Kyoko Oura

Diabetes mellitus and metformin in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the leading cause of cancer-related death worldwide. Diabetes mellitus, a risk factor for cancer, is also globally endemic. The clinical link between these two diseases has been the subject of investigation for a century, and diabetes mellitus has been established as a risk factor for HCC. Accordingly, metformin, a first-line oral anti-diabetic, was first proposed as a candidate anti-cancer agent in 2005 in a cohort study in Scotland. Several subsequent large cohort studies and randomized controlled trials have not demonstrated significant efficacy for metformin in suppressing HCC incidence and mortality in diabetic patients; however, two recent randomized controlled trials have reported positive data for the tumor-preventive potential of metformin in non-diabetic subjects. The search for biological links between cancer and diabetes has revealed intracellular pathways that are shared by cancer and diabetes. The signal transduction mechanisms by which metformin suppresses carcinogenesis in cell lines or xenograft tissues and improves chemoresistance in cancer stem cells have also been elucidated. This review addresses the clinical and biological links between HCC and diabetes mellitus and the anti-cancer activity of metformin in clinical studies and basic experiments.

Galectin-9: An anticancer molecule for gallbladder carcinoma.

Gallbladder cancer (GBC) is the most common and aggressive type of biliary tract cancer. There are various histological types of GBC, and the vast majority of GBC cases are adenocarcinomas. Squamous and adenosquamous carcinomas are rare GBC subtypes that are traditionally considered to be more aggressive and to be associated with a poorer prognosis than adenocarcinoma. Galectin-9 (Gal-9), a tandem-repeat-type galectin, has been reported to induce apoptosis-mediated elimination of various cancers, including hepatocellular carcinoma, cholangiocarcinoma, and hematologic malignancies. Therefore, we investigated the antitumor effects of Gal-9 on GBC in vitro and in vivo. In our in vitro experiments, Gal-9 suppressed cell proliferation in various GBC cell lines but not in the OCUG-1 cell line, which represents a poorly differentiated type of adenosquamous carcinoma. Gal-9 induced the apoptosis of Gal-9-sensitive GBC cells by increasing the levels of caspase-cleaved keratin 18 and phosphorylated p53. However, Gal-9 did not affect the expression of various cell cycle-related proteins. In addition, Gal-9 suppressed tumor growth by implanted human GBC cells in a xenograft model. Furthermore, Gal-9 induced the phosphorylation of the Ephrin type-B receptor, and the microRNA (miRNA) expression profile was markedly altered by Gal-9. Based on these results, various miRNAs might contribute to the suppression of tumor growth. Our data reveal that Gal-9 suppresses the growth of GBC, possibly by inducing apoptosis and altering miRNA expression. Thus, Gal-9 might serve as a therapeutic agent for the treatment of GBC.

Cancer Therapy Due to Apoptosis: Galectin-9

Dysregulation of apoptosis is a major hallmark in cancer biology that might equip tumors with a higher malignant potential and chemoresistance. The anti-cancer activities of lectin, defined as a carbohydrate-binding protein that is not an enzyme or antibody, have been investigated for over a century. Recently, galectin-9, which has two distinct carbohydrate recognition domains connected by a linker peptide, was noted to induce apoptosis in thymocytes and immune cells. The apoptosis of these cells contributes to the development and regulation of acquired immunity. Furthermore, human recombinant galectin-9, hG9NC (null), which lacks an entire region of the linker peptide, was designed to resist proteolysis. The hG9NC (null) has demonstrated anti-cancer activities, including inducing apoptosis in hematological, dermatological and gastrointestinal malignancies. In this review, the molecular characteristics, history and apoptosis-inducing potential of galectin-9 are described.

Angiotensin receptor blocker telmisartan inhibits cell proliferation and tumor growth of cholangiocarcinoma through cell cycle arrest

Cholangiocarcinoma (CCA) is at an advanced stage at the time of its diagnosis, and developing a more effective treatment of CCA would be desirable. Angiotensin II type 1 (AT1) receptor blocker (ARB), telmisartan may inhibit cancer cell proliferation, but the mechanisms by which telmisartan affects various cancers remain unknown. In this study, we evaluated the effects of telmisartan on human CCA cells and to assess the expression of microRNAs (miRNAs). We studied the effects of telmisartan on CCA cells using two cell lines, HuCCT-1 and TFK-1. In our experiments, telmisartan inhibited the proliferation of HuCCT-1 and TFK-1 cells. Additionally, telmisartan induced G0/G1 cell cycle arrest via blockade of the G0 to G1 cell cycle transition. Notably, telmisartan did not induce apoptosis in HuCCT-1 cells. This blockade was accompanied by a strong decrease in cell cycle-related protein, especially G1 cyclin, cyclin D1, and its catalytic subumits, Cdk4 and Cdk6. Telmisartan reduced the phosphorylation of EGFR (p-EGFR) and TIMP-1 by using p-RTK and angiogenesis array. Furthermore, miRNA expression was markedly altered by telmisartan in HuCCT-1. Telmisartan inhibits tumor growth in CCA xenograft model in vivo. In conclusion, telmisartan was shown to inhibit human CCA cell proliferation by inducing cell cycle arrest.

Metformin-suppressed differentiation of human visceral preadipocytes: Involvement of microRNAs

Visceral adipose tissue contributes to the pathophysiology of metabolic syndrome. Metformin has been reported to suppress lipogenesis in a murine preadipocyte cell line. However, the effect of metformin on the differentiation of human visceral adipose tissue remains unknown. MicroRNAs (miRNAs or miRs) have been suggested as therapeutic targets because of their involvement in the differentiation and maturation of fatty cells. The aim of this study was to determine whether metformin suppresses the differentiation of human preadipocytes and to identify miRNAs associated with the regulation of lipid metabolism. Human visceral preadipocytes (HPrAD-vis) were preincubated in growth media and then cultured with differentiation media containing metformin for 1 or 2 weeks. Adipogenic differentiation of the cells was assessed by Oil Red O staining, and soluble adiponectin in the culture media was measured using an enzyme-linked immunosorbent assay. Cell proliferation was assessed using a WST-8 assay, and the gene and protein expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT-enhancer-binding protein α (C/EBPα) was determined by RT-qPCR and western blot analysis, respectively. miRNAs were profiled using human miRNA Oligo chips after total RNA was extracted and labeled. Oil Red O staining showed that metformin suppressed the accumulation of lipid droplets in HPrAD-vis cells. The adiponectin concentration in the culture media was also decreased in metformin-treated cells. The WST-8 assay revealed no effect on proliferation or growth inhibition following metformin treatment, although metformin suppressed the expression of PPARγ and C/EBPα. miRNA profiling further revealed differences between the metformin-treated group and control HPrAD-vis cells. Thus, the findings of the present study demonstrated that metformin suppressed the differentiation of human preadipocytes in vitro and altered the miRNA profile of these cells. Thus, the miRNAs whose expression levels were altered by metformin may contribute to the observed suppression of HPrAD-vis cell differentiation.

Galectin‑9 ameliorates fulminant liver injury

Fulminant hepatitis is a severe liver disease resulting in hepatocyte necrosis. Galectin-9 (Gal-9) is a tandem-repeat-type galectin that has been evaluated as a potential therapeutic agent for various diseases that regulate the host immune system. Concanavalin A (ConA) injection into mice results in serious, immune-mediated liver injury similar to human viral, autoimmune and fulminant hepatitis. The present study investigated the effects of Gal-9 treatment on fulminant hepatitis in vivo and the effect on the expression of microRNAs (miRNAs), in order to identify specific miRNAs associated with the immune effects of Gal-9. A ConA-induced mouse hepatitis model was used to investigate the effects of Gal-9 treatment on overall survival rates, liver enzymes, histopathology and miRNA expression levels. Histological analyses, TUNEL assay, immunohistochemistry and miRNA expression characterization, were used to investigate the degree of necrosis, fibrosis, apoptosis and infiltration of neutrophils and macrophages. Overall survival rates following ConA administration were significantly higher in Gal-9-treated mice compared with control mice treated with ConA + PBS. Histological examination revealed that Gal-9 attenuated hepatocellular damage, reduced local neutrophil infiltration and prevented the local accumulation of macrophages and liver cell apoptosis in ConA-treated mice. In addition, various miRNAs induced by Gal-9 may contribute to its anti-apoptotic, anti-inflammatory and pro-proliferative effects on hepatocytes. The results of the present study demonstrate that Gal-9 may be a candidate therapeutic target for the treatment of fulminant hepatitis.

Telmisartan inhibits hepatocellular carcinoma cell proliferation in vitro by inducing cell cycle arrest

Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver and the third leading cause of cancer-related death. Telmisartan, a widely used antihypertensive drug, is an angiotensin II type 1 (AT1) receptor blocker (ARB) that might inhibit cancer cell proliferation, but the mechanisms through which telmisartan affects various cancers remain unknown. The aim of the present study was to evaluate the effects of telmisartan on human HCC and to assess the expression of microRNAs (miRNAs). We studied the effects of telmisartan on HCC cells using the HLF, HLE, HepG2, HuH-7 and PLC/PRF/5 cell lines. In our experiments, telmisartan inhibited the proliferation of HLF, HLE and HepG2 cells, which represent poorly differentiated types of HCC cells. However, HuH-7 and PLC/PRF/5 cells, which represent well-differentiated types of HCC cells, were not sensitive to telmisartan. Telmisartan induced G0/G1 cell cycle arrest of HLF cells by inhibiting the G0-to-G1 cell cycle transition. This blockade was accompanied by a marked decrease in the levels of cyclin D1, cyclin E and other cell cycle-related proteins. Notably, the activity of the AMP-activated protein kinase (AMPK) pathway was increased, and the mammalian target of rapamycin (mTOR) pathway was inhibited by telmisartan treatment. Additionally, telmisartan increased the level of caspase-cleaved cytokeratin 18 (cCK18), partially contributed to the induction of apoptosis in HLF cells and reduced the phosphorylation of ErbB3 in HLF cells. Furthermore, miRNA expression was markedly altered by telmisartan in vitro. In conclusion, telmisartan inhibits human HCC cell proliferation by inducing cell cycle arrest.

Circulating microRNA-636 is associated with the elimination of hepatitis C virus by ombitasvir/paritaprevir/ritonavir

Hepatitis C virus (HCV) infection causes sustained inflammation and fibrosis. Several oral direct-acting antivirals (DAAs) including ombitasvir/paritaprevir/ritonavir (OBV/PTV/r) were recently developed for HCV elimination. The combination of DAAs brought a higher sustained viral response (SVR) rate to anti-HCV therapy compared to interferon (IFN)-based regimens. However, 5% of hepatitis C patients who undergo DAA therapy still suffer from a sustained HCV infection. MicroRNA (miRNA) is essentially interfering, endogenous noncoding RNA that has been investigated as a new biomarker for the response to DAA in hepatitis C patients. Here we used a miRNA array and real-time polymerase chain reaction (PCR) to determine the targetable miRNA before and 12 weeks after OBV/PTV/r treatment for refractory hepatitis C. We used replicon cells, in which genotype 1b type HCV is stably transfected in Huh7 cells, to determine whether miRNA can inhibit HCV replication. Among 2,555 miRNAs, three were significantly up-regulated and eight miRNAs were down-regulated in serum 12 weeks after OBV/PTV/r treatment. An unsupervised hierarchical clustering analysis, using Pearsons correlation, showed that the miRNA profiles between before and 12 weeks after OBV/PTV/r treatment were clustered separately. At 12 weeks after OBV/PTV, miR-636 was targeted among the eight down-regulated miRNAs, and the expression level of circulating miR-636 was significantly diminished. The amount of HCV-RNA was significantly diminished 48 hours after miR-636 inhibitor transfection in HCV replicon cells. In conclusion, miR-636 might be one of the essential targetable molecules in HCV patients who undergo DAA therapy and still suffer from a sustained HCV infection.

Serum galectin-9 levels are correlated to liver fibrosis regardless of chronic inflammation or hepatocellular carcinoma complications.

Chronic liver diseases progress from chronic inflammation to fibrosis to tumorigenesis. Galectin‐9, a β‐galactoside‐specific animal lectin, is indicated to contribute to all three steps of progression. The aim of this study was to determine which of the three steps was most dominant in elevating the serum galectin‐9 concentration and to test the possibility of galectin‐9 as a serum biomarker.

Fibrosis Staging Using Direct Serum Biomarkers is Influenced by Hepatitis Activity Grading in Hepatitis C Virus Infection

Background: Chronic liver diseases (CLDs) generally progress from inflammation to fibrosis and finally to carcinogenesis. Staging of liver fibrosis progression is inevitable for the management of CLD patients. The purpose of this study was to compare the diagnostic abilities of Wisteria floribunda agglutinin-positive Mac-2 binding protein (WFA-M2BP), Enhanced liver fibrosis (ELF) score, Fibrosis-4 index, and AST to platelet ratio index (APRI) based on histopathological analysis of liver biopsy samples, from patients with positive Hepatitis C Virus (HCV) infection. Methods: Japanese patients with HCV infection who underwent liver biopsy examinations were enrolled in this study. WFA-M2BP levels and ELF scores were calculated using preserved serum samples. The fibrosis staging and activity grading were assessed using a modified METAVIR score. Results: A total of 122 patients were enrolled; the cohort included 27 patients with stage 1, 66 with stage 2, 20 with stage 3, and nine with stage 4 fibrosis. All four biomarkers distinguished stage 3 and stage 2 fibrosis. ROC curves revealed that all four fibrosis biomarkers presented AUC values greater than 0.8. Each of the four biomarkers in stage 2 was significantly different between the activity grade 1 and 2 groups. Conclusion: Fib-4 index and APRI were comparable with WFA-M2BP and ELF score in the diagnosis of advanced liver fibrosis in Japanese patients with HCV infection. All four biomarkers of liver fibrosis were influenced by histopathological activity grading, which implies that liver biopsy should be the gold standard to evaluate liver fibrosis staging even though several noninvasive biomarkers have been investigated well.