1. The BH3-mimetic GX synergizes with bortezomib in mantle cell lymphoma by enhancing Noxa-mediated activation of Bak
by Patricia Pérez-Galán, Gaël Roué, Neus Villamor, Elias Campo, and Dolors Colomer
Blood
Volume 109(10):
May 15, 2007
©2007 by American Society of Hematology

2. Cytotoxic effect of GX15-070 apoptosis in MCL cells and nomal lymphocytes.
Cytotoxic effect of GX apoptosis in MCL cells and nomal lymphocytes. (A) Dose response of GX in MCL cell lines at 20 hours (left panel) and 48 hours (right panel) analyzed by Annexin V–APC staining. (B) Cytofluorimetric analysis of Annexin V–positive cells after treatment of primary MCL cells with the indicated doses of GX for 20 hours. (C) PBMCs from healthy donors and CD19+ cells from reactive tonsils were incubated with the indicated doses of GX for 20 hours, and viability was analyzed with CD3-FITC/CD19-PE/Annexin V–APC. Results represent the mean ± SD of 3 independent experiments.
Patricia Pérez-Galán et al. Blood 2007;109:
©2007 by American Society of Hematology

3. Protein expression of Bcl-2 familly members in MCL cells.
Protein expression of Bcl-2 familly members in MCL cells. (A) Total protein extracts (50 μg) from 5 MCL cell lines were analyzed by Western blotting. Membranes were probed for Mcl-1, Bcl-XL, and Bcl-2 expression using suitable antibodies and α-tubulin was used to normalize protein loading. Western blot images are representative results from 3 independent experiments. (B) Relative protein quantification of Mcl-1, Bcl-XL, and Bcl-2 was performed using Image Gauge Fujifilm software.
Patricia Pérez-Galán et al. Blood 2007;109:
©2007 by American Society of Hematology

4. GX15-070 displaces Bak from Mcl-1 and Bcl-XL.
GX displaces Bak from Mcl-1 and Bcl-XL. MCL cell lines were treated for 5 hours with 5 μM (Jeko and Granta-519) or 0.5 μM (UPN-1) GX Mcl-1 and Bcl-XL immunoprecipitations were performed as described in “Patients, materials, and methods.” Immunoprecipitated (IP; bound) and nonimmunoprecipitated (unbound) fractions were analyzed by Western blotting for Bak, Mcl-1 and Bcl-XL proteins. Western blot (WB) images are representative results from 3 independent experiments.
Patricia Pérez-Galán et al. Blood 2007;109:
©2007 by American Society of Hematology

5. GX15-070 activates the intrinsic apoptotic pathway in a caspase-independent manner.
GX activates the intrinsic apoptotic pathway in a caspase-independent manner. UPN-1 cells were treated with 0.5 μM GX for 16 hours in the presence or absence of z-VAD-fmk (50 μM). Bax/Bak conformational changes, caspase-3 activation, loss of ΔΨm, and PS exposure were analyzed as described in “Patients, materials, and methods.” The percentages inside each chart refer to the population in black. These experiments have been performed twice with similar results and therefore 1 representative experiment is shown.
Patricia Pérez-Galán et al. Blood 2007;109:
©2007 by American Society of Hematology

6. Synergistic effect of GX15-070 and bortezomib in MCL cell lines.
Synergistic effect of GX and bortezomib in MCL cell lines. (A) Cells from 3 MCL cell lines were cotreated with 5 nM (UPN-1) or 10 nM (Jeko and Granta-519) bortezomib and increasing doses of GX (0.1-5 μM) for 18 hours. Cytotoxicity was evaluated by cytofluorimetric analysis of Annexin V–APC. *Doses needed to observe a synergistic effect between bortezomib and GX Results represent the mean ± SD of 3 independent experiments. (B) Mcl-1, Bak, and Noxa expression was analyzed by Western blot in 50 μg of total protein extracts from UPN-1, Jeko, and Granta-519 cells cotreated with GX and bortezomib for 18 hours. α-tubulin was also probed as an equal loading control. Western blot images are representative results from 3 independent experiments.
Patricia Pérez-Galán et al. Blood 2007;109:
©2007 by American Society of Hematology

7. GX15-070 and bortezomib combination enhances Bak-dependent apoptotic signaling in MCL cell lines.
GX and bortezomib combination enhances Bak-dependent apoptotic signaling in MCL cell lines. (A) Jeko cells were treated with 0.5 μM GX and/or 10 nM bortezomib for 5 hours. Mcl-1 immunoprecipitation was performed as described in “Patients, materials, and methods,” analyzing Mcl-1–bound and –unbound fractions by Western blotting for Mcl-1, Bak, and Noxa proteins. Western blot images are representative results from 3 independent experiments. (B) Jeko cells were treated with 0.5 μM GX and/or 10 nM bortezomib for 18 hours. Bax/Bak conformational changes, caspase-3 activation, loss of ΔΨm, and PS exposure were analyzed as described in “Patients, materials, and methods.” The percentage inside each chart refers to the population in black. These experiments have been performed twice with similar results, and therefore 1 representative experiment is shown. (C) NOXA siRNA and nonsilencing siRNA were introduced in Jeko cells by electroporation as described in “Patients, materials, and methods.” Total RNA was isolated 6 hours after transfection. NOXA mRNA levels were determined by quantitative RT-PCR (Taqman technology) using GUS as a housekeeping gene. The results showed are the mean ± SD of 2 different experiments. (D) Jeko cells transfected with nonsilencing siRNA (ns-siRNA) and with NOXA siRNA (NOXA-siRNA) were treated with 0.5 μM GX and/or 10 nM bortezomib for 18 hours. Loss of ΔΨm and Bak conformational change were analyzed as described in “Patients, materials, and methods.” The percentage inside each chart refers to the population in black.
Patricia Pérez-Galán et al. Blood 2007;109:
©2007 by American Society of Hematology

8. Synergistic interaction between GX15-070 and bortezomib in MCL primary cells.
Synergistic interaction between GX and bortezomib in MCL primary cells. (A) Primary cells from 4 representative patients with MCL were treated with 5 or 10 nM bortezomib and increasing doses of GX (0.1-1 μM) for 18 hours. Cytotoxicity was evaluated by cytofluorimetric analysis of Annexin V–APC. Linked arrows in patients no. 4 and no. 7 indicate equivalent cytotoxicities. Single arrows in patients no. 2 and no. 9 indicate the sensitizing effect of GX in these bortezomib-resistant patients. Results represent the mean ± SD of 3 independent experiments (B) Mcl-1, Bak, and Noxa expressions were analyzed by Western blot in 50 μg of total protein extracts from cells of patient no. 2 cotreated with 1 μM GX and/or 5 nM bortezomib for 18 hours. α-tubulin was also probed as an equal loading control. (C) Cells from patient no. 2 were treated with 1 μM GX and/or 5 nM bortezomib for 5 hours. Mcl-1, Bak, and Noxa proteins were analyzed in Mcl-1–immunoprecipitated and nonimmunoprecipitated fractions as described in “Patients, materials, and methods.” Western blot images are representative results from 3 independent experiments.
Patricia Pérez-Galán et al. Blood 2007;109:
©2007 by American Society of Hematology