Obatoclax

Manufactured by Selleck Chemicals

Sourced in United States, Germany

Obatoclax is a laboratory compound that functions as a pan-Bcl-2 inhibitor. It is used in research settings to study cellular processes related to apoptosis and cell survival.

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Obatoclax mesylate (8 citations) Obatoclax (GX15-070) (2 citations)

28 protocols using obatoclax

Fibroblast-Melanoma Crosstalk Modulation

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Fully confluent fibroblasts plated in T162 flasks were treated overnight with either fresh media or fresh media supplemented with 100 ng/ml human recombinant IL-1β (PeproTech). The next morning, the cells were incubated in fresh media for 5 h, which was subsequently added to melanoma cells plated in either 6- or 12-well plates for 48 h with various inhibitors. The reagents used for these experiments were 1% DMSO (Sigma-Aldrich), 1 µM PLX4032 (Selleck Chemicals), 1 µM RAF265 (Selleck Chemicals), or 0.5 µM both PLX4032 and selumetinib (Selleck Chemicals). When the duotherapy treatment (PLX4032 and selumetinib) was also used in combination with either MK-2206, SB 225002, Bay 11-7082, or obatoclax, the concentration of each drug used was: 1 µM MK-2206 (Selleck Chemicals), 0.5 µM SB 225002 (Alfa Aesar), 0.2 µM Bay 11-7082 (Sigma-Aldrich), and 0.2 µM obatoclax (Selleck Chemicals). These concentrations were also used when these inhibitors were used as single agents. For each drug treatment, melanoma cells were cultured in nonconditioned media, conditioned media taken from unstimulated fibroblasts, or conditioned media taken from fibroblasts previously stimulated with IL-1β. Then, cell survival was assayed by crystal violet staining (outlined in the next section).

Young H.L., Rowling E.J., Bugatti M., Giurisato E., Luheshi N., Arozarena I., Acosta J.C., Kamarashev J., Frederick D.T., Cooper Z.A., Reuben A., Gil J., Flaherty K.T., Wargo J.A., Vermi W., Smith M.P., Wellbrock C, & Hurlstone A. (2017). An adaptive signaling network in melanoma inflammatory niches confers tolerance to MAPK signaling inhibition. The Journal of Experimental Medicine, 214(6), 1691-1710.

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Cell Viability Assay with Apoptosis Inducers

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Cells were plated and incubated at 37°C and 5% CO₂ in DMEM containing 2% FBS. After 20 h, each compound was added at the indicated concentration and for the indicated times. Compounds used in vitro experiments were purchased from Selleckchem (Houston, TX): obatoclax; ABT-737; staurosporine; zVAD-FMK; zDEVD-FMK; and dinaciclib. For the in vivo test, ABT-737 was purchased from AstaTech Inc. (Bristol, PA).

Kim Y.J., Witwit H., Cubitt B, & de la Torre J.C. (2021). Inhibitors of Anti-apoptotic Bcl-2 Family Proteins Exhibit Potent and Broad-Spectrum Anti-mammarenavirus Activity via Cell Cycle Arrest at G0/G1 Phase. Journal of Virology, 95(24), e01399-21.

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Preparation and Storage of Reagents

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Obatoclax was purchased from Selleck Chemicals (Houston, TX, USA), prepared as a 20 mM stock solution in dimethyl sulfoxide (DMSO) (Sigma-Aldrich; St. Louis, MO, USA), and stored at 4°C until use. MG132 was obtained from AdooQ BioScience (Irvine, CA, USA). Polybrene was obtained from Sigma-Aldrich (St. Louis, MO, USA).

Or C.H., Huang C.W., Chang C.C., Lai Y.C., Chen Y.J, & Chang C.C. (2020). Obatoclax, a Pan-BCL-2 Inhibitor, Downregulates Survivin to Induce Apoptosis in Human Colorectal Carcinoma Cells Via Suppressing WNT/β-catenin Signaling. International Journal of Molecular Sciences, 21(5), 1773.

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Mitochondrial Calcium Uptake and Swelling Assay

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Two mg of isolated mitochondria were suspended in a total volume of 1 ml consisting KCl buffer, 1 mM Malic acid (Sigma-Aldrich), 7 mM Pyruvate (Sigma-Aldrich), and 50 nM Calcium Green 5N (Invitrogen) in a quartz cuvette, which was placed inside the fluorimeter (PTI QuantaMaster 800, Horiba Scientific). Calcium uptake was measured by fluorescent emission of Calcium Green 5N. Simultaneously, mitochondrial swelling was measured by transmittance light. Each BH3 mimetic was used at concentrations: 0.5, 1, 10, 50, 100 nM, and 1 µM. Final BH3 mimetics concentrations used throughout the study were 200 nM ABT-199 (Selleckchem.com, S8048), 100 nM A-1331852 (abcam, ab218112), 100 nM ABT-737 (EMD Millipore Sigma, 197333), 10 nM S63845 (Selleckchem.com, S8383), 10 nM Obatoclax (Selleckchem, GX15-070). For some experiments ADP (300 uM) (Sigma-Aldrich, A2754) and/or CsA (2 uM) (Sigma-Aldrich, 30024) were used to desensitize the MPTP. CaCl₂ (20 μM, 40 μM, and 80 µM) (Sigma-Adrich, C4901) was added into this system in succession until MPTP opening occurred, indicated by an upturn in Calcium Green 5N fluorescence, or when mitochondria were saturated with Ca²⁺ and were no longer able to take up further additions of CaCl₂, indicated by a stair-stacking in the Ca²⁺ uptake graphs.

Patel P., Mendoza A., Robichaux D.J., Wang M.C., Wehrens X.H, & Karch J. (2021). Inhibition of the Anti-Apoptotic Bcl-2 Family by BH3 Mimetics Sensitize the Mitochondrial Permeability Transition Pore Through Bax and Bak. Frontiers in Cell and Developmental Biology, 9, 765973.

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Pharmacological Evaluation of LCL161

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LCL161 was synthesized and provided by Novartis (Basel, Switzerland) under a Material Transfer Agreement (MTA). Obatoclax was purchased from Selleckchem (Houston, TX, USA). Pancaspase inhibitor (Q-VD-OPH), caspase 9 inhibitor (Ac-LEHD-CMK) and caspase 8 inhibitor (Z-IETD-FMK) were purchased from EMD Millipore (Billerica, MA, USA). Akti and chloroquine were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Ramakrishnan V., Gomez M., Prasad V., Kimlinger T., Painuly U., Mukhopadhyay B., Haug J., Bi L., Rajkumar S.V, & Kumar S. (2016). Smac mimetic LCL161 overcomes protective ER stress induced by obatoclax, synergistically causing cell death in multiple myeloma. Oncotarget, 7(35), 56253-56265.

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Pharmacological Agents in Autophagy Research

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7BIO came from Enzo Life Sciences (Farmingdale, NY, USA), 3-methyladenine (3-MA) and N-(2-Quinolyl)valyl-aspartyl-(2,6-difluorophenoxy)methyl ketone (Q-VD-OPh) were provided by Millipore/Calbiochem (MA, USA), obatoclax and staurosporine were from Selleck Chemicals (Houston, TX, USA). All compounds were dissolved in DMSO to 5–10 mM, stored in aliquots at −20 °C and further diluted in the appropriate medium. Microtubule-associated protein 1A/1B-light chain B (LC3B) antibodies were from Cell Signaling Technology (Danvers, MA, USA).

Broecker-Preuss M., Becher-Boveleth N., Gall S., Rehmann K., Schenke S, & Mann K. (2015). Induction of atypical cell death in thyroid carcinoma cells by the indirubin derivative 7-bromoindirubin-3′-oxime (7BIO). Cancer Cell International, 15, 97.

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Evaluating Drug Effects on Human PASMCs

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Human PASMCs purchased from ScienCell Research Laboratories (Carlsbad, CA, USA) were cultured in accordance with the manufacturer’s instructions in 5% CO₂ at 37 °C. Passages 3–6 were used. For siRNA knockdown, cells were transfected with siRNA Transfection Reagent and test siRNA or control siRNA with a scrambled sequence (Santa Cruz Biotechnology, Dallas, TX, USA) and used for experiments 2 days later. Cells were treated with ABT-263 (Navitoclax), ABT-199 (Venetoclax), ABT-737, or Obatoclax (Selleckchem, Houston, TX, USA) dissolved in dimethyl sulfoxide (DMSO). Preliminary dose–response experiments determined the dose that produced an approximately 50% decrease in the cell number. The same dose (1 µM) was tested for all the drugs for comparison. An equal amount of DMSO (0.1%) was included as a control. The number of viable cells was determined by counting on a hemocytometer or by using a Cell Counting Kit-8 (Dojindo Molecular Technologies, Rockville, MD, USA).

Rybka V., Suzuki Y.J, & Shults N.V. (2018). Effects of Bcl-2/Bcl-xL Inhibitors on Pulmonary Artery Smooth Muscle Cells. Antioxidants, 7(11), 150.

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Evaluation of ABT-737 and Obatoclax

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ABT-737 and obatoclax were from Selleck Chemicals (Houston, TX, USA). They were stored in 10 mM aliquots in DMSO at −20 °C and further diluted in the appropriate medium. Chemotherapeutic agents were from Merck-Millipore (Darmstadt, Germany).Antibodies were from Cell Signaling Technology (Danvers, MA, USA).

Broecker-Preuss M., Becher-Boveleth N., Müller S, & Mann K. (2016). The BH3 mimetic drug ABT-737 induces apoptosis and acts synergistically with chemotherapeutic drugs in thyroid carcinoma cells. Cancer Cell International, 16, 27.

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Evaluating Combination Therapy Cytotoxicity

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GSK1120212 and Obatoclax (Selleck Chemicals, Houston, TX) and doxorubicin (Sigma Aldrich) were added 24h after plating, in sextuplicate. After 48–72h, viability was assessed using the Wst-1 assay (Clontech, Mountain View, CA) and IC50s were determined using Prism software. Alternatively, cells were trypsinized and counted using a Z2 Coulter counter (Beckman Coulter, Indianapolis IN).

Champa D., Russo M.A., Liao X.H., Refetoff S., Ghossein R.A, & Di Cristofano A. (2014). Obatoclax overcomes resistance to cell death in aggressive thyroid carcinomas by countering Bcl2a1 and Mcl1 overexpression. Endocrine-related cancer, 21(5), 755-767.

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Compound Screening for Cellular Assays

Cited 1 time

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ABT-199, ABT-263, ABT-737, WEHI-539, A-1331852, A-1155463, obatoclax, and gemcitabine were purchased from Selleck Chemicals (Houston, TX, USA), Saliphenylhalamide (SaliPhe) was synthesized as described previously [24 (link)]. 10 mM solutions of the compounds were prepared in 100% dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO, USA) and stored at −80 °C. Lyophilized lipopolysaccharide (LPS, 10 mg/mL) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Plasmid pEGFP was from Clontech (Mountain View, CA, USA) (cat #: HLP309). Hoechst 33342 (20 mM solution; cat #: 62249) and ATP (10 mM solution; cat #: PV3227) were from Thermo Fisher Scientific (Waltham, MA, USA). Genomic RNA was isolated from influenza A/WSN/1933 strain as described previously [11 (link)].

Bulanova D., Ianevski A., Bugai A., Akimov Y., Kuivanen S., Paavilainen H., Kakkola L., Nandania J., Turunen L., Ohman T., Ala-Hongisto H., Pesonen H.M., Kuisma M.S., Honkimaa A., Walton E.L., Oksenych V., Lorey M.B., Guschin D., Shim J., Kim J., Than T.T., Chang S.Y., Hukkanen V., Kulesskiy E., Marjomaki V.S., Julkunen I., Nyman T.A., Matikainen S., Saarela J.S., Sane F., Hober D., Gabriel G., De Brabander J.K., Martikainen M., Windisch M.P., Min J.Y., Bruzzone R., Aittokallio T., Vähä-Koskela M., Vapalahti O., Pulk A., Velagapudi V, & Kainov D.E. (2017). Antiviral Properties of Chemical Inhibitors of Cellular Anti-Apoptotic Bcl-2 Proteins. Viruses, 9(10), 271.

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