Preclinical studies of Apogossypolone: a new nonpeptidic pan small-molecule inhibitor of Bcl-2, Bcl-XL and Mcl-1 proteins in Follicular Small Cleaved Cell Lymphoma model
© Arnold et al; licensee BioMed Central Ltd. 2008
Received: 28 November 2007
Accepted: 14 February 2008
Published: 14 February 2008
Elevated expression of anti-apoptotic Bcl-2 family proteins have been linked to a poor survival rate of patients with Follicular Lymphoma (FL). This prompted us to evaluate a very potent non-peptidic Small-Molecule Inhibitor (SMI) targeting Bcl-2 family proteins, Apogossypolone (ApoG2) using follicular small cleaved cell lymphoma cell line (WSU-FSCCL) and cell isolated from lymphoma patients. ApoG2 inhibited the growth of WSU-FSCCL significantly with a 50% growth inhibition of cells (IC50) of 109 nM and decreased cell number of fresh lymphoma cells. ApoG2 activated caspases-9, -3, and -8, and the cleavage of Poly (ADP-ribose) polymerase (PARP) and Apoptosis Inducing Factor (AIF). In the WSU-FSCCL-SCID xenograft model, ApoG2 showed a significant anti-lymphoma effect, with %ILS of 84% in the intravenous and 63% in intraperitoneal treated mice. These studies suggest that ApoG2 can be an effective therapeutic agent against FL.
Follicular Lymphoma (FL) is fifth leading diagnosed cancer estimated with over 63,000 new patients in 2007 within the United States. FL is the most common type of low grade lymphoma and the second most common subtype of lymphoma worldwide. The natural history of FL has not changed over the last 3 decades with median survival ranging from 7–10 years; the disease is considered incurable using various anti-cancer agents [1–3]. Current treatment strategies are aimed at producing remissions, preserving vital organ function and enhancing patients' quality of life . Phase II trials of CHOP followed by Tositumomab/Iodine I-131 demonstrated progression free survival of 67% of patients . Phase III trials of Rituximab shows improved progression free survival in relapsed/resistant FL and enhanced remission induction when used with CHOP , with these improvements in the treatment, to date there is not a cure except for a limited number of patients who present with localized disease. Therefore, developing targeted therapy to proteins such as Bcl-2 that prevent death of lymphoma cells is advantageous.
Bcl-2 plays an important role in the lymphomagenesis of FL. Bcl-2 was originally discovered in FL as a proto-oncogene involved in the t(14;18) chromosomal translocation [7–9]. This genetic event is found in more than 85% of FL. It has been shown that transfection of Bcl-2 into B-cell lines could increase cell viability and decrease apoptosis of lymphoma cells and additionally, confers resistance of these cells to chemotherapeutic drugs . Thus, interfering with Bcl-2 function is hypothesized to lead to apoptosis of lymphoma cells. Therefore, Bcl-2 is a rational therapeutic target because of its role in regulating the apoptotic pathway.
Structural analysis of the binding clefts in Bcl-2 and Bcl-XL using X-ray crystallography and NMR spectroscopy showed conserved similarity in the BH1, BH2, and BH3 domains. These domains create a hydrophobic surface pocket that may represent a binding site for pro-apoptotic members of the Bcl-2 family, such as Bax. The heterodimerization of Bcl-2 family proteins is believed to be pivotal to the anti-apoptotic function of these proteins. Furthermore, site-specific mutagenesis of BH1 and BH2 domains completely abrogrates the anti-apoptotic activity of these proteins [11–13]. These studies suggest that this region could be a promising target for the use of SMIs to induce apoptosis.
Previous studies in this lab using the SMI (-)-gossypol has shown significant growth inhibition in vitro and tumor growth inhibition in vivo in a diffuse large cell lymphoma model . With a structural based screening approach, TW-37 a more potent SMI to Bcl-2, was discovered . Subsequently, we have confirmed that TW-37 has anti-lymphoma properties in our diffuse large cell lymphoma model . More recently, we developed a new non-peptidic SMI, ApoG2, which binds like the previous SMIs but with a considerably lower Ki. ApoG2 is a derivative of (-)-gossypol that binds to the Bcl-2 family of proteins in the low nanomolar range with a Ki of 35 and 25 nmol/L for Bcl-2 and Mcl-1, respectively and a Ki of 660 nmol/L for Bcl-XL . Therefore, the new SMI, ApoG2, could in theory inhibit the anti-apoptotic function of Bcl-2, Bcl-XL and Mcl-1 more efficiently and induce apoptosis in FL cells. In this study, we evaluated the effect of ApoG2 on growth of malignant lymphoid cells in vitro, its ability to induce apoptosis as well as its anti-lymphoma activity in vivo using a SCID mouse xenograft model of FSCCL.
Materials and methods
Cell Culture and Reagents
The origin of human FL B cell line WSU-FSCCL was described previously . The cell line was maintained in RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum (FBS), 1% L-glutamine, 100 U/ml penicillin G and 100 μg/ml streptomycin. Cells were incubated at 37°C in a humidified incubator with 5% CO2. Fresh samples from patients with pre-B-acute lymphoblastic leukemia (Pre-B-ALL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and chronic lymphocytic leukemia (CLL) were isolated using Lymphoprep (Axis-Shield, Oslo, Norway). ApoG2 was synthesized by modifying (-)-gossypol's two aldehyde groups and prepared at a stock concentration of 1 mM.
Western Blot Analysis
Proteins obtained from extracts were resolved using 12% SDS-PAGE and transferred to Hybond C-extra membranes (Amersham Life Science, Arlington Heights, IL). Membranes were blocked with 5% milk in Tris Buffer Saline containing 0.05% Tween 20 (TBST) for 1 h at 25°C and then incubated with unlabeled primary antibodies in 2% Bovine Serum Albumin in TBST (1:1000 dilutions in BSA-TBST) overnight at 4°C. Following incubation, membranes were washed in TBST and incubated with corresponding horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA; 1:5000 dilution in 5% milk-TBST) for 1 h at 25°C and then washed before proteins were visualized using an ECL assay (Amersham Pharmacia Biotech, Inc., Piscataway, NJ). Primary antibodies specific for Bcl-2, Bcl-XL, Bax, and Mcl-1(Santa Cruz Biotechnology, Santa Cruz, CA) were used. Primary antibodies specific for caspase-3, -9, PARP and AIF were obtained from Cell Signaling, (Danvers, MA). Protein concentrations were determined using the Micro BCA protein assay (Pierce Chemical Company, Rockford, IL). Quantitation of bands: Values are fold increase of intensity over control based on percentage Integrated Density Value (IDV), using AlphaEaseFC (San Leandro, CA).
Detection of apoptosis
ApoG2 effectiveness to induce apoptosis was quantified using two DNA intercalating dyes. 3,6-bis [Dimethylamino] acridinium chloride hemi-[zinc chloride] (termed "acridine orange" or AO) and 3,8-diamino-5-ethyl-6-phenyl-phenanthridine bromide (termed "ethidium bromide" or "EB") were used (Invitrogen, Carlsbad, California). AO stains DNA bright green allowing visualization of the nuclear chromatin pattern. EB stains DNA orange but is excluded by viable cells. Dual staining allows separate enumeration of populations of viable-non-apoptotic, early-apoptotic, late-apoptotic and necrotic cells. The assay was performed by combining 100 μg/μl of AO and 100 μg/μl of EB in PBS. WSU-FSCCL cells and primary lymphocytes from patients were incubated for the indicated times and concentrations, centrifuged at 2000 g for 5 m at 4°C, resuspended in 50 μl of PBS, with a resultant of 20 μl of suspension being counted using a Nikon Fluorescent microscope. For each sample at least 200 cells were counted.
Flow cytometric analysis of apoptosis
Apoptosis was determined by the flow cytometric measurement of phosphatidylserine exposure using Annexin V FITC and Propidium Iodide stain. Cells were grown in the presence or absence of ApoG2 and then centrifuged at 2000 × g for 5 min. The cells were then resuspended in PBS and stained with fluorescent conjugates of Annexin V (BioVision, Mountain View, CA) for 1 hour and propidium iodide for 30 min, and then analyzed on a FACScan machine (BD, San Jose, CA).
Detection of Caspase Activity
WSU-FSCCL cells exposed to 0.35 and 3.50 μM ApoG2 for 0 to 72 hrs were incubated on ice for 30 minutes in cell lysis buffer (Sigma-Aldrich, St. Louis, MO). The supernatant after centrifugation at 14,000 g at 4°C was collected and proteins were quantified according to the bicinchoninic acid protein assay methodology (Pierce Chemical, Rockford, IL.). A total of 50 μg protein in a volume of 50 μl cell lysis mixture was resuspended on ice as triplicates in a 96-well plate; 50 μl of 2× reaction buffer containing 10 mM DTT was added to each sample (MBL International, Woburn, MA); 50 μM final concentration substrates for caspase-3 (DEVD-pNA) and caspase-9 (LEHD-pNA; MBL International, Woburn, MA) were added to each sample for a total volume of 100 μl and incubated for 180 m at 37°C. Free pNA released from the labeled synthetic substrate on cleavage by active caspase was measured on a fluorescence plate reader (Molecular Devices, Sunnyvale, CA) at 405 nm.
Female ICR SCID mice were obtained from Taconic Laboratories (Germantown, NY) and were housed and treated in the Wayne State University School of Medicine under an approved protocol. Four week old mice were injected intraperitoneally (i.p.) with 5 × 106 WSU-FSCCL cells. ApoG2 was injected 25 mg/kg QD × 5 days either IP or intravenously (i.v.). Mice were observed daily and euthanized when they appeared ill. Animals' activity, weight and survival were monitored three times a week; mice were sacrificed when they developed hind region paralysis, had decreased activity and weight loss of 15% or more, or death was felt to be imminent. Necropsy was carried out and the extent of macroscopic disease was identified with all major organs being taken for microscopic pathological examination. Major organs included the brain, femur (for bone marrow), heart, kidney, liver, lungs, pancreas, retroperitoneal fat, and spleen. Peripheral blood smears were examined for evidence of circulating lymphoma cells.
Survival curves were created using the product limit of Kaplan and Meier, and compared using the log-rank test. The end point for assessing anti-lymphoma activity was calculated by percent increase in host life span (%ILS). %ILS = 100 × MDD (median day of death of the treated tumor-bearing mice) – (MDD of tumor-bearing control mice)/MDD of the tumor bearing control mice.
Apoptosis induction by AnnexinV/PI stains and AO/EB were compared to control by the student t-test. Survival functions were estimated using the Kaplan-Meier method and compared by the log-rank test. P-values < 0.05 were considered statistically significant. All statistical analyses were evaluated using GraphPad Prism 4 (San Diego, CA)
Effect of ApoG2 on WSU-FSCCL Cells
Effect of ApoG2 on Primary Fresh Cases
Effect of ApoG2 on the activation of Caspases in WSU-FSCCL cells
ApoG2 Induced Activation of PARP and AIF in the Apoptotic Pathway
PARP can release apoptosis-inducing factor (AIF), which induces chromatin condensation and large-scale DNA fragmentation when released into the cytosol . Similar to PARP, AIF cleavage was shown at concentrations greater than 0.35 μM. Cleaved bands of AIF greater than 2-fold and (up to 4-fold) were shown at 3.5 μM at 24 hrs (Fig. 5B).
Determination of Anti-lymphoma Effect of ApoG2 in SCID Mice
FL has been increasing in incidence over the past three decades and is now the fifth most common malignancy in the United States . There are many approaches to the treatment of FL, but the goal of therapy has been to maintain the best quality of life and treat when a patient is at "high risk" or the disease progresses. Standard chemotherapy regimens directed towards these low-grade lymphomas still lack complete curative effects. This may be in part due to the overexpression of Bcl-2, a key molecule of resistance in indolent lymphoma. Overexpression of Bcl-2 has been implicated to play a significant role in the clinical outcome of FL patients . A number of approaches have been sought to target overexpression of Bcl-2 in FL, e.g. downregulation of Bcl-2 protein via antisense oligonucleotides [18, 22–24]. Most recently, hydrophobic groove of the Bcl-2 family of anti-apoptotic proteins has become a very attractive target for the design of SMIs. SMIs filling the hydrophobic groove mimic cognate proteins such as Bax and Bid. SMIs directed against BH3 domains have been categorized into at least eight different chemical classifications . Laboratories, including this one, have been in the search to find novel small-molecule inhibitors to Bcl-2 [15, 26–29]. Gossypol has been used as an anti-cancer agent against prostate cancer, metastatic adrenal cancer and many other cancers before the BH3 mimetic activity was discovered [30, 31]. This discovery has shown that (-)-gossypol, the active enantiomer of gossypol, binds to anti-apoptotic members of the Bcl-2 family, Bcl-2, Bcl-XL and Mcl-1, with nanomolar affinities and this active enantiomer has been tested in clinical trails for treatment of patients with advanced malignancies .
In clinical trials, gossypol has been associated with side effects, such as emesis and diarrhea, because of the two reactive aldehyde-groups . ApoG2 has been designed and synthesized with the reactive groups completely removed in order to minimize side effects. In addition, ApoG2 has superior stability under both stress and normal conditions compared to (-)-gossypol .
It is notable that our study is the first on ApoG2 in FL. The goals of new agents such as ApoG2, are to have higher binding affinity to its targets; ApoG2 has greater than 8-fold binding affinity to Bcl-2 over its predecessors TW-37 and (-)-gossypol [16, 17]. In this study, we have shown a potent anti-lymphoma effect on FL. ApoG2 shows an IC50 of 9- and 18-fold lower when compared to TW-37 or gossypol. When compared to HA14-1, which is a SMI to Bcl-2 used against leukemia cell lines HL60 and K562 , ApoG2 has a IC50 which is 200-fold lower. The SMI ABT-737 has a considerably lower IC50 (8 and 30 nM) when used against FL cell lines, but ABT-737 does not bind to Mcl-1 and thus Mcl-1 expression could result in resistance. In comparison, ApoG2 targets all these three anti-apoptotic proteins [27, 35, 36]. In our study, ApoG2 is effective against FL, pre-B-acute lymphoblastic leukemia, mantle cell lymphoma, marginal zone lymphoma, as well as chronic lymphocytic leukemia. Therefore, ApoG2 could potentially be a more effective drug in the lymphoma clinic spanning a greater array of patients.
With the binding of ApoG2 to Bcl-2 family of proteins, it would be expected that ApoG2 would lead to activation of downstream apoptotic proteins. The mechanism of action of ApoG2 has not been elucidated in FL. We show here that ApoG2 can activate the initiator caspase-9, and the effector caspase-3, and induce caspase cleavage in nanomolar concentrations. Moreover, ApoG2 can lead to the activation of caspase-8 which serves as amplification loop together with caspase-3 [37, 38]. PARP and AIF have been implicated in the final stages of apoptosis. They play a role in the chromatin condensation and DNA fragmentation. We show that ApoG2 activates PARP and AIF in the nanomolar range.
These findings clearly demonstrate that ApoG2 can activate the Bcl-2 apoptotic pathway in vitro. The exact mechanism of action of ApoG2 is unclear. Likely mechanisms are that ApoG2 binds to Bcl-2 (or Mcl-1, Bcl-XL, A1, Bcl-w) and prevents its association with BH3-only pro-apoptotic proteins, thus unleashing the pro-apoptotic proteins to participate in the apoptotic response. Work in our laboratory is being done to further elucidate the mechanism of ApoG2 action.
Many agents targeting the Bcl-2 family were shown to have activity in vitro. However, the main goal of our research endeavor is to find out if ApoG2 can make its way into clinical trials. Here, we tested the anti-lymphoma activity of ApoG2 in vivo. The endpoint for treatment efficacy is survival of the mice bearing the human FL cells. Our study showed that regardless of route of injection (i.p. or i.v.), ApoG2 could significantly increase the life span of lymphoma-bearing SCID mice by at least 42% (Fig. 6A). Moreover, ApoG2 was safe and well tolerated up to 800 mg/kg with no weight lose in all treated animals. ApoG2 has an undetermined MTD, and a large therapeutic window of 25 to 800 mg/kg; with effective dose of only 25 mg/kg; compared to ABT-737, that has a therapeutic window of 25 to 100 mg/kg, with an undetermined MTD .
In closing, we have shown that ApoG2 can be a potential novel agent against FL. Our data suggest that ApoG2 also could be used in several different types of lymphoid malignancies. ApoG2 in this study does show efficacy for treatment of FL as a single agent; it can prove to be even more effective when used in combination with standard chemotherapy.
concentration of agent with fifty percent of growth inhibition
increase in life span
- CHOP chemotherapy = C-cyclophosphamide:
H-doxorubicin Hydrochloride, O-oncovin, P-prednisone
Poly (ADP-ribose) polymerase
Apoptosis Inducing Factor
acute lymphoblastic leukemia
mantle cell lymphoma
marginal zone lymphoma
chronic lymphocytic leukemia.
Grant support: Leukemia and Lymphoma Society grant 6028-8 (R.M. Mohammad); National Institute of Health grant R01 CA109389 (R.M. Mohammad; Department of Defense Breast Cancer Program grant BC0009140 (S. Wang); National Institute of Health grant P30 CA22453-20; and NIH/National Institute of General Medical Sciences (NIGMS) grant GM058905B (J. Dunbar – Initiative for Maximizing Student Diversity (IMSD) – program director). We also thank Drs. Anton-Scott Goustin and Ben Chen for their valuable input on ApoG2.
- Rogers B: Looking at lymphoma and leukemia. Nursing. 2005, 35 (7): 56-63; quiz 63-4.View ArticlePubMedGoogle Scholar
- Ganti AK, Bociek RG, Bierman PJ, Enke CA, Vose JM, Armitage JO: Follicular lymphoma: expanding therapeutic options. Oncology (Williston Park). 2005, 19 (2): 213-28; discussion 228, 233-6, 239.Google Scholar
- Fisher RI, LeBlanc M, Press OW, Maloney DG, Unger JM, Miller TP: New treatment options have changed the survival of patients with follicular lymphoma. J Clin Oncol. 2005, 23 (33): 8447-8452.View ArticlePubMedGoogle Scholar
- Sacchi S, Pozzi S, Marcheselli L, Bari A, Luminari S, Angrilli F, Merli F, Vallisa D, Baldini L, Brugiatelli M: Introduction of rituximab in front-line and salvage therapies has improved outcome of advanced-stage follicular lymphoma patients. Cancer. 2007, 109 (10): 2077-2082.View ArticlePubMedGoogle Scholar
- Press OW, Unger JM, Braziel RM, Maloney DG, Miller TP, Leblanc M, Fisher RI: Phase II trial of CHOP chemotherapy followed by tositumomab/iodine I-131 tositumomab for previously untreated follicular non-Hodgkin's lymphoma: five-year follow-up of Southwest Oncology Group Protocol S9911. J Clin Oncol. 2006, 24 (25): 4143-4149.View ArticlePubMedGoogle Scholar
- van Oers MH, Klasa R, Marcus RE, Wolf M, Kimby E, Gascoyne RD, Jack A, Van't Veer M, Vranovsky A, Holte H, van Glabbeke M, Teodorovic I, Rozewicz C, Hagenbeek A: Rituximab maintenance improves clinical outcome of relapsed/resistant follicular non-Hodgkin lymphoma in patients both with and without rituximab during induction: results of a prospective randomized phase 3 intergroup trial. Blood. 2006, 108 (10): 3295-3301.View ArticlePubMedGoogle Scholar
- Raghoebier S, Broos L, Kramer MH, van Krieken JH, Kluin-Nelemans JC, van Ommen GJ, Kluin P: Histological conversion of follicular lymphoma with structural alterations of t(14;18) and immunoglobin genes. Leukemia. 1995, 9 (10): 1748-1755.PubMedGoogle Scholar
- Basu A, Haldar S: The relationship between BcI2, Bax and p53: consequences for cell cycle progression and cell death. Mol Hum Reprod. 1998, 4 (12): 1099-1109.View ArticlePubMedGoogle Scholar
- Staudt LM: A closer look at follicular lymphoma. N Engl J Med. 2007, 356 (7): 741-742.View ArticlePubMedGoogle Scholar
- Klasa RJ: Targeting the proapoptotic factor Bcl-2 in non-Hodgkin's lymphoma. Oncology (Williston Park). 2004, 18 (13 Suppl 10): 25-31.Google Scholar
- Muchmore SW, Sattler M, Liang H, Meadows RP, Harlan JE, Yoon HS, Nettesheim D, Chang BS, Thompson CB, Wong SL, Ng SL, Fesik SW: X-ray and NMR structure of human Bcl-xL, an inhibitor of programmed cell death. Nature. 1996, 381 (6580): 335-341.View ArticlePubMedGoogle Scholar
- Aritomi M, Kunishima N, Inohara N, Ishibashi Y, Ohta S, Morikawa K: Crystal structure of rat Bcl-xL. Implications for the function of the Bcl-2 protein family. J Biol Chem. 1997, 272 (44): 27886-27892.View ArticlePubMedGoogle Scholar
- Yin D, Zhou H, Kumagai T, Liu G, Ong JM, Black KL, Koeffler HP: Proteasome inhibitor PS-341 causes cell growth arrest and apoptosis in human glioblastoma multiforme (GBM). Oncogene. 2005, 24 (3): 344-354.View ArticlePubMedGoogle Scholar
- Mohammad RM, Wang S, Aboukameel A, Chen B, Wu X, Chen J, Al-Katib A: Preclinical studies of a nonpeptidic small-molecule inhibitor of Bcl-2 and Bcl-X(L) [(-)-gossypol] against diffuse large cell lymphoma. Mol Cancer Ther. 2005, 4 (1): 13-21.PubMedGoogle Scholar
- Wang G, Nikolovska-Coleska Z, Yang CY, Wang R, Tang G, Guo J, Shangary S, Qiu S, Gao W, Yang D, Meagher J, Stuckey J, Krajewski K, Jiang S, Roller PP, Abaan HO, Tomita Y, Wang S: Structure-based design of potent small-molecule inhibitors of anti-apoptotic Bcl-2 proteins. J Med Chem. 2006, 49 (21): 6139-6142.View ArticlePubMedGoogle Scholar
- Mohammad RM, Goustin AS, Aboukameel A, Chen B, Banerjee S, Wang G, Nikolovska-Coleska Z, Wang S, Al-Katib A: Preclinical studies of TW-37, a new nonpeptidic small-molecule inhibitor of Bcl-2, in diffuse large cell lymphoma xenograft model reveal drug action on both Bcl-2 and Mcl-1. Clin Cancer Res. 2007, 13 (7): 2226-2235.View ArticlePubMedGoogle Scholar
- Mohammad R: ApoG2, a potent, non-toxic small-molecule inhibitor of Bcl-2 family: A preclinical trial in lymphoma. AACR. 2006Google Scholar
- Mohammad RM, Mohamed AN, Smith MR, Jawadi NS, al-Katib A: A unique EBV-negative low-grade lymphoma line (WSU-FSCCL) exhibiting both t(14;18) and t(8;11). Cancer Genet Cytogenet. 1993, 70 (1): 62-67.View ArticlePubMedGoogle Scholar
- Susin SA, Lorenzo HK, Zamzami N, Marzo I, Snow BE, Brothers GM, Mangion J, Jacotot E, Costantini P, Loeffler M, Larochette N, Goodlett DR, Aebersold R, Siderovski DP, Penninger JM, Kroemer G: Molecular characterization of mitochondrial apoptosis-inducing factor. Nature. 1999, 397 (6718): 441-446.View ArticlePubMedGoogle Scholar
- Groves FD, Linet MS, Travis LB, Devesa SS: Cancer surveillance series: non-Hodgkin's lymphoma incidence by histologic subtype in the United States from 1978 through 1995. J Natl Cancer Inst. 2000, 92 (15): 1240-1251.View ArticlePubMedGoogle Scholar
- Schmitt C, Grundt A, Buchholtz C, Scheuer L, Benner A, Hensel M, Ho AD, Leo E: One single dose of rituximab added to a standard regimen of CHOP in primary treatment of follicular lymphoma appears to result in a high clearance rate from circulating bcl-2/IgH positive cells: Is the end of molecular monitoring near?. Leuk Res. 2006, 30 (12): 1563-1568.View ArticlePubMedGoogle Scholar
- Mohammad R, Abubakr Y, Dan M, Aboukameel A, Chow C, Mohamed A, Hamdy N, Al-Katib A: Bcl-2 antisense oligonucleotides are effective against systemic but not central nervous system disease in severe combined immunodeficient mice bearing human t(14;18) follicular lymphoma. Clin Cancer Res. 2002, 8 (4): 1277-1283.PubMedGoogle Scholar
- Smith MR, Abubakr Y, Mohammad R, Xie T, Hamdan M, al-Katib A: Antisense oligodeoxyribonucleotide down-regulation of bcl-2 gene expression inhibits growth of the low-grade non-Hodgkin's lymphoma cell line WSU-FSCCL. Cancer Gene Ther. 1995, 2 (3): 207-212.PubMedGoogle Scholar
- Ramanarayanan J, Hernandez-Ilizaliturri FJ, Chanan-Khan A, Czuczman MS: Pro-apoptotic therapy with the oligonucleotide Genasense (oblimersen sodium) targeting Bcl-2 protein expression enhances the biological anti-tumour activity of rituximab. Br J Haematol. 2004, 127 (5): 519-530.View ArticlePubMedGoogle Scholar
- Zhai D, Jin C, Satterthwait AC, Reed JC: Comparison of chemical inhibitors of antiapoptotic Bcl-2-family proteins. Cell Death Differ. 2006, 13 (8): 1419-1421.View ArticlePubMedGoogle Scholar
- Zhang Z, Jin L, Qian X, Wei M, Wang Y, Wang J, Yang Y, Xu Q, Xu Y, Liu F: Novel Bcl-2 inhibitors: Discovery and mechanism study of small organic apoptosis-inducing agents. Chembiochem. 2007, 8 (1): 113-121.View ArticlePubMedGoogle Scholar
- Bruncko M, Oost TK, Belli BA, Ding H, Joseph MK, Kunzer A, Martineau D, McClellan WJ, Mitten M, Ng SC, Nimmer PM, Oltersdorf T, Park CM, Petros AM, Shoemaker AR, Song X, Wang X, Wendt MD, Zhang H, Fesik SW, Rosenberg SH, Elmore SW: Studies leading to potent, dual inhibitors of Bcl-2 and Bcl-xL. J Med Chem. 2007, 50 (4): 641-662.View ArticlePubMedGoogle Scholar
- Oliver L, Mahe B, Gree R, Vallette FM, Juin P: HA14-1, a small molecule inhibitor of Bcl-2, bypasses chemoresistance in leukaemia cells. Leuk Res. 2007Google Scholar
- Skommer J, Wlodkowic D, Matto M, Eray M, Pelkonen J: HA14-1, a small molecule Bcl-2 antagonist, induces apoptosis and modulates action of selected anticancer drugs in follicular lymphoma B cells. Leuk Res. 2006, 30 (3): 322-331.View ArticlePubMedGoogle Scholar
- Tuszynski GP, Cossu G: Differential cytotoxic effect of gossypol on human melanoma, colon carcinoma, and other tissue culture cell lines. Cancer Res. 1984, 44 (2): 768-771.PubMedGoogle Scholar
- Flack MR, Pyle RG, Mullen NM, Lorenzo B, Wu YW, Knazek RA, Nisula BC, Reidenberg MM: Oral gossypol in the treatment of metastatic adrenal cancer. J Clin Endocrinol Metab. 1993, 76 (4): 1019-1024.PubMedGoogle Scholar
- Bayes M, Rabasseda X, Prous JR: Gateways to clinical trials. Methods Find Exp Clin Pharmacol. 2006, 28 (8): 533-591.PubMedGoogle Scholar
- Stein RC, Joseph AE, Matlin SA, Cunningham DC, Ford HT, Coombes RC: A preliminary clinical study of gossypol in advanced human cancer. Cancer Chemother Pharmacol. 1992, 30 (6): 480-482.View ArticlePubMedGoogle Scholar
- Zhang Y LY: A novel pan inhibitor of Bcl-2 and Mcl-1 apogossypolone (ApoG2) with superior stability and improved activity against human leukemia and lymphoma. Abstract. 2007, 98th AACR Annual Meeting, 2007 Los Angeles, CA-Google Scholar
- Del Gaizo Moore V, Brown JR, Certo M, Love TM, Novina CD, Letai A: Chronic lymphocytic leukemia requires BCL2 to sequester prodeath BIM, explaining sensitivity to BCL2 antagonist ABT-737. J Clin Invest. 2007, 117 (1): 112-121.PubMed CentralView ArticlePubMedGoogle Scholar
- Trudel S, Stewart AK, Li Z, Shu Y, Liang SB, Trieu Y, Reece D, Paterson J, Wang D, Wen XY: The Bcl-2 family protein inhibitor, ABT-737, has substantial antimyeloma activity and shows synergistic effect with dexamethasone and melphalan. Clin Cancer Res. 2007, 13 (2 Pt 1): 621-629.View ArticlePubMedGoogle Scholar
- Slee EA, Harte MT, Kluck RM, Wolf BB, Casiano CA, Newmeyer DD, Wang HG, Reed JC, Nicholson DW, Alnemri ES, Green DR, Martin SJ: Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner. J Cell Biol. 1999, 144 (2): 281-292.PubMed CentralView ArticlePubMedGoogle Scholar
- Tang D, Lahti JM, Kidd VJ: Caspase-8 activation and bid cleavage contribute to MCF7 cellular execution in a caspase-3-dependent manner during staurosporine-mediated apoptosis. J Biol Chem. 2000, 275 (13): 9303-9307.View ArticlePubMedGoogle Scholar
- Oltersdorf T, Elmore SW, Shoemaker AR, Armstrong RC, Augeri DJ, Belli BA, Bruncko M, Deckwerth TL, Dinges J, Hajduk PJ, Joseph MK, Kitada S, Korsmeyer SJ, Kunzer AR, Letai A, Li C, Mitten MJ, Nettesheim DG, Ng S, Nimmer PM, O'Connor JM, Oleksijew A, Petros AM, Reed JC, Shen W, Tahir SK, Thompson CB, Tomaselli KJ, Wang B, Wendt MD, Zhang H, Fesik SW, Rosenberg SH: An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature. 2005, 435 (7042): 677-681.View ArticlePubMedGoogle Scholar
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