Spreds, inhibitors of the Ras/ERK signal transduction, are dysregulated in human hepatocellular carcinoma and linked to the malignant phenotype of tumors.

Aberrant activation of the Ras/Raf-1/extracellular-regulated kinase (ERK) pathway has been shown to be involved in the progression of human hepatocellular carcinoma (HCC). However, the mechanism of dysregulation of ERK activation is poorly understood. Recently, we identified Sprouty-related protein with Ena/vasodilator-stimulated phosphoprotein homology-1 domain (Spred) as a physiological inhibitor of the Ras/Raf-1/ERK pathway. In this study, we found that the expression levels of Spred-1 and -2 in human HCC tissue were frequently decreased, comparing with those in adjacent non-tumorous tissue. Moreover, Spred expression levels in HCC tissue were inversely correlated with the incidence of tumor invasion and metastasis. Forced expression of Spred-1 inhibited HCC cell proliferation in vitro and in vivo, which was associated with reduced ERK activation. Spred-1 overexpression also reduced the secretion of matrix metalloproteinase-9 (MMP-9) and MMP-2, which play important roles in tumor invasion and metastasis. In addition, Spred-1 inhibited growth factor-mediated HCC cell motility. These data indicate that the reduction of Spred expression in HCC is one of the causes of the acquisition of malignant features. Thus, Spred could be not only a novel prognostic factor but also a new therapeutic target for human HCC.

The herbal medicine sho-saiko-to inhibits proliferation of cancer cell lines by inducing apoptosis and arrest at the G0/G1 phase.

Water-soluble ingredients of the herbal medicine sho-saiko-to dose-dependently inhibited the proliferation of a human hepatocellular carcinoma cell line (KIM-1) and a cholangiocarcinoma cell line (KMC-1). Fifty % effective doses on day 3 of exposure to sho-saiko-to were 353.5 +/- 32.4 micrograms/ml for KIM-1 and 236.3 +/- 26.5 micrograms/ml for KMC-1. However, almost no suppressive effects were detected in normal human peripheral blood lymphocytes or normal rat hepatocytes. Sho-saiko-to suppressed the proliferation of the carcinoma cell lines significantly more strongly than did each of its major ingredients, i.e., saikosaponin a, c, and d, ginsenoside Rb1 and Rg1, glycyrrhizin, baicalin, baicalein, and wogonin, or another herbal medicine, juzen-taiho-to (P < 0.05 or 0.005). Because such ingredients are barely soluble in water, there could be synergistic or additive effects of the ingredients in sho-saiko-to. Morphological, DNA, and cell cycle analyses revealed two possible modes of action of sho-saiko-to to suppress the proliferation of carcinoma cells; (a) it induces apoptosis in the early period of exposure and (b) it induces arrest at the G0/G1 phase in the late period of exposure.

Epidermoid cyst of the spleen with CA19-9 or carcinoembryonic antigen productions: report of three cases.

True splenic cyst is a relatively rare disease, and the majority of the cases are classified as epidermoid cysts. Three cases of epidermoid cysts in the spleen or accessory spleen were studied using an immunohistochemical technique and staining for mucin. In case 1, serum carcinoembryonic antigen (CEA) and CA19-9, and in cases 2 and 3, serum CA19-9, before surgery were markedly elevated, and these levels decreased postoperatively. This strongly indicates the relationship between the increase of tumor marker levels and the presence of the epidermoid cyst. In addition, stratified squamous epithelium in the resected tissues of cases 1 and 2 was positive for anti-CEA antibody and anti-CA19-9 antibody, and that of case 3 was positive for anti-CA19-9 antibody. This strongly supports CEA or CA19-9 production in the squamous epithelium.

Kinetics of apoptotic cells in experimental autoimmune uveoretinitis.

PURPOSE: To investigate the role of apoptosis in immunopathogenic mechanisms of experimental autoimmune uveoretinitis (EAU), the kinetics of apoptotic cells and expression of Fas and Fas ligand (FasL) in the eye with EAU were studied. METHODS: Male inbred Lewis rats were immunized with S-antigen (40 microg/rat), and eyes were examined to detect apoptotic cells on days 1, 4, 8, and 10 post-immunization and days 0, 2, 4, 6, and 8 after the onset of EAU. The clinical and pathologic scores were used for estimating EAU. Apoptotic cells were analyzed by TdT-mediated dUTP nick-end labeling, electron microscopic and immunohistologic examinations, and agarose gel electrophoresis. The anti-rat Fas and anti-rat FasL antibodies were used to examine the expression of Fas and FasL. RESULTS: Apoptotic cells were detected in the infiltrating cells in the aqueous humor, the vitreous body, the iris-ciliary body, and the retina. Apoptotic cells were observed as early as the day of EAU onset and reached a peak on day 2 after the disease onset. Fas and FasL were expressed on the infiltrating cells in the aqueous humor and the vitreous. FasL was expressed on resident cells of the ciliary body. The kinetics of the expression of FasL corresponded with the kinetics of apoptotic cells. CONCLUSIONS: Fas-FasL-mediated apoptosis is considered to occur in the eye with EAU and plays a role in the immunopathogenic mechanisms to eliminate ocular infiltrating cells, thereby down-regulating the inflammatory processes.

Expression of Fas and anti-Fas-mediated apoptosis in human renal cell carcinoma.

We investigated Fas expression and Fas-mediated apoptosis on 25 surgically resected renal cell carcinoma (RCC) tissues and 8 RCC cell lines. Immunohistochemistry demonstrated that cancer cells in the 25 tissues expressed Fas (100%). Fas expression was confirmed in all cell lines at protein and mRNA levels, but the 7 cell lines were resistant or low sensitive to anti-Fas induced apoptosis. In addition, cancer cells in all of the RCC tissues and all cell lines expressed an anti-apoptotic proto-oncogene, bcl-2 product. In conclusion, i) Fas/Fas-ligand system may be often impaired in RCCs despite their frequent expression; and ii) their frequent Bcl-2 expression may further contribute to the resistance to various apoptotic stimuli.

Expression and function of interleukin-8 in human hepatocellular carcinoma.

Expression and functions of interleukin (IL)-8, a pro-inflammatory cytokine with angiogenesis action, was examined in 23 surgically resected hepatocellular carcinoma (HCC) specimens and 7 HCC cell lines. In all HCC tissues, IL-8 expression was confirmed with reverse-transcription polymerase chain reaction method and enzyme-linked immunosorbent assay, and immunohistochemistry showed HCC cells were the major producer of IL-8 in the tissues. Microvessel density was measured by the double immunohistochemical staining of muscular vessels in HCC tissues, but the density was not related to the level of IL-8 in the HCC tissues. On the other hand, in the co-culture of human umbilical vein endothelial cells (HUVEC) and a HCC cell line (KIM-1), IL-8 produced by KIM-1 significantly accelerated the proliferation of HUVEC. In addition, cases with a high IL-8 level in cancerous tissue had a significantly higher frequency of portal vein invasion, venous invasion and bile duct invasion (p<0.05). In the cultures of 7 HCC cell lines IL-8 secretion into culture medium increased with the treatment of IL-1beta or tumor necrosis factor-alpha. This showed IL-8 expression is regulated by inflammatory cytokines. IL-8 produced by HCC is an angiogenesis factor of HCC, but it could have a much more important role in the invasion and metastasis of HCC.

Clinicopathologic comparison of hepatitis B virus-related and hepatitis C virus-related hepatocellular carcinoma.

137 cases of hepatocellular carcinoma associated with cirrhosis that had undergone resection between 1991-95 have been analyzed. One hundred and three had hepatitis C (positive for anti HCV alone) and 34 hepatitis B (positive for HBsAg alone). The hepatitis C cases were older and were associated with more severe cirrhosis. The tumors from hepatitis B cases were on average larger and histologically less-differentiated, and were more likely to occur in the background of macronodular cirrhosis than hepatitis C cases.

Establishment and characterization of a new human hepatocellular carcinoma cell line, HAK-3, and its response to growth factors.

A new human hepatocellular (HCC) cell line, HAK-3, was established from a resected HCC of a Japanese, female patient. HAK-3 retains morphologic features of the original HCC, and proliferates in a monolayered sheet (doubling time: 26 h). HAK-3 is a single aneuploid cell population with a DNA index of 2.42, the karyotype is human, chromosomes are 80-85 (mode: 83), and secretes fibronectin and tissue polypeptide antigen. Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) dose-dependently accelerated the cell proliferation, while deletion-type hepatocyte growth factor (dHGF) tended to suppress the proliferation, and transforming growth factor (TGF)-alpha showed almost no influence. dHGF induced the decrease of cell adhesiveness, changed the cell morphology to spindle-shaped cells, increased cell movement, and showed chemotactic effects with the increase of its concentration gradient in cultures. HAK-3 would be useful in studies on the acceleration mechanisms of cancer cell proliferation by growth factors and of chemotaxis by dHGF.

Expressions of epidermal growth factor family and its receptor in hepatocellular carcinoma cell lines: relationship to cell proliferation.

In 6 HCC cell lines, clear expressions of EGFR and TGF-alpha were found in flow cytometry, while expressions of EGF, HB-EGF and AR were quite low. TGF-alpha secretion into culture supernatants became measurable when TPA 0.5 microM was added. TPA accelerated the proliferation of KYN-3 cells, and anti-TGF-alpha neutralizing antibody suppressed this proliferation in a dose-dependent manner. Addition of exogenous TGF-alpha, EGF, AR, or HB-EGF with heparin accelerated cell proliferation. In non-stimulated cultures, cell proliferation was suppressed by anti-EGFR neutralizing antibody, but not by the antibodies for EGF, TGF-alpha, AR and HB-EGF. HCC may possess a paracrine system regulated by these 4 ligands, and an autocrine system, under a certain condition, via TGF-alpha and EGFR.

An alpha-fetoprotein-producing human gallbladder carcinoma cell line (KMG-C) shows more aggressive biologic behavior than an alpha-fetoprotein-non-producing cell line.

We have established a new human alphafetoprotein (AFP)-producing gallbladder carcinoma (GBC) cell line, termed KMG-C, from a 67-year-old Japanese male. KMG-C and its reconstituted tumors in nude mice showed the morphological features of an adenocarcinoma. Functionally, KMG-C secreted AFP, carcinoembryonic antigen, and carbohydrate antigen 19-9, as well as 7 serum proteins, including albumin and C-reactive protein. KMG-C showed more malignant biological behavior than an AFP-nonproducing GBC cell line, KMG-A, established originally from the tumor of the same patient; KMG-C had a shorter doubling time, higher tumorigenicity, and an aneuploid DNA index. Our results suggest that AFP-producing GBC cells may have more malignant biological characteristics than AFP-non-producing GBC cells in GBCs having both of these components.

p53 overexpression in small hepatocellular carcinomas containing two different histologic grades.

There is evidence to suggest that a focus of less-differentiated hepatocellular carcinoma (HCC) may arise within a pre-existing well-differentiated HCC, eventually replacing it. In the present study, the p53 tumor suppressor gene was analyzed by immunohistochemistry in 31 hepato-cellular carcinomas (HCCs) containing two or more regions in the same nodule with different histologic grades. p53 was overexpressed in the nucleus in 13 of 31 HCCs (42%), in seven of which p53 overexpression was seen only in the less-differentiated area of the tumor. This suggests that overexpression of presumed mutant p53 may have contributed to dedifferentiation during the development of HCC.

Deletion type hepatocyte growth factor has different effects on growth and c-met expression in 10 different human hepatocellular carcinoma cell lines.

We examined expression of c-met protein, and the mitogenic and morphologic effects of deletion type hepatocyte growth factor (dHGF) by using 10 human hepatocellular carcinoma (HCC) cell lines having different morphologic and biologic features. c-met protein was detected at varying levels in all cells, regardless of the histological grades. Among the 7 lines expressing c-met at high levels, mitogenic effects of dHGF were stimulative for 2 lines; suppressive for 3 lines; and not distinguishable for the other 2 lines. Furthermore, mitogenic effects of dHGF were different in two clonally related cell lines, having different morphologic and biologic features, even though expression of c-met protein was comparable. dHGF induced scattering of cells and morphologic changes in two lines with suppressing and unaffected growth. In the 3 lines expressing c-met at relatively low levels, no remarkable mitogenic or morphogenic effects were detected. These results suggest that the expression levels of c-met protein were not related to the differentiation levels of HCC cells, and dHGF may cause different biological effects on the cells with almost identical c-met protein expression.

Evaluation of curability and prediction of prognosis after surgical treatment for hepatocellular carcinoma by lens culinaris agglutinin-reactive alpha-fetoprotein.

The clinical significance of serum lens culinaris agglutinin-reactive alpha-fetoprotein (AFP-L3), which can distinguish between hepatocellular carcinoma and hepatitis by detecting a sugar chain micro heterogeneity, was evaluated for its possible ability to recognize previously undetectable residual tumors, and for increasing the accuracy of prognosis after surgical treatment for hepatocellular carcinoma. Serum lens culinaris agglutinin-reactive alpha-fetoprotein was measured pre- and post-operatively in 130 patients who underwent curative surgical treatment for hepatocellular carcinoma. The preoperative AFP-L3 positive rate was 35.4%. AFP-L3 remained positive postoperatively in 28 of the 46 preoperative AFP-L3 positive patients, and converted to positive in 4 of the 84 preoperative AFP-L3 negative patients. Regardless to preoperative AFP-L3, the postoperative AFP-L3 positive patients had a poorer recurrence-free rate (p<0.0001). The postoperative L3 positive patients had a high incidence of recurrence due to metastasis, but did not have recurrence due to multicentric origin. Multivariate analysis revealed that AFP-L3 (p<0.0001) was the most independently significant factor for predicting survival after surgery among several conventional prognostic factors. Thus, AFP-L3 is a valuable marker for evaluation of curability of surgical treatment and for improving the accuracy of prognosis.

High prevalence of hepatitis C virus infection in schistosomiasis japonica patients associated with hepatocellular carcinoma.

Schistosomiasis japonica (SCJ) patients frequently develop hepatocellular carcinoma (HCC). This study investigated relationship between SCJ infection, hepatitis virus infection, and incidence of HCC, in 25 patients with chronic SCJ infection and HCC (SCJ with HCC group), 51 patients with chronic SCJ infection without HCC (SCJ group) and 65 HCC patients without SCJ (HCC group). Number of patients who were positive to HBsAg or hepatitis B virus DNA were 4 (16.0%) in the SCJ with HCC group, none (0%) in the SCJ group, and 5 (7.9%) in the HCC group; while number of patients who were positive to anti-hepatitis C virus antibody were 21 (87.5%) in the SCJ with HCC group, 3 (5.9%) in the SCJ group, and 58 (84.6%) in the HCC group. Biopsy was performed for all patients, and background histological features of each specimen were evaluated based on the histological activity index scoring system. Mean scores of inflammatory changes in both the portal area and parenchyma in the SCJ with HCC group were significantly higher than those in the SCJ group. Nodular formation which is common in post-viral hepatitis was frequently observed in the SCJ with HCC group, and histological changes in non-cancerous area of the SCJ with HCC group showed the characteristics of chronic viral hepatitis. We conclude that infection of hepatitis virus, particularly hepatitis C virus, affects synergistically on the hepatocarcinogenesis in patients having SCJ infection.

Nodules of less-differentiated tumor within or adjacent to hepatocellular carcinoma: relative expression of transforming growth factor-alpha and its receptor in the different areas of tumor.

Expression of transforming growth factor-alpha (TGF-alpha) and its receptor, the epidermal growth factor receptor (EGFR), in hepatocellular carcinomas (HCCs) and adjacent nontumorous livers from 25 Japanese patients were examined using immunoperoxidase staining of paraffin-embedded sections. TGF-alpha was detected in 24 of 25 (96%) HCCs and 23 of 24 (96%) available adjacent nontumorous livers. EGFR was detected in 16 of 25 (64%) HCCs and 17 of 24 (71%) adjacent nontumorous livers. TGF-alpha and EGFR were not detected by immunohistochemical staining in normal livers. Fifteen of 25 HCCs contained an apparent area of a second tumor (two of the 15 also contained a third tumor) that had a less-differentiated histological grade developing within or adjacent to the first tumor. In those cases, staining in the less-differentiated area of tumor was usually less intense than in the more highly differentiated area (80% of cases for TGF-alpha; 91% for EGFR). These data confirm that increased expression of TGF-alpha and EGFR occur frequently in human HCC. Furthermore, the detection of greater staining in more highly differentiated portions of the tumors suggests that increased expression of TGF-alpha and EGFR may be events of the early stages of human hepatocarcinogenesis.

Histologic growth pattern of hepatocellular carcinoma: relationship to orcein (hepatitis B surface antigen)-positive cells in cancer tissue.

The histologic pattern of tumor growth at tumor-nontumor boundaries was studied in 60 livers bearing hepatocellular carcinoma. Three growth patterns, arbitrarily described as "sinusoidal," "replacing," and "encapsulated," were distinguished. Cancer cells growing in the sinusoids between liver cell cords (sinusoidal pattern) were anaplastic; those growing in an expansile fashion and acquiring a fibrous capsule (encapsulated pattern) were most differentiated; and those growing into the cord of liver cells and replacing them (replacing pattern) were differentiated to an intermediate degree. There was certain relation between the histologic growth patterns and gross morphologic features of the tumors. Test results for 20 of the 60 cases were positive for serum hepatitis B surface antigen (HBsAg), and the livers also contained orcein-positive cells. Orcein-positive cells were frequently seen at the border between tumor and parenchyma. Cells containing HBsAg as an orcein-positive inclusion were present in cancer tissue in three cases. When serial sections were made from such areas and stained alternately with hematoxylin and eosin and orcein, it was found that these cells were hepatocytes blended with cancer cells. This phenomenon was related to the growth pattern of tumor cells. Orcein-positive cells were never found in metastatic lesions.

Hepatocellular carcinoma with sarcomatous change: a special reference to the relationship with anticancer therapy.

Among 579 autopsy cases of hepatocellular carcinoma (HCC), 55 cases (9.4%) exhibited a sarcomatous appearance. The incidence of HCC with a sarcomatous appearance has been increasing over the past 17 years. A sarcomatous appearance was found in 20 out of 335 autopsy cases of HCC (5.9%) during the 12 years from 1969 to 1980, and in 35 out of 244 autopsy cases of HCC (14.3%) during the last 6 years, when effective anticancer therapies, such as the one-shot injection of anticancer agents into the hepatic artery (one-shot therapy) and transcatheter arterial embolization (TAE), have become popular. A sarcomatous appearance was found in 20.9% of the cases undergoing anticancer therapy and in 4.2% of the cases not undergoing anticancer therapy. Among the various anticancer therapies, the sarcomatous appearance was most frequent (27.6%) in cases with repeated TAE. Thus, a close relationship between the sarcomatous appearance in HCC and anticancer therapies was suggested. Regarding the development of the sarcomatous appearance, we presume that it may be caused by the phenotypic change of HCC cells caused by anticancer therapy, or that a number of factors, including anticancer therapy, may accelerate the proliferation of the sarcomatous cells existing in the original tumor as one of the histological components. In order to clarify the true nature of sarcomatous lesions in HCC, further histological and biological studies are required.

Apoptosis in the pancreas of genetically diabetic rats with a disrupted cholecystokinin (CCK-A) receptor gene.

In a previous study, we reported that the pancreatic wet weight in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, cholecystokinin-A (CCK-A) receptor-defective because of a congenital gene abnormality, was significantly lower than in control rats (Long-Evans Tokushima Otsuka; LETO) from 3 weeks of age. In this study we examined apoptosis of pancreatic acinar cells in OLETF rats at 5 to 6 weeks of age in comparison with that in LETO rats. We present here direct morphologic evidence of apoptosis in OLETF rats, using a 3'-OH nick end-labeling method for detecting cells with DNA strand breaks and electron microscopy. Nick end-labeling revealed a small number of positively labeled acinar cells in OLETF rats. On electron microscopic examination, small numbers of apoptotic cells were seen in the lobules in OLETF rats but not in LETO rats. These results suggest that apoptosis plays an important role in the destruction of acinar cells of OLETF rats and induces atrophy of the pancreas.

Clinicopathologic study on cholangiolocellular carcinoma.

We clinicopathologically studied 6 resected cases of cholangiolocarcinoma (CLC) including 2 referred cases from other hospitals. The frequency of CLC was 0.56% of the 708 consecutively resected cases of primary liver cancer and the mean age of CLC cases was 66 years. Three of the 6 cases (50%) were hepatitis C virus antibody (HCVab) positive, one (17%) was hepatitis B virus surface antigen (HBsAg) positive, and 2 (33%) were negative to both HCVAb and HBsAg. Serum levels of alpha-fetoprotein were slightly elavated only in 1 case. Clinically, 4 cases were diagnosed as hepatocellular carcinoma (HCC) and 2 cases as cholangiocellular carcinoma (CCC). Grossly, CLCs were whitish in color and solid, not encapsulated, and resembled CCC. Histologically, the tumor cells had eosinophilic cytoplasm with ovoid nuclei, and mild atypia. The tumor proliferated in an anastomosing pattern of Hering's canal-like small glands with an abundant fibrous stroma. Four of the 6 tumors (83%) consisted of only CLC and other 2 tumors contained CCC-like area and HCC-like area in a part of the nodules, respectively. Immunohistochemically, all tumors were positive to cytokeratin (CK) 7. CK8 were also positive in all of 6 cases. These results revealed that CLC had the clinical features resembling HCC but the morphologic features resembling CCC. It is suggested that CLC cells might be derived from Hering's canal or stem cells which have the intermediate features between hepatocytes and bile duct epithelium.

Metachronous multicentric occurrence of hepatocellular carcinoma after surgical treatment - clinicopathological comparison with recurrence due to metastasis.

This study investigated clinicopathological features of patients with recurrence of metachronous multicentric occurrence by comparison with patients with recurrence due to metastasis. In 177 patients, recurrences after curative surgical treatment were classified into recurrence due to metastasis according to criteria based on imaging findings. This group consisted of 35 patients. Among the rest of the patients, 59 underwent fine needle biopsies for recurrent tumor and, in these patients, a classification of recurrence of metachronous multicentric occurrence was made based on the histological findings of primary and recurrent tumor. This group consisted of 33 patients. The estimated incidence for recurrence of metachronous multicentric occurrence was 44.8% to total total patients. Metachronous multicentric occurrence frequently developed in patients with anti-HCV antibody and an early stage of primary tumor. In 80% of the patients who had recurrent tumor of multicentric origin, the recurrence developed within 3 postoperative years. The survival rate in patients with metachronous multicentric occurrence was significantly higher than that in patients with recurrence due to metastasis. Conclusively, the incidence of patients with recurrence of metachronous multicentric occurrence was high, but the prognosis for these patients was significantly better than that for patients with recurrence due to metastasis.