Ayako Kimura

Resveratrol induces apoptosis via ROS-triggered autophagy in human colon cancer cells

Resveratrol (Res; 3,4′,5-trihydroxy-trans-stilbene), which is a polyphenol found in grapes, can block cell proliferation and induce growth arrest and/or cell death in several types of cancer cells. However, the precise mechanisms by which Res exerts anticancer effects remain poorly understood. Res blocked both anchorage-dependent and -independent growth of HT-29 and COLO 201 human colon cancer cells in a dose- and time-dependent manner. Annexin V staining and Western blot analysis revealed that Res induced apoptosis accompanied by an increase in Caspase-8 and Caspase-3 cleavage. In HT-29 cells, Res caused autophagy as characterized by the appearance of autophagic vacuoles by electron microscopy and elevation of microtubule-associated protein 1 light chain 3 (LC3)-II by immunoblotting, which was associated with the punctuate pattern of LC3 detected by fluorescein microscopy. Inhibition of Res-induced autophagy by the autophagy inhibitor 3-methyladenine caused a significant decrease in apoptosis accompanied by decreased cleavage of Casapse-8 and Caspase-3, indicating that Res-induced autophagy was cytotoxic. However, inhibition of Res-induced apoptosis by the pan-caspase inhibitor Z-VAD(OMe)-FMK did not decrease autophagy but elevated LC3-II levels. Interestingly, Res increased the intracellular reactive oxygen species (ROS) level, which correlated to the induction of Casapse-8 and Caspase-3 cleavage and the elevation of LC3-II; treatment with ROS scavenger N-acetyl cysteine diminished this effect. Therefore, the effect of Res on the induction of apoptosis via autophagy is mediated through ROS in human colon cancer cells.

N -ethyl- N -nitrosourea induces retinal photoreceptor damage in adult rats.

Seven-week-old male Lewis rats received a single intraperitoneal injection of N-ethyl-N-nitrosourea (ENU) (100, 200, 400 or 600 mg/kg), and retinal damage was evaluated 7 days after the treatment. Sequential morphological features of the retina and retinal DNA damage, as determined by a TUNEL assay and phospho-histone H2A.X (γ-H2AX), were analyzed 3, 6, 12, 24 and 72 hr, 7 days, and/or 30 days after 400 mg/kg ENU treatment. Activation of the nuclear enzyme poly (ADP-ribose) polymerase (PARP) was analyzed immunohistochemically by poly (ADP-ribose) (PAR) expression in response to DNA damage of the retina. All rats that received ≥ 400 mg/kg of ENU developed retinal degeneration characterized by the loss of photoreceptor cells in both the central and peripheral retina within 7 days. In the 400 mg/kg ENU-treated rats, TUNEL-positive signals were only located in the photoreceptor cells and peaked 24 hr after ENU treatment. The γ-H2AX signals in inner retinal cells appeared at 24 hr and peaked at 72 hr after ENU treatment, and the PAR signals selectively located in the photoreceptor cell nuclei appeared at 12 hr and peaked at 24 hr after ENU treatment. However, degeneration was restricted to photoreceptor cells, and no degenerative changes in inner retinal cells were seen at any time points. Retinal thickness and the photoreceptor cell ratio in the central and peripheral retina were significantly decreased, and the retinal damage ratio was significantly increased 7 days after ENU treatment. In conclusion, ENU induced retinal degeneration in adult rats that was characterized by photoreceptor cell apoptosis through PARP activity.

Histopathological and immunohistochemical characterization of spontaneously occurring uterine deciduomas in young adult rats.

Uterine deciduomas were found in two female virgin rats, a 15-week-old Lewis rat and a 7-week-old Sprague-Dawley rat. The firm white nodules were located at the base of unilateral uterine horns and were approximately 6 mm and 4 mm in diameter. Histopathologically, the nodules were composed of three areas, each with a distinct type of proliferating cells: large epithelioid decidual cells with round nuclei, prominent nucleoli and abundant eosinophilic cytoplasm (antimesometrial region); compact spindle-shaped cells with oval nuclei and vacuolar cytoplasm (transitional region); and pleomorphic and spiny cells with round to oval nuclei and compact eosinophilic cytoplasm (mesometrial region). These cells proliferated in sheet-like arrangements and transformed into the other types of cells located in surrounding regions. Immunohistochemically, proliferating cells in all regions were strongly positive for proliferating cell nuclear antigen. The proliferating cells were positive for vimentin, and large decidual cells were positive for common acute lymphoblastic leukemia antigen 10, a marker of uterine interstitial cells. Large decidual cells were positive for α-smooth muscle actin and desmin, suggesting differentiation into muscular cells. Progesterone receptor was expressed in all cell types; however, estrogen receptor α was not expressed in the antimesometrial region. These extremely rare tumor-like nodules represent nonneoplastic lesions referred as decidual reactions of endometrial interstitial cells, and their biological behavior is that of a space-occupying benign tumor in young rats. Our cases might provide information as a historical control in toxicity and pharmacological studies in rats.

Two cases of malignant peritoneal mesothelioma without asbestos exposure: cytologic and immunohistochemical features

Approximately half a century has passed since asbestos was first reported to be the main cause of malignant mesothelioma; yet the incidence of this disease continues to increase worldwide. Twenty percent of cases occur without prior asbestos exposure, and in these patients, malignant peritoneal mesothelioma is more common than malignant pleural mesothelioma. Here, we report the cytomorphologic and immunohistochemical features of 2 cases of malignant peritoneal mesothelioma where there was no history of asbestos exposure. Ascitic cytology showed that most cells were isolated and that clusters were rarely observed, but the findings were consistent with malignant mesothelioma in both cases. Immunohistochemical analysis for epithelial membrane antigen, calretinin, vimentin, β-catenin, melan-A, glucose transporter-1, cytokeratin CAM5.2, Wilms tumor antigen-1, D2-40, CD146, progesterone receptor, estrogen receptor, and cytokeratin 5/6 was indicative of malignant mesothelioma. In malignant mesothelioma without prior asbestos exposure, the etiology and prognostic significance is still unclear. Further study is needed to clarify this point.

A case report of lipid-rich carcinoma of the breast including histological characteristics and intrinsic subtype profile.

A 57-year-old Japanese woman with schizophrenia, who had received long-term treatment with neuroleptics, noticed a painless, pea-sized lump in her right breast. She was admitted to our hospital and a malignant tumor was diagnosed. The patient underwent a conservative radical mastectomy (Patey’s operation). The excised tumor measured 2.0 × 1.2 × 1.1 cm in diameter, and its cut surface was grayish-white. Histologically, tumor cells with clear to foamy cytoplasm were invariably Oil Red O-positive and periodic acid Schiff-negative with or without diastase digestion. The tumor was diagnosed as a lipid-rich carcinoma accompanied by an in situ component. Neuroleptics increase serum prolactin levels by interfering with dopaminergic inhibition of prolactin secretion. Immunohistochemical analysis revealed that, although prolactin was not detected, the tumor cells expressed prolactin receptor, indicating prolactin as the genesis of this neoplasm. In immunohistochemical intrinsic subtype analysis, the tumor was negative for estrogen receptor, progesterone receptor, human epidermal growth factor receptor 1 and 2, and basal cytokeratins (CK5, CK6, and CK14), indicating an unclassified (all-marker negative) subtype. Axillary lymph nodes were free of metastasis (stage I), and the patient has been well for 20 years without any evidence of recurrence.

Arachidonic acid supplementation does not affect N-methyl-N-nitrosourea-induced renal preneoplastic lesions in young Lewis rats

Arachidonic acid (AA) is naturally found in human breast milk. AA, together with docosahexaenoic acid, is commonly added as a functional food ingredient to commercial infant formula worldwide, in accordance with the international standards of Codex Alimentarius. However, few studies of the possible renal carcinogenic effects of AA supplementation during neonatal life have been performed. The effect of dietary AA supplementation in dams during gestation and lactation was investigated on N-methyl-N-nitrosourea (MNU)-induced preneoplastic lesions in the kidneys of young Lewis rats. Dams were fed a 2.0% AA diet or a basal diet (<0.01% AA). At birth (postnatal day 0), male and female pups received a single intraperitoneal injection of 35 mg/kg MNU or vehicle. Renal morphology was examined after 7, 14, 21, 28 and 60 days. Histopathologically, renal preneoplastic lesions, such as nephroblastomatosis and mesenchymal cell proliferation, were found on day 60 in both the MNU-treated groups. There was no significant difference in lesion incidence of 38% in the basal diet group and 31% in the AA diet group. In conclusion, an AA-rich diet for dams during gestation and lactation does not modify MNU-induced renal preneoplastic lesions in their offspring.

Pulmonary and Meningeal Cryptococcosis after Corticosteroid Therapy for Autoimmune Hepatitis: Coexistence of Cryptococci within Pulmonary Cancer Nodule

A case of autoimmune hepatitis complicated with pulmonary and meningeal cryptococcosis during long-term treatment with corticosteroid is reported. An 84-year-old woman who received long-term corticosteroid therapy (40 mg/day prednisolone for two years) for autoimmune hepatitis developed a headache, slight fever, and anorexia and was diagnosed with cryptococcal meningitis two months prior to hospital admission. Due to deterioration of her condition, the patient was transferred to our university hospital. After admission, a pulmonary nodule 1 cm in diameter was noticed in the patients right lower lobe. Cryptococcal meningitis was diagnosed as positive for cryptococcal antigen from both serum and cerebrospinal fluid (CSF) as well as the growth of Cryptococcus neoformans (C. neoformans) in fungal culture. A combination therapy of amphotericin B and flucytosine was started, and the corticosteroid therapy was gradually reduced and finally discontinued. In addition to continuous cryptococcal infection, complications of Pseudomonas aeruginosa and methicillin-resistance Staphylococcus aureus infection caused death after a 2-month hospitalization. Autopsy disclosed encapsulated yeast in the lungs and subarachnoid space characteristic of Cryptococcus. The pulmonary nodule was found to be squamous cell carcinoma coexisting with C. neoformans within and around the cancer cell nests.

Spontaneously occurring intracranial lipomatous hamartoma in a young BALB/c mouse and a literature review.

An intracranial lipomatous hamartoma was found in the third ventricle of a 7-week-old female BALB/cAnNCrlCrlj mouse. The nodule was composed of mature white adipose cells, which contained one large fat droplet, and there was no evidence of cytological atypia. The brain parenchyma at the retrosplenial granular cortex and the hippocampus in the cerebrum were slightly compressed, and the choroid plexus was dislocated downward. Scattered capillary vessels penetrated the nodule from the surrounding tissue. Based on these findings, the lesion was diagnosed as a lipomatous hamartoma that occurred from the roof of the third ventricle. This extremely rare tumor-like nodule represents an overgrowth of the mature adipocyte population as a malformation rather than a true neoplasm.

N-Methyl-N -nitrosourea-induced Renal Tumors in Rats: Immunohistochemical Comparison to Human Wilms Tumors

N-Methyl-N-nitrosourea (MNU)-induced renal tumors in rats and Wilms tumors in humans were compared. Renal mesenchymal tumors (RMTs) and nephroblastomas (blastemal and epithelial components) in female Lewis rats treated with a single intraperitoneal injection of 50 mg/kg MNU at birth and Wilms tumors (blastemal, epithelial and mesenchymal components) in humans were analyzed for the expression of pancytokeratin (CK), vimentin, p63, α-smooth muscle actin (SMA), desmin, S-100, CD57, CD117/c-kit, Wilms tumor 1 protein (WT1) and β-catenin. The mesenchymal components of rat RMTs and human Wilms tumors expressed vimentin, SMA and β-catenin. The blastemal components of rat nephroblastomas and human Wilms tumors expressed vimentin, CD117/c-kit and β-catenin. The epithelial components of rat nephroblastomas and human Wilms tumors expressed vimentin and β-catenin. WT1 was expressed in different cellular components of rat tumors as compared with human Wilms tumors; the expression was seen in mesenchymal tumors and blastemal components of nephroblastomas in rats and epithelial components in human Wilms tumors. CK, p63 and CD57 were not expressed in rat RMTs or nephroblastomas, while CK and WT1 were expressed in epithelial components and CD57 was expressed in blastemal and epithelial components of human Wilms tumors. Rat and human tumors were universally negative for the expression of desmin and S-100. The immunohistochemical characteristics of rat renal tumors and human Wilms tumors may provide valuable information on the differences in renal oncogenesis and biology between the two species.