concanamycin-a and Multiple-Myeloma

The specific 26S proteasome inhibitor bortezomib (BZ) potently induces autophagy, endoplasmic reticulum (ER) stress and apoptosis in multiple myeloma (MM) cell lines (U266, IM-9 and RPMI8226). The macrolide antibiotics including concanamycin A, erythromycin (EM), clarithromycin (CAM) and azithromycin (AZM) all blocked autophagy flux, as assessed by intracellular accumulation of LC3B-II and p62. Combined treatment of BZ and CAM or AZM enhanced cytotoxicity in MM cell lines, although treatment with either CAM or AZM alone exhibited almost no cytotoxicity. This combination also substantially enhanced aggresome formation, intracellular ubiquitinated proteins and induced the proapoptotic transcription factor CHOP (CADD153). Expression levels of the proapoptotic genes transcriptionally regulated by CHOP (BIM, BAX, DR5 and TRB3) were all enhanced by combined treatment with BZ plus CAM, compared with treatment with each reagent alone. Like the MM cell lines, the CHOP+/+ murine embryonic fibroblast (MEF) cell line exhibited enhanced cytotoxicity and upregulation of CHOP and its transcriptional targets with a combination of BZ and one of the macrolides. In contrast, CHOP-/- MEF cells exhibited resistance against BZ and almost completely canceled enhanced cytotoxicity with a combination of BZ and a macrolide. These data suggest that ER stress-mediated CHOP induction is involved in pronounced cytotoxicity. Simultaneously targeting two major intracellular protein degradation systems such as the ubiquitin-proteasome system by BZ and the autophagy-lysosome system by a macrolide antibiotic enhances ER stress-mediated apoptosis in MM cells. This result suggests the therapeutic possibility of using a macrolide antibiotic with a proteasome inhibitor for MM therapy.

Topics: Animals; Apoptosis; Autophagy; Azithromycin; Boronic Acids; Bortezomib; Cell Line, Tumor; Clarithromycin; Endoplasmic Reticulum Stress; Erythromycin; Fibroblasts; Gene Expression Regulation, Neoplastic; Humans; Macrolides; Mice; Multiple Myeloma; Pyrazines; Transcription Factor CHOP

Because WT1 is a universal tumor antigen, we examined the sensitivity of myeloma cells to WT1-specific cytotoxic T lymphocyte (CTL)-mediated cytotoxicity.. WT1 expression in hematologic malignant cells was examined by quantitative reverse transcription-polymerase chain reaction. The cytotoxicity of a WT1-specific CTL clone against hematologic malignant cells, including myeloma cells, was examined by standard chromium-51 release assays. The extent of membrane damage induced by purified perforin was examined. Induction of WT1-specific CTLs from the patients with multiple myeloma (MM) was attempted, and we examined their function against myeloma cells.. The expression levels of WT1 mRNA in myeloma and lymphoma cells were significantly lower than that in acute leukemia cells. Although the WT1 expression levels in myeloma and lymphoma cells were almost same, only myeloma cells were lysed efficiently by WT1-specific CTLs in a HLA-restricted manner. The amounts of interferon-gamma produced by WT1-specific CTLs in response to stimulation with myeloma cells and with lymphoma cells were almost the same, suggesting that WT1 protein is processed and expressed in the context of HLA class I molecules similarly on both myeloma and lymphoma cells. The extent of membrane damage induced by purified perforin appeared to be significantly higher in myeloma cells than in lymphoma cells. WT1-specific CTLs appeared to be present in patients with MM.. The present study has shown that susceptibility of membranes to perforin is an important factor determining the sensitivity of target cells to CTL-mediated cytotoxicity and that WT1 is an ideal target antigen for cellular immunotherapy of MM.

Topics: Antigens, Neoplasm; Bone Marrow Cells; Calcium; Cell Line; Cell Line, Tumor; Chromium; Cytoplasmic Granules; Dose-Response Relationship, Drug; Egtazic Acid; Enzyme-Linked Immunosorbent Assay; Exocytosis; Flow Cytometry; Genes, MHC Class I; Humans; Immunotherapy; Interferon-gamma; Lymphoma; Macrolides; Membrane Glycoproteins; Multiple Myeloma; Peptides; Perforin; Pore Forming Cytotoxic Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; T-Lymphocytes, Cytotoxic; Treatment Outcome; WT1 Proteins

Accumulating evidence indicates that a graft-vs.-myeloma effect (GVM) and its associated clinical remission of the disease can be induced by donor lymphocyte infusion in myeloma patients who have relapsed after allogeneic bone marrow transplantation. Although it is believed that GVM is induced by allospecific T cells, T-cell subsets and the mechanisms involved in the killing of myeloma cells by donor T cells have not been studied. In this study, we generated allospecific cytotoxic T lymphocyte (CTL) lines against three different myeloma cell lines, ARK, ARP-1 and U266, from unmatched healthy donors and examined their cytotoxicity against the target cells. Our results demonstrate that the allospecific CTLs efficiently lysed myeloma cells. The observed cytotoxicity was mediated mainly by CD8+ T cells and inhibited by MHC class I-blocking antibody. Furthermore, the CTLs lysed the target cells via the perforin-mediated pathway, as concanamycin A, but not brefeldin A (the selective inhibitors for perforin- or Fas-mediated pathways respectively) or tumour necrosis factor-alpha (TNF-alpha)-blocking antibody, abrogated the cytolytic activity of the cells. These CTLs expressed and produced predominantly TNF-alpha and interferon-gamma (IFN-gamma), indicating that they belong to the type 1 T-cell subsets. Taken together, these results indicate that CD8+ allospecific T cells may be responsible for mediating GVM and that the granule-mediated lysis of target cells is the major pathway in the CD8+ T-cell response against myeloma cells.

Topics: Adoptive Transfer; Anti-Bacterial Agents; Antibodies, Monoclonal; Brefeldin A; CD8-Positive T-Lymphocytes; Cell Degranulation; Cytotoxicity Tests, Immunologic; Enzyme Inhibitors; Flow Cytometry; Graft vs Tumor Effect; Histocompatibility Antigens Class I; Histocompatibility Antigens Class II; Humans; Lymphocyte Culture Test, Mixed; Macrolides; Multiple Myeloma; Protein Synthesis Inhibitors; Proton-Translocating ATPases; T-Lymphocytes, Cytotoxic; Transplantation, Homologous; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha