Nobuaki Shikata

Resveratrol inhibits human breast cancer cell growth and may mitigate the effect of linoleic acid, a potent breast cancer cell stimulator

Abstract Resveratrol is a naturally occurring product found in grapes and wine. The effect of synthetic resveratrol on the growth of estrogen receptor (ER)-positive (KPL-1 and MCF-7) and -negative (MKL-F) human breast cancer cell lines was examined. Resveratrol at low concentrations caused cell proliferation in ER-positive lines (KPL-1, ≤22 μM; MCF-7, ≤4 μM) whereas at high concentrations (≥44 μM) it caused suppression of cell growth in all three cell lines examined. Growth suppression was due to apoptosis as seen by the appearance of a sub-G1 fraction. The apoptosis cascade up-regulated Bax and Bak protein, down-regulated Bcl-xL protein, and activated caspase-3. Resveratrol (52–74 μM) antagonized the effect of linoleic acid, a potent breast cancer cell stimulator, and suppressed the growth of both ER-positive and -negative cell lines. Thus, resveratrol could be a promising anticancer agent for both hormone-dependent and hormone-independent breast cancers, and may mitigate the growth stimulatory effect of linoleic acid in the Western-style diet.

Morphologic characteristics of retinal degeneration induced by sodium iodate in mice

Purpose. Retinal degeneration induced by sodium iodate (NaIO3) in mice was evaluated morphologically. Methods. Male and female ICR and C57BL mice were intraperitoneally administered 100 mg/kg NaIO3 at 7 weeks of age, and were killed 6, 12, 24 hrs, and 3, 7 and 28 days after the treatment. Retinas were examined histologically, ultrastructurally, immunohistochemically, and by the TUNEL method. Results. Retinal degeneration was evoked in all NaIO3- treated mice. The primary site of damage appeared in the retinal pigment epithelial (RPE) cells followed by photoreceptor cell degeneration. Initially, the RPE cells showed necrosis starting 6 hrs post-NaIO3, followed by photoreceptor outer segment disruption and photoreceptor cell apoptosis at 24 hrs; photoreceptor cell apoptosis peaked at day 3 and was completed by day 7. At day 3, Müller cell proliferation, macrophage migration within the retina, and regeneration of damaged RPE cells occurred. Finally at day 7 and day 28, the retina showed a mosaic pattern of relatively normal retina and areas lacking RPE cells and photoreceptor cells. Conclusions. RPE cell necrosis followed by photoreceptor cell apoptosis and the resulting mosaic pattern of the retina phenotypically resembles gyrate atrophy of the choroid and retina.

Bax and Bcl-2 expressions predict response to radiotherapy in human cervical cancer

AbstractPurpose: The ratio of Bcl-2 to Bax expression determines survival or death following an apoptotic stimulus. In order to establish a new predictor of the outcome of treatment for human cervical carcinoma, we investigated the relationship between the expressions of the Bax and Bcl-2 proteins and the response to radiotherapy after the administration of 10.8 Gy. Methods: A total of 44 patients with histologically proven carcinoma of the uterine cervix, including three with recurrent cervical stump carcinomas, were treated with definitive radiotherapy. The presence of mutations in exons 5–8 of the p53 gene was analyzed by a single-strand conformation polymorphism analysis and DNA sequencing. Results: Forty patients were found to have wild-type p53, and the remaining four had mutant p53. The Bax and Bcl-2 protein expressions prior to radiotherapy did not correlate with response and survival. However, the Bax and Bcl-2 protein expressions after radiotherapy correlated with both response and survival. Bax-positive tumors showed significantly better responses than the Bax-negative tumors after 10.8 Gy radiation (P = 0.0002). In contrast, the Bcl-2-positive tumors showed significantly poorer responses than the Bcl-2-negative tumors after radiation (P = 0.002). Increased Bax expression after the 10.8 Gy radiotherapy was found to be correlated with good survival (P = 0.04). In contrast, increased Bcl-2 expression after such radiotherapy was correlated with poor survival (P = 0.002). Conclusion: The levels of Bax and Bcl-2 expression after 10.8 Gy radiotherapy are useful prognostic markers in patients with human cervical carcinoma.

Perillyl Alcohol Inhibits Human Breast Cancer Cell Growth in vitro and in vivo

The effect of monoterpene perillyl alcohol (POH) on cell growth, cell cycle progression, and expression of cell cycle-regulatory proteins in estrogen receptor (ER)-positive (KPL-1 and MCF-7) and ER-negative (MKL-F and MDA-MB-231) human breast cancer cell lines was examined. POH inhibited cell proliferation in a dose-dependent manner in all cell lines tested. POH at a dose of 500 µM had a cytostatic effect, in which growth inhibition was due to accumulation of cells in G1-phase. Cell cycle progression was preceded by a decrease in G1 cyclins (cyclin D1 and E), followed by an increase in p21Cip1/Waf1 and a decrease in proliferating cell nuclear antigen level. Levels of p53 and cyclin A were unchanged. POH at a dose of 75 mg/kg administered intraperitoneally three times a week throughout the entire 6-week experimental period suppressed orthotopically transplanted KPL-1 tumor cell growth and regional lymph node metastasis in a nude mouse system. POH inhibited both ER-positive and -negative human breast cancer cell growth in vitro, and suppressed growth and metastasis in vivo.

Immunohistochemical staining patterns of keratins in normal oesophageal epithelium and carcinoma of the oesophagus

To clarify the keratin staining patterns of invasive carcinoma of the oesophagus, 22 cases of formalin‐fixed paraffin‐embedded surgical specimens were examined immunohistochemically with the labelled streptavidin biotin method using a panel of six different monoclonal anti‐keratin antibodies. The antibody reacted adequately when antigen was retrieved in a microwave oven, and the relationship between morphological characteristics and keratin reaction patterns was analyzed in carcinomas and compared with adjacent histologically normal epithelium. In the normal oesophageal epithelium, AE3 and CK8.12 labelled all layer of cells, KS‐1A3, E3 and KL1 labelled suprabasal cells, and LL002 selectively labelled the basal cells. In squamous cell carcinomas, AE3, CK8.12, KL1 and LL002 labelled almost all the tumour cells regardless of their differentiation, E3 only labelled keratinized cells, while marked decrease or loss of KS‐1A3 staining was seen in all cases examined. Therefore, the characteristic profile of squamous cell carcinoma was a strong and diffuse expression of keratin 14 and 16, strong but localized expression of keratin 17, and loss of keratin 13 expression. Undifferentiated carcinoma totally lacked all keratin reactivity. The findings suggested that the neoplastic epithelial cells showed different keratin reactivity and distribution compared to normal oesophageal epithelium. In addition, histologically normal epithelium, dysplasia and carcinoma‐in‐situ adjacent to or overlying carcinoma expressed keratin 14.

Expression of Ku80 in cervical cancer correlates with response to radiotherapy and survival.

To reveal the genes relevant for prediction of cervical cancer after radiotherapy, we previously carried out cDNA microarray experiments on primary cervical cancer comparing patients with a complete response (CR) and those with no change (NC). Some of these genes had already been associated with the radiation response, such as x-ray repair cross-complementing 5 (XRCC5), which was found more in radioresistant tumors than in radiosensitive ones. The aim of this study was to confirm the possible roles of XRCC5 mRNA levels by a real-time polymerase chain reaction method in 20 cervical cancers, and Ku80 protein, which is the gene product of XRCC5, using a histopathologic method of formalin-fixed sections of tumor biopsies in determining tumor response to radiotherapy and survival in 89 patients with cervical cancer. The levels of XRCC5 mRNA were 104.82 ± 100.2 copies/&mgr;g total RNA in tumor tissues in the CR group (mean ± standard deviation) and 104.95 ± 100.32 copies/&mgr;g total RNA in those in the NC group. The levels of XRCC5 mRNA were not significantly different between the CR and NC groups. Histopathologic methods revealed 29.2% (26 of 89) of the patients to be Ku80-negative, with Ku80-positive findings in 70.8% (63 of 89). Of the Ku80-negative patients, 19 had CR, 3 had a partial response (PR), and 4 had NC. Of the Ku80-positive patients, 25 had CR, 22 had PR, and 16 had NC. Ku80-negative tumors showed significantly better responses than Ku80-positive ones, comparing CR and PR/NC responses (p = 0.01). In addition, overall survival was significantly better in the Ku80-negative patients as compared with those who were Ku80-positive (p = 0.04). The results of this study suggest that a low expression of Ku80 protein leads to radiosensitivity in cervical cancer and that Ku80 might play a role in treatment outcome.

Immunohistochemical localization of myoepithelial cells and basement membrane in normal, benign and malignant human breast lesions

Distributions of actin and type IV collagen were investigated immunohistochemically as markers for myoepithelial cells and basement membranes. Carnoys and Methacarn-fixed, paraffin-embedded tissues from 103 human breast lesions from 103 patients were examined; 65 with carcinomas, 27 with mastopathies, 9 with fibroadenomas and 2 with phyllodes tumours. Fifty-five samples of the normal mammary gland tissue adjacent to tumours were also included for comparison. In normal breast and benign breast diseases, type IV collagen was identified around the mammary glandular cells and actin-positive cells were demonstrated to attach to basement membranes. In noninvasive carcinomas, type IV collagen was found as a continuous lining around a cell nest, while actin-positive cells were usually absent in ductal but quite numerous in lobular carcinomas. In invasive carcinomas, type IV collagen was fragmented or absent and actin-positive cells were very uncommon around the fragmentary basement membranes. These results suggest that the different distributions of myoepithelial cells and basement membrane material is useful in the differential diagnosis of surgical pathology of the breast.

Involvement of inducible costimulator- and interleukin 10-positive regulatory T cells in the development of IgG4-related autoimmune pancreatitis.

Objectives: Immunoglobulin G4 (IgG4)-related autoimmune pancreatitis (AIP) is a new clinical entity of pancreatic disorder. There are immunologic and histological abnormalities, including increased serum IgG4 levels and the infiltration of IgG4-positive plasmacytes. However, the role of IgG4 is unclear. Recently, regulatory T cells (Tregs) were reported to contribute to the development of various autoimmune diseases as well as in B-cell shifting to IgG4-producing plasmacytes. We studied Tregs in the pancreas and peripheral blood. Methods: We recruited 44 patients with IgG4-related AIP. For comparison, we recruited 37 patients with other pancreatic diseases and 27 healthy subjects as controls. We studied infiltrating cells in the pancreas by immunohistochemistry and analyzed inducible costimulator-positive Tregs and interleukin 10-positive Tregs in the peripheral blood by flow cytometry. Results: The ratio of Foxp3-positive cells to infiltrated mononuclear cells (Foxp3/Mono) in AIP patients was significantly higher than in patients with alcoholic chronic pancreatitis. In AIP, Foxp3/Mono and IgG4/Mono were positively correlated. Inducible costimulator-positive Tregs were significantly higher in AIP patients than in the patients with other pancreatic diseases and the healthy control group. Interleukin 10-positive Tregs were significantly higher in AIP patients than in the healthy control group. Conclusions: Increased quantities of inducible costimulator-positive Tregs may influence IgG4 production in IgG4-related AIP.

Trichilemmoma: an immunohistochemical study of cytokeratins

Summary Background The histogenesis of trichilemmoma remains unclear.

MORPHOLOGIC CHARACTERISTICS OF N-METHYL-N-NITROSOUREA-INDUCED RETINAL DEGENERATION IN C57BL MICE

Morphologic characteristics of retinal degeneration induced by a single systemic administration of Methyl‐Knitro‐sourea (MNU) in mice was Investigated. The aim was to characterize the MNU‐Induced retinal lesions In mice and compare them with human retinitis pigmentosa. A dose of 60 mg/kg body weight MNU, Injected intraperttoneally into male and female C57BL mice, evoked progressive retinal degeneration in all treated mice, while control mice re mined normal. An early change was photoreceptor apoptosis followed by Infiltration of macrophages and swelling of the pigment epithellal cells with phagosomal inclusions for apoptotic photoreceptor cell removal. Loss of the majority of photoreceptor cells occurred within a week Then, Feulgenpositive corpuscies, indicative of an aggregation of degenerative photoreceptor elements, vitread the outer limiting membrane were surrounded by Müller cell processes, and the duplication of the pigment epithelial cells sciurid the outer limiting membrane were seen 2 and 3 weeks after the treatment. Finally, the Feulgen‐positive corpuscles disappeared and Müller cell processes were in direct contact with the continuous lining of the single layer of pigment epithellal cells. As in retinitis pigmentosa in humans, the primary event was loss of photoreceptor cells by apoptosis, but the migration of the pigment epithellal cells within the retina was not seen in the present model.