Immunohistochemical Analyses of Mammalian Target of Rapamycin (mTOR) Expression in Pituitary Neuroendocrine Tumors (PitNETs): mTOR as a Therapeutic Target for Functional PitNETs.

Current therapeutic modalities for pituitary neuroendocrine tumors (PitNETs) include medication, surgery, and radiotherapy. Some patients have tumors that are refractory to current modalities. Therefore, novel treatment options are needed for patients with intractable diseases. Consequently, we examined the pathological data of PitNETs to study medical therapies. We retrospectively studied 120 patients with histologically diagnosed PitNETs. We used the data for the histopathological examination of hormones, such as growth hormone (GH), prolactin (PRL), adrenocorticotropic hormone, thyroid stimulating hormone, luteinizing hormone, follicle-stimulating hormone, and α-subunit, together with the immunohistochemical studies of the phospho-mammalian target of rapamycin (mTOR), cytokeratin (CAM5.2), somatostatin receptor (SSTR) type 2 and 5, Pit-1 (POU1F1/GHF-1), steroidogenic factor-1 (SF-1), and Tpit. GH-, PRL-, and SSTR5-immunopositive PitNETs had significantly higher percentage of mTOR-positivity, compared with GH-, PRL-, and SSTR5-immunonegative Pit NETs. Our results show that activation of the AKT/phosphatidylinositol-3-kinase pathway, including mTOR activation, might be related the development of PitNETs, especially GH- and PRL-producing PitNETs. Thus, mTOR is a potential target for treating functional PitNETs.

Carbon tetrachloride-induced hepatic injury through formation of oxidized diacylglycerol and activation of the PKC/NF-κB pathway.

Protein kinase C (PKC) participates in signal transduction, and its overactivation is involved in various types of cell injury. PKC depends on diacylglycerol (DAG) for its activation in vivo We have previously reported that DAG peroxides (DAG-O(O)H) activate PKC in vitro more strongly than unoxidized DAG, suggesting that DAG-O(O)H, if generated in vivo under oxidative stress, would act as an aberrant signal transducer. The present study examined whether DAG-O(O)H are formed in carbon tetrachloride (CCl(4))-induced acute rat liver injury in association with activation of the PKC/nuclear factor (NF)-κB pathway. A single subcutaneous injection of CCl(4) resulted in a marked increase in hepatic DAG-O(O)H content. At the molecular level, immunohistochemistry and subcellular fractionation combined with immunoblotting localized PKCα, ßI, ßII and δ isoforms to cell membranes, while immunoblotting showed phosphorylation of the p65 subunit of NF-κB, and immunoprecipitation using isoform-specific anti-PKC antibodies revealed specific association of PKCα and p65. In addition, expression of tumor necrosis factor α (TNFα) and neutrophil invasion increased in the CCl(4)-treated rats. Furthermore, we demonstrated that Vitamin E, one of the most important natural antioxidants that suppresses peroxidation of membrane lipids, significantly inhibited the CCl(4)-induced increase in hepatic DAG-O(O)H content and TNFα expression as well as phosphorylation of PKCα and p65. These data demonstrate for the first time that DAG-O(O)H are generated in the process of CCl(4)-induced liver injury, resulting in activation of the PKC/NF-κB pathway and TNFα-mediated aggravation of liver injury.

Analysis of mTOR inhibition-involved pathway in ovarian clear cell adenocarcinoma.

This study was designed to clarify the mechanism of the mammalian target of rapamycin (mTOR)-hypoxia inducible factor-1 (HIF-1) pathway using the cultured cell strain derived from human ovarian clear cell adenocarcinoma (CCA). Everolimus (a derivative of rapamycin)-treated cells and non-treated cells did not show any difference in mTOR expression. But, phosphorylated-mTOR (p-mTOR) expression significantly decreased in the treated cells, and mTOR-related factors such as phosphorylated-4E-BP1 (p-4E-BP1), HIF-1α, and vascular endothelial growth factor (VEGF) in the downstream region of mTOR revealed a marked decrease in expression. The analysis of influences of the drug on the HIF-1α degradation system showed an increase in von-Hippel Lindau (VHL) expression in the treated cells. Increase of cleaved caspase-3, one of key factors involved in apoptosis, was also shown in the treated cells. In the next step, using nude mice implanted with RMG-1 cells, a decrease in tumor size was demonstrated in 4 of the 7 mice which were orally administered with everolimus. As a result, it was suggested that everolimus administration would be helpful as an anti-tumor therapy for CCA not only via down-regulation of p-mTOR but also degradation of HIF-1α by VHL and induction of apoptosis by cleaved caspase-3.

Quercetin enhances melanogenesis by increasing the activity and synthesis of tyrosinase in human melanoma cells and in normal human melanocytes.

Quercetin (3,3',4',5,7-pentahydroxyflavone) is a diphenyl propanoid widely distributed in edible plants. In this study, we examined the effect of quercetin on melanogenesis in human HMVII melanoma cells and in normal human epidermal melanocytes (NHEM) in the absence of ultraviolet radiation. Upon the addition of quercetin to the culture medium, the melanin content in melanoma cells (HMVII) increased remarkably in time- and dose-dependent manners. In addition, quercetin induced melanogenesis in cultured NHEM. As compared with controls, melanin content was increased about sevenfold by treatment with 20 microM (HMVII) or 1 microM (NHEM) quercetin for 7 d. Tyrosinase activity was also increased, to 61.8-fold higher than the control. The expression of tyrosinase protein was slightly increased by the addition of quercetin. However, quercetin did not affect the expression of tyrosinase mRNA. Tyrosinase activation by quercetin was blocked by actinomycin-D or by cycloheximide demonstrating that its actions in stimulating melanogenesis may involve both transcriptional and translational events. Tyrosinase activity was increased dramatically whereas the level of melanogenic inhibitor was remarkably decreased following quercetin treatment. Taken together, these results demonstrate that in human melanoma cells and in NHEM, quercetin stimulates melanogenesis by increasing tyrosinase activity and decreasing other factors such as melanogenic inhibitors.

Lupus erythematosus cells in pleural fluid cytologic diagnosis in two patients.

Lupus erythematosus (LE) cells were demonstrated inpleural fluids from two patients with systemic lupus erythematosus (SLE). They were observed in preparations stained with the Giemsa and the Papanicolaou stains. The finding of LE cells in serous fluid has rarely been reported. Our results suggest that it is imporant to search for LE cells in serous fluid in patients in whom the diagnosis of SLE has not been established.