Rational Design and Real Time, In-Cell Detection of the Proapoptotic Activity of a Novel Compound Targeting Bcl-XL

Becattini, Barbara; Kitada, Shinichi; Leone, Marilisa; Monosov, Edward; Chandler, Sharon; Zhai, Dayong; Kipps, Thomas J.; Reed, John C.; Pellecchia, Maurizio

doi:10.1016/j.chembiol.2004.02.020

Cited by 152 publications

(103 citation statements)

References 34 publications

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“…These data suggest that, in tumors with Bcl-2 inhibitor-driven down-regulation of Bcl-2 function, combination therapy would prevent this avenue of escape. In general, studies involving small-molecule inhibitors of Bcl-2 or Bcl-x L have indeed shown increasing efficacy in tumor types that present upregulated Bcl-2 expression (38,(43)(44)(45)(46)(47). However, in the present study, we investigate the therapeutic potential of targeting Bcl-2-related angiogenic functions in endothelial cells.…”

Section: Discussionmentioning

confidence: 96%

Antiangiogenic Effect of TW37, a Small-Molecule Inhibitor of Bcl-2

Zeitlin¹,

Joo²,

Dong³

et al. 2006

Cancer Research

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Bcl-2 is an antiapoptotic protein that is up-regulated in several tumor types, and its expression levels have strong correlation to development of resistance to therapy and poor prognosis. We have shown recently that Bcl-2 also functions as a proangiogenic signaling molecule that activates a nuclear factor-KB-mediated pathway resulting in up-regulation of the angiogenic chemokines CXCL1 and CXCL8 by neovascular endothelial cells. Here, we evaluate the antiangiogenic effect of the novel small-molecule inhibitor of Bcl-2 (TW37) developed using a structure-based design strategy. We observed that TW37 has an IC 50 of 1.8 Mmol/L for endothelial cells but showed no cytotoxic effects for fibroblasts at concentrations up to 50 Mmol/L. The mechanism of TW37-induced endothelial cell death was apoptosis, in a process mediated by mitochondrial depolarization and activation of caspase-9 and caspase-3. The effect of TW37 on endothelial cell apoptosis was not prevented by coexposure to the growth factor milieu secreted by tumor cells. Inhibition of the angiogenic potential of endothelial cells (i.e., migration and capillary sprouting assays) and expression of the angiogenic chemokines CXCL1 and CXCL8 were accomplished at subapoptotic TW37 concentrations (0.005-0.05 Mmol/L). Notably, administration of TW37 i.v. resulted in a decrease in the density of functional human microvessels in the severe combined immunodeficient mouse model of human angiogenesis. In conclusion, we describe functionally separate proapoptotic and antiangiogenic mechanisms for a smallmolecule inhibitor of Bcl-2 and show the potential for Bcl-2 inhibition as a target for antiangiogenic therapy. (Cancer Res 2006; 66(17): 8698-706)

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Section: Discussionmentioning

confidence: 96%

Antiangiogenic Effect of TW37, a Small-Molecule Inhibitor of Bcl-2

Zeitlin¹,

Joo²,

Dong³

et al. 2006

Cancer Research

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“…3,20 Since Bcl-2 family proteins are highly expressed in CLL lymphocytes, we determined whether these cells were susceptible to gossypol treatment ( Figure 1Ai,ii). When untreated CLL lymphocytes were incubated for 24 hours, 22% apoptotic cells (spontaneous apoptosis); increased to 63% after gossypol treatment.…”

Section: Gossypol Induced Apoptosis In Cll Lymphocytesmentioning

confidence: 99%

“…1 It exhibits a type of enantiomerism that arises from restricted rotation: the (Ϫ)-gossypol isomer showed greater cytotoxicity than the (ϩ)-isomer in several human cancer cell lines. 2 To reduce toxicity, structural modifications in the (Ϫ)-gossypol isomer led to the analog apogossypol, which lacks the reactive aldehydic groups and displays proapoptotic activity comparable with that of gossypol 3 ; another derivative, gossypolone, demonstrates lower cytotoxicity than the parent compound. 4 The success of these agents resulted in the development of additional analogs, such as the l-isomer of gossypol, AT-101, 5 and ABT-737, which was designed on the basis of structure activity-based studies.…”

Section: Introductionmentioning

confidence: 99%

Gossypol, a BH3 mimetic, induces apoptosis in chronic lymphocytic leukemia cells

Balakrishnan¹,

Wierda

Keating

et al. 2008

Blood

117

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Gossypol, a cottonseed extract derivative, acts as a BH3-mimetic, binding to the BH3 pocket of antiapoptotic proteins and displacing pro-death partners to induce apoptosis. However, knowledge on the molecular underpinnings of its downstream effects is limited. Since chronic lymphocytic leukemia (CLL) cells express high levels of antiapoptotic proteins that act as a survival mechanism for these replicationally quiescent lymphocytes, we investigated whether gossypol induces apoptosis in these cells and what mechanism underlies gossypol-mediated cytotoxicity. Gossypol induced cell death in a concentration-and time-dependent manner; 24-hour incubation with 30 M gossypol resulted in 50% cell death (median; range, 10%-80%; n ‫؍‬ 47) that was not abrogated by pan-specific caspase inhibitor. Starting at 4 hours, the mitochondrial outer membrane was significantly permeabilized (median, 77%; range, 54%-93%; n ‫؍‬ 15). Mitochondrial outer membrane permeabiliztaion (MOMP) was concurrent with increased production of reactive oxygen species (ROS); however, antioxidants did not abrogate gossypolinduced cell death. Mitochondrial membrane permeabilization was also associated with loss of intracellular adenosine triphosphate (ATP), activation of BAX, and release of cytochrome c and apoptosisinducing factor (AIF), which was translocated to the nucleus. Blocking AIF translocation resulted in a decreased apoptosis, suggesting that AIF contributes to gossypol-mediated cytotoxicity in CLL lymphocytes. (Blood. 2008;112:1971-1980 Introduction Gossypol, a natural product derived from cottonseed extracts, was originally extensively investigated in China as a male contraceptive agent. 1 It exhibits a type of enantiomerism that arises from restricted rotation: the (Ϫ)-gossypol isomer showed greater cytotoxicity than the (ϩ)-isomer in several human cancer cell lines. 2 To reduce toxicity, structural modifications in the (Ϫ)-gossypol isomer led to the analog apogossypol, which lacks the reactive aldehydic groups and displays proapoptotic activity comparable with that of gossypol 3 ; another derivative, gossypolone, demonstrates lower cytotoxicity than the parent compound. 4 The success of these agents resulted in the development of additional analogs, such as the l-isomer of gossypol, AT-101, 5 and ABT-737, which was designed on the basis of structure activity-based studies. 6,7 The gossypol isomers and analogs exhibited inhibitory activity against a wide range of human carcinoma cell lines and in tumor xenograft models. [8][9][10][11] However, these and other investigations illustrated different actions of this agent, and knowledge on the molecular underpinnings of its biologic effects is still limited.Early studies in HeLa cells demonstrated that the cytotoxicity of gossypol was based on its inhibitory activity on DNA synthesis through DNA polymerases alpha and beta 12 or on topoisomerases. 13 Gossypol's effect on DNA synthesis, leading to S-phase arrest, was specific: no effects on RNA and protein syntheses were observed. 14 In concordance...

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“…14,15 Other BCL2 antagonists also bind MCL1, and obatoclax (GX15-070) disrupts BAK binding to MCL1. 16 In this study, we investigated the mechanism of cell death induction by gossypol, 17 apogossypol, 18 chelerythrine, 19 obatoclax, 16 EM20-25, 20 and ABT-737. 11 Gossypol, isolated from cotton seeds and used as a male contraceptive, was recently recognized as binding and interacting with antiapoptotic BCL2 family members and inducing apoptosis.…”

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confidence: 99%

Different forms of cell death induced by putative BCL2 inhibitors

et al. 2009

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Several inhibitors of BCL2 proteins have been identified that induce apoptosis in a variety of tumor cells, indicating their potential in cancer therapy. We investigated the specificity of six putative BCL2 inhibitors (obatoclax, gossypol, apogossypol, EM20-25, chelerythrine and ABT-737). Using cells deficient either for Bax/Bak or caspase-9, we found that only ABT-737 specifically targeted BCL2 proteins and induced apoptosis by activation of caspase-9, as only ABT-737 induced apoptosis was completely inhibited in cells deficient for Bax/Bak or caspase-9. Our data show that only ABT-737 is a specific BCL2 inhibitor and all other compounds investigated were not specific for BCL2 proteins. Furthermore, investigations of the effects of these compounds in primary chronic lymphocytic leukemic cells showed that all compounds induced certain biochemical hallmarks of apoptosis, such as release of cytochrome c and caspase cleavage. However, they all caused strikingly different ultrastructural changes. ABT-737 induced all the characteristic ultrastructural changes of apoptosis together with early rupture of the outer mitochondrial membrane, whereas obatoclax, chlelerythrine and gossypol induced pronounced mitochondrial swelling with formation of phospholipid inclusions. Therefore, we conclude that biochemical measurements used earlier to define apoptosis like mitochondrial release of cytochrome c and caspase cleavage, are insufficient to distinguish between classic apoptosis and other forms of cell death.

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Rational Design and Real Time, In-Cell Detection of the Proapoptotic Activity of a Novel Compound Targeting Bcl-XL

Cited by 152 publications

References 34 publications

Antiangiogenic Effect of TW37, a Small-Molecule Inhibitor of Bcl-2

Antiangiogenic Effect of TW37, a Small-Molecule Inhibitor of Bcl-2

Gossypol, a BH3 mimetic, induces apoptosis in chronic lymphocytic leukemia cells

Different forms of cell death induced by putative BCL2 inhibitors

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