elafin and Urinary-Bladder-Neoplasms

Citrus unshiu peel has been used to prevent and treat various diseases in traditional East-Asian medicine including in Korea. Extracts of C. unshiu peel are known to have various pharmacological effects including antioxidant, anti-inflammatory, and antibacterial properties. Although the possibility of their anti-cancer activity has recently been reported, the exact mechanisms in human cancer cells have not been sufficiently studied. In this study, the inhibitory effect of ethanol extract of C. unshiu peel (EECU) on the growth of human bladder cancer T24 cells was evaluated and the underlying mechanism was investigated. The present study demonstrated that the suppression of T24 cell viability by EECU is associated with apoptosis induction. EECU-induced apoptosis was found to correlate with an activation of caspase-8, -9, and -3 in concomitance with a decrease in the expression of the inhibitor of apoptosis family of proteins and an increase in the Bax:Bcl-2 ratio accompanied by the proteolytic degradation of poly(ADP-ribose) polymerase. EECU also increased the generation of reactive oxygen species (ROS), collapse of mitochondrial membrane potential, and cytochrome c release to the cytosol, along with a truncation of Bid. In addition, EECU inactivated phosphatidylinositol 3-kinase (PI3K) as well as Akt, a downstream molecular target of PI3K, and LY294002, a specific PI3K inhibitor significantly enhanced EECU-induced apoptosis and cell viability reduction. However, N-acetyl cysteine, a general ROS scavenger, completely reversed the EECU-induced dephosphorylation of PI3K and Akt, as well as cell apoptosis. Taken together, these findings suggest that EECU inhibits T24 cell proliferation by activating intrinsic and extrinsic apoptosis pathways through a ROS-mediated inactivation of the PI3K/Akt pathway.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Cell Culture Techniques; Cell Line, Tumor; Cell Survival; Citrus; Elafin; Ethanol; Humans; Plant Extracts; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Signal Transduction; Urinary Bladder Neoplasms

Gemcitabine (2'-deoxy-2', 2'-difluorocytidine; Gem) is a nucleoside anti-metabolite and is commonly used for treating various human cancers including human bladder carcinoma. Gemcitabine not only functions as a suicide nucleoside analog but also inhibits DNA polymerase activity and results in the termination of chain elongation. Using 2-dimensional gel electrophoresis analysis, a Gem-induced protein was identified as UBE2M (a.k.a. UBC12), a NEDD8 conjugation E2 enzyme which contributes to protein degradation. Gem induced UBE2M expression at both RNA and protein levels in several human cancer cell lines. The induction of UBE2M by Gem was accompanied by a reduction in p27(Kip1) protein levels, which could be restored by silencing UBE2M expression with siRNA or by treating cells with the proteasome inhibitor MG132, indicating that UBE2M mediates Gem-induced p27(Kip1) protein degradation. The induction of UBE2M and reduction of p27(Kip1) by Gem were prevented by the PI3K inhibitor LY294002. These results indicate that PI3K activity is necessary for Gem-induced UBE2M expression and that UBE2M facilitates degradation of p27(Kip1). Notably, silencing of UBE2M expression reduced Gem sensitivity in NTUB1 cells, suggesting that UBE2M mediates in part cell sensitivity to Gem, possibly by degradation of p27(Kip1). Analysis of Gem-resistant sub lines also showed that loss of UBE2M and increased p27(Kip1) expression were associated with the acquisition of drug resistance. In conclusion, our results demonstrate a role for UBE2M in mediating cytotoxicity of gemcitabine in human urothelial carcinoma cells while also suggesting a potential function of p27(Kip1) in drug resistance.

Topics: Antimetabolites, Antineoplastic; Blotting, Western; Carcinoma, Transitional Cell; Cell Line, Tumor; Cell Survival; Chromones; Cyclin-Dependent Kinase Inhibitor p27; Deoxycytidine; Drug Screening Assays, Antitumor; Elafin; Formazans; Gemcitabine; Gene Expression Regulation; Gene Silencing; Humans; Morpholines; RNA, Small Interfering; Spectrometry, Mass, Electrospray Ionization; Tetrazolium Salts; Ubiquitins; Urinary Bladder Neoplasms