (EMT). The biguanide metformin is reported to prevent transforming growth factor-ß (TGF-ß)-induced EMT and proliferation of cancer. This study examined whether IR induces EMT and promotes the invasive potential of TE-9 esophageal squamous cell carcinoma cells and the effect of metformin on IR-induced EMT. After IR exposure, TE-9 cells showed a spindle-shaped morphology and lost cell-cell adhesion. Immunoblotting showed that IR induced expression of mesenchymal markers (vimentin and N-cadherin), transcription factors (Slug, Snail, and Twist), and matrix metalloproteinases. A scratch wound assay and Matrigel invasion assay showed that IR enhanced the invasive potential and migratory capacity of TE-9 cells. Expression of hypoxia-related factor-1α and TGF-ß was increased after IR. IR also induced phosphorylation of Smad2 and Smad3. Metformin inhibited radiation-induced EMT-like morphological changes, and enhanced invasion and migration of TE-9 cells. Metformin inhibited IR-induced phosphorylation of Smad2 and Smad3. Although phosphorylation of AMP-activated protein kinase was enhanced by IR and metformin, phosphorylation of mammalian target of rapamycin was enhanced by IR and suppressed by metformin. These results indicated that metformin suppressed IR-induced EMT via suppression of the TGF-ß-Smad phosphorylation pathway, and a part of the non-Smad pathway. Metformin might be useful to prevent IR-induced invasion and metastasis of esophageal squamous cell carcinoma.

Proteomic Profiling in the Spinal Cord and Sciatic Nerve in a Global Cerebral Ischemia-Induced Mechanical Allodynia Mouse Model.

Central post-stroke pain (CPSP) is one of the complications of cerebral ischemia and neuropathic pain syndrome. At present, there are few studies of pain in regions such as the spinal cord or sciatic nerve in cerebral ischemic animal models. To identify proteomic changes in the spinal cord and sciatic nerve in global cerebral ischemic model mice, in the present study we performed an investigation using proteomic methods. In a comparison between the intensity of protein spots obtained from a sham and that from a bilateral carotid artery occulusion (BCAO) in spinal cord and sciatic nerve, the levels of 10 (spinal cord) and 7 (sciatic nerve) protein spots were altered. The protein levels in the spinal cord were significantly increased in N(G),N(G)-dimethylarginine dimethylaminohydrolase 1 (DDAH1), 6-phosphogluconolactonase isoform 1, and precursor apoprotein A-I and decreased in dihydropyrimidinase-related protein 2 (CRMP-2), enolase 1B, rab guanosine 5'-diphosphate (GDP) dissociation inhibitor beta, septin-2 isoform a, isocitrate dehydrogenase subunit alpha, cytosolic malate dehydrogenase, and adenosine triphosphate synthase. The protein levels in the sciatic nerve were significantly increased in a mimecan precursor, myosin light chain 1/3, and myosin regulatory light chain 2 (MLC2), and decreased in dihydropyrimidinase-related protein 3 (CRMP-4), protein disulfide-isomerase A3, 3-hydroxy-3-methylglutaryl-coenzyme A synthase 1, and B-type creatine kinase. In addition, CRMP-2 and CRMP-4 protein levels were decreased, and DDAH1 and MLC2 protein levels were increased on day 1 after BCAO using Western blotting. These results suggested that changes in these proteins may be involved in the regulation of CPSP.