Asahiro Morishita

miRNA in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths worldwide. Despite improvements in HCC therapy, the prognosis for HCC patients remains poor due to a high incidence of recurrence. An improved understanding of the pathogenesis of HCC development would facilitate the development of more effective outcomes for the diagnosis and treatment of HCC at earlier stages. miRNA are small, endogenous, non‐coding, ssRNA that are 21–30 nucleotides in length and modulate the expression of various target genes at the post‐transcriptional and translational levels. Aberrant expression of miRNA is common in various human malignancies and modulates cancer‐associated genomic regions or fragile sites. As for the relationship between miRNA and HCC, several studies have demonstrated that the aberrant expression of specific miRNA can be detected in HCC cells and tissues. However, little is known about the mechanisms of miRNA‐related cell proliferation and development. In this review, we summarize the central and potential roles of miRNA in the pathogenesis of HCC and elucidate new possibilities that may be useful as diagnostic and prognostic markers, as well as novel therapeutic targets in HCC.

Effect of the anti-diabetic drug metformin in hepatocellular carcinoma in vitro and in vivo.

Metformin is a commonly used oral anti-hyperglycemic agent of the biguanide family. Recent studies suggest that metformin may reduce cancer risk and improve prognosis. However, the antitumor mechanism of metformin in several types of cancers, including hepatocellular carcinoma (HCC), has not been elucidated. The goal of the present study was to evaluate the effects of metformin on HCC cell proliferation in vitro and in vivo, and to study microRNAs (miRNAs) associated with the antitumor effect of metformin in vitro. We used the cell lines Alex, HLE and Huh7, and normal hepatocytes to study the effects of metformin on human HCC cells. In an in vivo study, athymic nude mice bearing xenograft tumors were treated with metformin or left untreated. Tumor growth was recorded after 4 weeks, and the expression of cell cycle-related proteins was determined. Metformin inhibited the proliferation of Alex, HLE and Huh7 cells in vitro and in vivo. Metformin blocked the cell cycle in G0/G1 in vitro and in vivo. This blockade was accompanied by a strong decrease of G1 cyclins, especially cyclin D1, cyclin E and cyclin-dependent kinase 4 (Cdk4). In addition, microRNA (miRNA) expression was markedly altered by the treatment with metformin in vitro and in vivo. In addition, various miRNAs induced by metformin also may contribute to the suppression of tumor growth. Our results demonstrate that metformin inhibits the growth of HCC, possibly by inducing G1 cell cycle arrest through the alteration of microRNAs.

Targeting receptor tyrosine kinases in gastric cancer

Molecularly targeted therapeutic agents are constantly being developed and have been shown to be effective in various clinical trials. One group of representative targeted oncogenic kinases, the receptor tyrosine kinases (RTKs), has been associated with gastric cancer development. Trastuzumab, an inhibitor of ERBB2, has been approved for the treatment of gastric cancer, although other receptor tyrosine kinases, such as epidermal growth factor receptor, vascular endothelial growth factor, platelet-derived growth factor receptor, c-Met, IGF-1R and fibroblast growth factor receptor 2, are also activated in gastric cancer. The promising results of the trastuzumab clinical trial for gastric cancer resulted in the approval of trastuzumab-based therapy as a first-line treatment for human epidermal growth factor receptor 2-positive patients. On the other hand, the trial examining bevacizumab in combination with conventional chemotherapy did not meet its primary goal of increasing the overall survival time of gastric cancer patients; however, a significantly higher response rate and a longer progression-free survival were observed in the bevacizumab arm of the trial. Other clinical trials, especially phase III trials that have tested drugs targeting RTKs, such as cetuximab, panitumumab, gefitinib, erlotinib, figitumumab, sorafenib, sunitinib and lapatinib, have shown that these drugs have modest effects against gastric cancer. This review summarizes the recent results from the clinical trials of molecularly targeted drugs and suggests that further improvements in the treatment of advanced gastric cancer can be achieved through the combination of conventional drugs with the new molecularly targeted therapies.

Reduced expression of cell cycle regulator p18INK4C in human hepatocellular carcinoma

Cyclins, cyclin‐dependent kinases (Cdks), and Cdk inhibitors (CdkIs) are frequently altered in human cancer. p18INK4C, a member of the INK4 family of CdkIs, is a potential tumor‐suppressor gene product. However, the expression of p18INK4C in hepatocellular carcinoma (HCC) remains unknown. The aim of this study was to examine the expression of p18INK4C in various liver diseases including HCC and to assess its clinical significance in HCC. To that end, we examined the expression of p18INK4C by immunohistochemistry in various liver diseases, including 51 HCCs, and also studied the relationship between p18INK4C expression, the phosphorylation of retinoblastoma protein (pRb), and the activity level of Cdk4 and Cdk6. Immunohistochemical analysis revealed the frequent loss of p18INK4C expression in HCC, especially in poorly differentiated HCC. The loss of p18INK4C expression was shown to be associated with a poor prognosis compared with that associated with p18INK4C‐ positivity. Further, the kinase activity of Cdk4 was found to be higher in p18INK4C‐negative HCCs than in p18INK4C‐ positive HCCs. However, the level of Cdk6 activity was similar in the 2 groups of HCCs. In p18INK4C‐ positive HCCs, p18INK4C dominantly interacted with Cdk4 rather than with Cdk6. pRb phosphorylated at serine(Ser) 780 was detected more frequently in p18INK4C ‐ negative than in p18INK4C ‐ positive HCCs. In conclusion, the loss of p18INK4C expression may play a role in the differentiation and development of HCC through the up‐regulation of Cdk4 activity. (HEPATOLOGY 2004;40:677–686.)

Antidiabetic drug metformin inhibits esophageal adenocarcinoma cell proliferation in vitro and in vivo.

Esophageal carcinoma is the eighth most common cancer worldwide and the sixth leading cause of cancer-related deaths, with one of the worst prognoses of any form of cancer. Treatment with the anti-diabetic drug metformin has been associated with reduced cancer incidence in patients with type 2 diabetes. This study therefore evaluated the effects of metformin on the proliferation, in vitro and in vivo, of human esophageal adenocarcinoma cells, as well as the microRNAs associated with the antitumor effects of metformin. Metformin inhibited the proliferation of the esophageal adenocarcinoma cell lines OE19, OE33, SK-GT4 and OACM 5.1C, blocking the G0 to G1 transition in the cell cycle. This was accompanied by strong reductions in G1 cyclins, especially cyclin D1, cyclin-dependent kinase (Cdk)4, and Cdk6, and decreases in retinoblastoma protein phosphorylation. In addition, metformin reduced the phosphorylation of epidermal growth factor receptor and insulin-like growth factor and insulin-like growth factor-1 receptor, as well as angiogenesis-related proteins, such as vascular endothelial growth factor, tissue inhibitor of metalloproteinases (TIMP)-1, and TIMP-2. Metformin also markedly altered microRNA expression. Treatment with metformin of athymic nude mice bearing xenograft tumors reduced tumor proliferation. These findings suggest that metformin may have clinical use in the treatment of esophageal adenocarcinoma.

Molecular Mechanism Underlying the Detection of Colorectal Cancer by 18F-2-Fluoro-2-Deoxy- d -Glucose Positron Emission Tomography

BackgroundBiological imaging by positron emission tomography (PET) using 18F-2-fluoro-2-deoxy-d-glucose (FDG) has been widely used clinically for the detection of primary tumors and for early prediction of response to chemotherapy. In this study, we examined the molecular mechanism underlying the detection of colorectal cancers by FDG-PET.Material and MethodsIn all, 37 patients with colorectal cancer were examined with FDG-PET, and the maximal standardized uptake value (SUV) was calculated. Using surgical tissue samples, we examined the expression levels of hypoxia-inducible factor alpha (HIF1α), a marker of tissue hypoxia; proliferative cellular nuclear antigen (PCNA), a marker of proliferation; and glucose transporter (GLUT)1 and hexokinase (HK)2, protein of glucose uptake by using reverse transcriptase-polymerase chain reaction.ResultsAll except two colorectal cancer lesions showed increased uptake of FDG. The mean SUV of FDG-PET was 12.0 ± 1.2 (±SEM). The mean mRNA expression levels of GLUT1 and HK2 were significantly higher in cancer tissues than in the surrounding normal mucosa. Moreover, to promote the upregulation of glucose uptake, the expressions of HIF1α and PCNA were induced to 2.6 and 3.3 times higher than that in the normal mucosa. However, the quantitative correlation analysis showed SUV was correlated with HIF1α expression but not with PCNA expression.ConclusionOur molecular-based analysis suggested that FDG accumulation due to induction of glucose uptake proteins might be associated with the hypoxic environment in tumors rather than the tumor growth. Therefore, for assessing the efficacy of chemotherapy using FDG-PET, we must keep in mind that SUV does not indicate the tumor growth directly.

Galectin-9 suppresses the growth of hepatocellular carcinoma via apoptosis in vitro and in vivo

Galectin-9, a soluble β-galactoside-binding animal lectin, evokes apoptosis in various human cancer cell lines. The galectin-9 antitumor effect against hepatocellular carcinoma (HCC) is, however, unknown. We investigated whether galectin-9 suppresses HCC growth in vitro and in vivo. We assessed the antitumor effect of galectin-9 on HCC cells by conducting WST-8 assay in vitro and xenograft model analysis in vivo. Galectin-9-induced apoptosis was evaluated by FACS and ELISA in vitro and by TUNEL stain in vivo. Cell cycle alteration was profiled by FACS. Caspases were profiled by colorimetry. MicroRNAs related to the galectin-9 antitumor effects were determined using microarrays, and their antitumor effect was confirmed in a transfection study in vitro. The expression levels of the target proteins of the miRNAs extracted above were analyzed by western blot analysis. To summarize the results, galectin-9 inhibited the growth of the HCC cell lines HLE and Li-7 in vitro and Li-7 in vivo inducing apoptosis. Cell cycle turnover was not arrested in HLE and Li-7 cells in vitro. miR-1246 was similarly extracted both in vitro and in vivo, which sensitized Li-7 cells to apoptosis when transfected into the cells. DYRK1A, a target protein of miR-1246 was downregulated in Li-7 cells. Caspase-9 was upregulated in Li-7 cells in vitro and in vivo. In conclusion, galectin-9 inhibited the growth of HCC cells by apoptosis, but not cell cycle arrest, in vitro and in vivo. miR-1246 mediated signals of galectin-9, possibly through miR-1246-DYRK1A-caspase-9 axis. Galectin-9 might be a candidate agent for HCC chemotherapy.

Galectin-9 suppresses cholangiocarcinoma cell proliferation by inducing apoptosis but not cell cycle arrest

Cholangiocarcinoma is the most common biliary malignancy and the second most common hepatic malignancy after hepatocellular carcinoma (HCC). Galectin-9 (Gal-9) is a tandem-repeat-type galectin that has recently been shown to exert antiproliferative effects on cancer cells. Therefore, the present study evaluated the effects of Gal-9 on the proliferation of human cholangiocarcinoma cells in vitro as well as the microRNAs (miRNAs) associated with the antitumor effects of Gal-9. Gal-9 suppressed the proliferation of cholangiocarcinoma cell lines in vitro and the growth of human cholangiocarcinoma cell xenografts in nude mice. Our data further revealed that Gal-9 increased caspase‑cleaved keratin 18 (CCK18) levels, and the expression of cytochrome c increased in Gal-9-treated cholangiocarcinoma cell lines. These data suggested that Gal-9 induced cholangiocarcinoma cell apoptosis via the intrinsic apoptosis pathway mediated by caspase-dependent or -independent pathways. In addition, Gal-9 reduced the phosphorylation of the epidermal growth factor receptor (EGFR), insulin-like growth factor and insulin-like growth factor-1 receptor (IGF-1R), hepatocyte growth factor receptor and fibroblast growth factor receptor 3 (FGFR3). These findings suggest that Gal-9 can be a candidate of therapeutic target in the treatment of cholangiocarcinoma.

Exacerbation of Insulin Resistance and Hepatic Steatosis Deriving from Zinc Deficiency in Patients with HCV-Related Chronic Liver Disease

The role of zinc (Zn) in hepatic steatosis of patients with HCV-related chronic liver disease (CLD-C) remains uncertain, although persistent HCV infection often evokes hepatic steatosis. The primary purpose of this study was to elucidate the contribution of Zn deficiency to hepatic steatosis in patients with CLD-C. Fifty nondiabetic patients with CLD-C were enrolled. Hepatic 4-hydroxy-2-nonenal (4-HNE) expression was examined using an immunohistochemical procedure as a marker for lipid peroxidation. Serum ferritin levels were assessed for iron overload. Insulin resistance was evaluated using the values of the homeostasis model for assessment of insulin resistance (HOMA-IR). The severity of hepatic steatosis was graded on the classification system proposed by Brunt and colleagues. Serum Zn levels were inversely correlated with serum ferritin levels in the patients with CLD-C (r = −0.382, p = 0.0062). Serum ferritin levels were strongly associated with the HOMA-IR values (r = 0.476, p = 0.0005). Therefore, Zn deficiency resulted in insulin resistance through iron overload. Moreover, serum Zn levels were significantly decreased in proportion to the level of hepatic 4-HNE expression, which was enhanced as hepatic steatosis developed. Then, Zn deficiency eventually seemed to exacerbate hepatic steatosis by way of an increase in lipid peroxidation. However, the serum Zn levels were not associated with either loads of HCV-RNA or HCV genotypes. These data suggest that, in patients with CLD-C, Zn deficiency promotes insulin resistance by exacerbating iron overload in the liver and induces hepatic steatosis by facilitating lipid peroxidation.

Current Innovations in Endoscopic Therapy for the Management of Colorectal Cancer: From Endoscopic Submucosal Dissection to Endoscopic Full-Thickness Resection

Endoscopic submucosal dissection (ESD) is accepted as a minimally invasive treatment for colorectal cancer. However, due to technical difficulties and an increased rate of complications, ESD is not widely used in the colorectum. In some cases, endoscopic treatment alone is insufficient for disease control, and laparoscopic surgery is required. The combination of laparoscopic surgery and endoscopic resection represents a new frontier in cancer treatment. Recent developments in advanced polypectomy and minimally invasive surgical techniques will enable surgeons and endoscopists to challenge current practice in colorectal cancer treatment. Endoscopic full-thickness resection (EFTR) of the colon offers the potential to decrease the postoperative morbidity and mortality associated with segmental colectomy while enhancing the diagnostic yield compared to current endoscopic techniques. However, closure is necessary after EFTR and natural transluminal endoscopic surgery (NOTES). Innovative methods and new devices for EFTR and suturing are being developed and may potentially change traditional paradigms to achieve minimally invasive surgery for colorectal cancer. The present paper aims to discuss the complementary role of ESD and the future development of EFTR. We focus on the possibility of achieving EFTR using the ESD method and closing devices.