leupeptins and pifithrin

The E6 oncoprotein can interfere with the ability of infected cells to undergo programmed cell death through the proteolytic degradation of proapoptotic proteins such as p53, employing the proteasome pathway. Therefore, inactivation of the proteasome through MG132 should restore the activity of several proapoptotic proteins. We investigated whether in HPV16 E6-expressing keratinocytes (KE6 cells), the restoration of p53 levels mediated by MG132 and/or activation of the CD95 pathway through apoptosis antigen-1 (APO-1) antibody are responsible for the induction of apoptosis. We found that KE6 cells underwent apoptosis mainly after incubation for 24 h with MG132 alone or APO-1 plus MG132. Both treatments activated the extrinsic and intrinsic apoptosis pathways. Autophagy was also activated, principally by APO-1 plus MG132. Inhibition of E6-mediated p53 proteasomal degradation by MG132 resulted in the elevation of p53 protein levels and its phosphorylation in Ser46 and Ser20; the p53 protein was localized mainly at nucleus after treatment with MG132 or APO-1 plus MG132. In addition, induction of its transcriptional target genes such as p21, Bax and TP53INP was observed 3 and 6 h after treatment. Also, LC3 mRNA was induced after 3 and 6 h, which correlates with lipidation of LC3B protein and induction of autophagy. Finally, using pifithrin alpha we observed a decrease in apoptosis induced by MG132, and by APO-1 plus MG132, suggesting that restoration of APO-1 sensitivity occurs in part through an increase in both the levels and the activity of p53. The use of small molecules to inhibit the proteasome pathway might permit the activation of cell death, providing new opportunities for CC treatment.

Topics: Antibodies; Apoptosis; Autophagy; bcl-2-Associated X Protein; Benzothiazoles; Carrier Proteins; Cyclin-Dependent Kinase Inhibitor p21; fas Receptor; Female; Heat-Shock Proteins; Humans; Keratinocytes; Leupeptins; Oncogene Proteins, Viral; Proteasome Endopeptidase Complex; Proteolysis; Repressor Proteins; Toluene; Tumor Suppressor Protein p53; Uterine Cervical Neoplasms

Scopadulciol (1), a scopadulan-type diterpenoid, was isolated from Scoparia dulcis along with three other compounds (2-4) by an activity-guided approach using the TCF reporter (TOP) luciferase-based assay system. A fluorometric microculture cytotoxicity assay (FMCA) revealed that compound 1 was cytotoxic to AGS human gastric adenocarcinoma cells. The treatment of AGS cells with 1 decreased β-catenin levels and also inhibited its nuclear localization. The pretreatment of AGS cells with a proteasome inhibitor, either MG132 or epoxomicin, protected against the degradation of β-catenin induced by 1. The 1-induced degradation of β-catenin was also abrogated in the presence of pifithrin-α, an inhibitor of p53 transcriptional activity. Compound 1 inhibited TOP activity in AGS cells and downregulated the protein levels of cyclin D1, c-myc, and survivin. Compound 1 also sensitized AGS cells to tumor necrosis factor-related apoptosis ligand (TRAIL)-induced apoptosis by increasing the levels of the death receptors, DR4 and DR5, and decreasing the level of the antiapoptotic protein Bcl-2. Collectively, our results demonstrated that 1 induced the p53- and proteasome-dependent degradation of β-catenin, which resulted in the inhibition of TCF/β-catenin transcription in AGS cells. Furthermore, 1 enhanced apoptosis in TRAIL-resistant AGS when combined with TRAIL.

Topics: Abietanes; Adenocarcinoma; Apoptosis; Benzothiazoles; beta Catenin; Down-Regulation; Humans; Leupeptins; Molecular Structure; Receptors, TNF-Related Apoptosis-Inducing Ligand; Scoparia; Stomach Neoplasms; TNF-Related Apoptosis-Inducing Ligand; Toluene; Tumor Necrosis Factor-alpha

Decreased proteasome activity is an important pathology in Parkinson's disease (PD), which is related to cell death and Lewy body formation. In this study, we show that p53-activity may correlate with neuronal death via the mitochondrial pathway in PD model. The proteasome inhibitor, MG132, induced the accumulation of p53 in human dopaminergic neuroblastoma SH-SY5Y cells. The increased stabilization of p53 upregulated the level of Bax and mitochondrial depolarization. These events were inhibited by the p53 inhibitor, pifithrin-alpha (PFT). Cell viability analyzes demonstrated that PFT partially prevented MG132-induced cell death. These results suggest that p53 is a candidate as an intermediary between the proteasome system and mitochondria-related neuronal death in PD.

Topics: bcl-2-Associated X Protein; Benzimidazoles; Benzothiazoles; Blotting, Western; Carbocyanines; Cell Death; Cell Line, Tumor; Cell Survival; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dopamine; Dose-Response Relationship, Drug; Drug Interactions; Fluorescent Antibody Technique; Humans; Leupeptins; Membrane Potentials; Mitochondria; Multienzyme Complexes; Neuroblastoma; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Thiazoles; Time Factors; Toluene; Tumor Suppressor Protein p53

Protein-NLS-coated gold particles up to approximately 250 A in diameter are transported through the nuclear pores in normal, proliferating BALB/c 3T3 cells. This size can increase or decrease, depending on cellular activity. It has been suggested that increases in functional pore size are related to a reduction in the amount of available p53. To further test this hypothesis, we investigated the effects of cycloheximide and pifithrin-alpha, which inhibits p53-dependent transcriptional activation, on nuclear transport. After 3 hours in cycloheximide, there was a significant increase in the size of the gold particles that entered the nucleoplasm. When the incubation period was extended to 6 hours or longer, transport capacity returned to the control level. By using proteasome inhibitors, it was shown that the cycloheximide-dependent increase in functional pore size was due to the inhibition of protein synthesis, consistent with the fact that p53 is a short-lived protein, and requires the activity of at least two different factors. Although cycloheximide increases the functional diameter of the channel available for signal-mediated transport by approximately 60 A, it had no significant effect on either the import rate of small NLS-containing substrates (FITC-BSA-NLS), or passive diffusion of fluorescent-labeled proteins across the envelope. This suggests that changes in transport capacity were not caused by an increase in overall pore diameter but instead are due to a transient increase in pore size that accompanies signal-mediated transport. Pifithrin-alpha also caused an increase in functional pore diameter without altering the import rate of FITC-BSA-NLS, providing further support for the view that p53 can initiate changes in nuclear transport capacity.

Topics: 3T3 Cells; Active Transport, Cell Nucleus; Animals; Benzothiazoles; Cycloheximide; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Diffusion; Fibroblasts; Gold Colloid; Leupeptins; Mice; Multienzyme Complexes; Nuclear Localization Signals; Nuclear Pore; Proteasome Endopeptidase Complex; Thiazoles; Toluene; Tumor Suppressor Protein p53