Efficacy of daily abdominal self-massage for the prevention of recurrent common bile duct stones after endoscopic biliary sphincterotomy.

Recurrent common bile duct stones (CBDS) can occur after endoscopic biliary sphincterotomy (EST). Bile flow through the papilla of Vater could be improved by means of abdominal massage. We report the results of self-abdominal massage in four patients who had previously undergone cholecystectomy and experienced multiple CBDS recurrences after EST.

EUS-guided fine-needle tissue acquisition by using a 19-gauge needle in a selected patient population: a prospective study.

BACKGROUND: The ability to obtain tissue samples for histological examination during EUS has theoretical advantages over cytology alone. OBJECTIVE: To prospectively evaluate the feasibility and yield of EUS-guided fine-needle tissue acquisition (EUS-FNTA) with a large-gauge needle in patients in whom we expected histology to be more useful than cytology to reach a definitive diagnosis. DESIGN: Prospective cohort study. SETTING: Tertiary care academic medical center. PATIENTS: Consecutive patients with subepithelial lesions, esophagogastric wall thickening, mediastinal and abdominal masses/lymphadenopathy of unknown origin, or pancreatic lesions after nondiagnostic FNA. INTERVENTIONS: EUS-FNTA with a 19-gauge needle. MAIN OUTCOME MEASUREMENTS: Feasibility and yield of EUS-FNTA. RESULTS: A total of 120 patients with a mean age of 61 ± 14.6 years and mean lesion size of 38 ± 25 mm (range 8-140 mm) were enrolled. FNTA was successfully performed in all but 1 patient (98.9%), and adequate samples for histological examination were obtained in 116 of the 119 patients (97.5%) in whom EUS-FNTA was technically successful. A mean of 2.8 ± 0.8 passes per patient were performed. At the time of current follow-up, a definitive diagnosis was available in 117 of the 120 patients (97.5%), with only 8 false-negative results. The sensitivity, specificity, positive and negative predictive values, and diagnostic accuracy of EUS-FNTA in the 117 patients with a definitive diagnosis were 91.8%, 100%, 100%, 71.4%, and 93.2%, respectively. LIMITATIONS: Single-center study with limited power. CONCLUSIONS: EUS-FNTA by using a large-gauge needle has a high yield and promising diagnostic accuracy and could be used when histology may be more useful than cytology to reach a definitive diagnosis.

Importance of mediastinal screening-based observation during endoscopic ultrasound to examine gastrointestinal pathologies.

In all endoscopic ultrasound (EUS) examinations performed at our hospital, the heart, vasculature, and mediastinal lymph nodes from the esophagus are observed after checking for gastrointestinal pathologies. Since the introduction of EUS using a convex linear-array echoendoscope at our hospital in April 2015, EUS examinations have been performed in 371 cases for examining pancreaticobiliary diseases, submucosal tumors, and other pathologies during the 3-year period, till March 2018. We diagnosed 2 patients with asymptomatic cardiovascular disease while observing the mediastinum during EUS examination to examine identified pancreaticobiliary disease. No subjective symptoms associated with cardiovascular disease were observed and the respective conditions had not been identified previously in either case. One case involved a left atrial myxoma while the other involved a saccular aortic aneurysm in the thoracic aorta. A left atrial tumor resection and aortic replacement surgery were performed in each case. Their postoperative courses have been favorable. As cardiovascular diseases are often life-threatening, as in the present 2 cases, observational screening of the cardiovascular system from the esophagus should also be performed during EUS examinations just as the pharyngeal region is examined during upper gastrointestinal endoscopy.

Annexin A2 expression and phosphorylation are up-regulated in hepatocellular carcinoma.

Annexins (ANXs) constitute a family of Ca2+-dependent membrane-binding proteins; at least 20 of them have been described to date. Among these, Annexin A2 (ANXA2) has been revealed as a multi-functional protein in vitro. Its actual role in vivo, however, requires further investigation. We already reported that ANX-I (ANXA1) was up-regulated in hepatocellular carcinoma (HCC). The role of ANXA2 in various liver diseases including HCC remains obscure. In the present study, the protein and mRNA levels of ANXA2, as well as its localization, were determined for the normal human liver, chronic hepatitis liver, and non-tumorous and tumorous portions of HCC tissues. ANXA2 was rarely detected in either normal or chronic hepatitis liver tissues, whereas it was overexpressed at both the transcriptional and translational levels in tumorous and non-tumorous regions of HCC. In addition, in many cases, more ANXA2 was expressed in the tumorous portion than in the non-tumorous portion of HCC. The expression of ANXA2 was mainly localized in cancer cells, especially in poorly differentiated HCC. Furthermore, ANXA2 was tyrosine-phosphorylated in HCC. These data suggest that overexpression and tyrosine phosphorylation of ANXA2 play important roles in the malignant transformation process leading to HCC and are related to the histological grade of HCC.

How many cytological examinations should be performed for the diagnosis of malignant biliary stricture via an endoscopic nasobiliary drainage tube?

BACKGROUND AND AIM: The sensitivity of bile cytology is recognized as being low. Repeating cytological sampling is likely to improve the sensitivity. The aim of this study is to determine the optimal number of repeated cytological sampling of bile obtained via an endoscopic nasobiliary drainage (ENBD) tube for the diagnosis of malignant biliary stricture. METHODS: Ninety-eight patients with malignant biliary stricture who underwent ENBD were enrolled. Diagnoses included bile duct carcinoma (n = 53), pancreatic carcinoma (n = 28), carcinoma of the major papilla (n = 8), gallbladder carcinoma (n = 6), and hepatocellular carcinoma (n = 3). Bile was aspirated via an ENBD tube once a day and immediately evaluated cytologically. RESULTS: The median number of cytological samplings via an ENBD tube was 2.8 times (range, 1-10). In 40 of 98 patients with malignant biliary stricture, cytology was positive at the first cytological sampling (sensitivity 40.8%). Cytology was cumulatively positive in 71 of 98 patients (sensitivity 72.4%) from which repeated samples were taken. In 71 patients with positive cytology, correlation of the positive rate and the number of cytological samplings performed was investigated. In 68 of 71 (95.8%) patients with positive cytology, positive results were obtained by or at the sixth examination. CONCLUSIONS: Bile cytology via an ENBD tube is an easy method, and has been shown to have relatively high sensitivity. The optimal number of repeated cytological samplings using bile obtained via an ENBD tube for the diagnosis of malignant biliary stricture was concluded to be six.

Long-Term Management of Recurrent Cholecystitis after Initial Conservative Treatment: Endoscopic Transpapillary Gallbladder Stenting.

Background: Endoscopic transpapillary gallbladder stenting (ETGBS) is an effective procedure for treating high-risk patients with acute cholecystitis and severe comorbidities. However, the efficacy of ETGBS for recurrent cholecystitis (RC) remains unclear. This study aimed to explore its efficacy in patients with RC for whom cholecystectomy is contraindicated because of its high surgical risk. Methods: Data on 19 high-risk patients who had undergone ETGBS for RC after initial conservative therapy in our institution between June 2006 and May 2012 were retrospectively examined. The primary outcome was the clinical success rate, which was defined as no recurrences of acute cholecystitis after ETGBS until death or the end of the follow-up period. Secondary outcomes were technical success rate and adverse events (AEs). Results: The clinical success rate of ETGBS was 100%, the technical success rate 94.7%, and AE rate 5%: one patient developed procedure-related mild acute pancreatitis. The clinical courses of all patients were as follows: four died of nonbiliary disease, and the remaining 15 were subsequently treated conservatively. The median duration of follow-up was 14.95 months (range 3-42 months). Conclusions: ETGBS is an effective alternative for managing RC in high-risk patients with severe comorbidities.

Vitamins K2, K3 and K5 exert antitumor effects on established colorectal cancer in mice by inducing apoptotic death of tumor cells.

Although a number of studies have shown that vitamin K possesses antitumor activities on various neoplastic cell lines, there are few reports demonstrating in vivo antitumor effects of vitamin K, and the antitumor effect on colorectal cancer (CRC) remains to be examined. Therefore, antitumor effects of vitamin K on CRC were examined both in vitro and in vivo. Vitamins K2, K3 and K5 suppressed the proliferation of colon 26 cells in a dose-dependent manner, while vitamin K1 did not. On flow cytometry, induction of apoptosis by vitamins K2, K3 and K5 was suggested by population in sub-G1 phase of the cell cycle. Hoechst 33342 staining and a two-color flow cytometric assay using fluorescein isothiocyanate-conjugated annexin V and propidium iodide confirmed that vitamins K2, K3 and K5 induced apoptotic death of colon 26 cells. Enzymatic activity of caspase-3 in colon 26 cells was significantly up-regulated by vitamins K2, K3 and K5. The pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, substantially prevented vitamin K-mediated apoptosis. In vivo study using syngeneic mice with subcutaneously established colon 26 tumors demonstrated that intravenous administration of vitamins K2, K3 and K5 significantly suppressed the tumor growth. The number of apoptotic tumor cells was significantly larger in the vitamin K-treated groups than in the control group. These results suggest that vitamins K2, K3 and K5 exerted effective antitumor effects on CRC in vitro and in vivo by inducing caspase-dependent apoptotic death of tumor cells, suggesting that these K vitamins may be promising agents for the treatment of patients with CRC.

Identification of c-Yes expression in the nuclei of hepatocellular carcinoma cells: involvement in the early stages of hepatocarcinogenesis.

It is thought that the subcellular distribution of Src-family tyrosine kinases, including c-Yes binding to the cellular membrane, is membranous and/or cytoplasmic. c-Yes protein tyrosine kinase is known to be related to malignant transformation. However, the expression patterns of c-Yes in hepatocellular carcinoma (HCC) remains unknown. In the present study, we report that c-Yes is expressed not only in the membrane and cytoplasm, but also in the nuclei of cancer cells in some human HCC tissues and in a human HCC cell line. We examined the expression and localization of c-Yes in human HCC cell lines (HLE, HLF, PLC/PRF/5 and Hep 3B) by Western blotting and immunohistochemical analyses; we also examined the expression of c-Yes by immunohistochemistry and Western blotting in the tissues of various liver diseases, including 39 samples from HCC patients. We used an antibody array to detect proteins that bind to nuclear c-Yes in PLC/PRF/5 cell line. c-Yes was found to be expressed in the membranes and cytoplasm of HLE, HLF and Hep 3B HCC cells; it was also detected in the nuclei in addition to the membranes and cytoplasm of PLC/PRF/5 HCC cells. HCC with nuclear c-Yes was detected in 5 of 39 cases (13.0%), and nuclear c-Yes expression was not detected in normal, chronic hepatitis or cirrhotic livers. All HCCs with nuclear c-Yes expression were well-differentiated, small tumors at the early stages. In the PLC/PRF/5 cell line, the nuclear localization of c-Yes with cyclin-dependent kinase 1 was confirmed by a protein antibody array. In conclusion, nuclear c-Yes expression was found in cancer cells at the early stages of hepatocarcinogenesis, suggesting that nucleus-located c-Yes may be a useful marker to detect early-stage HCC.

Usefulness of single and repetitive percutaneous transhepatic gallbladder aspiration for the treatment of acute cholecystitis.

BACKGROUND: The aim of this study was to evaluate the safety and usefulness of single and repetitive percutaneous transhepatic gallbladder aspiration (PTGBA) for the treatment of acute cholecystitis. METHODS: PTGBA was performed in patients with acute cholecystitis who showed no improvement after treatment with broad-spectrum antibiotics. PTGBA was carried out at bedside. When the bile was too thick to be aspirated through a 21-gauge needle, an 18-gauge needle was used. Aspiration of the gallbladder contents and injection of antibiotics into the gallbladder were performed without the placement of a drainage catheter. When improvement was not observed after the first attempt, PTGBA was repeated. RESULTS: Single PTGBA achieved improvement in 32 of 45 patients. In 11 of the remaining 13 patients, the second PTGBA was effective. In the remaining two patients, repetitive PTGBA was not carried out because of advanced cancer. In two of 45 patients, 18-gauge needles were necessary for PTGBA because of the high viscosity of the bile. PTGBA was carried out in three patients with blockage of the cystic duct by a stent, and it was effective in all three. Two patients whose conditions improved with a single PTGBA experienced a recurrence at 4 and 31 months, respectively, after PTGBA. No other severe complications related to PTGBA were observed in any patients. CONCLUSIONS: For the treatment of acute cholecystitis that does not react to conservative therapies, PTGBA is a safe, simple, and effective treatment modality that can be performed at bedside without any severe complications.

Utility of pancreatic duct brushing for diagnosis of pancreatic carcinoma.

BACKGROUND: The aim of this study was to evaluate the usefulness of pancreatic duct brushing for diagnosis of pancreatic carcinoma. METHODS: Brush cytology was attempted in 58 patients suspected of having pancreatic malignancy because of stricture of the main pancreatic duct, confirmed by endoscopic retrograde cholangiopancreatography. Thirty-eight patients were finally diagnosed by an operation or the clinical course as having pancreatic carcinoma, and the remaining 20 patients as having chronic pancreatitis. The usefulness of brush cytology for diagnosis of pancreatic carcinoma was estimated. We interpreted failures of pancreatic duct brushing to be false negatives when the lesion was malignant. RESULTS: In 48 of 58 patients (82.8%), brushing was successfully performed and satisfactory specimens were obtained. Brush cytology was positive in 25 of 38 patients with pancreatic carcinoma (sensitivity 65.8%) and negative in all patients without malignancy (specificity 100%). Overall accuracy was 76.4%. During 2001-2005, the number of back-and-forth motions of the brush was increased to more than 30 times. The sensitivity significantly improved from 43.8% in 1997-2000 to 81.8% in 2001-2005 (P < 0.05). The increased success rate of brushing by improvement of skill in manipulating the guidewire and increased number of cells smeared on glass slides by increased back-and-forth motion of the brush may account for this improvement over time. Moreover, the sensitivity in 2001-2005 was 85.7% if failures of brushing with pancreatic carcinoma are excluded. No major complications occurred, except for two patients with a moderate grade of acute pancreatitis. CONCLUSIONS: Although further studies with a large number of patients are needed, our results suggest that with recent improvements of the brushing technique, pancreatic duct brushing is a useful and safe method for the differential diagnosis of malignancy from benign diseases of the pancreas.

Sequential assessment of the intrahepatic expression of epidermal growth factor and transforming growth factor-beta1 in hepatofibrogenesis of a rat cirrhosis model.

Responses of the liver to chronic injury include inflammation, regeneration and fibrosis, which finally lead to cirrhosis. The cause of liver cirrhosis appears to be impaired proliferative capability of hepatocytes caused by continuous hepatic damage, and subsequent accumulation of extracellular matrix produced by hepatic stellate cells (HSCs). Epidermal growth factor (EGF) and transforming growth factor-beta1 (TGF-beta1) play a crucial role in hepatocyte proliferation and hepatofibrogenesis, respectively. However, sequential analyses of the intrahepatic expression of EGF and TGF-beta1 in the course of cirrhosis development have not been examined fully. In the present study, liver cirrhosis was produced in rats by intraperitoneal administration of dimethylnitrosamine (DMN), and intrahepatic mRNA expression levels of proliferating cell nuclear antigen (PCNA), EGF and TGF-beta1 were quantitatively estimated by a real-time reverse transcription-polymerase chain reaction method. Histological and semiquantitative densitometric examination of liver sections revealed that the accumulation of extracellular matrix components was increased according to the period of DMN treatment. Histological examination of liver sections of rats treated with DMN for 4 and 6 weeks revealed pre-cirrhosis and cirrhosis, respectively. Intrahepatic mRNA expression levels of PCNA and EGF correlated well. Expression levels of both molecules were increased significantly during the course of cirrhosis development, but decreased significantly at the time of complete cirrhosis manifestation. In contrast, intrahepatic TGF-beta1 expression was increased significantly according to the period of DMN treatment, and reached a peak at the time of cirrhosis manifestation. These results suggest that proliferative capability of hepatocytes was impaired by continuous liver damage due, in part, to the decrease of a hepatocyte mitogen EGF, and that increased intrahepatic TGF-beta1 activated HSCs to retrieve space lost by hepatocyte destruction, resulting in complete cirrhosis manifestation.

Relationship between the proliferative capability of hepatocytes and the intrahepatic expression of hepatocyte growth factor and c-Met in the course of cirrhosis development in rats.

Liver cirrhosis is the fatal end stage of various chronic liver diseases. One of the most important causes of liver cirrhosis appears to be an impaired proliferative capability of hepatocytes caused by continuous hepatic damage. Hepatocyte growth factor (HGF) and its specific receptor, c-Met, play a pivotal role in hepatocyte proliferation. However, the relationship between the proliferative capability of hepatocytes and the intrahepatic expression of HGF and c-Met during the course of cirrhosis development has not been studied fully. In the present study, liver cirrhosis was produced in rats by intraperitoneally administering dimethylnitrosamine (DMN) and intrahepatic expression levels of HGF and c-Met were quantitatively estimated using a real-time reverse transcription-polymerase chain reaction (RT-PCR) method. Histological examination of liver sections and biochemical estimation of serum levels of transaminase revealed that the degree of hepatocyte destruction was elevated gradually during cirrhosis development in the DMN-induced rat cirrhosis model. The proliferative capability of hepatocytes estimated immunohistochemically by proliferating cell nuclear antigen staining was markedly increased at an early stage of cirrhosis development. However, it was gradually decreased thereafter and suppressed substantially at the time of cirrhosis manifestation. Intrahepatic HGF expression was also increased significantly during the course of cirrhosis development but decreased significantly at the time of cirrhosis manifestation. Conversely, there was a tendency for the intrahepatic expression of c-Met to decrease from an early stage of cirrhosis development and intrahepatic c-Met expression was decreased significantly at the time of cirrhosis manifestation. These results suggest that the highly proliferative capability of hepatocytes at an early stage of cirrhosis development is induced by increased intrahepatic HGF expression. However, both HGF and c-Met expression levels in the liver are decreased significantly there-after. Accordingly, the proliferative capability of hepatocytes is severely impaired and extracellular matrix components are deposited to retrieve space lost by the destruction of hepatic parenchyma, resulting in the establishment of liver cirrhosis.

Extracorporeal abdominal massage may help prevent recurrent bile duct stones after endoscopic sphincterotomy.

BACKGROUND AND STUDY AIMS: Endoscopic sphincterotomy (EST) is effective, but recurrent bile duct stones are a common late complication. Because there are still no effective therapies for preventing this complication, some patients have experienced bile duct stone recurrence many times. We describe herein a method of abdominal massage to treat patients with prior cholecystectomy who have experienced recurrence of bile duct stones.

Antitumor effects of vitamins K1, K2 and K3 on hepatocellular carcinoma in vitro and in vivo.

A number of studies have shown that various K vitamins, specifically vitamins K2 and K3, possess antitumor activity on various types of rodent- and human-derived neoplastic cell lines. In the present study, we examined the antitumor effects of vitamins K1, K2 and K3 on PLC/PRF/5 human hepatocellular carcinoma (HCC) cells in vitro and in vivo. Furthermore, we examined the mechanisms of antitumor actions of these vitamins in vitro and in vivo. Although vitamin K1 did not inhibit proliferation of PLC/PRF/5 cells at a 90-microM concentration (the highest tested), vitamins K2 and K3 suppressed proliferation of the cells at concentrations of 90 and 9 microM, respectively. By flow cytometric analysis, it was shown that not only vitamin K1, but also vitamin K2 did not induce apoptosis or cell cycle arrest on PLC/PRF/5 cells. In contrast, vitamin K3 induced G1 arrest, but not apoptosis on PLC/PRF/5 cells. Subsequent in vivo study using subcutaneous HCC-bearing athymic nude mice demonstrated that both vitamins K2 and K3 markedly suppressed the growth of HCC tumors to similar extent. Protein expression of cyclin D1 and cyclin-dependent kinase 4 (Cdk4), but not p16INK4a Cdk inhibitor in the tumor was significantly reduced by vitamin K2 or K3 treatment, indicating that vitamins K2 and K3 may induce G1 arrest of cell cycle on PLC/PRF/5 cells in vivo. Taken collectively, vitamins K2 and K3 were able to induce potent antitumor effects on HCC in vitro and in vivo, at least in part, by inducing G1 arrest of the cell cycle. The results indicate that vitamins K2 and K3 may be useful agents for the treatment of patients with HCC.

Vitamins K2, K3 and K5 exert in vivo antitumor effects on hepatocellular carcinoma by regulating the expression of G1 phase-related cell cycle molecules.

A number of studies have shown that various vitamins K, specifically vitamin K2, possessed antitumor activity on various types of rodent- and human-derived neoplastic cell lines. However, there are only a small number of reports demonstrating in vivo antitumor effects of vitamins K. Furthermore, the mechanism of antitumor effects of vitamins K still remains to be examined. In the present study, we examined the antitumor effects of vitamins K2, K3 and K5 on PLC/PRF/5 human hepatocellular carcinoma (HCC) cells in vivo. Furthermore, to examine the mechanism of antitumor actions of these vitamins K, mRNA expression levels of various G1 phase-related cell cycle molecules were evaluated by using a real-time reverse transcription-polymerase chain reaction (RT-PCR) method. HCC-bearing animals were produced by implanting PLC/PRF/5 cells subcutaneously into athymic nude mice, and drinking water containing vitamin K2, K3 or K5 was given to the animals. Treatments with vitamins K2, K3 and K5 were shown to markedly inhibit the growth of HCC tumors. To examine the mechanism of in vivo antitumor effects of vitamins K, total RNA was extracted from HCC tumors, and the expression of G1 phase-related cell cycle molecules was quantitatively examined. Real-time RT-PCR demonstrated that the expression of the cell cycle-driving molecule, cyclin-dependent kinase 4 (Cdk4), in HCC was significantly reduced by the treatments with vitamin K2, K3 and K5. Conversely, the expression of the cell cycle-suppressing molecules, Cdk inhibitor p16INK4a and retinoblastoma, in HCC was significantly enhanced by the treatments with vitamins K2, K3 and K5. These results indicate that vitamins K2, K3 and K5 exert antitumor effects on HCC by regulating the expression of G1 phase-related cell cycle molecules. These results also indicate that vitamins K2, K3 and K5 may be useful agents for the treatment of patients with HCC.

Analyses of autoantibodies against tumor-associated antigens in patients with hepatocellular carcinoma.

Autoantibodies against tumor-associated antigens (TAAs) such as insulin-like growth factor II mRNA-binding proteins (IMPs), p53, c-myc, and survivin were analyzed in patients with hepatocellular carcinoma (HCC), using recombinant proteins of these antigens. Eight of 86 (9.3%) HCC patients had one or more of these autoantibodies. However, serum alpha-fetoprotein (AFP) levels ranged within normal limits in HCC patients with anti-TAAs except for one case with anti-IMP1. One of the HCC patients had autoantibodies against IMP1, IMP3 and p53 before the diagnosis of HCC. These findings may indicate that anti-TAAs seem to be supplementary serological markers for the diagnosis of HCC in AFP-negative cases and that autoantibodies against IMP1, IMP3 and p53 are candidates for predictive markers of HCC development.

Enhanced expression of p46 Shc in the nucleus and p52 Shc in the cytoplasm of human gastric cancer.

Shc protein is known to be related to cell proliferation and carcinogenesis. However, the involvement of Shc in gastric cancer remains unknown. In the present study, we examined the expression and localization of Shc in human gastric cancer and the adjacent normal mucosa by Western blotting and immunohistochemical analyses. Furthermore, the expression of Shc in a gastric cancer cell line, MKN 28, was also studied. p66 Shc was not detected at all in gastric cancer or the adjacent normal mucosa. In contrast, immunohistochemical and Western blot analyses revealed that p52 Shc was detected in the cytoplasmic fractions obtained from gastric normal mucosa and cancer, while p46 Shc expression was observed in the nuclear fractions from gastric normal mucosa and cancer. Furthermore, not only p52 Shc expression in the cytoplasm but also p46 Shc expression in the nucleus was much higher in gastric cancer than in the adjacent normal mucosa. Subsequent in vitro experiments with MKN 28 gastric cancer cells also revealed that p52 Shc was expressed solely in the cytoplasm and p46 Shc was solely in the nucleus. These results suggest that the enhancement of p46 Shc and p52 Shc expression may be related to the occurrence of gastric cancer. Furthermore, unlike p52 Shc, p46 Shc was shown to be expressed solely in the nucleus, suggesting that p46 Shc expressed in the nucleus may play an important role in gastric carcinogenesis.

In vitro and in vivo antitumor effects of vitamin K5 on hepatocellular carcinoma.

Although a number of studies have shown that vitamins K1, K2 and K3 exerted antitumor effects on various types of rodent- and human-derived neoplastic cell lines, it has not been examined whether or not vitamin K5 also possesses antitumor activity. In the present study, we examined the antitumor effects of vitamin K5 on PLC/PRF/5 human hepatocellular carcinoma (HCC) cells in vitro and in vivo. Furthermore, we examined the mechanisms of antitumor actions of vitamin K5 not only in vitro but also in vivo. Vitamin K5 was shown to suppress the proliferation of PLC/PRF/5 cells at a concentration of 30 microM. By a flow cytometric analysis, it was shown that although vitamin K5 did not induce apoptosis on PLC/PRF/5 cells, it did induce G1 arrest on PLC/PRF/5 cells. Subsequent in vivo study using subcutaneous HCC-bearing athymic nude mice demonstrated that vitamin K5 markedly suppressed the growth of HCC tumors. Although protein expression levels of cyclin D1 and p16INK4a cyclin-dependent kinase (Cdk) inhibitor in HCC tumors were not decreased by vitamin K5 treatment, those of Cdk4 were reduced significantly by the treatment. Taken collectively, vitamin K5 could induce potent antitumor effects on HCC not only in vitro but also in vivo, at least in part by inducing G1 arrest of cell cycle through downregulation of Cdk4 expression. The results demonstrated here indicate that vitamin K5 may be a useful agent for the treatment of patients with HCC.

Comparison study of the expressions of myristoylated alanine-rich C kinase substrate in hepatocellular carcinoma, liver cirrhosis, chronic hepatitis, and normal liver.

The myristoylated alanine-rich C kinase substrate (MARCKS) is a prominent substrate for protein kinase C (PKC) in a variety of cells. The aim of this study was not only to evaluate the expression and localization of MARCKS in various pathological liver tissues, including HCC, but also to analyze the difference in MARCKS expression between hepatitis virus-induced HCC and cirrhosis. The level of MARCKS and its phosphorylated proteins, as well as its localization, were determined using Western blot and/or immunohistochemistry in HCC and other pathological liver tissues. We also analyzed the change of MARCKS localization on the influence of MARCKS phosphorylation in the HLF cancer cell line by phosphorylation study. In addition, the relationship between MARCKS expression and proliferative activity was studied in HCC. In the immunohistochemical study, a very small amount of MARCKS protein was found along the contour of the hepatocellular membrane in normal liver and in cases of chronic hepatitis. MARCKS was up-regulated in liver cirrhosis tissue and was localized in the cytoplasm of hepatocytes. The expression of MARCKS was down-regulated in HCC tissues, as compared with non-tumorous liver cirrhosis tissues from the same patients. Furthermore, MARCKS was serine-phosphorylated in liver cirrhosis and HCC, and phosphorylated MARCKS was detected in a cytosolic fraction of these tissues. In a phosphorylation study using the HLF HCC cell line, MARCKS was displaced from the plasma membrane to the cytosol following the activation of protein kinase C (PKC) by phorbol 12-myristrate 13-acetate (PMA). Furthermore, the activity of cyclin D1 and cyclin E kinases was found to be higher in HCCs with low MARCKS expression than in HCCs with high MARCKS expression. These results suggest that up-regulation of MARCKS might be essential in the generation of cirrhotic nodules through chronic hepatitis from normal liver, and that the phosphorylation and/or down-regulation of MARCKS might play an important role in the development and progression of HCC from liver cirrhosis.