Changes in Coagulation and Fibrinolytic Factors in Patients With Cirrhotic Refractory Ascites Undergoing Cell-free and Concentrated Ascites Reinfusion Therapy: A Retrospective Observational Study in Japan.

BACKGROUND/AIM: The management of refractory ascites is critical for the treatment of patients with decompensated cirrhosis. This study aimed to evaluate the feasibility and safety of cell-free and concentrated ascites reinfusion therapy (CART) in patients with cirrhosis and refractory ascites, with a focus on changes in coagulation and fibrinolytic factors in ascitic fluid following CART. PATIENTS AND METHODS: This was a retrospective cohort study including 23 patients with refractory ascites undergoing CART. Serum endotoxin activity (EA) before and after CART and the levels of coagulation and fibrinolytic factors and proinflammatory cytokines in original and processed ascitic fluid were measured. The Ascites Symptom Inventory-7 (ASI-7) scale was used for subjective symptom assessment before and after CART. RESULTS: Body weight and waist circumference significantly decreased after CART, whereas serum EA did not significantly change after CART. Similar to the previous reports, ascitic fluid concentrations of total protein, albumin, high-density lipoprotein cholesterol, γ-globulin, and immunoglobulin G levels were significantly increased after CART; mild elevations in body temperature and interleukin 6 and tumor necrosis factor-alpha levels in ascitic fluid were also observed. Importantly, the levels of antithrombin-III, factor VII, and X, which are useful for patients with decompensated cirrhosis, were markedly increased in the reinfused fluid during CART. Finally, the total ASI-7 score was significantly lower following CART, compared with the pre-CART score. CONCLUSION: CART is an effective and safe approach for the treatment of refractory ascites that allows the intravenous reinfusion of coagulation and fibrinolytic factors in the filtered and concentrated ascites.

Evaluation of DNA/RNA quality from cell block of malignant mesothelioma and lung adenocarcinoma.

Malignant mesothelioma (MM) is a rare and highly lethal tumor that arises from mesothelial tissue on the surface of the chest and abdominal cavity. Cytological examination of body fluids, including pleural fluid and ascites, is essential for the differentiation of malignant mesothelioma from other carcinomas, such as lung and gastrointestinal carcinomas and metastatic tumors. To evaluate the effectiveness of cell block preparation procedures, which are used for immunocytochemical staining and genetic panel analysis of tumor-specific gene mutations, we used various fixatives. We also evaluated the effects of immunostaining, and the quality of nucleic acids for genetic analysis. METHODS: Cell blocks were prepared using the malignant mesothelioma cell lines MESO4 and H226 and non-small cell lung carcinoma cell line HCC78. The cells were fixed using 10% neutral buffered formalin and four different fixatives for liquid cytology. Fixed cells were formed into cell clusters using sodium alginate or centrifugation, and paraffin-embedded cell blocks were prepared. RESULTS: Cell blocks were morphologically evaluated by hematoxylin and eosin and immunocytological staining, and the nucleic acid quality was evaluated by DNA/RNA extraction, qPCR, and next-generation sequence analysis. D2-40 and WT1 staining differed depending on the fixation solution and the cell cluster formation method; however, the degree of nucleic acid degradation was not impaired by any method. CONCLUSION: Although the morphological evaluation of cytology specimens is affected by the method of cell block preparation, it is still useful for nucleic acid extraction and gene panel analysis, as long as there are sufficient amounts of tumor cells.

Molecular Analysis of Liquid-Based Cytological Specimen Using Virtually Positive Sputum with Adenocarcinoma Cells.

Liquid-based cytology (LBC) analysis of sputum is a useful diagnostic and prognostic tool for detecting lung cancer. DNA and RNA derived from lung cancer cells can be used for this diagnosis. However, the quality of cytological material is not always adequate for molecular analysis due to the effect of formalin in the commercially available fixation kits. In this study, we examined DNA and RNA extraction methods for LBC analysis with formalin fixation, using lung carcinoma cell lines and sputum. The human non-small cell lung cancer cell lines were fixed with LBC fixation reagents, such as CytoRich red preservative. Quantification of thyroid transcription factor-1 (TTF-1) and actin mRNA, epidermal growth factor receptor (EGFR) DNA in HCC827, H1975, and H1299 cells, and mutation analysis of EGFR in HCC827 and H1975 cells were performed by quantitative PCR (qPCR) and fluorescence resonance energy transfer (FRET)-based preferential homoduplex formation assay (F-PHFA) method, respectively. mRNA and DNA extracted from cell lines using RNA and/or DNA extraction kits for formalin-fixed paraffin-embedded (FFPE) fixed with various LBC solutions were efficiently detected by qPCR. The detection limit of EGFR mutations was at a rate of 5% mutated positive cells in LBC. The detection limit of the EGFR exon 19 deletion in HCC827 was detected in more than 1.5% of the positive cells in sputum. In contrast, the detection limit of the T790M/L858R mutation in H1975 was detected in more than 13% of the positive cells. We also detected EGFR mutations using next generation sequencing (NGS). The detection limit of NGS for EGFR mutation was lower than that of the F-PHFA method. Furthermore, more than 0.1% of positive cells could be cytomorphologically detected. Our results demonstrate that LBC systems are powerful tools for cytopathological and genetic analyses. However, careful attention should be paid to the incidence of false negative results in the genetic analysis of EGFR mutations detected by LBC.

Hemostatic function in hyperfibrinolytic disseminated intravascular coagulation.

BACKGROUND: Global hemostatic mechanism(s) in patients with disseminated intravascular coagulation (DIC) are poorly understood. There are few diagnostic criteria of DIC based on overall or global hemostatic mechanisms. METHODS: We have assessed in detailed the dynamic global hemostatic changes using thrombin and plasmin generation assay (T/P-GA), clot fibrinolytic waveform analysis (CFWA) and not-activated rotational thromboelastometry (NATEM), in a young girl with DIC associated with acute myeloid leukemia (AML). The ratios of endogenous thrombin potential (T-EP) and plasmin lag time (P-LT) relative to normal plasma was sourced from pooled normal plasma from healthy volunteers on T/P-GA. RESULTS: The inverse P-LT ratio prior to tranexamic acid (TXA) treatment was greater than the T-EP ratio (1.1-2.8 and 0.83-1.2, respectively). Significant reduction in inverse P-LT ratio (0.084-1.3) was observed after TXA treatment. The interval from clotting to the initiation of fibrinolysis (fibrinolysis lag time: FLT) in CFWA was significantly shorter than the control at onset (74.2-91.6 s vs 109 s), indicating enhanced fibrinolysis. Data from an adult with acute promyelocytic leukemia-associated DIC also supportively showed a high inverse P-LT ratio (2.1) and shortened FLT (83.7 s). The clotting time in patient whole blood using NATEM-mode during an episode of severe epistaxis markedly shortened beyond control, but returned to normal after the addition of an anti-tissue factor (TF) monoclonal antibody. CONCLUSION: The release of intravascular TF contributed to sustained activation of coagulation and subsequent fibrinolytic activity in this patient with AML-associated DIC, and T/P-GA could provide better quantitative data than conventional assays in these circumstances.

NACC1, as a Target of MicroRNA-331-3p, Regulates Cell Proliferation in Urothelial Carcinoma Cells.

The nucleus accumbens-associated protein 1 (NACC1) is a transcription factor constitutively expressed in the urothelium, where it regulates cell growth, senescence, autophagy, and epithelial-mesenchymal transition. microRNA (miRNA) constitutes a class of small non-coding RNAs which are involved in cell proliferation, differentiation, and progression of tumors. miRNAs and their target molecules are utilized for molecular diagnosis of urothelial carcinoma. NACC1 is one of several putative target molecules of miR-331-3p, and is associated with cell proliferation in cancers such as prostate and cervical cancer. Functional experiments involving miR-331-3p and its target molecule NACC1 were conducted using the urothelial carcinoma (UC) cell lines, T24, UMUC6, and KU7. Furthermore, quantitative reverse transcription polymerase chain reaction and immunostaining were performed to evaluate the expression of NACC1 in UC derived from transurethral resection of bladder tumor (TUR-Bt) specimens. The methane thiosulfonate (MTS) assay revealed that cell proliferation was significantly reduced after transient transfection of miR-331-3p precursor and/or NACC1 siRNA in UC cells. Cell senescence via cell cycle arrest at the G1 phase was induced by NACC1 inhibition. On the other hand, suppression of NACC1 induced cell migration and invasion abilities. Immunohistochemical analysis of TUR-Bt specimens revealed that over 70% of UC cells presented strongly positive results for NACC1. In contrast, normal urothelial cells were weakly positive for NACC1. It was also found that NACC1 expression was lower in invasive UC cells than in non-invasive UC cells. Loss of NACC1 induced vessel invasion in invasive UC tissues. The present results indicate that NACC1 regulated by miR-331-3p contributes to cell proliferation, and is involved in cell migration and invasion. This suggests that NACC1 can serve as a potential target molecule for the prediction and prognosis of UC, and can contribute to effective treatment strategies.

MicroRNA-331-3p Suppresses Cervical Cancer Cell Proliferation and E6/E7 Expression by Targeting NRP2.

Aberrant expression of microRNAs (miRNAs) is involved in the development and progression of various types of cancers. In this study, we investigated the role of miR-331-3p in cell proliferation and the expression of keratinocyte differentiation markers of uterine cervical cancer cells. Moreover, we evaluated whether neuropilin 2 (NRP2) are putative target molecules that regulate the human papillomavirus (HPV) related oncoproteins E6 and E7. Cell proliferation in the human cervical cancer cell lines SKG-II, HCS-2, and HeLa was assessed using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay. Cellular apoptosis was measured using the TdT-mediated dUTP nick end labeling (TUNEL) and Annexin V assays. Quantitative RT-PCR was used to measure the messenger RNA (mRNA) expression of the NRP2, E6, E7, p63, and involucrin (IVL) genes. A functional assay for cell growth was performed using cell cycle analyses. Overexpression of miR-331-3p inhibited cell proliferation, and induced G2/M phase arrest and apoptosis in SKG-II, HCS-2 and HeLa cells. The luciferase reporter assay of the NRP2 3'-untranslated region revealed the direct regulation of NRP2 by miR-331-3p. Gene expression analyses using quantitative RT-PCR in SKG-II, HCS-2, and HeLa cells overexpressing miR-331-3p or suppressing NRP2 revealed down-regulation of E6, E7, and p63 mRNA and up-regulation of IVL mRNA. Moreover, miR-331-3p overexpression was suppressed NRP2 expression in protein level. We showed that miR-331-3p and NRP2 were key effectors of cell proliferation by regulating the cell cycle, apoptosis. NRP-2 also regulates the expression of E6/E7 and keratinocyte differentiation markers. Our findings suggest that miR-331-3p has an important role in regulating cervical cancer cell proliferation, and that miR-331-3p may contribute to keratinocyte differentiation through NRP2 suppression. miR-331-3p and NRP2 may contribute to anti-cancer effects.

[Measures Against Hepatitis B Virus Reactivation Where Specialist Teams Collaborate].

Recently, hepatitis B virus (HBV) reactivation has attracted attention as a complication of cancer chemo- therapy or immunosuppressive therapy. To prevent hepatitis B due to HBV reactivation, practical guide- lines were issued in 2009. The guidelines include the relevant diagnostic algorithms for HBV markers (HBsAg, anti-HBc, anti-HBs, and HBV-DNA). Nonetheless, cases of acute liver failure due to HBV reacti- vation have occurred in Japan since 2009, likely because many of the physicians prescribing anti-cancer or immunosuppressive agents have not acted in conformity with the guidelines. The reasons for this non- conformance are considered to be as follows: First, the incidence of HBV reactivation varies markedly be- tween anti-cancer or immunosuppressive agents, and many physicians are simply not aware of this risk. Second, establishing a system for assessing compliance to the guidelines is complicated because it requires integrating both prescription data and HBV marker data, and then feeding back this information to physicians. Several medical faculties have established a survey system by establishing specialist teams comprising a hepatologist, pharmacist, laboratory technician, medical information manager, and other specialists. The multidisciplinary nature of these teams means that the actions of individuals are complemented and supported by the team as a whole and problems are resolved through teamwork. The role of clinical laboratory special- ists is likely to become more important, as their commitment to teamwork means that they are highly capable of supporting the development of clinical risk management initiatives. [Review].

A case of severe acalculous cholecystitis associated with sorafenib treatment for advanced hepatocellular carcinoma.

Sorafenib, a multikinase inhibitor, is the first and only drug, which improves significantly the overall survival in patients with advanced hepatocellular carcinoma (HCC). However, many patients experience diverse side effects, some of them severe and unexpected. To date, acute acalculous cholecystitis has not been documented in association with a HCC patient treated with sorafenib. Here, we report the case of a 43-year-old woman with hepatitis C virus-related advanced HCC. She received sorafenib, and later complained of a sudden onset of severe right hypocondrial pain with rebound tenderness and muscle defense. Laboratory examination showed mild elevation of transaminases, biliary enzymes, bilirubin, inflammation markers, and a marked peripheral eosinophilia. Abdominal computed tomography (CT) revealed a swollen gallbladder with exudate associated with severe inflammation without stones or debris. Consequently, sorafenib treatment was stopped immediately, and steroid-pulse therapy was performed. Steroid therapy drastically improved all clinical manifestations along with normalization of CT findings, eosinophilia, and liver functions. In summary, we herein report a rare case of acute severe acalculous cholecystitis associated with sorafenib in the patient with advanced HCC.

A non-invasive form of lipid-secreting carcinoma of the breast.

Lipid-secreting carcinoma is a rare variant of breast carcinoma. The tumor cells possess abundant vacuolated cytoplasm containing neutral fat. A 68-year-old Japanese female patient presented with a left breast tumor, which was detected by mass screening, and she was admitted to our hospital. The physical examination revealed an elastic hard lump in the left lateral quadrant of the left breast. The tumor size was 1.2 × 1.0 cm in diameter and the borderline was unclear. There were no palpable axillary lymph nodes or supraclavicular nodes. Mammography showed a polygonal mass with microcalcification. Ultrasonography indicated a hypoechoic lesion measuring 9 × 4 mm in diameter, with an irregularly shaped, slightly indistinct surface. The internal echoic level of the mass was heterogenous. Enhanced magnetic resonance imaging revealed a mass of high intensity in the left breast, and the connection of the intraductal spread was not detected. The time-intensity curve showed a peak-and-plateau pattern. Fine-needle aspiration cytology suggested a malignant tumor. The patient underwent a partial resection of the left breast (breast-conserving therapy) and a left axillary lymphadenectomy. Macroscopically, the resected specimen revealed a white tumor measuring approximately 0.6 × 0.5 cm. Histopathologically, the tumor measured up to approximately 0.9 × 0.7 cm because of additional components of intraductal spread and therefore was diagnosed as an extensive ductal carcinoma in situ with focal mass formation; the tumor also had abundant foamy cytoplasm. Oil-red-O staining confirmed the presence of marked cytoplasmic lipid droplets. These droplets were periodic acid-Schiff (PAS) negative even after diastase digestion, and negative with PAS-Alcian blue staining. In immunohistochemistry, these carcinoma cells were positive for E-cadherin. Thus, the pathological diagnosis was a non-invasive form of lipid-secreting carcinoma. The tumors were negative for both estrogen receptors and progesterone receptors. There were no metastases in the left axillary lymph nodes. The patient has remained well for 8 years without any evidence of recurrence.

Combination of branched-chain amino acids and angiotensin-converting enzyme inhibitor suppresses the cumulative recurrence of hepatocellular carcinoma: a randomized control trial.

Insulin resistance (IR) is reportedly involved in the progression of hepatocellular carcinoma (HCC). Since neovascularization plays an important role in hepatocarcinogenesis and IR, an angiostatic therapy may be considered as one of the promising approaches for chemoprevention against HCC. The aim of the current study was to examine the combination effect of a clinically used branched-chain amino acid (BCAA) and an angiotensin-converting enzyme inhibitor (ACE-I), both reportedly possess anti-angiogenic and IR-improving activities, on the cumulative recurrence after curative therapy. BCAA granules (Livact; 12 g/day) and/or ACE-I (perindopril; 4 mg/day) were administered after the curative therapy for HCC, and several indices were analyzed. A 48-month follow-up revealed that the combination treatment with BCAA and ACE-I markedly inhibited the cumulative recurrence of HCC under IR conditions, whereas neither single treatment exerted a significant inhibition. The soluble form of the vascular endothelial growth factor (VEGF; a central angiogenic factor) receptor-2 (sVEGFR2) was significantly decreased only three months after the treatment without recurrence. We also observed that IR, determined by the homeostasis model assessment (HOMA-IR), was significantly improved by this regimen, indicating that an inhibitory effect was achieved, at least partly, by coordinated effects of anti-angiogenesis and IR improvement. In conclusion, since both BCAA and ACE-I are widely used in clinical practice with safety, this combination therapy may represent a potential new strategy for chemoprevention against IR-based HCC recurrence in the future. Moreover, sVEGFR2 may become a useful clinical predictive marker of this combination treatment.

Apocrine carcinoma of the breast.

Apocrine carcinoma is a rare variant of breast carcinoma, and accounts for 0.3 to 1.0% of all breast cancers. A 55-year-old Japanese female patient presented with a right breast tumor, which had been detected by mass-screening, and she was admitted to our hospital. The physical examination revealed an elastic hard lump in the upper lateral quadrant of the right breast. The tumor size was approximately 1.0 cm in diameter, and the border was clear. There were no palpable axillary lymph nodes nor supraclavicular nodes. Fine-needle aspiration cytology revealed invasive ductal carcinoma. The patient underwent a partial resection of the right breast (breast conserving therapy) and a right axillary lymphadenectomy. Macroscopically, the resected specimen revealed a white tumor measuring 1.2 x 1.2 x 1.0 cm. The TNM classification was diagnosed as T1cN0M0 stage I. Histopathologically, the tumor revealed a proliferation of atypical epithelial cells with apocrine differentiation, arranged in a papillotubular or cribriform growth pattern with stromal invasion. The tumor cells showed irregular round-shaped nuclei often containing prominent nucleoli, and had particularly abundant eosinophilic granular cytoplasm. In the immunohistochemical analysis, these carcinoma cells were positive for Gross Cystic Disease Fluid Protein 15 and the androgen receptor, whereas they were negative for the estrogen and progesterone receptors. Immunohistochemical staining for Her2 using the HercepTest was found to be negative (score 0). Thus, the pathological diagnosis was apocrine carcinoma. There were no metastases in the axillary lymph nodes. The patient has had no recurrence in 8 years after surgery.

Selective aldosterone blocker ameliorates the progression of non-alcoholic steatohepatitis in rats.

Although non-alcoholic steatohepatitis (NASH) may progress to cirrhosis and hepatocellular carcinoma (HCC), no effective therapeutic modalities have been fully established yet. Recent studies have shown that the renin-angiotensin-aldosterone-system plays an important role in NASH. The aim of our current study was to elucidate the effects of aldosterone (Ald) inhibition on the progression of NASH. In the choline-deficient L-amino acid-defined diet-induced rat NASH model, the effects of a clinically used selective Ald blocker (SAB) were elucidated in conjunction with the activated hepatic stellate cells (HSC) and neovascularization, which are both known to play important roles in liver fibrosis development and hepatocarcinogenesis, respectively. Liver fibrosis development and the glutathione-S-transferase placental form-positive pre-neoplastic lesions were both markedly attenuated by SAB along with the suppression of the activated HSC and neovascularization. SAB inhibited the hepatic expression of transforming growth factor-beta 1 and also that of the vascular endothelial growth factor. Our in vitro study showed that SAB also inhibited the Ald-induced HSC proliferation and in vitro angiogenesis in a dose-dependent manner. These results indicated that Ald plays a pivotal role in the progression of NASH. Considering that SAB is already widely used in clinical practice, this drug could represent a potential new strategy against NASH in the future.

Selective aldosterone blocker, eplerenone, attenuates hepatocellular carcinoma growth and angiogenesis in mice.

AIM: The renin-angiotensin-aldosterone system (RAAS) has become known as a prerequisite for tumor angiogenesis, including hepatocellular carcinoma (HCC). Although angiotensin II is known to play an important role in tumor growth and angiogenesis, the role of aldosterone (Ald) is still obscure. The aim of our current study was to elucidate the effect of eplerenone, a clinically used selective Ald blocker (SAB), on murine HCC development especially in conjunction with angiogenesis. METHODS: To create an allograft model, we injected 1 x 10(6) of BNL-HCC cells into the flanks of BALB/c mice. After the tumor was established, SAB was administrated at dose of 100 mg/kg per day. RESULTS: Administration of SAB significantly suppressed HCC development along with inhibition of angiogenesis and expression of the vascular endothelial growth factor (VEGF), a potent angiogenic factor. SAB treatment resulted in a marked increase of apoptosis in the tumor, whereas tumor cell proliferation was not altered. Our in vitro study showed that SAB significantly suppressed the Ald-induced endothelial proliferation and tubular formation through inhibition of phosphorylation of the extracellular signal-regulated kinase 1/2. On the contrary, neither Ald nor SAB affected the proliferation of HCC cells in vitro. CONCLUSION: Ald plays a pivotal role in HCC development through VEGF-mediated tumor angiogenesis, and SAB may be a potential new strategy in HCC therapy in the future.

[Three cases of spindle cell carcinoma of the breast].

Spindle cell carcinoma of the breast is a rare variant of breast cancer composed of spindle-shaped sarcomatoid cancer cells. We report three females with spindle cell carcinoma of the breast who underwent resection, with a review of the literature. The females were 26, 52, and 58 years old, and the tumor diameters were 3.5, 3.5, and 9.0 cm, respectively. All three patients underwent Auchincloss method mastectomy. A cystic lesion accompanied by necrosis was observed in the tumor of two of the three patients. Axillary lymph node metastasis was pathologically diagnosed in one of the three patients. All three tumors were estrogen receptor- and progesterone receptor- negative, showed a proliferation of severely atypical spindle or polygonal epithelial cells, and were diagnosed as spindle cell carcinoma. One patient died of pulmonary, bone, and brain metastases 2 years after the operation, but the other two have followed a favorable course without recurrence for 5 years since the surgical resection.

Synergistic inhibitory effect of gemcitabine and angiotensin type-1 receptor blocker, losartan, on murine pancreatic tumor growth via anti-angiogenic activities.

Pancreatic cancer is one of the leading causes of cancer death, and represents a challenging chemotherapeutic problem. The crucial role of angiogenesis in tumor growth has been widely recognized, and several reports have revealed that the combination treatment of the conventional chemotherapeutic drugs and anti-angiogenic agents exerted synergistic anti-cancerous effects. It has been reported that the clinically used angiotensin type-1 receptor blocker (ARB) exerted potent anti-angiogenic activity. The aim of our current study was to examine the combination effect of gemcitabine (GEM), a widely used conventional chemotherapeutic drug against pancreas cancer, and losartan (Lo), an ARB, on murine pancreatic tumor growth, especially in conjunction with angiogenesis. When used individually, GEM and Lo at clinically comparable low doses moderately suppressed pancreatic tumor development. The combination treatment with GEM and Lo exerted a marked inhibitory effect as compared with single agent treatments even after the tumor was fully established. Neovascularization and the expression of the vascular endothelial growth factor (VEGF), a central angiogenic factor, in the tumor were both markedly suppressed in a magnitude similar to the inhibitory effects against the tumor growth. Since both agents are widely used in the clinical practice, the combination regimen of GEM and Lo may represent a potential new therapeutic strategy for pancreatic cancer in the future.

Combination of vitamin K2 and angiotensin-converting enzyme inhibitor ameliorates cumulative recurrence of hepatocellular carcinoma.

BACKGROUND/AIMS: No chemopreventive agent has been approved against hepatocellular carcinoma (HCC) yet. Since neovascularization plays a pivotal role in HCC, an angiostatic agent is considered as one of the promising approaches. The aim of this study was to elucidate the combined effect of the clinically used vitamin K(2) (VK) and angiotensin-converting enzyme inhibitor (ACE-I) on cumulative recurrence after curative treatment on a total of 87 patients, especially in consideration of neovascularization. METHODS: VK (menatetrenone; 45 mg/day) and/or ACE-I (perindopril; 4 mg/day) were administered for 36-48 months after curative therapy for HCC. The cumulative recurrence and several indices were analyzed. RESULTS: A 48-month follow-up revealed that the combination treatment with VK and ACE-I markedly inhibited the cumulative recurrence of HCC in association with suppression of the serum level of the vascular endothelial growth factor (VEGF); a central angiogenic factor. The serum level of lectin-reactive alpha-fetoprotein was also suppressed almost in parallel with VEGF. These beneficial effects were not observed with single treatment using VK or ACE-I. CONCLUSIONS: The combination treatment of VK and ACE-I may suppress the cumulative recurrence of HCC after the curative therapy, at least partly through suppression of the VEGF-mediated neovascularization.

Branched-chain amino acids suppress insulin-resistance-based hepatocarcinogenesis in obese diabetic rats.

BACKGROUND: Branched-chain amino acids (BCAAs) reportedly inhibit the incidence of hepatocellular carcinoma (HCC) in patients with liver cirrhosis and obesity that is frequently associated with insulin resistance (IR). However, the possible mechanism is still obscure. The aim of the present study was to examine the effect of BCAAs, especially in conjunction with angiogenesis, on hepatocarcinogenesis under the condition of IR. METHODS: The effect of BCAAs on the development of liver enzyme-altered preneoplastic lesions and angiogenesis was examined in obese diabetic Otsuka Long-Evans Tokushima Fatty rats. We also performed an in vitro study to elucidate the possible mechanisms involved. RESULTS: Treatment with BCAAs markedly inhibited glutathione-S-transferase placental form (GST-P)-positive preneoplastic lesions along with suppression of neovascularization in the liver. The hepatic expression of vascular endothelial growth factor (VEGF), a potent angiogenic factor, was also attenuated. BCAA treatment significantly suppressed glucose- and insulin-induced in vitro angiogenesis in the presence of VEGF. CONCLUSIONS: In obese diabetic rats BCAAs exerted a chemopreventive effect against HCC, associated with the suppression of VEGF expression and hepatic neovascularization. Since BCAA preparations are widely used in clinical practice for patients with chronic liver diseases, this agent may represent a new strategy for chemoprevention against HCC in the future.

Development of hepatocellular carcinoma in a patient 13 years after sustained virological response to interferon against chronic hepatitis C: a case report.

BACKGROUND: Although several recent reports have shown that hepatocellular carcinoma (HCC) developed in patients with chronic hepatitis C (CH-C) even after having a sustained virological response (SVR) to interferon (IFN) therapy, it is not common for HCC to develop more than 10 years after SVR. CASE PRESENTATION: A 73-year-old Japanese man with CH-C who achieved SVR to IFN therapy 13 years ago was admitted into our hospital because of huge multiple liver tumors along with marked elevation of the tumor markers. Several diagnostic modalities strongly suggested HCC, and we performed histopathological examination. After confirming the diagnosis as well-differentiated HCC, we successfully treated these tumors with intensive combination therapies. CONCLUSION: Our report highlights the need for careful follow-up for more than 10 years even if the patients with CH-C achieve SVR to IFN therapy.

Impact of insulin resistance on the progression of chronic liver diseases.

Recent studies have revealed a close relationship between insulin resistance (IR) and the progression of chronic liver diseases, although relatively little is known regarding the possible mechanisms involved. The aim of this study was to elucidate the impact of IR on the development of liver fibrosis and hepatocarcinogenesis using obese diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Liver fibrosis development and glutathione-S-transferase placental form (GST-P)-positive pre-neoplastic lesions were both markedly accelerated in OLETF rats, being induced by pig serum and diethylnitrosamine (DEN), respectively. In the fibrosis experiment, alpha-smooth muscle actin-positive activated hepatic stellate cells (HSCs) also significantly increased in OLETF rats along with augmentation of the hepatic collagen content and transforming growth factor-beta1. Our in vitro study showed that both glucose and insulin stimulated the proliferation of activated HSCs, and the combination treatment exerted an additive effect. In the DEN model, neovascularization, which plays a pivotal role in hepatocarcinogenesis, was up-regulated in OLETF rats almost in parallel with pre-neoplastic lesion development and a potent angiogenic factor, vascular endothelial growth factor. High glucose and insulin also significantly augmented the in vitro neovascularization via extracellular signal-regulated kinase 1/2 phosphorylation. Similar to the effect on the activated HSCs, co-existence of both factors exerted a more potent effect than either single factor. In conclusion, these results indicated that the IR status directly accelerated liver fibrosis development and hepatocarcinogenesis at least partly through the stimulation of activated HSC proliferation and hepatic neovascularization, respectively, in the rat.

Different tumoricidal effects of interferon subclasses and p53 status on hepatocellular carcinoma development and neovascularization.

Interferon (IFN) is known as a multifunctional cytokine. The aim of this study was to examine the different effects of IFN subclass; namely, IFN-alpha and IFN-beta, on hepatocellular carcinoma (HCC) growth especially in conjunction with angiogenesis that is known to play a pivotal role in the tumor growth. Furthermore, we also examined whether the p53 status in the tumor would alter the anti-tumoral effect of IFN against HCC growth since the p53 status reportedly affected the therapeutic effect of anti-angiogenic agents against cancer. When compared with IFN-alpha, IFN-beta exerted a more potent inhibitory effect on HCC growth, even after the tumor was established, along with suppression of neovascularization in the tumor. A single treatment with clinically comparable low doses of IFN-beta significantly inhibited HCC growth whereas the same dose of IFN-alpha did not. IFN-beta also significantly suppressed the tumor growth both in the p53-wild and p53-mutant HCC cells. Our in vitro study revealed that IFN-beta showed a more potent inhibitory effect on the endothelial cell proliferation than IFN-alpha as in the in vivo study. Collectively, IFN may be an alternative anti-angiogenic agent against HCC since it exerted a significant tumoricidal effect regardless of the host p53 status even at a low dose. A cautious approach may be also required in the clinical practice since even in a same IFN subclass (class-I), IFN-alpha and IFN-beta exert tumoricidal effects of different magnitudes on HCC.