benzyloxycarbonylleucyl-leucyl-leucine-aldehyde and Urinary-Bladder-Neoplasms

The ubiquitin-proteasome pathway degrades ubiquitinated proteins to remove damaged or misfolded protein and thus plays an important role in the maintenance of many important cellular processes. Because the pathway is also crucial for tumor cell growth and survival, proteasome inhibition by specific inhibitors exhibits potent antitumor effects in many cancer cells. xCT, a subunit of the cystine antiporter system xc (-), plays an important role in cellular cysteine and glutathione homeostasis. Several recent reports have revealed that xCT is involved in cancer cell survival; however, it was unknown whether xCT affects the cytotoxic effects of proteasome inhibitors. In this study, we found that two stress-inducible transcription factors, Nrf2 and ATF4, were upregulated by proteasome inhibition and cooperatively enhance human xCT gene expression upon proteasome inhibition. In addition, we demonstrated that the knockdown of xCT by small interfering RNA (siRNA) or pharmacological inhibition of xCT by sulfasalazine (SASP) or (S)-4-carboxyphenylglycine (CPG) significantly increased the sensitivity of T24 cells to proteasome inhibition. These results suggest that the simultaneous inhibition of both the proteasome and xCT could have therapeutic benefits in the treatment of bladder tumors.

Topics: Activating Transcription Factor 4; Amino Acid Transport System y+; Antineoplastic Agents; Boronic Acids; Bortezomib; Cell Line, Tumor; Cysteine; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Glutathione; Glycine; HEK293 Cells; HeLa Cells; Humans; Leupeptins; NF-E2-Related Factor 2; Oligopeptides; Proteasome Endopeptidase Complex; Pyrazines; RNA, Small Interfering; Sulfasalazine; Urinary Bladder Neoplasms

Celecoxib is a selective cyclooxygenase-2 (COX-2) inhibitor that has been reported to elicit anti-proliferative response in various tumors. In this study, we aim to investigate the antitumor effect of celecoxib on urothelial carcinoma (UC) cells and the role endoplasmic reticulum (ER) stress plays in celecoxib-induced cytotoxicity. The cytotoxic effects were measured by MTT assay and flow cytometry. The cell cycle progression and ER stress-associated molecules were examined by Western blot and flow cytometry. Moreover, the cytotoxic effects of celecoxib combined with glucose-regulated protein (GRP) 78 knockdown (siRNA), (-)-epigallocatechin gallate (EGCG) or MG132 were assessed. We demonstrated that celecoxib markedly reduces the cell viability and causes apoptosis in human UC cells through cell cycle G1 arrest. Celecoxib possessed the ability to activate ER stress-related chaperones (IRE-1α and GRP78), caspase-4, and CCAAT/enhancer binding protein homologous protein (CHOP), which were involved in UC cell apoptosis. Down-regulation of GRP78 by siRNA, co-treatment with EGCG (a GRP78 inhibitor) or with MG132 (a proteasome inhibitor) could enhance celecoxib-induced apoptosis. We concluded that celecoxib induces cell cycle G1 arrest, ER stress, and eventually apoptosis in human UC cells. The down-regulation of ER chaperone GRP78 by siRNA, EGCG, or proteosome inhibitor potentiated the cytotoxicity of celecoxib in UC cells. These findings provide a new treatment strategy against UC.

Topics: Apoptosis; Catechin; Celecoxib; Cell Line, Tumor; Cell Survival; Cyclooxygenase 2 Inhibitors; Down-Regulation; Drug Synergism; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; G1 Phase Cell Cycle Checkpoints; Heat-Shock Proteins; Humans; Indoles; Leupeptins; Pyrazoles; RNA, Small Interfering; Stress, Physiological; Sulfonamides; Unfolded Protein Response; Urinary Bladder Neoplasms

The tumor suppressor p53 is activated in response to many forms of cellular stress leading to cell cycle arrest, senescence or apoptosis. Appropriate sub-cellular localization is essential for modulating p53 function. We recently showed that p53 localizes to the nucleolus after proteasome inhibition with MG132 and this localization requires sequences within its carboxyl terminus. In the present study, we found that after treatment with MG132, p53 associates with a discrete sub-nucleolar component, the fibrillar center (FC), a region mainly enriched with RNA polymerase I. Moreover, we now demonstrate that this localization is an energy-dependent process as reduction of ATP levels prevents nucleolar localization. In addition, p53 sub-nucleolar accumulation is abolished when cells are subjected to various types of genotoxic stress. Furthermore, we show that monoubiquitination of p53, which causes it to localize to the cytoplasm and nucleoplasm, does not prevent the association of p53 with the nucleolus after MG132 treatment. Importantly, we demonstrate that p53 nucleolar association occurs in lung and bladder carcinomas.

Topics: Amino Acid Sequence; Carcinoma; Cell Nucleus; Cysteine Proteinase Inhibitors; Cytoplasm; Humans; Leupeptins; Lung Neoplasms; Molecular Sequence Data; Mutant Proteins; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53; Ubiquitin; Ubiquitination; Urinary Bladder Neoplasms

The possibility that bacteria may have evolved strategies to overcome host cell apoptosis was explored by using Rickettsia rickettsii, an obligate intracellular Gram-negative bacteria that is the etiologic agent of Rocky Mountain spotted fever. The vascular endothelial cell, the primary target cell during in vivo infection, exhibits no evidence of apoptosis during natural infection and is maintained for a sufficient time to allow replication and cell-to-cell spread prior to eventual death due to necrotic damage. Prior work in our laboratory demonstrated that R. rickettsii infection activates the transcription factor NF-kappa B and alters expression of several genes under its control. However, when R. rickettsii-induced activation of NF-kappa B was inhibited, apoptosis of infected but not uninfected endothelial cells rapidly ensued. In addition, human embryonic fibroblasts stably transfected with a superrepressor mutant inhibitory subunit Ikappa B that rendered NF-kappa B inactivatable also underwent apoptosis when infected, whereas infected wild-type human embryonic fibroblasts survived. R. rickettsii, therefore, appeared to inhibit host cell apoptosis via a mechanism dependent on NF-kappa B activation. Apoptotic nuclear changes correlated with presence of intracellular organisms and thus this previously unrecognized proapoptotic signal, masked by concomitant NF-kappa B activation, likely required intracellular infection. Our studies demonstrate that a bacterial organism can exert an antiapoptotic effect, thus modulating the host cell's apoptotic response to its own advantage by potentially allowing the host cell to remain as a site of infection.

Topics: Apoptosis; Base Sequence; Binding Sites; Cell Nucleus; Cell Survival; Cells, Cultured; Consensus Sequence; Cysteine Proteinase Inhibitors; DNA Fragmentation; Embryo, Mammalian; Fibroblasts; Humans; Kinetics; Leupeptins; Microscopy, Electron; Necrosis; NF-kappa B; Oligodeoxyribonucleotides; Rickettsia rickettsii; Time Factors; Tumor Cells, Cultured; Umbilical Veins; Urinary Bladder Neoplasms