Katsuya Shiraki

Phosphorylation of CPI-17, an inhibitory phosphoprotein of smooth muscle myosin phosphatase, by Rho-kinase

Phosphorylation of CPI‐17 by Rho‐associated kinase (Rho‐kinase) and its effect on myosin phosphatase (MP) activity were investigated. CPI‐17 was phosphorylated by Rho‐kinase to 0.92 mol of P/mol of CPI‐17 in vitro. The inhibitory phosphorylation site was Thr38 (as reported previously) and was identified using a point mutant of CPI‐17 and a phosphorylation state‐specific antibody. Phosphorylation by Rho‐kinase dramatically increased the inhibitory effect of CPI‐17 on MP activity. Thus, CPI‐17 as a substrate of Rho‐kinase could be involved in the Ca2+ sensitization of smooth muscle contraction as a downstream effector of Rho‐kinase.

Survivin initiates procaspase 3/p21 complex formation as a result of interaction with Cdk4 to resist Fas-mediated cell death

Caspase 3 is an essential death factor for the Fas-mediated cell death, and its inactivation in cells is initiated by an interaction with p21 on mitochondria or with IAP family member ILP. Survivin is also a member of IAP family and is specifically expressed during embryogenesis and in tumor cells and suppresses cell death signaling. In our current study, we demonstrated that Survivin translocation into the nucleus is dependent on Fas stimulation and cell proliferation. Survivin also interacts with the cell cycle regulator Cdk4, leading to Cdk2/Cyclin E activation and Rb phosphorylation. As a result of Survivin/Cdk4 complex formation, p21 is released from its complex with Cdk4 and interacts with mitochondrial procaspase 3 to suppress Fas-mediated cell death. Here, we propose that Survivin supports procaspase 3/p21 complex formation as a result of interaction with Cdk4 resulting in suppression of cell death signaling.

Survivin initiates cell cycle entry by the competitive interaction with Cdk4/p16(INK4a) and Cdk2/cyclin E complex activation.

Survivin is observed uniquely in tumor cells and developmental cells, which undergo either inappropriate or programmed cell growth. In the current study, we investigated the influence of Survivin on cell cycle. Overexpression of Survivin resulted in accelerated S phase shift, resistance to G1 arrest, and activated Cdk2/Cyclin E complex leading Rb phosphorylation. In addition, nuclear translocation of Survivin followed by an interaction with Cdk4 was detected. Interestingly, Survivin nuclear translocation coincided with S phase shift, and prevention of nuclear transport suppressed Survivin nuclear translocation and S phase shift. Further, we also observed that Survivin competitively interacted with the Cdk4/p16INK4a complex in a cell free system and in vivo. These results suggest that Survivin initiates the cell cycle entry as a result of nuclear translocation followed by an interaction with Cdk4.

Radiofrequency ablation combined with chemoembolization in hepatocellular carcinoma: treatment response based on tumor size and morphology.

PURPOSE To evaluate local therapeutic efficacy of radiofrequency (RF) ablation after chemoembolization for hepatocellular carcinoma (HCC) based on tumor size and morphology. MATERIALS AND METHODS Sixty-four patients underwent RF ablation under ultrasonographic or real-time computed tomographic (CT) fluoroscopic guidance within 2 weeks after chemoembolization. One hundred eight lesions were treated. Sixty-five lesions were small (<or=3 cm), 32 were intermediate in size (3.1-5 cm), and 11 were large (5.1-12 cm). Seventy-four of the HCCs were nodular lesions and the other 34 were nonnodular (multinodular and infiltrative) lesions. Response to treatment was evaluated by dynamic enhanced CT and magnetic resonance imaging. Tumor necrosis was considered to be complete when no foci of enhancement were seen within the tumor and at its periphery. RESULTS Complete necrosis was achieved in all lesions regardless of tumor size or morphology. There have been no local recurrences in small and intermediate-sized lesions regardless of tumor morphology during a mean follow-up of 12.5 months. In large HCCs, nodular lesions showed no recurrence, but two of six of nonnodular lesions recurred beyond the thermal lesions created around the tumor. The estimated 1-year survival rate was 98.0% in all patients. CONCLUSIONS This combined therapy has a therapeutic effect on small and intermediate-sized HCCs regardless of tumor morphology and is a promising treatment option for large nodular lesions. Control of large nonnodular lesions is still a challenging problem.

Early-stage hepatocellular carcinoma: radiofrequency ablation combined with chemoembolization versus hepatectomy.

PURPOSE To retrospectively evaluate the long-term results of radiofrequency (RF) ablation combined with chemoembolization (combination therapy) as compared with hepatectomy for the treatment of early-stage hepatocellular carcinoma (HCC). MATERIALS AND METHODS The study was approved by the institutional review board, and informed consent was waived. Patients with early-stage HCC were included if they underwent either combination therapy or hepatectomy and met the following inclusion criteria: no previous treatment for HCC, three or fewer tumors with a maximum diameter of 3 cm or less each or a single tumor with a maximum diameter of 5 cm or less, Child-Pugh class A liver profile, no vascular invasion, and no extrahepatic metastases. The primary endpoint was overall survival, and the secondary endpoint was recurrence-free survival. RESULTS One hundred four patients (mean age, 66.5 years +/- 8.7 [standard deviation]; 79 men, 25 women) underwent combination therapy, and 62 patients (mean age, 64.5 years +/- 9.6; 51 men, 11 women) underwent hepatectomy. The 1-, 3-, and 5-year overall survival rates following combination therapy (98%, 94%, and 75%, respectively) were similar (P = .87) to those following hepatectomy (97%, 93%, and 81%, respectively). The 1-, 3-, and 5-year recurrence-free survival rates were also comparable (P = .70) for combination therapy (92%, 64%, and 27%, respectively) and hepatectomy (89%, 69%, and 26%, respectively). CONCLUSION RF ablation combined with chemoembolization in patients with early-stage HCC provides overall and disease-free survival rates similar to those achieved by hepatectomy.

Cellular FLICE/Caspase-8–Inhibitory Protein as a Principal Regulator of Cell Death and Survival in Human Hepatocellular Carcinoma

Human hepatocellular carcinomas (HCCs) show resistance to apoptosis mediated by several death receptors. Because cellular FLICE/caspase-8–inhibitory protein (cFLIP) is a recently identified intracellular inhibitor of caspase-8 activation that potently inhibits death signaling mediated by all known death receptors, including Fas, TNF-receptor (TNF-R), and TNF-related apoptosis-inducing ligand receptors (TRAIL-Rs), we investigated the expression and function of cFLIP in human HCCs. We found that cFLIP is constitutively expressed in all human HCC cell lines and is expressed more in human HCC tissues than in nontumor liver tissues. Metabolic inhibitors, actinomycin D (ActD) or cycloheximide (CHX), dramatically rendered HCC cells sensitive to Fas-mediated apoptosis. Neither caspase-8 nor caspase-3 was activated by agonistic anti-Fas antibody alone, but both caspases were activated by Fas stimulation in the presence of ActD or CHX, indicating the importance of caspase-8 inhibitors that are sensitive to metabolic inhibitors. Actually, cFLIP expression was decreased in ActD or CHX treatment. cFLIP down-regulation induced by cFLIP antisense oligodeoxynucleotides sensitized HLE cells to Fas, TNF-R, and TRAIL-R–mediated apoptosis. Furthermore, cFLIP over-expression activated nuclear factor (NF)-κB and cFLIP down-regulation attenuated NF-κB activation induced by TNF-α or TRAIL. Pretreatment with pan-caspase-inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD-fmk), restored NF-κB activity attenuated by cFLIP down-regulation. cFLIP expression was increased by TNF-α, TRAIL, or vascular endothelial growth factor but decreased by wortmannin, indicating that cFLIP expression is regulated by both the NF-κB and phosphatidylinostiol-3 kinase (PI-3)/Akt pathways. These results suggest that cFLIP plays an important role in cell survival not simply by inhibiting death-receptor–mediated apoptosis but also by regulating NF-κB activation in human HCCs.

Complications of Partial Splenic Embolization in Cirrhotic Patients

In recent years, partial splenic embolization (PSE) has been widely used in patients with cirrhosis and hypersplenism caused by portal hypertension. We investigated the complications associated with PSE cases seen in our hospital. Seventeen cases of liver cirrhosis that had undergone PSE were examined to investigate the complications associated with it. Mean infarcted area of the spleen was 66.2%. Leukocyte and platelet counts in 16 of 17 patients were seen to improve after PSE and persisted for at least one year. The most frequent side effects were abdominal pain (82.4%) and fever (94.1%). Severe side effects were seen in two of those 17 patients. One patient died from acute on chronic liver failure. The other patients contracted bacterial peritonitis and splenic abscess and needed drainage of splenic abscess before recovery. These two cases were in Child-Pugh class B. In conclusions, PSE is a useful treatment for patients with cirrhosis and hypersplenism caused by portal hypertension. However, the possibility of severe complications, especially in patients with noncompensated cirrhosis, should be kept in mind.

B-mode ultrasound with algorithm based on statistical analysis of signals: evaluation of liver fibrosis in patients with chronic hepatitis C.

OBJECTIVE The purpose of this study was to evaluate the degree of liver fibrosis in patients with chronic hepatitis C by use of a method in which the homogeneity of the tissue texture of the liver on B-mode ultrasound images is analyzed on the basis of results of a statistical chi-square test of the echo amplitudes. The method includes an algorithm for removing small structures, such as cross sections of the thin vessels, in the background texture to minimize differences in analysis results between users. SUBJECTS AND METHODS Analysis was performed on images of 148 patients with histologically proven chronic hepatitis C without cirrhosis. The peak value of the C(m)(2) (modified chi-square distribution) histogram was calculated from B-mode ultrasound images, and the resulting value was compared with the histologic fibrosis grade. RESULTS The peak C(m)(2) histogram value for grade F3 fibrosis was higher than that for grades F0 and F1 (p < 0.0001) and F2 (p = 0.0003). The value for grade F2 was higher than that for grades F0 and F1 (p = 0.0027). The values gradually increased with an increase in liver fibrosis grade, although no difference was found between grades F0 and F1. CONCLUSION The grades of liver fibrosis in patients with chronic hepatitis C are well discriminated with the B-mode ultrasound-based analysis algorithm without discrimination between grades F0 and F1. Findings on conventional ultrasound images may reflect progression of liver fibrosis even in the absence of cirrhosis.

Expression of survivin during liver regeneration.

Survivin functions to suppress cell death and regulate cell division, and is observed uniquely in tumor cells and developmental cells. However, the expression and regulation of survivin in non-transformed cells are not well elucidated. Therefore, we investigated the expression of survivin in a murine liver regeneration model after partial hepatectomy and intraperitoneal carbon tetrachloride (CCl(4)) injection. We found that the expression of survivin transcript and protein were markedly elevated with the onset of DNA synthesis and remained elevated during G2 and M phases during liver regeneration. In a normal mouse liver cell line, over-expression of survivin resulted in a decrease in the G0/G1 phase and an increase in the S and G2/M phases, resulting in Rb phosphorylation. These findings suggest that survivin is dramatically expressed in a cell cycle-dependent manner during liver regeneration and provide a new insight into the regulation of cell proliferation and differentiation.

Radiofrequency Ablation Combined with Chemoembolization for the Treatment of Hepatocellular Carcinomas Larger than 5 cm

PURPOSE To evaluate survival, recurrence-free survival, technical success, technique effectiveness, and safety of radiofrequency (RF) ablation combined with chemoembolization in patients with hepatocellular carcinomas (HCCs) larger than 5 cm. MATERIALS AND METHODS Patients with Child-Pugh class A or B cirrhosis and three or fewer HCCs with a maximum tumor diameter of 5.1-10 cm were included. Twenty patients with 32 HCCs were included. There were 16 men and four women with mean age of 69 years +/- 7.4 (range, 46-79 years).The maximum mean tumor diameter was 6.2 cm (range, 5.1-9.5 cm). RF ablation was performed under computed tomographic (CT) fluoroscopic guidance 1-2 weeks after chemoembolization. The primary endpoint of this study was survival. RESULTS RF electrodes were placed in the planned sites, and RF ablation was completed with a planned protocol (technical success rate, 100%). Tumor enhancement was eradicated in all patients after 32 RF sessions. The primary and secondary technique effectiveness rates were 40% and 100%, respectively. There were two major complications in the 32 RF sessions (6%)--hepatic abscess and diaphragm perforation. Local tumor progression developed in five of the 20 patients (25%) during the mean follow-up of 30 months. The overall and recurrence-free survival rates were, respectively, 100% and 74% at 1 year, 62% and 28% at 3 years, and 41% and 14% at 5 years. The serum bilirubin level of 1.0 mg/dL (17.1 micromol/L) or less was a significantly better prognostic factor in the univariate analysis. CONCLUSIONS This combination therapy may enhance survival in patients with HCCs larger than 5 cm.