Rapamycin enhances docetaxel-induced cytotoxicity in a androgen-independent prostate cancer xenograft model by survivin downregulation

BackgroundDocetaxel is a first-line treatment choice in castration-resistant prostate cancer (CRPC). However, the management of CRPC remains an important challenge in oncology. There have been many reports on the effects of rapamycin, which is an inhibitor of the mammalian target of rapamycin (mTOR), in the treatment of carcinogenesis. We assessed the cytotoxic effects of the combination treatment of docetaxel and rapamycin in prostate cancer cells. Furthermore, we examined the relationship between these treatments and survivin, which is a member of the inhibitory apoptosis family.MethodsProstate cancer cells were cultured and treated with docetaxel and rapamycin. The effects on proliferation were evaluated with the MTS assay. In addition, we evaluated the effect on proliferation of the combination treatment induced knockdown of survivin expression by small interfering RNA transfection and docetaxel. Protein expression levels were assayed using western blotting. PC3 cells and xenograft growth in nude mice were used to evaluate the in vivo efficacy of docetaxel and its combination with rapamycin.ResultsIn vitro and in vivo, the combination of rapamycin with docetaxel resulted in a greater inhibition of proliferation than treatment with rapamycin or docetaxel alone. In addition, in vitro and in vivo, rapamycin decreased basal surviving levels, and cotreatment with docetaxel further decreased these levels. Transfection siRNA against survivin enhanced the cytotoxicity of docetaxel in PC3 cells.ConclusionThe rapamycin-dependent enhancement of the cytotoxic effects of docetaxel was associated with the downregulation of survivin expression. Our results suggest that the combination of docetaxel and rapamycin is a candidate for the improved treatment of advanced prostate cancer.     

Hepatic macrophage iron aggravates experimental alcoholic steatohepatitis

One prime feature of alcoholic liver disease (ALD) is iron accumulation in hepatic macrophages/Kupffer cells (KC) associated with enhanced NF-kappaB activation. Our recent work demonstrates a peroxynitrite-mediated transient rise in intracellular labile iron (ILI) as novel signaling for endotoxin-induced IKK and NF-kappaB activation in rodent KC. The present study investigated the mechanism of KC iron accumulation and its effects on ILI response in experimental ALD. We also tested ILI response in human blood monocytes. Chronic alcohol feeding in rats results in increased expression of transferrin (Tf) receptor-1 and hemochromatosis gene (HFE), enhanced iron uptake, an increase in nonheme iron content, and accentuated ILI response for NF-kappaB activation in KC. Ex vivo treatment of these KC with an iron chelator abrogates the increment of iron content, ILI response, and NF-kappaB activation. The ILI response is evident in macrophages derived from human blood monocytes by PMA treatment but not in vehicle-treated monocytes, and this differentiation-associated phenomenon is essential for maximal TNF-alpha release. PMA-induced macrophages load iron dextran and enhance ILI response and TNF-alpha release. These effects are reproduced in KC selectively loaded in vivo with iron dextran in mice and more importantly aggravate experimental ALD. Our results suggest enhanced iron uptake as a mechanism of KC iron loading in ALD and demonstrate the ILI response as a function acquired by differentiated macrophages in humans and as a priming mechanism for ALD.     

Involvement of Gicerin in the Extension of Microvilli

Gicerin is a cell adhesion molecule belonging to the immunoglobulin superfamily. To study the functional differences between l- and s-gicerin, we first examined the distribution of endogenous gicerin in B16 cells and found that l-gicerin was densely localized in microvilli. To clarify the relationship between gicerin and the microvilli, we established independent stable cell lines expressing l- and s-gicerin in L cells and found that l-gicerin localized to the microvilli. Scanning electron microscopic analysis revealed that the microvilli of l-gicerin-transfected cells were longer than those of s-gicerin and control transfectants. This suggested that l-gicerin might participate in the elongation of the microvilli. When cells were double-stained with antibodies to gicerin and moesin, a microvilli-specific protein, the staining of l-gicerin corresponded to that of moesin in the elongated microvilli. Moesin was coprecipitated with glutathione S-transferase-fusion proteins of the l-gicerin cytoplasmic domain but not with the s-gicerin cytoplasmic domain. To determine the region involved in the extension of microvilli, we generated transfectants of two truncated forms of l-gicerin cytoplasmic domain, and we found that only the transfectants of the longer mutant had the longer microvilli, while the shorter mutant exhibited short microvilli. These results suggested that l-gicerin-specific amino acid residues, especially amino acids 16-39, within the cytoplasmic domain of l-gicerin might be involved in the extension of microvilli.     

Visual detection of filaria-specific IgG4 in urine using red-colored high density latex beads

The use of urine for the immunodiagnosis of lymphatic filariasis has a definite advantage: the sample collection is not invasive and thus well accepted by people. Urine-based ELISA to detect filaria-specific IgG4 has been used successfully. However, ELISA requires equipment such as a microplate reader, which is often not available in most endemic areas. We have developed a new visual immunodiagnosis that detects urinary IgG4 using red-colored latex beads (bead test). The sensitivity was 87.2% when ICT antigen test positive people were regarded as the standard (136/156), and the specificity was 97.2% with the non-endemic people in Japan and Bangladesh, and the urine ELISA negatives in Sri Lanka (1264/1300). In a prevalence study, the bead test could detect filarial infection more effectively than ICT test among young children in Sri Lanka, indicating the usefulness of the visual test in epidemiological studies.     

Techniques for universal evaluation of astringency of green tea infusion by the use of a taste sensor system

A practical method for universal evaluation of the astringency of green tea infusion by a taste sensor system was established. The use of EGCg aqueous solution as a standard enabled analysis with high accuracy and reproducibility. The sensor output was converted into taste-intensity on the basis of Weber's and Weber-Fechner laws, which was named the "EIT(ast)" value ("EIT" and "ast" are abbreviations for "Estimated Intensity of Taste" and "astringency" respectively). It was clarified that green tea infusion is to be classified into eight grades on the EIT(ast) scale. Furthermore, the high correlation of the EIT(ast) value with the human gustatory sense and the high stability of the taste sensor were proved.     

Suppressive oligodeoxynucleotides protect mice from lethal endotoxic shock

Endotoxic shock is a life-threatening condition caused by exposure to bacterial LPS. LPS triggers the release of acute phase, proinflammatory, and Th1 cytokines that facilitate the development of endotoxic shock. Synthetic oligodeoxynucleotides (ODN) expressing suppressive TTAGGG motifs effectively down-regulate the production of proinflammatory and Th1 cytokines elicited by a variety of immune stimuli. The current results demonstrate that suppressive ODN protect mice from LPS-induced endotoxic shock. Underlying this protective effect is the ability of suppressive ODN to bind to and prevent the phosphorylation of STAT1 and STAT4, thereby blocking the signaling cascade mediated by LPS-induced IFN-beta and IL-12. These findings suggest that suppressive ODN might be of use in the treatment of endotoxic shock.     

Anionic sites in glomerular basement membrane of rats with serum sickness nephritis: quick-freezing and deep-etching study

Summary Serum sickness nephritis was induced in male Fisher 344/JCL rats by injecting egg albumin into the foot pads and peritoneal cavity. The alteration of anionic sites in the glomerular basement membrane (GBM) of the rats with significant proteinuria was studied with a quick-freezing and deep-etching method using polyethyleneimine as a cationic probe. In control rats, anionic sites were located around the fibrils of the lamina rara externa, which radiated perpendicularly from the lamina densa to podocyte cell membranes. In the glomeruli of proteinuric rats, many electron-dense deposits were observed in the subepithelial side of the GBM, where the fibrils of the lamina rara externa were usually obscured and anionic sites around them could not be recognized. However, in some areas, a clear boundary could be observed between deposits and the lamina densa. Electron micrographs of freeze-fractured deposits showed that the fibrils radiated perpendicularly from the lamina densa and that anionic sites around them had been preserved. These results suggest that some of the deposits simply passed through the GBM and masked transiently the fibril structures of the GBM, but others probably destroyed these fibril structures, including anionic sites.