Cerulenin overcomes the protective effects of IL-6, IGF-1 and BMSCs on MM cell growth

(A, B, C) MM.1S and U266 cells were cultured for 48 h with the indicated concentrations [0 μmol/l (□), 3·15 μmol/l () 6·25 μmol/l (), 12·5 μmol/l ()] of Cerulenin, in the presence or absence of IL-6 (1 or 10 ng/ml: A), IGF-1 (10 or 50 ng/ml: B), or BMSC (C). Cell growth was assessed by [H]-thymidine uptake. Cerulenin inhibits MM cell growth and overcomes the stimulating effect of IL-6 (A) or IGF-1 (B) ( < 0·05), and BMSC ( < 0·05) (C). Values represent mean ± SD of quadruplicate cultures.<p><b>Copyright information:</b></p><p>Taken from "Retraction: Fatty acid synthase is a novel therapeutic target in multiple myeloma"</p><p></p><p>British Journal of Haematology 2008;141(5):659-671.</p><p>Published online Jan 2008</p><p>PMCID:PMC2408665.</p><p>Journal compilation © 2008 Blackwell Publishing Ltd</p

Cerulenin induces apoptosis via activation of caspase-independent pathway

(A) MM.1S cells were cultured for 24 h with Cerulenin at the indicated doses. Induction of apoptosis by Cerulenin was determined by Apo2·7 staining and flow-cytometric analysis. (B) MM.1S cells were cultured with Cerulenin (50 μmol/l) for the indicated times (left panel), and preincubated with or without Z-VAD-FMK (50 μmol/l) for 1 h prior to treatment with Cerulenin for 12 h at the indicated doses (right panel). Total cell lysates (20 μg /lane) were subjected to Western blotting using anti-caspase -8, -9, -3, PARP, and α-tubulin Abs. FL, CF indicate the full length and cleaved form, respectively. (C, D) MM.1S cells were treated with the indicated dose of Cerulenin for 24 h, with or without Z-VAD-FMK (25 μmol/l or 50 μmol/l) 1 h pretreatment. Cytotoxicity was determined by MTT assay (C). Values represent mean ± SD of quadruplicate cultures. The percentage of apoptotic cells was determined by flow-cytometric analysis for APO2·7 staining (D). (E) Mitochondrial proteins AIF and Endo G were released into the cytosolic fraction from mitochondria after Cerulenin (50 μmol/l) treatment in MM.1S cells. Total cell lysates (20 μg/lane) were subjected to Western blotting using anti-AIF, Endo G, VDAC and α-tubulin Abs.<p><b>Copyright information:</b></p><p>Taken from "Retraction: Fatty acid synthase is a novel therapeutic target in multiple myeloma"</p><p></p><p>British Journal of Haematology 2008;141(5):659-671.</p><p>Published online Jan 2008</p><p>PMCID:PMC2408665.</p><p>Journal compilation © 2008 Blackwell Publishing Ltd</p

Downregulation of RAS inhibits growth and enhances cytotoxicity of doxorubicin and bortezomib in <i>RAS</i>-mutated MM cell lines.

<p>(A) INA6 cells were transiently transfected with non-targeting or <i>NRAS</i> siRNA, and RPMI-8226 and RPMI-8226 DOX40 cells were transiently transfected with non-targeting or <i>KRAS</i> siRNA. The cell number and viability 48 h later were assessed with trypan blue exclusion. Whole-cell lysates were subjected to western blotting to confirm the downregulation of NRAS and KRAS expression using NRAS, KRAS, HRAS, and β-actin Abs. Data are the mean ± SD of triplicate wells. (B) INA6 and NCI-H929 cells were transiently transfected with non-targeting or <i>NRAS</i> siRNA, and RPMI-8226 and RPMI-8226 DOX40 cells were transiently transfected with non-targeting or <i>KRAS</i> siRNA. After 48 h, whole-cell lysates were subjected to western blotting using p27, cyclin D1, NRAS and β-actin Abs. (C) INA6 cells were transiently transfected with non-targeting or <i>NRAS</i> siRNA and then treated with or without bortezomib (5 nM) for 48 h. RPMI-8226 cells were transiently transfected with non-targeting or <i>KRAS</i> siRNA and then treated with or without doxorubicin (0.1 μM) for 48 h. In each case, cell viability was assessed with the MTT assay of triplicate cultures and expressed as the percentage of the untreated control. Data are the mean ± SD. (D) RPMI-8226 DOX40 cells were transiently transfected with non-targeting or <i>KRAS</i> siRNA and then treated with or without doxorubicin (1 μM) for 24 h. Cell viability was assessed with the MTT assay of triplicate cultures and expressed as the percentage of the untreated control. Data are the mean ± SD.</p

TAS-116 effects on cell viability and RAS-RAF-MEK-ERK signaling in <i>RAS</i>-mutated MM cell lines.

<p>(A) NCI-H929, INA6, MM.1S, and RPMI-8226 MM cell lines were cultured with TAS-116 (0–5 μM) for 24, 48, or 72 h. In each case, cell viability was assessed with the MTT assay of triplicate cultures and expressed as the percentage of the untreated control. Data are the mean ± SD. (B) NCI-H929 and RPMI-8226 cells were treated with the indicated doses of TAS-116 for 24 h. Whole-cell lysates were subjected to western blotting using p-B-Raf, B-Raf, p-C-Raf, C-Raf, p-MEK1/2, MEK1/2, p-ERK, ERK, p-Akt (S473), Akt, PARP, and β-actin Abs. FL, full-length; CF, cleaved form.</p

RAS pathway inhibitors induce cytotoxicity and apoptosis in <i>RAS</i>-mutated MM cell lines.

<p>(A) NCI-H929, INA6, MM.1S, and RPMI-8226 MM cell lines were cultured with tipifarnib (0–20 μM), dabrafenib (0–20 μM), or AZD6244 (0–20 μM) for 72 h. In each case, cell viability was assessed with the MTT assay of triplicate cultures and expressed as the percentage of the untreated control. Data are the mean ± SD. (B) NCI-H929 and RPMI-8226 cells were treated with TAS-116 (3 μM), tipifarnib (10 μM), dabrafenib (20 μM), or AZD6244 (20 μM) for 24 h. Whole-cell lysates were subjected to western blotting using NRAS, KRAS, p-B-Raf, B-Raf, p-C-Raf, C-Raf, p-MEK1/2, MEK1/2, p-ERK, ERK, p-Akt (S473), Akt, PARP, and β-actin Abs. FL, full-length; CF, cleaved form. (C) NCI-H929, INA6, MM.1S, and RPMI-8226 cells were treated with tipifarnib (0–20 μM), dabrafenib (0–20 μM), or AZD6244 (0–20 μM) for 48 h. Apoptotic cells were analyzed with flow cytometry using annexin V/PI staining. Each treatment was tested in triplicate wells, and apoptosis was assessed as the percentage of annexin V-positive cells.</p

Combination of TAS-116 plus tipifarnib, dabrafenib, or AZD6244 blocks the growth stimulatory effect of the bone marrow microenvironment.

<p>(A) NCI-H929, INA6, MM.1S, and RPMI-8226 cells were treated with TAS-116 (1 μM) either alone or in combination with tipifarnib (NCI-H929: 2 μM, INA6: 0.5 μM, MM.1S: 2 μM, RPMI-8226: 2 μM), dabrafenib (NCI-H929: 10 μM, INA6: 2 μM, MM.1S: 10 μM, RPMI-8226: 10 μM), or AZD6244 (NCI-H929: 10 μM, INA6: 2 μM, MM.1S: 20 μM, RPMI-8226: 20 μM) for 48 h. Apoptotic cells were analyzed with flow cytometry using annexin V/PI staining. Each treatment was tested in triplicate wells, and apoptosis was assessed as the percentage of annexin V-positive cells. TAS, TAS-116; Tipi, tipifarnib; Dabra, dabrafenib; AZD, AZD6244. (B) MM.1S and NCI-H929 cells were cultured with TAS-116 (2 μM), AZD6244 (20 μM), or TAS-116 plus AZD6244 for 24 h in the presence or absence of BMSC supernatant. Whole-cell lysates were subjected to western blotting using PARP, caspase 3, and β-actin Abs. FL, full-length; CF, cleaved form; SN, supernatant. (C) MM.1S cells were cultured with TAS-116 (1 μM), AZD6244 (20 μM), or TAS-116 plus AZD6244; and NCI-H929 cells were cultured with TAS-116 (1 μM), AZD6244 (10 μM), or TAS-116 plus AZD6244 for 48 h in the presence or absence of BMSC supernatant. Apoptotic cells were analyzed with flow cytometry using annexin V/PI staining. Each treatment was tested in triplicate wells, and apoptosis was assessed as the percentage of annexin V-positive cells. TAS, TAS-116; AZD, AZD6244; SN, supernatant (*: <i>P</i> < 0.05; **: <i>P</i> < 0.01).</p

TAS-116 induces synergistic cytotoxicity with dabrafenib in the BRAF-mutated U266 MM cell line.

<p>U266 cells were cultured with TAS-116 (0–5 μM) (A) or dabrafenib (0–5 μM) (B) for 24, 48, or 72 h. Cell viability was assessed with the MTT assay of triplicate cultures and expressed as the percentage of the untreated control. Data are the mean ± SD. (C) U266 cells were treated with the indicated concentrations of TAS-116, dabrafenib, or TAS-116 plus dabrafenib for 48 h, and then the viability was analyzed with the MTT assay. Isobologram analysis shows the synergistic cytotoxic effect of TAS-116 and dabrafenib. The graph (left) is derived from the values given in the table (right). The numbers 1–4 in the graph correspond to the combinations shown in the table. CI values < 1.0 indicate synergism; CI = 1.0 indicates an additive effect; and CI > 1.0 indicates antagonism. (D) U266 cells were treated with TAS-116 (0.5 μM), dabrafenib (1 μM), or TAS-116 plus dabrafenib for 48 h. Apoptotic cells were analyzed with flow cytometry using annexin V/PI staining. Each treatment was tested in triplicate wells, and apoptosis was assessed as the percentage of annexin V-positive cells. (E) U266 cells were treated with TAS-116 (1 μM), dabrafenib (2 μM), or TAS-116 plus dabrafenib for 24 h. Whole-cell lysates were subjected to western blotting using p-B-Raf, B-Raf, p-C-Raf, C-Raf, p-MEK1/2, MEK1/2, p-ERK, ERK, p-Akt (S473), Akt, PARP, caspase 3, and β-actin Abs. FL, full-length; CF, cleaved form.</p