benzyloxycarbonylleucyl-leucyl-leucine-aldehyde and Skin-Neoplasms

We have previously discovered that Notch1 is expressed on malignant T cells in cutaneous T-cell lymphoma (CTCL), and is required for survival of CTCL cell lines. Notch can be inhibited by γ-secretase inhibitors (GSIs), which differ widely in their ability to induce apoptosis in CTCL.. To investigate whether GSI-I, in addition to inhibiting Notch, induces apoptosis in CTCL by proteasome inhibition, as GSI-I is very potent and has structural similarity to the proteasome inhibitor MG-132.. Cell lines derived from CTCL (MyLa, SeAx, JK, Mac1 and Mac2a) were treated with GSI-I and two other proteasome inhibitors (MG-132 and bortezomib). The effects on cell viability, apoptosis and proteasome activity were measured, as was the impact on the prosurvival, nuclear factor κB (NF-κB) pathway.. In CTCL, GSI-I had proteasome-blocking activity with a potency comparable to the proteasome inhibitors MG-132 and bortezomib. Proteasome inhibition was the main mechanism responsible for GSI-I-induced cell death, as tiron, a compound known to reverse the effect of MG-132, restored proteasome activity and largely abrogated the cytotoxic effect of GSI-I. Although inactivation of NF-κB is an important mechanism of action for proteasome inhibitors, we demonstrated an apparent activation of NF-κB. Furthermore, we showed that while the tumour suppressor protein p53 was induced during proteasome inhibition, it was dispensable for CTCL apoptosis, as both SeAx cells, which harbour p53 mutations that attenuate the apoptotic capacity, and HuT-78 cells, which have a deleted p53 gene, demonstrated potent apoptotic response.. GSI-I represents an interesting drug with a dual mechanism of action comprising inhibition of both Notch and the proteasome.

Topics: Amyloid Precursor Protein Secretases; Antineoplastic Agents; Apoptosis; Boronic Acids; Bortezomib; Cell Line, Tumor; Enzyme Inhibitors; Humans; Leupeptins; Lymphoma, T-Cell, Cutaneous; NF-kappa B; Oligopeptides; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Pyrazines; Receptor, Notch1; Skin Neoplasms; Tumor Suppressor Protein p53

SKI and SnoN proteins have been shown to inhibit TGF-β signaling, acting both as transcriptional co-repressors in the cell nucleus, and as sequestrators of SMAD proteins in the cytoplasm. TGF-β, on the other hand, induces rapid, proteasome-mediated, degradation of both proteins. How elevated SKI and SnoN protein levels co-exist with active autocrine TGF-β signaling in cancer cells is yet to be understood.. In this study, we found elevated SKI and SnoN protein levels in a panel of melanoma cell lines, as compared to normal melanocytes. There was no correlation between SKI protein content and the capacity of melanoma cells to invade Matrigel™, to form subcutaneous tumors, or to metastasize to bone after intracardiac inoculation into nude mice. Nor did we find a correlation between SKI expression and histopathological staging of human melanoma. TGF-β induced a rapid and dose-dependent degradation of SKI protein, associated with SMAD3/4 specific transcriptional response and induction of pro-metastatic target genes, partially prevented by pharmacologic blockade of proteasome activity. SKI knockdown in 1205Lu melanoma cells did not alter their invasive capacity or transcriptional responses to TGF-β, and did not allow p21 expression in response to TGF-β or reveal any growth inhibitory activity of TGF-β.. Despite high expression in melanoma cells, the role of SKI in melanoma remains elusive: SKI does not efficiently interfere with the pro-oncogenic activities of TGF-β, unless stabilized by proteasome blockade. Its highly labile nature makes it an unlikely target for therapeutic intervention.

Topics: Animals; Cell Line; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; DNA-Binding Proteins; Gene Knockdown Techniques; Humans; Intracellular Signaling Peptides and Proteins; Leupeptins; Melanoma; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Proteasome Inhibitors; Proto-Oncogene Proteins; RNA Interference; Skin Neoplasms; Smad Proteins; Transcriptional Activation; Transforming Growth Factor beta; Up-Regulation

The NF-kappa B family of transcription factors is an important regulator of genes expressed during inflammatory responses, immunoglobulin (Ig) class switching, cellular differentiation, and apoptosis. Recently, members of the NF-kappaB family, including p65(Rel A), have been implicated in promoting survival of various hematopoeitic neoplasms, including T cell malignancies such as adult T cell leukemia-lymphoma. We investigated the expression of active NF-kappa B p65(Rel A) in cases of mycosis fungoides (MF) and the effect of chemical inhibitors of NF-kappa B on apoptosis in cutaneous T cell lymphoma (CTCL) cell lines. Paraffin-embedded tissues from 23 cutaneous lesions and a single lymph node biopsy from patients diagnosed with MF were evaluated for p65(Rel A) expression by using a monoclonal mouse antibody that detects the activated form of p65(Rel A). Apoptosis after treatment with the NF-kappa B inhibitors gliotoxin, MG132, BAY 11-7082, and BAY 11-7085 was quantitatively measured in the CTCL cell lines HuT-78 and HH by propidium iodide (PI)/cell cycle analysis for detection of a hypodiploid (sub-G(0)) population and by determination of increased Annexin V/7-amino-actinomycin D (7-AAD) expression. Nuclear extracts from CTCL cells before and after chemical inhibition were analyzed for NF-kappa B nuclear DNA-binding activity by electrophoretic mobility shift assay (EMSA) with quantitative densitometry. Nuclear expression of p65(Rel A) before and after treatment with the various inhibitory compounds was measured by immunofluorescence staining in each CTCL cell line. Neoplastic T lymphocytes from 22 of 24 cases of MF showed strong nuclear and cytoplasmic expression of active p65(Rel A). Compared with untreated control cells, a marked increase in apoptosis, a significant decrease in NF-kappa B DNA-binding activity, and a marked decrease in nuclear p65(Rel A) expression were seen in cells from both CTCL cell lines after chemical NF-kappa B inhibition. These data show that the active form of NF-kappa B p65(Rel A) is commonly expressed in neoplastic T lymphocytes in patients with MF. In CTCL cell lines, the significant decrease in nuclear NF-kappa B expression and the marked increase in spontaneous apoptosis caused by chemical NF-kappa B inhibition suggest a critical role for NF-kappa B in the pathogenesis and tumor cell maintenance of CTCLs. HUM PATHOL 31:1482-1490.

Topics: Anti-Infective Agents; Apoptosis; DNA, Neoplasm; Electrophoresis, Polyacrylamide Gel; Flow Cytometry; Gliotoxin; Humans; Immunohistochemistry; Leupeptins; Mycosis Fungoides; NF-kappa B; Nitriles; Skin Neoplasms; Sulfones; T-Lymphocytes; Transcription Factor RelA; Tumor Cells, Cultured