A Serine Palmitoyltransferase Inhibitor Blocks Hepatitis C Virus Replication in Human Hepatocytes

Background & AimsHost cell lipid rafts form a scaffold required for replication of hepatitis C virus (HCV). Serine palmitoyltransferases (SPTs) produce sphingolipids, which are essential components of the lipid rafts that associate with HCV nonstructural proteins. Prevention of the de novo synthesis of sphingolipids by an SPT inhibitor disrupts the HCV replication complex and thereby inhibits HCV replication. We investigated the ability of the SPT inhibitor NA808 to prevent HCV replication in cells and mice.MethodsWe tested the ability of NA808 to inhibit SPT’s enzymatic activity in FLR3-1 replicon cells. We used a replicon system to select for HCV variants that became resistant to NA808 at concentrations 4- to 6-fold the 50% inhibitory concentration, after 14 rounds of cell passage. We assessed the ability of NA808 or telaprevir to inhibit replication of HCV genotypes 1a, 1b, 2a, 3a, and 4a in mice with humanized livers (transplanted with human hepatocytes). NA808 was injected intravenously, with or without pegylated interferon alfa-2a and HCV polymerase and/or protease inhibitors.ResultsNA808 prevented HCV replication via noncompetitive inhibition of SPT; no resistance mutations developed. NA808 prevented replication of all HCV genotypes tested in mice with humanized livers. Intravenous NA808 significantly reduced viral load in the mice and had synergistic effects with pegylated interferon alfa-2a and HCV polymerase and protease inhibitors.ConclusionsThe SPT inhibitor NA808 prevents replication of HCV genotypes 1a, 1b, 2a, 3a, and 4a in cultured hepatocytes and in mice with humanized livers. It might be developed for treatment of HCV infection or used in combination with pegylated interferon alfa-2a or HCV polymerase or protease inhibitors

Expression of HER2 and MUC1 in Advanced Colorectal Cancer: Frequency and Clinicopathological Characteristics

There have been many reports on the overexpression of human epidermal growth factor receptor 2 (HER2) in patients with colon cancer. However, the role and frequency of HER2 overexpression have not been clearly defined. Anti-HER2 therapy has been shown to improve the prognosis of HER2-positive patients with breast and stomach cancers. In this study, we explored HER2 expression in patients with colon cancer at stages II and III by immunohistochemistry (IHC) and dual-color in situ hybridization (DISH), and examined the correlation between HER2 expression and clinicopathological factors. Moreover, we examined the correlation between HER2 expression and mucin 1 (MUC1) expression. The subjects were 121 patients with colon cancer at stages II and III who underwent surgery in our hospital during the period from 2007 to 2009. Sections containing the deepest part of a lesion were subjected to immunostaining for HER2 and MUC1. HER2 expression was assessed in accordance with Ventana\u27s Guidelines for HER2 Testing in Stomach Cancer, with sections comprising less than 10% of weakly to moderately stained tumor cells scored as 1 > 2. HER2 expression scored as 2 was defined with sections comprising more than 10% of the weakly to moderately stained tumor cells. Patients with a score of 1 > 2 and 2 were also subjected to DISH using a Dual ISH HER2 kit. MUC1 expression was scored according to the percentage of stained area as follows: 0, 0 to 5%; 1, 5 to 50%; and 2, 50% and higher. Patients with a score of 1 and 2 were defined as MUC1-positive. The analysis of HER2 by IHC yielded the following scores: 45 patients (37.2%), 0; 38 patients (31.4%), 1; 14 patients (11.6%); 1 > 2; 24 patients (19.8%), 2; and 0 patients (0%), 3. For the 38 patients with a score of 1 > 2 and 2, DISH returned ratios of HER2 to Chr17 expression (HER2: Chr17 ratio) from 1.13 to 1.93 (mean = 1.46). There was no significant correlation between HER2 expression and clinicopathological factors. The numbers of MUC1-positive patients according to HER2 score were as follows: 22 patients (48.9%) in the score 0 group (45 patients); 25 patients (65.8%) in the score 1 group (38 patients); 10 patients (71.4%) in the score 1 > 2 group (14 patients), and 22 patients (91.7%) in the score 2 group (24 patients). There was a positive correlation between HER2 expression and MUC1 expression. Specifically, MUC1 expression levels increased with HER2 expression level, and the percentage of MUC1-positive patients was significantly higher in the HER2 score 2 group than in the HER2 score 0 group (P < 0.01). Rates of HER2 positivity by DISH or fluorescence in situ hybridization (FISH) in patients who had an HER2 score of 2+ by IHC were 45% and 24% in the patients with stomach and breast cancers, respectively. However, the positivity rate was 0% in the patients with colon cancer in this study. This result indicates that patients with colon cancer who have an IHC HER2 score of 2+ are more likely to be HER2 negative by DISH than patients with breast and stomach cancers, although larger cohort studies are required before a definitive conclusion can be made. There was a positive correlation between HER2 expression and MUC1 expression in this study, although further examination is required because there were no patients who had an HER2 score of 3+ or 2+ by IHC and were HER2 positive by DISH in this study. HER2 expression in colon cancer should be cautiously assessed by both IHC and DISH

Clinicopathological Significance of FOXP3 Expression in Esophageal Squamous Cell Carcinoma

The expression of transcription factor forkhead box protein 3 (FOXP3), a master control gene for regulatory T cells, has been reported to influence patient survival. However, there have been few reports of the relationship between FOXP3 positive cells and esophageal squamous cell carcinoma (ESCC). The aim of this study was to clarify the prognostic value of FOXP3 expression in ESCC. Ninety-five patients who were diagnosed with primary ESCC and underwent subtotal esophagectomy during 2009 and 2010 were retrospectively analyzed. Deepest sections from each tumor were selected for immunohistochemistry and the number of FOXP3 positive cells was counted. The median number was used as a cutoff to divide into FOXP3 positive and FOXP3 negative subgroups. Relationships between FOXP3 expression and clinicopathological features, disease-free survival (DFS) and overall survival (OS) were determined. Statistical values of p < 0.05 were considered significant. FOXP3 positive cells were found in all 95 cases and the number of FOXP3 positive cells was significantly higher in the peri-tumor compartment than in the intra-tumor compartment (p = 0.0006). For this reason, the peri-tumor compartment numbers were used for all of the association studies. Results showed that the FOXP3 positive group had a significantly larger mean tumor size (43.8 ± 4.1mm vs 29.1 ± 4.0mm, p = 0.0055), and the FOXP3 negative group had a significantly higher percentage of deep invasion (T2, T3, T4)(p = 0.0399). There was no significant association for DFS, however, for OS the FOXP3 positive group demonstrated a significantly better prognosis (p = 0.0024). Multivariate analysis showed that peri-tumor FOXP3 expression is an independent prognostic factor for OS (p = 0.0035). Peri-tumoral FOXP3 expression is an independent and favorable prognostic factor for ESCC

Poly ADP-ribose Polymerase (PARP) Staining for Immunohistological Investigation of Primary Breast Cancer

Given that clinical trials of poly ADP-ribose polymerase (PARP) 1 inhibitors are underway, in the present study we investigated the prevalence of triple-negative breast cancer and PARP1 expression in patients with primary invasive breast cancer. Immunohistological studies plus PARP staining were performed on samples from 206 primary breast cancer patients undergoing surgery at Showa University Hospital between January 2010 and May 2011. Fifteen patients (7.3%) were found to have triple-negative breast cancer. Hormone receptor-positive patients were significantly more likely to be PARP1 negative. There were no PARP1-negative patients in the triple-negative group. However, there was no significant difference in the rate of PARP1 negativity between patients with triple-negative breast cancer and those with other breast cancer subtypes. There were no PARP1-negative patients in the triple-negative breast cancer group. Given that the effectiveness of PARP inhibitors has not been sufficiently established in clinical trials, a more in-depth analysis is required to determine the factors contributing to effective treatment. Future studies should include more subjects with triple-negative breast cancer and those with BRCA mutations

Self-Enhancement of Hepatitis C Virus Replication by Promotion of Specific Sphingolipid Biosynthesis

<div><p>Lipids are key components in the viral life cycle that affect host-pathogen interactions. In this study, we investigated the effect of HCV infection on sphingolipid metabolism, especially on endogenous SM levels, and the relationship between HCV replication and endogenous SM molecular species. We demonstrated that HCV induces the expression of the genes (<em>SGMS1</em> and <em>2</em>) encoding human SM synthases 1 and 2. We observed associated increases of both total and individual sphingolipid molecular species, as assessed in human hepatocytes and in the detergent-resistant membrane (DRM) fraction in which HCV replicates. SGMS1 expression had a correlation with HCV replication. Inhibition of sphingolipid biosynthesis with a hepatotropic serine palmitoyltransferase (SPT) inhibitor, NA808, suppressed HCV-RNA production while also interfering with sphingolipid metabolism. Further, we identified the SM molecular species that comprise the DRM fraction and demonstrated that these endogenous SM species interacted with HCV nonstructural 5B polymerase to enhance viral replication. Our results reveal that HCV alters sphingolipid metabolism to promote viral replication, providing new insights into the formation of the HCV replication complex and the involvement of host lipids in the HCV life cycle.</p> </div

Relationship between the SGMS genes and HCV infection.

<p>(<b>A, B</b>) The correlation between SGMS1/2 and liver HCV-RNA of HCV infected humanized chimeric mice (n = 7). (<b>C</b>) The effect of silencing HCV genome RNA with siRNA (siE-R7: 1 nM) on HCV in HCV-infected cells. (<b>D</b>) The effect of silencing HCV genome RNA with siRNA (siE-R7: 1 nM) on the expression of SGMS1/2 mRNA measured by RTD-PCR. (<b>E</b>) The effect of silencing SGMS1/2 mRNA with siRNA (3 nM each) measured by RTD-PCR. (<b>F</b>) The effect of silencing SGMS1/2 mRNA with siRNA (3 nM) on HCV replication in FLR 3-1. In all cases, error bars indicate SDs. *<i>p</i><0.05 and **<i>p</i><0.01.</p