Lactate promotes survival and hepatocyte differentiation of human induced pluripotent stem cells in a medium without glucose and supplemented with galactose.

Human induced pluripotent stem (iPS) cells initiate hepatocyte differentiation in a medium without glucose and supplemented with galactose, oncostatin M and small molecules [hepatocyte differentiation inducer (HDI)]. To clarify the metabolic differences between iPS cells in HDI and ReproFF (undifferentiated state), a metabolome analysis was performed. iPS cells were cultured in a medium without glucose and supplemented with galactose, as well as 1 mM of calcium lactate, sodium lactate or lactic acid. After 7 days of culture, the cells were subjected to reverse transcription-quantitative PCR analysis. The galactose-1-phosphate concentration was significantly higher in cells cultured in HDI than in those cultured with ReproFF. The lactate concentration in the HDI group was significantly lower than that in the ReproFF group. The expression levels of α-feto protein and albumin were significantly higher in the groups cultured with calcium lactate, sodium lactate and lactic acid as compared with ReproFF. It was suggested that lactate promoted the survival of iPS cells cultured in a medium without glucose and supplemented with galactose. Under these conditions, iPS cells begin to differentiate into a hepatocyte lineage. Lactate may be applied to produce hepatocytes from iPS cells more efficiently.

Serum cytokine profiles and Mac-2 binding protein glycosylation isomer (M2BPGi) level in patients with autoimmune hepatitis.

Autoimmune hepatitis (AIH) is an autoimmune liver disease that is characterized by a progressive destruction of the liver parenchyma and the development of liver fibrosis. We aimed to examine the relationship between circulating cytokines/chemokines and the Mac-2 binding protein glycosylation isomer (M2BPGi) levels in Japanese patients with autoimmune hepatitis (AIH).We investigated the relationship between circulating cytokines/chemokines and M2BPGi levels in Japanese patients with AIH. Seventy-seven patients with well-documented AIH were enrolled in the National Hospital Organization (NHO)-AIH-liver-network database. We measured the serum levels of 20 cytokines in 31 selected AIH patients before and after steroid treatment using multisuspension cytokine array.Eleven cytokines and soluble adhesion molecules were increased in untreated AIH patients compared with treated AIH patients. Among these cytokines and soluble adhesion molecules, soluble intercellular adhesion molecule-1 (sICAM-1) and interferon-γ-inducible protein 10 (IP-10) were most downregulated by steroid therapy in AIH patients. We measured serum sICAM-1 and IP-10 by ELISA and found the levels were significantly higher in AIH patients (n = 77) compared with chronic viral hepatitis C patients (n = 32). Furthermore, there was a positive correlation between sICAM-1 or IP-10 and alanine aminotransferase, total bilirubin, and circulating M2BPGi levels. M2BPGi levels were increased in AIH patients with high stages of liver fibrosis. Additionally, M2BPGi levels were correlated with the histological grade of inflammation in AIH. Circulating M2BPGi levels were significantly reduced by steroid treatment in AIH patients.sICAM-1 and IP-10 are useful markers to assess immune-mediated hepatitis activity in AIH and they correlate with circulating M2BPGi. Serum M2BPGi levels increased in untreated AIH patients with active hepatitis and were decreased by steroid therapy. M2BPGi reflects autoimmune-mediated hepatic inflammation as well as liver fibrosis.

Oct3/4 is potentially useful for the suppression of the proliferation and motility of hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) cells are immature compared with healthy mature hepatocytes. Transcription factors serve a role in hepatocyte differentiation. The expression levels of transcription factors in HCC cell lines have been investigated to determine potential therapeutic targets. In the present study, the HLE, HLF, PLC/PRF/5, Huh-7, Hep3B, Huh-6 and HepG2 HCC cell lines were subjected to reverse-transcription polymerase chain reaction (RT-PCR) of transcription factors, including NANOG, Oct3/4, GATA binding protein 4 (GATA4), GATA6 and hematopoietically expressed homeobox (HHEX). In addition, these cell lines were analyzed using RT-quantitative PCR (RT-qPCR) of NANOG and Oct3/4. The 201B7 human induced pluripotent stem cells were evaluated as a model of pluripotent cells. The HLF cells were transfected with Oct3/4 small interfering RNA (siRNA) and used in an MTS colorimetric assay and a scratch assay. NANOG was not expressed in any of the cell lines. However, GATA4, GATA6 and HHEX were expressed in the majority of the HCC cell lines. In addition, NANOG and Oct3/4 were expressed in 201B7 cells. Oct3/4 was expressed in HLE, HLF and Hep3B cells; however, its expression levels were significantly reduced compared with those in 201B7 cells. RT-qPCR demonstrated that the expression of Oct3/4 siRNA suppressed the proliferation and motility of HLF cells. Oct3/4 siRNA may be a potentially effective therapy for the suppression of the proliferation and motility of HCC cells.

Association of a single nucleotide polymorphism in TNIP1 with type-1 autoimmune hepatitis in the Japanese population.

Several studies reported that autoimmune diseases share a number of susceptibility genes. Of these genes, a SNP rs7708392 in TNIP1 was reported to be associated with systemic lupus erythematosus (SLE). Autoimmune hepatitis (AIH), a rare chronic progressive liver disease, shares some clinical features with SLE. Therefore, we investigated whether the SNP is associated with Japanese AIH. An association study of rs7708392 was conducted in 343 Japanese AIH patients and 828 controls. We found that rs7708392 is associated with AIH (P = 0.0236, odds ratio (OR) 1.26, 95% confidence interval (CI): 1.03-1.54), under the allele model for C allele. Significant differences of clinical characteristics of the AIH patients with or without G allele of rs7708392 were not detected. Of interest, the association was stronger in AIH without HLA-DRB1*04:05 allele (P = 0.0063, Q = 0.0127, OR 1.48, 95% CI: 1.12-1.96), though the association was not detected in AIH with DRB1*04:05. The C allele of rs7708392 was associated with AIH, especially AIH without DRB1*04:05, an already established risk factor.

Comparison of DWIBS/T2 image fusion and PET/CT for the diagnosis of cancer in the abdominal cavity.

Fusion images of diffusion-weighted whole-body imaging with background body signal suppression and T2-weighted image (DWIBS/T2) demonstrate a strong signal for malignancies, with a high contrast against the surrounding tissues, and enable anatomical analysis. In the present study, DWIBS/T2 was compared with 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) for diagnosing cancer in the abdomen. Patient records, including imaging results of examination conducted between November 2012 and May 2014, were analyzed retrospectively. In total, 10 men (age, 73.6±9.6 years) and 8 women (age, 68.9±7.1 years) were enrolled into the current study. Of the enrolled patients, 2 were diagnosed with hepatocellular carcinoma, 1 with cholangiocellular carcinoma, 1 with liver metastasis, 2 with pancreatic ductal adenocarcinoma, 1 with renal cell carcinoma and 1 with malignant lymphoma. Benign lesions were also analyzed, including adenomyomatosis of the gallbladder (5 patients), intraductal papillary mucinous neoplasm (4 patients) and right adrenal adenoma (1 case). All the patients with cancer showed positive results on DWIBS/T2 images. However, only 7 out of 8 patients were positive with PET/CT. One patient with right renal cellular carcinoma was positive with DWIBS/T2, but negative with PET/CT. All the patients with benign lesions were negative with DWIBS/T2 and PET/CT. In conclusion, DWIBS/T2 was more sensitive in diagnosing cancer of organs in the abdominal cavity compared with PET/CT. Furthermore, negative results with DWIBS/T2 and PET/CT were useful for the diagnosis of benign lesions, such as adenomyomatosis of the gallbladder and intraductal papillary mucinous neoplasm.

Diffusion-weighted whole-body magnetic resonance imaging with background body signal suppression/T2 image fusion for the diagnosis of acute cholecystitis.

Prompt and accurate diagnosis is critical in the treatment of acute cholecystitis. Diffusion-weighted whole-body magnetic resonance imaging with background body signal suppression/T2 image fusion (DWIBS/T2) identifies areas with high signal intensity, corresponding to inflammation. In the present study, the records and images of patients with acute cholecystitis who underwent DWIBS/T2 between January 2013 and March 2014 were retrospectively analyzed. A total of 11 patients with acute cholecystitis were enrolled. In one patient, DWIBS/T2 identified a thickened wall and high signal intensity, with high signal intensity in the pericholecystic space that suggested localized peritonitis. Positive DWIBS/T2 results indicating acute cholecystitis were obtained in 10/11 patients, with a sensitivity of 90.9%. In addition, wall thickening and high signal intensity were absent in DWIBS/T2 images when wall thickening was not detected by computed tomography. Wall thickening and high signal intensity was attenuated when patients with acute cholecystitis were clinically treated. These data suggest that a thickened gallbladder wall and high signal intensity are indicative of acute cholecystitis and that DWIBS/T2 may be a useful technique in evaluating the severity of acute cholecystitis.

Diagnosis of complications associated with acute cholecystitis using computed tomography and diffusion-weighted imaging with background body signal suppression/T2 image fusion.

In a clinical setting, it is important to diagnose complications of acute cholecystitis accurately. Diffusion-weighted whole body imaging with background body signal suppression/T2-weighted image fusion (DWIBS/T2) provides high signal intensity with a strong contrast against surrounding tissues in anatomical settings. In the present study, patients who were being treated for acute cholecystitis and underwent DWIBS/T2 in the National Hospital Organization Shimoshizu Hospital between December 2012 and August 2015 were enrolled. A total of 10 men and 4 women underwent DWIBS/T2. Records, including DWIBS/T2 and computed tomography (CT) imaging, were retrospectively analyzed for patients with acute cholecystitis. CT images revealed thickened gallbladder walls in patients with acute cholecystitis, and high signal intensity was observed in DWIBS/T2 images for the thickened gallbladder wall. Inflammation of the pericholecystic space and the liver resulted in high intensity signals with DWIBS/T2 imaging, whereas CT imaging revealed a low-density area in the cholecystic space. Plain CT scanning identified a low-density area in the liver, which became more obvious with contrast-enhanced CT. DWIBS/T2 imaging showed the inflammation of the liver and pericholesyctic space as an area of high signal intensity. Detectability of inflammation of the pericholecystic space and the liver was the same for DWIBS/T2 and CT, which suggests that DWIBS/T2 has the same sensitivity as CT scanning for the diagnosis of complicated acute cholecystitis. However, the strong contrast shown by DWIBS/T2 allows for easier evaluation of acute cholecystitis than CT scanning.

Hepatocyte selection medium-enriched hepatocellular carcinoma cells are positive for α-fetoprotein and CD44.

Tissues surrounding hepatocellular carcinomas (HCCs) lack glucose. Hepatocyte selection medium (HSM) is deficient in glucose and is supplemented with galactose. HCC cells were cultured in HSM to investigate the stem cell markers α-fetoprotein (AFP) and cluster of differentiation 44 (CD44). HCC cells (HLF and PLC/PRF/5 cells) were cultured in HSM. Viable cell numbers were determined on days 0 and 7 following culture in HSM. RNA was isolated and subjected to reverse transcription-quantitative PCR (RT-qPCR) to analyze the mRNA expression levels of AFP and CD44. Immunostaining was performed to analyze the protein levels of AFP and CD44. The number of viable cells was significantly decreased on day 7 following culture in HSM. The expression levels of AFP and CD44 increased on day 7 as assessed using RT-qPCR. Immunostaining confirmed the results of RT-qPCR analysis. The number of viable HCC cells was decreased in HSM, whereas the expression levels of AFP and CD44 increased. Therefore, HSM is potentially useful for the enrichment of HCC cells with cancer stem cell characteristics.

Immunosuppressive agents are associated with peptic ulcer bleeding.

Peptic ulcer bleeding can be fatal. Non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids and immunosuppressive agents are administered for long-term usage. The present study assessed the association between peptic ulcer bleeding and administration of NSAIDs, corticosteroids and immunosuppressive agents. Furthermore, the efficacy of lowering the risk of peptic ulcer bleeding with proton pump inhibitors (PPI) and histamine 2 receptor antagonists (H2RA) was evaluated. Medical records were retrospectively analyzed for patients subjected to an upper gastrointestinal (GI) endoscopy performed at the National Hospital Organization Shimoshizu Hospital (Yotsukaido, Japan) from October 2014 to September 2015. During this period, a total of 1,023 patients underwent an upper GI endoscopy. A total of 1,023 patients, including 431 males (age, 68.1±12.9 years) and 592 females (age, 66.4±12.3 years), who had been administered NSAIDs, corticosteroids, immunosuppressive agents, PPIs and H2RAs, were respectively enrolled. Endoscopic findings of the patients were reviewed and their data were statistically analyzed. Logistic regression analysis was used to determine the odds ratio of peptic ulcer bleeding for each medication; immunosuppressive agents had an odds ratio of 5.83, which was larger than that for NSAIDs (4.77). The Wald test was applied to confirm the correlation between immunosuppressive agents and peptic ulcer bleeding. Furthermore, χ2 tests were applied to the correlation between peptic ulcer bleeding and administration of PPIs or H2RAs. Immunosuppressive agents had the largest χ2, and the P-value was 0.03. Administration of PPIs was significantly correlated with non-peptic ulcer bleeding (P=0.02); furthermore, a tendency toward non-peptic ulcer bleeding with administration of H2RA was indicated, but it was not statistically significant (P=0.12). In conclusion, immunosuppressive agents were correlated with peptic ulcer bleeding and PPIs were effective at lowering the risk of peptic ulcer bleeding.

Abdominal ultrasonography for patients with abdominal pain as a first-line diagnostic imaging modality.

The utility and limitations of abdominal ultrasonography (US) were retrospectively evaluated as a first-line diagnostic imaging modality in patients with abdominal pain. Hospital records from patients subjected to abdominal US as a first-line diagnostic imaging examination at the National Hospital Organization Shimoshizu Hospital (Yotsukaido, Japan) from April 2010 to April 2015 were analyzed. Only those patients who underwent abdominal US to diagnose abdominal symptoms were included in the present study. All patients with prior diagnostic imaging examination findings were excluded from the study in order to reduce bias of results. The analyzed patients included 39 males with an average (mean ± standard deviation) age of 65.8±18.8 years and 37 females with an average age of 53.7±19.3 years. Diagnosis with abdominal US was in agreement with the final diagnosis in 66 of the 76 patients. Final diagnosis of symptoms by abdominal US was not successful in the remaining 10 patients who required further investigation. Acute cholangitis, acute cholecystitis, acute pancreatitis, acute appendicitis, colonic diverticulitis and spleen rupture were correctly diagnosed. Different types of cancer, including colorectal cancer, were also successfully diagnosed. Bile duct cancer and sigmoid colon volvulus could not be diagnosed by abdominal US due to the presence of intestinal gas. Abnormal findings were detected using abdominal US, but the diagnosis required additional consultation with gynecologists. Abdominal US was suitable for patients with abdominal symptoms. It is recommended that patients undergo further diagnostic imaging or consultation with gynecologists when large gas bubbles are present or gynecological conditions are suspected.

Comparison of acute cholangitis with or without common bile duct dilatation.

To improve the management of patients with acute cholangitis, the present study compared laboratory test variables between acute cholangitis patients with or without common bile duct (CBD) dilatation [CBDdil(+) and CBDdil(-), respectively]. The medical records of patients diagnosed with acute cholangitis and subjected to endoscopic retrograde cholangiopancreatography between February 2008 and May 2015 were retrospectively analyzed. The present study consisted of 40 men (aged 69.4±8.8 years) and 37 women (aged 68.8±11.6 years). It was observed that CBDdil(-) patients were slightly younger than CBDdil(+) patients (P=0.0976), and levels of C-reactive protein (CRP) were significantly higher in CBDdil(-) patients than in CBDdil(+) patients (P=0.0392). In addition, logistic regression analysis indicated that CRP levels were associated with the presence of CBD dilatation (P=0.0392). These data indicate that patients with acute cholangitis without CBD dilatation tend to be younger and have higher levels of CRP. Thus, in acute cholangitis patients without CBD dilatation, diagnosis should be determined using clinical symptoms and laboratory data.

Diagnostic accuracy of diffusion-weighted whole-body imaging with background body signal suppression/T2-weighted image fusion for the detection of abdominal solid cancer.

Diffusion-weighted whole-body imaging with background body signal suppression (DWIBS) images show significant contrast for cancer tissues against non-cancerous tissues. Fusion of a DWIBS and a T2-weighted image (DWIBS/T2) can be used to obtain functional, as well as anatomic, information. In the present study, the performance of DWIBS/T2 in the diagnosis of abdominal solid cancer was evaluated. The records of 14 patients were retrospectively analyzed [5 patients with hepatocellular carcinoma (HCC), 4 with metastatic liver cancer, 3 with pancreatic cancer, 1 with renal cellular carcinoma and 1 with malignant lymphoma of the para-aortic lymph node]. T1WI and T2WI scans did not detect pancreatic cancer in certain cases, whereas DWIs and DWIBS/T2 clearly demonstrated pancreatic cancer in all cases. In addition, metastatic liver cancer and HCC were successfully detected with abdominal US and CECT; however, US did not detect pancreatic cancer in 1 case, while CECT and DWIBS/T2 detected pancreatic cancer in all cases. In conclusion, the diagnostic performance of DWIBS/T2 was the same as that of abdominal US and CECT in detecting primary and metastatic liver cancer. DWIBS/T2 enabled the diagnosis of pancreatic cancer in cases where it was not detected with US, T1WI or T2WI.

CCAAT/enhancer-binding protein α decreases the viability of gastric cancer cells.

CCAAT/enhancer-binding protein (C/EBP) α, C/EBPß and C/EBPδ are involved in inflammation and cell differentiation. In the present study, their roles in human gastric cancer cells were investigated. The human gastric cancer cell lines MKN45 and MKN74 were subjected to the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to analyze the expression levels of C/EBPα, C/EBPß and C/EBPδ. The cells were transfected with expression plasmids for either C/EBPα or C/EBPδ, and subjected to a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay and RT-qPCR for analysis of cyclin D1 expression. Expression levels of C/EBPα and C/EBPδ were decreased in MKN45 and MKN74 cells compared with in normal gastric tissue. Expression levels of C/EBPß were decreased in MKN45 cells and increased in MKN74 cells. Viability of MKN45 cells was decreased by C/EBPα and C/EBPδ. Viability of MKN74 cells was decreased by C/EBPα, but increased by C/EBPδ. Expression levels of cyclin D1 were decreased in association with C/EBPα and C/EBPδ overexpression in MKN45 cells. Expression levels of cyclin D1 were decreased in association with C/EBPα overexpression, but increased in association with C/EBPδ overexpression, in MKN74 cells. The results of the present study indicate that C/EBPα is potentially useful for the treatment of gastric cancer.

Introduction of plasmids into gastric cancer cells by endoscopic ultrasound.

Short hairpin RNA of frizzled-2 (shRNA-Fz2) suppresses the cell proliferation of gastric cancer cells. Endoscopic ultrasound (EUS) is considered a suitable method for the introduction of therapeutic plasmids into cells, since the device enables the access and real-time monitoring of gastric cancer tissues. In the present study, plasmids were introduced into cells by sonoporation, as evidenced by the production of H2O2. The production of H2O2 was measured by absorbance of a potassium-starch solution irradiated with EUS. Luciferase activity was analyzed in the cells irradiated with EUS after the addition of a pMetLuc2-control in the media, and cell proliferation was analyzed using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt assay after irradiation with EUS following the addition of shRNA-Fz2. Absorbance levels corresponding to free radical levels were found to be higher in the cells irradiated with EUS. Luciferase activities were found to be significantly higher in the transfected cells (plasmid with Lipofectamine LTX) than in untreated cells and were furthermore found to be higher in MKN45 cells irradiated for 0.5 min than in cells not subjected to irradiation. Luciferase activity was also found to be higher in MKN74 cells irradiated for 2 min than in cells that were not irradiated. Although the cell proliferation of the MKN45 cells tended to be suppressed by irradiation with EUS, this was non-significant suppression, while the cell proliferation of MKN74 cells was found to be suppressed by irradiation with 12 MHz for 2 min (P<0.05). In conclusion, plasmids were introduced into cultured gastric cancer cells by irradiation with EUS due to sonoporation, as evidenced by the production of H2O2; however, the efficiency of the plasmid introduction was low compared with a traditional transfection approach.

Differentiation of human induced pluripotent stem cells in William's E initiation medium supplemented with 3­bromopyruvate and 2­deoxy­d­glucose.

Hepatocyte selection medium (HSM) is deprived of glucose and supplemented with galactose, and is based on Leibovitz's­15 (L15) medium. HSM may promote the differentiation of human induced pluripotent stem (iPS) cells towards hepatocyte lineage. These culture conditions result in increased expression of galactokinase (GALK)­1 and GALK2. However, iPS cells do not survive in HSM. Two potential alternatives to glucose deprivation are treatment with 3­bromopyruvate (3BP), an analogue of pyruvate, and 2­deoxy­d­glucose (2DG), an analogue of glucose. The promoters of GALK1 and GALK2 were subcloned using the pMetLuc2 reporter plasmid to make pMetLuc2/GALK1 and pMetLuc2/GALK2, respectively. 201B7 human iPS cells were transfected with the reporter plasmids, cultured in HSM and analyzed by luciferase assay. Furthermore, 201B7 cells were cultured in L15, William's E (WE) or Dulbecco's modified Eagle's medium/nutrient mixture F­12 Ham (DF12) supplemented with 3BP, 2DG or a combination of the two, for 15 days, and subjected to reverse transcription­quantitative polymerase chain reaction to measure the levels of α­fetoprotein (AFP) mRNA expression. Metridia luciferase activity was significantly higher in cells cultured in HSM compared with those in ReproFF medium (P<0.05). 3BP and 2DG treatment, alone or in combination, decreased AFP expression levels in cells cultured in L15 and DF12. The combination of 3BP+2DG increased the expression levels of AFP in WE. Without 3BP or 2DG, AFP expression was higher in L15 compared with WE or DF12. The promoters of GALK1 and GALK2 were activated in 201B7 cells cultured in HSM, enabling survival using galactose as an energy source. 3BP and 2DG supplementation in WE medium may promote the differentiation of iPS cells to the hepatocyte lineage.

Proliferation and motility of hepatocellular, pancreatic and gastric cancer cells grown in a medium without glucose and arginine, but with galactose and ornithine.

Human primary hepatocytes are able to survive in a medium without glucose and arginine, but supplemented with galactose and ornithine (hepatocyte selection medium; HSM). To address the possibility of the application of HSM in cancer therapy, hepatocellular carcinoma cells, pancreatic cancer cells and gastric cancer cells were cultured in HSM. Cell proliferation was analyzed using an MTS assay. Morphological changes were analyzed using hematoxylin and eosin staining. Apoptosis was analyzed using a TUNEL assay and cell motility was assessed with a scratch assay. Cell proliferation was significantly suppressed in cell lines grown in HSM (P<0.01 in all the cell lines). Hematoxylin and eosin staining revealed pyknotic nuclei, suggesting that these cells had undergone apoptosis. The number of TUNEL-positive cells was significantly increased in HSM. In the scratch assay, the distance between the growing edge and the scratched edge was significantly lower (P<0.01 in all the cell lines) in cells cultured in HSM, compared with those grown in Dulbecco's modified Eagle's medium or RPMI-1640. Therefore, the proliferation and motility of hepatocellular carcinoma cells, pancreatic cancer cells and gastric cancer cells was suppressed, and these cells subsequently underwent apoptosis in a medium without glucose and arginine, but containing galactose and ornithine.

Proliferation of sphere-forming hepatocellular carcinoma cells is suppressed in a medium without glucose and arginine, but with galactose and ornithine.

Resistance to sorafenib in hepatocellular carcinoma (HCC) cells exhibiting stemness was evaluated using a sphere formation assay. A hepatocyte selection medium (HSM) deficient in glucose and arginine was used to suppress the proliferation of cell spheres composed of HLF and PLC/PRF/5 HCC cells, which were subjected to a sphere formation assay. Cell spheres were cultured with sorafenib and subjected to a cell proliferation assay and the expression levels of cytochrome P450 (CYP3A4) were analyzed in RNA extracted from sphere-forming cells using reverse transcription-quantitative polymerase chain reaction. Sphere-forming PLC/PRF/5 cells were more resistant to sorafenib, as compared with control cells, exhibiting higher expression levels of CYP3A4. When cultured in HSM, suppressed proliferation was observed in the sphere-forming PLC/PRF/5 cells and in the control cells, with no significant variation between them. The results suggest that deprivation of glucose and arginine is a potential novel treatment for HCC.

2-Deoxyglucose and sorafenib synergistically suppress the proliferation and motility of hepatocellular carcinoma cells.

Cancer cells consume more glucose than normal cells, mainly due to their increased rate of glycolysis. 2-Deoxy-d-glucose (2DG) is an analogue of glucose, and sorafenib is a kinase inhibitor and molecular agent used to treat hepatocellular carcinoma (HCC). The present study aimed to demonstrate whether combining 2DG and sorafenib suppresses tumor cell proliferation and motility more effectively than either drug alone. HLF and PLC/PRF/5 HCC cells were incubated with sorafenib with or without 1 µM 2DG, and subjected to a proliferation assay. A scratch assay was then performed to analyze cell motility following the addition of 2DG and sorafenib in combination, and each agent alone. RNA was isolated and subjected to reverse transcription-quantitative polymerase chain reaction to analyze the expression of cyclin D1 and matrix metalloproteinase-9 (MMP9) following the addition of 2DG and sorafenib in combination and each agent alone. Proliferation was markedly suppressed in cells cultured with 1 µM 2DG and 30 µM sorafenib compared with cells cultured with either agent alone (P<0.05). In addition, levels of Cyclin D1 expression decreased in cells exposed to 3 µM sorafenib and 1 µM 2DG compared with cells exposed to 2DG or sorafenib alone (P<0.05). Scratch assay demonstrated that the distance between the growing edge of the cell sheet and the scratched line was shorter in cells cultured with sorafenib and 2DG than in cells cultured with 2DG or sorafenib alone (P<0.05). Levels of MMP9 expression decreased more in cells treated with both sorafenib and 2DG than in cells treated with 2DG or sorafenib alone (P<0.05). Therefore, 2DG and sorafenib in combination suppressed the proliferation and motility of HCC cells more effectively than 2DG or sorafenib alone, and a cancer treatment combining both drugs may be more effective than sorafenib alone.

Diffusion-weighted whole-body imaging with background body signal suppression/T2 image fusion for the diagnosis of colorectal polyp and cancer.

Diffusion-weighted whole-body imaging with background body signal suppression/T2 image fusion (DWIBS/T2) is useful for the diagnosis of cancer as it presents a clear contrast between cancerous and non-cancerous tissue. The present study investigated the limitations and advantages of DWIBS/T2 with regards to the diagnosis of colorectal polyp (CP) or cancer (CRC). The current study included patients diagnosed with CP or CRC following colonoscopy, who were subjected to DWIBS/T2 between July 2012 and March 2015. Patient records were analyzed retrospectively. Patients were subjected to DWIBS/T2 when they presented with abdominal cancers or inflammation. Colonoscopy was performed as part of screening, or if patients had suspected colon cancer or inflammatory bowel disease. A total of 8 male and 7 female patients were enrolled in the present study. All patients, with the exception of one who had been diagnosed with CRC following colonoscopy, had positive results and all patients diagnosed with CP following a colonoscopy, with the exception of one, had negative results on DWIBS/T2. Thus, CRC was detected by DWIBS/T2, while CP was not (P=0.0028). The diameter of CRC lesions was significantly larger than that of CP (P<0.0001) and that of lesions positive on DWIBS/T2 was significantly larger than that of negative lesions (P=0.0004). The depth of invasion tended to be greater for lesions positive on DWIBS/T2 compared with that of negative ones. This indicated that DWIBS/T2 may be suitable for the detection of CRC but not for detection of CP. The results of DWIBS/T2 may also be affected by lesion diameter and depth of invasion.

Oncostatin M in William's E medium is suitable for initiation of hepatocyte differentiation in human induced pluripotent stem cells.

William's E (WE) is a suitable medium for the differentiation of human induced pluripotent stem (iPS) cells to the hepatocyte lineage. The aim of the present study was to investigate various growth factors in their ability to promote hepatocyte differentiation of iPS cells in WE medium. Human iPS 201B7 cells were cultured in WE medium supplemented with growth factors, and mRNA expression levels and promoter activities of α­fetoprotein (AFP) and albumin were examined by reverse transcription­quantitative polymerase chain reaction and luciferase assay, respectively. In addition, time course analysis of AFP mRNA expression was performed in 201B7 cells cultured in WE medium supplemented with oncostatin M. The results demonstrated that mRNA expression levels of AFP were significantly elevated by most growth factors tested as supplements in WE medium, except all­trans retinoic acid, compared with cells cultured in ReproFF (a medium that maintains pluripotency). The highest increase in AFP mRNA expression levels was observed by oncostatin M stimulation. Albumin mRNA expression levels were increased by all­trans retinoic acid and insulin­transferrin­selenium supplementation in WE medium compared with cells cultured in ReproFF. Oncostatin M supplementation significantly stimulated the promoter activity of the AFP gene, but no growth factor tested significantly stimulated the promoter activity of the albumin gene. By time course analysis, significant increase of AFP mRNA expression was observed on the sixth day post­stimulation, compared with cells cultured in WE medium alone. In conclusion, the present study demonstrated that oncostatin M supplementation in WE medium was sufficient to initiate hepatocyte differentiation in iPS cells.