Hirohisa Yano

Luteolin promotes degradation in signal transducer and activator of transcription 3 in human hepatoma cells: an implication for the antitumor potential of flavonoids.

In this study, we have investigated the underlying molecular mechanism for the potent proapoptotic effect of luteolin on human hepatoma cells both in vitro and in vivo, focusing on the signal transducer and activator of transcription 3 (STAT3)/Fas signaling. A clear apoptosis was found in the luteolin-treated HLF hepatoma cells in a time- and dosage-dependent manner. In concert with the caspase-8 activation by luteolin, an enhanced expression in functional Fas/CD95 was identified. Consistent with the increased Fas/CD95 expression, a drastic decrease in the Tyr(705) phosphorylation of STAT3, a known negative regulator of Fas/CD95 transcription, was found within 20 minutes in the luteolin-treated cells, leading to down-regulation in the target gene products of STAT3, such as cyclin D1, survivin, Bcl-xL, and vascular endothelial growth factor. Of interest, the rapid down-regulation in STAT3 was consistent with an accelerated ubiquitin-dependent degradation in the Tyr(705)-phosphorylated STAT3, but not the Ser(727)-phosphorylated one, another regulator of STAT3 activity. The expression level of Ser(727)-phosphorylated STAT3 was gradually decreased by the luteolin treatment, followed by a fast and clear down-regulation in the active forms of CDK5, which can phosphorylate STAT3 at Ser(727). An overexpression in STAT3 led to resistance to luteolin, suggesting that STAT3 was a critical target of luteolin. In nude mice with xenografted tumors using HAK-1B hepatoma cells, luteolin significantly inhibited the growth of the tumors in a dosage-dependent manner. These data suggested that luteolin targeted STAT3 through dual pathways-the ubiquitin-dependent degradation in Tyr(705)-phosphorylated STAT3 and the gradual down-regulation in Ser(727)-phosphorylated STAT3 through inactivation of CDK5, thereby triggering apoptosis via up-regulation in Fas/CD95.

Histological Diversity in Cholangiocellular Carcinoma Reflects the Different Cholangiocyte Phenotypes

Cholangiocellular carcinoma (CC) originates from topographically heterogeneous cholangiocytes. The cylindrical mucin‐producing cholangiocytes are located in large bile ducts and the cuboidal non–mucin‐producing cholangiocytes are located in ductules containing bipotential hepatic progenitor cells (HPCs). We investigated the clinicopathological and molecular features of 85 resected CCs (14 hilar CCs [so‐called Klatskin tumor], 71 intrahepatic CCs [ICCs] including 20 cholangiolocellular carcinomas [CLCs], which are thought to originate from HPCs]) and compared these with the different cholangiocyte phenotypes, including HPCs. Immunohistochemistry was performed with biliary/HPC and hepatocytic markers. Gene expression profiling was performed in different tumors and compared with nonneoplastic different cholangiocyte phenotypes obtained by laser microdissection. Invasion and cell proliferation assay were assessed using different types of CC cell lines: KMC‐1, KMCH‐1, and KMCH‐2. Among 51 ICCs, 31 (60.8%) contained only mucin‐producing CC features (muc‐ICCs), whereas 39.2% displayed histological diversity: focal hepatocytic differentiation and ductular areas (mixed‐ICCs). Clinicopathologically, muc‐ICCs and hilar CCs showed a predominantly (peri‐)hilar location, smaller tumor size, and more lymphatic and perineural invasion compared with mixed‐ICCs and CLCs (predominantly peripheral location, larger tumor size, and less lymphatic and perineural invasion). Immunoreactivity was similar in muc‐ICCs and hilar CCs and in mixed‐ICCs and CLCs. S100P and MUC1 were significantly up‐regulated in hilar CCs and muc‐ICCs compared with mixed‐ICCs and CLCs, whereas NCAM1 and ALB tended to be up‐regulated in mixed‐ICCs and CLCs compared with other tumors. KMC‐1 showed significantly higher invasiveness than KMCH‐1 and KMCH‐2. Conclusion: Muc‐ICCs had a clinicopathological, immunohistochemical, and molecular profile similar to that of hilar CCs (from mucin‐producing cholangiocytes), whereas mixed‐ICCs had a profile similar to that of CLCs (thought to be of HPC origin), possibly reflecting their respective cells of origin. (HEPATOLOGY 2012;55:1876–1888)

Self-assembled micellar nanocomplexes comprising green tea catechin derivatives and protein drugs for cancer therapy

In designing drug carriers, the drug-to-carrier ratio is an important consideration because using high quantities of carriers can cause toxicity resulting from poor metabolism and elimination of the carriers1. However, these issues would be of less concern if both the drug and carrier possess therapeutic effects. (-)-Epigallocatechin-3-O-gallate (EGCG), which is a major ingredient of green tea, has been shown to possess anticancer effects2-7, anti-HIV effects8, neuroprotective effects9, DNA-protective effects10, etc. Here we show that sequential self-assembly of the EGCG derivative with anticancer proteins forms stable micellar nanocomplexes (MNCs), which have greater anticancer effects in vitro and in vivo than the free protein. The MNC is obtained by complexation of oligomerized EGCG with the anticancer protein, Herceptin, to form the core, followed by complexation of poly(ethylene glycol)-EGCG to form the shell. When injected into mice, the Herceptin-loaded MNC showed better tumour selectivity and growth reduction, and longer blood-half-life than free Herceptin.

Vascular endothelial growth factor C and vascular endothelial growth factor receptor 2 are related closely to the prognosis of patients with ovarian carcinoma

The vascular endothelial growth factor (VEGF) family and VEGF receptors (VEGFR) play an essential role in the angiogenesis of both pathologic and nonpathologic conditions. However, the prognostic significance of VEGF and VEGFR expression in ovarian carcinoma is unclear.

Application of a new histological staging and grading system for primary biliary cirrhosis to liver biopsy specimens: Interobserver agreement

Recently the authors proposed a new staging and grading system for primary biliary cirrhosis (PBC) that takes into account necroinflammatory activity and histological heterogeneity. Herein is proposed a convenient version of this system. Scores for fibrosis, bile duct loss, and chronic cholestasis were combined for staging: stage 1, total score of 0; stage 2, score 1–3; stage 3, score 4–6; and stage 4, score 7–9. Cholangitis activity (CA) and hepatitis activity (HA) were graded as CA0–3, and HA0–3, respectively. Analysis of interobserver agreement was then conducted. Digital images of 62 needle liver biopsy specimens of PBC were recorded as virtual slides on DVDs that were sent to 28 pathologists, including five located overseas. All participants were able to apply this version in all 62 cases. For staging, kappa was 0.385 (fair agreement) and the concordance rate was 63.9%. For necroinflammatory activity, the kappa and concordance rate were 0.110 (slight agreement) and 36.9% for CA, and 0.197 (slight agreement) and 47% for HA, respectively. In conclusion, this new staging and grading system for PBC seems to be more convenient and practical than those used at present, but more instruction and guidance are recommended for the grading of necroinflammatory activity in practice.

Tumor-infiltrating lymphocytes are important pathologic predictors for neoadjuvant chemotherapy in patients with breast cancer

Neoadjuvant chemotherapy or preoperative systemic therapy is increasingly considered for patients with operable breast cancer. Patients with breast cancer were examined for pathologic factors predictive of response to neoadjuvant chemotherapy, using an anthracycline-based regimen. For clinical histomorphology and biomarkers, factors were compared among 16 pathologically complete responses and 52 nonpathologically complete responses, using univariate analysis and multivariate regression analysis of principal components, using preneoadjuvant chemotherapy needle biopsy samples as follows: degree of tumor-infiltrating lymphocytes, histologic grade, biology-based tumor type (hormone receptors and HER2 [human epidermal growth factor receptor type 2]), age, clinical TNM stage, and TNM staging. In univariate analysis, high tumor-infiltrating lymphocyte, high histologic grade, and hormone receptors(-)/HER2(+) were significantly associated with pathologically complete responses (93.7%, P < .0001; 81.3%, P = .0206; 43.7%, P = .014, respectively). In multivariate principal component regression analysis, high tumor-infiltrating lymphocytes were the best independent predictor for pathologically complete responses (odds ratio, 4.7; confidence interval, 2.2-10.06; P < .0001). Among tumor-infiltrating lymphocytes and biology-based tumor types, patients with high tumor-infiltrating lymphocytes had pathologically complete responses more than nonpathologically complete responses, especially in the hormone receptors(-)/HER2(+) group. Among high tumor-infiltrating lymphocyte cases, T lymphocytes showed more predominant tendency than B lymphocytes in the pathologically complete responses cases, compared with nonpathologically complete responses cases. These findings indicate that high tumor-infiltrating lymphocytes are important predictors of pathologically complete responses to neoadjuvant chemotherapy, especially in the hormone receptors(-)/HER2(+) group.

Obstruction of st jude medical valves in the aortic position: histology and immunohistochemistry of pannus

OBJECTIVE This study aims to reveal the morphological, histological, and immunohistochemical mechanism of pannus formation using resected pannus tissue from patients with prosthetic valve dysfunction. METHOD Eleven patients with prosthetic valve (St Jude Medical valve) dysfunction in the aortic position who underwent reoperation were studied. We used specimens of resected pannus for histological staining (hematoxylin and eosin, Grocotts, azan, elastica van Gieson) and immunohistochemical staining (transforming growth factor-beta, transforming growth factor-beta receptor 1, alpha-smooth muscle actin, desmin, epithelial membrane antigen, CD34, factor VIII, CD68KP1, matrix metalloproteinase-1, matrix metalloproteinase-3, and matrix metalloproteinase-9). RESULTS Pannus without thrombus was observed at the periannulus of the left ventricular septal side; it extended into the pivot guard, interfering with the movement of the straight edge of the leaflet. The histological staining demonstrated that the specimens were mainly constituted with collagen and elastic fibrous tissue accompanied by endothelial cells, chronic inflammatory cells infiltration, and myofibroblasts. The immunohistochemical findings showed significant expression of transforming growth factor-beta, transforming growth factor-beta receptor 1, CD34, and factor VIII in the endothelial cells of the lumen layer; strong transforming growth factor-beta receptor 1, alpha-smooth muscle actin, desmin, and epithelial membrane antigen in the myofibroblasts of the media layer; and transforming growth factor-beta, transforming growth factor-beta receptor 1, and CD68KP1 in macrophages of the stump lesion. CONCLUSIONS Pannus appeared to originate in the neointima in the periannulus of the left ventricular septum. The structure of the pannus consisted of myofibroblasts and an extracellular matrix such as collagen fiber. The pannus formation after prosthetic valve replacement may be associated with a process of periannular tissue healing via the expression of transforming growth factor-beta.

A new human hepatocellular carcinoma cell line (KYN-1) with a transformation to adenocarcinoma

SummaryA human hepatocellular carcinoma (HCC) cell line (KYN-1) has been established from a resected HCC of a 58-yr-old Japanese, male patient with HCC. Original resected HCC was moderately differentiated and proliferated in a solid pattern with vague trabecular structure in part. This cell line has been maintained for 10 mo. through 50 passages. Morphological features of KYN-1 cells demonstrated one or more large, round-to-oval nuceli with prominent nucleoli and eosinophilic polygonal-to-spindle abundant cytoplasm. In addition, some of these cells contained mucicarmin-positive materials in the cytoplasm. The cells exhibited a typical epithelial feature with pavementlike cell arrangement, and lacked contact inhibition. The doubling times of the cells grown in a serum-containing and a serum-fre, medium were about 31 h and 10 to 11 d, respectively. Functonally, KYN-1 cells produced albumin, α-fetoprotein (AFP), carcinoembryonic antigen (CEA), ferritin, β2-microglobulin (BMG), and α1-anti-trypsin (AAT). Positive reactions for albumin, AFP, CEA, and ferritin were identified in the cells by immunohistochemical techniques. Chromosome study revealed the chromosome number in a range from 61 to 74 without mode. The tumorigenicity of KYN-1 cells was identified by the tumor formation after subcutaneous inoculation of the cells into nude mice. The developed tumor showed compact growth of the tumor cells with gland formations containing mucicarmin-positive materials. Features of adenocarcinoma were identified by electron microscopy. The tumor cells were also identified to contain albumin, AFP, CEA, ferritin, and AAT by immunohistochemical techniques. AFP, CEA, and BMG were detected in the sera of nude mice. Thus, KYN-1 cells represented the morphologic features of adenocarcinoma, retaining some characteristics of original HCC. These findings suggest that KYN-1 is a new human HCC cell line with transformation to adenocarcinoma, which will provide useful information to clarify the histogenesis of combined hepatocellular and cholangiocellular carcinoma.

β-Catenin expression in hepatocellular carcinoma: A possible participation of β-catenin in the dedifferentiation process

Background: β‐Catenin is known as a multifunctional protein acting as a regulator of the cadherin‐mediated cell–cell adhesion system and in the Wingless/Wnt signal transduction pathway. Recent studies reported mutation of the β‐catenin gene in some tissues of hepatocellular carcinoma (HCC).

A NEW HUMAN PLEOMORPHIC HEPATOCELLULAR CARCINOMA CELL LINE, KYN‐2

A new human hepatocellular carcinoma (HCC) cell line, KYN‐2, has been established from a surgical specimen obtained from a 52‐year‐old Japanese male HCC patient. The originally resected HCC was classified as pleomorphic HCC corresponding to Edmondson‐Steiners grade III with a thick trabecular to solid arrangement. The cell line has been maintained for 17 months through 35 passages. Morphologically, the KYN‐2 cells have retained the characteristics of the original HCC, being pleomorphic and composed of various types such as cells with relatively small, polygonal, eosinophilic cytoplasm and oval‐shaped nuclei with a marked tendency to pile up, flat cells with abundant clear cytoplasm and oval‐shaped nuclei, and many multinucleated giant cells, proliferating in a pavement‐like cell arrangement. Some junctional complexes and a number of microvilli are evident between the cells by electron microscopy. Functionally, these cells were found to secrete albumin, α,‐acid glycoprotein, α1‐antitrypsin, ceruloplasmin, transferrin, complement C, fibrinogen, fibronectin, prothrombin, retinol‐binding protein (serum type), α‐fetoprotein (AFP), carcinoembryonic antigen (CEA), ferritin and β2‐microglobulin in chemically defined medium (CDM). The secretion of AFP and CEA is apparently dependent upon culture medium and passage. The doubling time of cells growing in serum‐containing medium at the 14 th passage was 84 h, and those of cells in serum‐containing medium, HB101 (serum‐free medium) and CDM at late passage were 28,68, and 42 h, respectively. Chromosome analysis revealed that the chromosome number ranged from 56 to 69 without a mode, and the presence of marker chromosomes. HB virus DNA sequence was not detected by hybridization analysis. The tumorigenicity of KYN‐2 cells was identified by development of tumors in nude mice after subcutaneous injection of the cells; the tumors showed an appearance basically similar to that of the original HCC. Thus, these findings suggest that the KYN‐2 cell line is available as a new human HCC cell line and should be useful for various studies on HCC. ACTA PATHOL JPN 38: 953‐966, 1988.