Repositioning of a cyclin-dependent kinase inhibitor GW8510 as a ribonucleotide reductase M2 inhibitor to treat human colorectal cancer.

Colorectal cancer (CRC) is the second leading cause of cancer-related death in males and females in the world. It is of immediate importance to develop novel therapeutics. Human ribonucleotide reductase (RRM1/RRM2) has an essential role in converting ribonucleoside diphosphate to 2'-deoxyribonucleoside diphosphate to maintain the homeostasis of nucleotide pools. RRM2 is a prognostic biomarker and predicts poor survival of CRC. In addition, increased RRM2 activity is associated with malignant transformation and tumor cell growth. Bioinformatics analyses show that RRM2 was overexpressed in CRC and might be an attractive target for treating CRC. Therefore, we attempted to search novel RRM2 inhibitors by using a gene expression signature-based approach, connectivity MAP (CMAP). The result predicted GW8510, a cyclin-dependent kinase inhibitor, as a potential RRM2 inhibitor. Western blot analysis indicated that GW8510 inhibited RRM2 expression through promoting its proteasomal degradation. In addition, GW8510 induced autophagic cell death. In addition, the sensitivities of CRC cells to GW8510 were associated with the levels of RRM2 and endogenous autophagic flux. Taken together, our study indicates that GW8510 could be a potential anti-CRC agent through targeting RRM2.

Radiation induces senescence and a bystander effect through metabolic alterations.

Cellular senescence is a state of irreversible growth arrest; however, the metabolic processes of senescent cells remain active. Our previous studies have shown that radiation induces senescence of human breast cancer cells that display low expression of securin, a protein involved in control of the metaphase-anaphase transition and anaphase onset. In this study, the protein expression profile of senescent cells was resolved by two-dimensional gel electrophoresis to investigate associated metabolic alterations. We found that radiation induced the expression and activation of glyceraldehyde-3-phosphate dehydrogenase that has an important role in glycolysis. The activity of lactate dehydrogenase A, which is involved in the conversion of pyruvate to lactate, the release of lactate and the acidification of the extracellular environment, was also induced. Inhibition of glycolysis by dichloroacetate attenuated radiation-induced senescence. In addition, radiation also induced activation of the 5'-adenosine monophosphate-activated protein kinase (AMPK) and nuclear factor kappa B (NF-κB) pathways to promote senescence. We also found that radiation increased the expression of monocarboxylate transporter 1 (MCT1) that facilitates the export of lactate into the extracellular environment. Inhibition of glycolysis or the AMPK/NF-κB signalling pathways reduced MCT1 expression and rescued the acidification of the extracellular environment. Interestingly, these metabolic-altering signalling pathways were also involved in radiation-induced invasion of the surrounding, non-irradiated breast cancer and normal endothelial cells. Taken together, radiation can induce the senescence of human breast cancer cells through metabolic alterations.

Liver transplantation for acute hepatic failure.

INTRODUCTION: The mortality rate of acute hepatic failure (AHF) with conservative treatment is 40% to 90%, depending on the etiology. Hepatitis B infection is the major cause of AHF in Asia. In this study, we examined the role of liver transplantation for adult patients with AHF. METHODS: Sixteen patients with AHF received liver transplants in the past 6 years. Eight patients received cadaveric donor and another 8 living-related donor grafts. Fifteen patients suffered from hepatitis B-related disease and 1 had drug-induced AHF. Extracorporeal charcoal hemoperfusion was used as a bridge to liver transplantation in the first 2 patients and plasma exchange was used in the following patients. RESULTS: One patient died 1 month after the operation due to primary nonfunction. The other 15 patients are alive with good graft function at 2 months to 6 years follow-up. The success rate is 94%. Postoperative complications included infection in 10 patients (62.5%), acute rejection in 4 patients (25%), and biliary complication in 2 patients (12.5%). No neurological complications were noted. CONCLUSION: Liver transplantation is the most effective treatment for patients with AHF. Living donors may be considered due to the organ shortage and the critical patient disease.

Surgical complications and outcome of living related liver transplantation.

INTRODUCTION: Living donor liver transplantation (LDLT) is now widely performed for patients to resolve the critical shortage of organs from cadavers. Despite rapid implementation and expansion of the procedure, both outcome and complication analyses of LDLT are still incomplete. OBJECTIVES: To analyze the outcome of LDLT, with particular reference to complications of those in need of surgical or radiological intervention. METHODS: Forty-eight LDLTs performed at National Taiwan University Hospital between December 1997 and April 2003 were reviewed retrospectively. RESULTS: Forty-two (87.5%) patients survived the operation. The 1-year graft and patient survival rate was 81.5%. Seventeen of the 48 LDLT patients had at least one postoperative complication, which needed surgical or radiological intervention. The complications included bile leakage (n = 3), biliary stricture (n = 4), internal bleeding (n = 7), intra-abdominal abscess (n = 2), liver abscess (n = 1), hepatic artery thrombosis (n = 2), duodenal ulcer bleeding (n = 1), jejunal perforation (n = 1), adhesion ileus (n = 1), and intracranial hemorrhage (n = 1). Nine of the 17 patients with complications died. In contrast, only 2 of the other 31 patients died. Seven of the mortalities were related to the complications. All survivors received only one definite intervention early after the complications were diagnosed. However, the others received an average of 1.71 +/- 0.95 (0 to 3) interventions. CONCLUSIONS: Complications requiring surgical or radiological treatment caused major mortality of LDLT. Early and definite treatment of these complications is important to improve the patient's outcome.

Liver transplantation for patients with hepatocellular carcinoma.

BACKGROUND: Liver transplantation (LT) has been advocated as a salvage treatment for unresectable hepatocellular carcinoma (HCC). Selection criteria still need to be developed in Taiwan. OBJECTIVES: The purpose of our study was to assess the clinical findings and outcome of cirrhotic patients with HCC undergoing liver transplantation. METHODS: Our study consisted of 13 HCC patients who underwent liver transplantation during October 1996 to March 2003. The medical records and pathologic reports were analyzed retrospectively. RESULTS: Overall survival rates at 1 and 3 years were 86% and 61%, respectively. HCC recurrences occurred in three patients, one of whom is still alive with HCC recurrence 2 years after LT. The other two patients died of HCC recurrence 1 and 2 years after LT, respectively. Pretransplant alpha-fetoprotein (AFP) levels of >200 ng/mL were noted in all three patients with HCC recurrence. In contrast, only one of the ten patients without HCC recurrence had pretransplant AFP >200 ng/mL (P = .003). Four patients did not meet Milan criteria, two of whom had HCC recurrence. However, the other two patients with microscopic vascular invasion survived and were free of HCC. The only one patient, who had histologic grade 4 HCC, died of recurrence, although his tumor was AJCC stage 1. CONCLUSIONS: High AFP level is a risk factor for HCC recurrence after LT. In addition to Milan criteria, histologic tumor grading should be considered in patient selection. Microscopic vascular invasion may not affect the outcome of the patients with early HCC.

T-cell antigenic determinants within hepatitis C virus nonstructural protein 3 and cytokine production profiles in hepatitis C.

The aim of this study was to further investigate the role of T-helper cells in hepatitis C virus (HCV) infection, focusing on the T-cell antigenic determinants and cytokine profiles of nonstructural 3 (NS3) protein-stimulated peripheral blood mononuclear cells (PBMCs) of HCV patients. A total of 12 recombinant proteins of theNS3 region were purified and used to test T-cell proliferative response and antigenic determinants of HCV-seropositive patients. In addition, cytokines produced by antigen stimulated PBMCs were measured. Our data showed that PBMCs from 55.7% (34/61) of HCV patients proliferated to at least one antigen, but PBMCs of HCV seronegative patients did not. In addition, PBMCs from about 82.0% (32/39) HCV-seropositive patients produced significant amounts of cytokines (10 pg/mL). Interestingly, PBMCs from 66% of patients produced TH2-related cytokines such as interleukin (IL)-4 and IL-5. In mappingexperiments, the data showed multiple T-cell antigenic determinants. Our data demonstrated that NS3 antigen-stimulated PBMCs of HCV patients recognized multiple T-cell antigenic determinants and produced significant amounts of TH0 or TH2-related cytokines, which might play a critical role in the chronicity of HCV infection.

Ribavirin enhances interferon-gamma levels in patients with chronic hepatitis C treated with interferon-alpha.

Some patients with chronic hepatitis C respond to interferon (IFN)-alpha treatment, and the efficiency can be improved by combining it with ribavirin. The mechanism of this improvement is unknown. To investigate the effects of these two regimens on the immune responses in 51 patients with chronic hepatitis C, we examined the hepatitis C core antigen-specific proliferative response and cytokine production profiles, natural killer (NK) cell cytotoxicity and cytotoxic T cell function during treatment. The results are as follows: (1) both viral clearance and biochemical normalization occurred more frequently in patients receiving combination therapy; (2) the function of NK cells increased after treatment in the responders of both groups (p < 0.05); (3) the level of IFN-gamma produced by hepatitis C core antigen-stimulated peripheral blood mononuclear cells was higher in patients receiving combination therapy, especially in responders; (4) the core antigen-specific proliferative response decreased after treatment, and (5) in addition, the core-specific cytotoxic T cell activities of five responder patients also increased significantly after therapy. In conclusion, enhancement of immune responses, especially those related to type-1 T helper cell activity, may contribute to better efficacy in combining ribavirin with IFN-alpha for treatment of chronic hepatitis C.

Less reversal of liver fibrosis after prolonged carbon tetrachloride injection.

BACKGROUND/AIMS: It was generally believed, but not proved, that early cirrhosis may be reversible, while advanced cirrhosis may not. This present study is to compare in mice the spontaneous regression of liver fibrosis between early and more advanced stage. METHODOLOGY: Liver fibrosis in mice was induced by intraperitoneal injection of carbon tetrachloride for 4, 10, and 16 weeks. After the last dose of each schedule, mice were sacrificed 1 day later (progression model) or left untreated for 10, 20, and 60 days (regression model). Tissue sections were stained by Sirius red. Liver hydroxyproline levels were determined to assess severity of fibrosis. Gelatinases in tissue extracts were assayed by zymography. RESULTS: During regression, diminution of fibrotic bands was more prominent in the 4-week group than in the others. Liver hydroxyproline levels in the progression model increased and resolution of liver fibrosis in the regression model decreased as carbon tetrachloride injection was prolonged. Liver matrix metalloproteinase-2 and -9 activities in the progression model also decreased as the injection was prolonged. CONCLUSIONS: These data demonstrated that reversibility of liver fibrosis would be gradually lost as liver injuries were prolonged. Gradual loss of the expression of matrix metalloproteinases may be responsible for the loss of reversibility.

Functional measurement of hepatitis C virus core-specific CD8(+) T-cell responses in the livers or peripheral blood of patients by using autologous peripheral blood mononuclear cells as targets or stimulators.

As is widely recognized, CD8(+) cytotoxic T lymphocytes (CTLs) play a crucial role in hepatitis C virus (HCV) infection, both in pathogenesis of liver injury and in clearing the virus. CTL studies with HCV-infected patients have been difficult because of the relatively low frequency of CTL precursors in the peripheral blood and because the targeted epitopes vary depending on the human leukocyte antigen (HLA) types of the individuals. This study attempts to overcome these problems by assessing the feasibility of using autologous peripheral blood mononuclear cells (PBMCs) expressing viral antigens as stimulators or targets in order to monitor the CTL responses. Primary PBMCs were transduced using a retroviral vector pseudotyped with a vesicular stomatitis virus G glycoprotein expressing the HCV core gene. Additionally, the vector-transduced PBMCs were used as targets of CTL assays to measure the HCV core-specific CTL activities from the liver-infiltrating lymphocytes of six different HLA-type patients with chronic HCV infection. The core-expressing PBMCs also served as stimulators, allowing us to measure core-specific CD8(+) T-cell responses by intracellular gamma interferon staining of the peripheral blood of hepatitis C patients who had received treatment with alpha interferon plus ribavirin. This approach provides an efficient means of measuring antigen-specific CTL responses without HLA constraints.

Expressions of cancer-testis antigens in human hepatocellular carcinomas.

Though regular sonographic examination can early detect small hepatocellular carcinoma, the therapeutic results remains unsatisfactory. Antigen-specific immunotherapy is an alternative approach for controlling tumors. The prerequisite for antigen-specific cancer immunotherapy is the identification of appropriate tumor antigens. Recently, a new category of tumor-specific shared antigens, called cancer-testis antigens, has been identified. The cancer-testis antigens have been found in a variety of cancers. However, the expression of cancer-testis antigens in human hepatocellular carcinomas is unknown. The aim of this current study is to investigate the expression of cancer-testis antigens in human hepatocellular carcinomas. Reverse-transcription polymerase chain reaction (RT-PCR) was used to investigate the expression of the SSX-1,-2,-4,-5, SCP-1, NY-ESO-1 genes in tumorous and corresponding non-tumorous liver tissues. In the 30 hepatocellular carcinomas studied, SSX-1,-2,-4,-5, SCP-1, and NY-ESO-1 mRNA expressions were detected in 24 (80%), 14 (46.7%), 22 (73.3%), 10 (33.3%), 2 (6.7%), and 11 (36.7%), respectively. Expressions of these genes were detected in few non-tumor liver tissues. The cancer-testis antigens are expressed in a high percentage of hepatocellular carcinomas. These cancer-testis antigen gene products are potential targets for antigen-specific immunotherapy of hepatocellular carcinoma.

Recurrent hepatocellular carcinoma presenting with superior vena cava syndrome.

A 45-year-old male received wedge resection for his small hepatocellular carcinoma in April 1989 and extended right lobectomy for tumor recurrence 8 months later. Unfortunately, recurrent hepatic tumor with lung metastases were found 18 months after the second operation. Both the hepatic and pulmonary recurrent tumors were resected and transcatheter arterial embolization was added for the residual hepatic tumors. He remained symptom free for another 18 months. However, mediastinal lymphadenopathy, superior vena cava thrombus with superior vena cava syndrome, cardiac and brain metastases developed subsequently. He died of increased intracranial pressure. It is rare for hepatocellular carcinoma to have mediastinal metastases, superior vena cava thrombus and superior vena cava syndrome.

Sequential Doppler sonographic studies of embolization in a patient with hepatic involvement in hereditary hemorrhagic telangiectasia: correlation with angiographic findings.

A 41-year-old man was admitted for symptoms of progressive congestive heart failure. His family history and the results of a physical examination were highly suggestive of Osler-Weber-Rendu disease (hereditary hemorrhagic telangiectasia, HHT). Cardiac catheterization and hepatic angiography demonstrated HHT with left-to-right shunting from the liver. The patient underwent transcatheter arterial embolization (TAE) of the right hepatic artery. We performed both Doppler sonography and angiography before and after TAE. The treatment improved the clinical manifestations of congestive heart failure, including the edema of the leg and dyspnea. Doppler sonographic studies also showed an increased resistive index in the right hepatic artery and decreased flow volumes and velocities in the right and middle hepatic veins, respectively, after treatment. Corresponding changes on angiography after TAE showed decreased right hepatic arterial flow and nonopacified branches distal to the coils, disappearance of the mottled hepatogram in the right lobe, reduction of contrast agent staining, and enhanced calibers in the right and middle hepatic veins. This case illustrates that qualitative and quantitative studies with duplex and color Doppler ultrasound can be used to detect or define the extent of hepatic involvement in HHT patients before TAE, monitor hemodynamic changes of the intrahepatic vasculature after TAE, evaluate the efficacy of treatment, and possibly obviate the need for repeated angiography for diagnosis only.

Bacterial infections associated with hepatic arteriography and transarterial embolization for hepatocellular carcinoma: a prospective study.

Sepsis and liver abscess are serious complications following transarterial embolization (TAE) for hepatocellular carcinoma (HCC). However, the exact incidence and the necessity of antibiotic prophylaxis remain undetermined. Between November 1996 and November 1997, we prospectively studied bacterial infections in 231 HCC patients who underwent 287 angiographic procedures without antibiotic prophylaxis, including 176 TAEs and 111 hepatic arteriographies (HAs). Four of the 111 HAs were complicated by transient asymptomatic bacteremia. Of the 176 TAEs, 2 were associated with asymptomatic bacteremia, and 7 (4%) were associated with symptomatic bacterial infection, including 3 cases of sepsis, 2 of liver abscess, and 2 of infected biloma. For patients with HCC, TAE was associated with a higher risk of developing symptomatic bacterial infections than was HA (4% vs. 0, respectively; P = .03). Previous gastrectomy was the only possible risk factor for liver abscess. Finally, early diagnosis and treatment of these infectious complications usually result in successful outcome.

Intrahepatic bilomas associated with hepatic arteriovenous malformation.

A 26 year-old female presented with progressive intermittent right upper quadrant pain. Hepatic arteriovenous malformation with small intrahepatic bilomas were found. She underwent hepatic artery ligation for control of her abdominal pain. Though the abdominal pain subsided after the hepatic artery ligation, the intrahepatic bilomas progressed. It is possible that the hepatic arteriovenous malformation (AVM) might reduce blood flow to the bile duct and then induce ischemia in the peribiliary capillary plexus, thus leading to bile duct necrosis and formation of bilomas, which could be further aggravated by hepatic artery ligation.

High frequency of expression of MAGE genes in human hepatocellular carcinoma.

AIMS/BACKGROUND: Activation of human MAGE genes leads to the expression of a set of tumor rejection antigens, which are recognized by cytotoxic T lymphocytes. The antigens may become the targets of immunotherapy. The expression of MAGE genes was originally found in, but is not restricted, to melanomas. The aim of this study was to investigate the expression of MAGE genes in human hepatocellular carcinomas. METHODS: The expression of MAGE-1, -2, -3, -4 genes in tumorous and corresponding non-tumorous liver tissue was studied using a reverse-transcription polymerase chain reaction. RESULTS: In the 50 hepatocellular carcinomas studied, MAGE-1, -2, -3, -4 mRNA expression was detected in 23 (46%), 17 (34%), 21 (42%) and 8 (16%), respectively. Seventy-four percent of the hepatocellular carcinomas expressed at least one of the MAGE genes. MAGE mRNAs were not detected in the corresponding non-tumor liver tissues. MAGE gene expression was not significantly correlated with clinicopathological factors. CONCLUSIONS: The MAGE genes are expressed in a high percentage of hepatocellular carcinomas; the MAGE gene products are potential targets for tumor-specific immunotherapy.

Characterization of monoclonal antibodies against hepatitis C virus nonstructural protein 3: different antigenic determinants from human B cells.

The nonstructural (NS3) region protein of hepatitis C virus (HCV) possesses major B-cell epitopes that induce antibodies after infection. To elucidate further the characteristics of these B cells and their role in the immune regulation of HCV infection, T9 (portion of NS3 region, amino acids [a.a.] 1188-1493)-specific monoclonal antibodies were derived and mapped for B-cell antigenic determinants with recombinant proteins. A total of 10 T9-specific hybridomas were generated and tested for B-cell antigenic determinants. To analyze the B-cell antigenic determinants, eight recombinant proteins including NS3-e (a.a. 1175-1334), NS3-a' (a.a. 1175-1250), NS3-a (a.a. 1251-1334), NS3-b (a.a. 1323-1412), NS3-c (a.a. 1407-1499), NS3-a/b (a.a. 1251-1412), NS3-bc (a.a. 1323-1499), and NS3-abc (a.a. 1251-1499) encoded by NS3-region internal clones were expressed and tested for immunoblotting. The data suggested IgG hybridomas recognized NS3-a, NS3-a', or NS3-b protein by immunoblotting. By contrast, the NS3-e protein bears the major antigenic determinant recognized by human sera. Half of the hybridomas were found to react with protein NS3-a', which is not a major B-cell antigenic determinant in humans. These data suggested that conformational epitopes in vivo may be important for B-cell recognition.

A different experience of hepatic adenoma in Taiwan with emphasis on clinical manifestations and ultrasonographic features.

BACKGROUND/AIMS: Hepatic adenoma is a rare benign hepatic tumor and it is difficult to differentiate it from other focal hepatic tumors. Ultrasonography has become the choice of methods to detect focal hepatic lesions. The study aims at analyzing ultrasonographic features of hepatic adenoma. METHODOLOGY: A total of 8 patients with pathologically proven hepatic adenoma were studied retrospectively during an 8-year period. The ultrasound scanners used were Toshiba SSA-100A, Toshiba SSA-240 and Aloka 630. The ultrasonographic features and clinical data were analyzed. RESULTS: There were 7 males and 1 female. The mean age was 50 years. Of the 8 cases, 2 symptomatic cases had a tumor larger than 10 cm. The remaining 6 cases were asymptomatic and had tumors smaller than 5 cm. The echogenicity was variable in these tumors. An irregular sonolucent was only noted in a 15 cm tumor and was histologically proven to be internal bleeding. All the tumors were well-defined, however, a hypoechoic rim was obvious only in the isoechoic and hyperechoic tumors. CONCLUSIONS: Ultrasonographic features of some hepatic adenomas are different from those of hepatocellular carcinomas and hemangiomas, although the differential diagnosis cannot be made in small hypoechoic tumors. When ultrasonography is used more widely, more asymptomatic patients with small-sized hepatic adenoma will be detected, even in male subjects. The concept about the pathogenesis of hepatic adenomas may be changed.

[Effect of PIC-BE on the expression of MDR-1 and bcl-2 genes in a multidrug resistance cell variant K562/ADM].

The effect of PIC-BE on the expression of mdr-1, bcl-2 and bax genes and their protein products (P-gp, Bcl-2 and Bax) was observed respectively in a multidrug resistance (MDR) cell variant K562/ADM. The results showed that PIC-BE could significantly inhibit the expression of mdr-1 and bcl-2 genes at both mRNA and protein levels in K562/ADM cell line, and the effect was dose- and time-dependent within limited range. Under same condition, although PIC-BE could increase the expression of Bax slightly, there was no statistically significant difference. These results suggest that the reversal of the MDR of K562/ADM cell line by PIC-BE may result from its effect on the expression of mdr-1, bcl-2 genes and their protein products.