Shun Fujimaki

DNA Damage Sensor γ-H2AX Is Increased in Preneoplastic Lesions of Hepatocellular Carcinoma

Background. Phosphorylated histone H2AX (γ-H2AX) is a potential regulator of DNA repair and is a useful tool for detecting DNA damage. To evaluate the clinical usefulness of γ-H2AX in hepatocellular carcinoma (HCC), we measured the level of γ-H2AX in HCC, dysplastic nodule, and nontumorous liver diseases. Methods. The level of γ-H2AX was measured by immunohistochemistry in fifty-eight HCC, 18 chronic hepatitis, 22 liver cirrhosis, and 19 dysplastic nodules. Appropriate cases were also examined by fluorescence analysis and western blotting. Results. All cases with chronic liver disease showed increased levels of γ-H2AX expression. In 40 (69.9%) of 58 cases with HCC, the labeling index (LI) of γ-H2AX was above 50% and was inversely correlated with the histological grade. Mean γ-H2AX LI was the highest in dysplastic nodule (74.1 ± 22.1%), which was significantly higher than HCC (P < 0.005). Moreover, γ-H2AX was significantly increased in nontumorous tissues of HCC as compared with liver cirrhosis without HCC (62.5 ± 24.7%, from 5.1 to 96.0%, P < 0.005). Conclusions. γ-H2AX was increased in the preneoplastic lesions of HCC and might be a useful biomarker for predicting the risk of HCC.

Blockade of ataxia telangiectasia mutated sensitizes hepatoma cell lines to sorafenib by interfering with Akt signaling

Sorafenib is a multi-kinase inhibitor applicable to hepatocellular carcinoma (HCC), but its limited therapeutic effects are a major problem to be solved. Here, we show that blockade of ataxia telangiectasia mutated (ATM) improves the antitumor effects of sorafenib. When hepatoma cell lines HepG2 and PLC/PRF/5 were treated with sorafenib plus ATM small inhibitory RNAs, ATM inhibitor KU55933 or caffeine, Akt signaling was suppressed and the cytotoxic effects were significantly potentiated. Moreover, ATM inhibition effectively suppressed the sorafenib-induced cell migration. Taken together, manipulation of ATM activity might be a useful strategy for improving sorafenib treatment of HCC.

p21-activated kinase-2 is a critical mediator of transforming growth factor-β-induced hepatoma cell migration

Transforming growth factor‐β (TGF‐β) has been shown to play a central role in the promotion of cell motility, but its functional mechanism has remained unclear. With the aim of investigating the diagnostic and treatment modalities for patients with hepatocellular carcinoma (HCC), the signaling pathway that may contribute to TGF‐β‐mediated cell invasion in hepatoma cells was evaluated.

Clinical significance of cell cycle inhibitors in hepatocellular carcinoma

It is well accepted that cell cycle regulators are strongly implicated in the progression of cancer development. p16 and p27 are potent cyclin-dependent kinase (CDK) inhibitors involved in G1 phase progression, and are regarded as adverse prognostic biomarkers for various types of cancers. It has been reported that the main mechanism for p16 inactivation is aberrant DNA methylation, while p27 is exclusively inactivated by proteasome-mediated protein degradation. We have found that p27 is decreased in around half of hepatocellular carcinomas (HCCs), and in some cases p27 is inactivated by inappropriate interaction with cyclin D1/CDK4 complexes. In such cases, p16 is concomitantly inactivated through DNA methylation. Taking into consideration the complex interaction between p16 and p27, a comprehensive analysis including p16 and p27 would be useful for predicting the prognosis of HCC patients.

Mycotoxins are conventional and novel risk biomarkers for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a common malignant disease with poor prognosis. To improve the clinical outcome, early diagnosis of HCC arising from nonviral agents and hepatitis virus is important. Among several etiological factors, mycotoxins defined as carcinogens by the International Agency for Research in Cancer (IARC) might be one of the critical risk factors for nonviral HCC. Aflatoxin B1 is the most well-known carcinogenic mycotoxin for HCC, but the role of the other types of mycotoxin remains unclear. Several studies have reported that a chromatographic separation technique based on high-performance liquid chromatography can successfully detect the concentration of mycotoxins in plasma. Recently, serum level of ochratoxin A (OTA), a widely distributed mycotoxin classified as Group 2B by IARC, was evaluated in HCC patients in Egypt. The results suggested that serum OTA levels might be a good biomarker for HCC. In this article, we review recent studies of OTA, and discuss its possible significance as a biomarker of HCC.

p27 Is a Critical Prognostic Biomarker in Non-Alcoholic Steatohepatitis-Related Hepatocellular Carcinoma

Non-alcoholic steatohepatitis (NASH) is a recently identified chronic liver disease, which progresses to liver cirrhosis and hepatocellular carcinoma (HCC). As the number of patients studied to date has been limited, clinically useful prognostic biomarkers of NASH-related HCC have not been available. In this study, we investigated the status of a cell-cycle regulator, p27, in NASH-related HCC. p27 has been regarded as a prognostic factor in various types of cancer patients. A total of 22 cases with NASH-related HCC were analyzed for p27 protein expression, and phosphorylation at threonine 157 (T157) and serine 10 (S10) by immunohistochemical analysis. The correlation of p27 with tumor characteristics, disease-free survival (DFS), and overall survival was analyzed. p27 expression was decreased in 13 HCCs (59%), and was significantly correlated with enlarged tumor size (p = 0.01) and increased cell proliferation (p < 0.01). Phospho-p27 at T157 and S10 was detected in four (18%) and seven (32%) cases, respectively, and patients positive for phospho-p27 (S10) showed reduced DFS (hazard ratio 7.623, p = 0.016) by univariate analysis. Further studies with more patients are required to verify the usefulness of p27 as a biomarker for predicting tumor recurrence in NASH patients.

Hepatitis B virus X induces cell proliferation in the hepatocarcinogenesis via up‐regulation of cytoplasmic p21 expression

Hepatitis B virus X protein (HBx) has been shown to induce hepatocarcinogenesis by disrupting the functions of intracellular molecules. Cyclin‐dependent kinase inhibitor p21 (Cip1/WAF1), known as a tumour‐suppressor gene, has been reported to have paradoxical function, that is, acting as an oncogene, particularly when expressed in the cytoplasm. The effects of HBx on the expression and function of p21 also remain controversial.

Sorafenib-Inhibited Signaling: Emerging Evidence of RAFIndependent Pathways as Potential Therapeutic Targets in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide [Serag et al., 2007, Liovet et al., 2003, Yang et al., 2010]. More than 500,000 people are diagnosed with HCC every year, and it remains the leading cause of death among patients with hepatitis B virus (HBV), hepatitis C virus (HCV) and alcohol-induced liver cirrhosis. One of the main obstacles for treating HCC is late diagnosis of patients. Many unresectable HCC patients are treated with loco-regional therapies such as radiofrequency ablation and transarterial chemo‐ embolization (TACE), but the prognosis remains poor [Bruix et al., 2005]. A recent study in multiple clinical facilities in Japan reported that 5-year survival of patients treated with TACE was less than 30% [Takayasu et al., 2006]. Moreover, HCC is poorly responsive to chemother‐ apeutic drugs and radiotherapy [Arii et al., 2000, Kuwahara et al., 2009]; thus, effective therapeutic tools for HCC are long-awaited.

Effects of rapamycin on granulation formation in response to centrally doubled coiled stents as a tracheal substitute

INTRODUCTION In experiments involving tracheal wall defects in rabbits, metallic coil stents inevitably induce granulation formation in the defects. We examined the involvement of the mammalian target of rapamycin (mTOR) signaling pathway in granulation formation and examined the effects of rapamycin. METHODS The anterior half of the tracheal wall was removed for a longitudinal length of six tracheal rings. Metallic coils were placed into the tracheal lumen through a wall defect. The rabbits were sacrificed two months after undergoing an endoscopic examination, and the granulation tissue in the tracheal defects was removed for a Western blot analysis and immunohistochemical analysis. Rapamycin (0.5 mg kg(-1) day(-1)) was administered three times per week intramuscularly. The data were expressed as the relative expression versus the expression of actin. RESULTS The level of mTOR phosphorylation in the resected trachea was 0.72±0.45, and it significantly increased in the granulation tissue to 11.6±5.2, with concomitant increases in the phosphorylation levels of p70S6K and S6RP in all five rabbits. Although the systemic administration of rapamycin significantly decreased the levels of phosphorylated mTOR to 4.0±2.4 in the five treated rabbits, the clinical outcomes were unsatisfactory. Three of the five treated rabbits exhibited signs of wound complications, and wet granulation tissue that caused respiratory symptoms was found inside and outside of the coils in four rabbits. CONCLUSIONS Although rapamycin effectively reduced the mTOR activity in the granulation tissue, the granulation formation process seemed to be disturbed, most likely owing to the immunosuppressive effects of rapamycin.