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SMI of Bcl-2 TW-37 is active across a spectrum of B-cell tumors irrespective of their proliferative and differentiation status.

Al-Katib AM, Sun Y, Goustin AS, Azmi AS, Chen B, Aboukameel A, Mohammad RM - J Hematol Oncol (2009)

Bottom Line: Multiple cytochemical and molecular approaches such as acridine orange/ethidium bromide assay for apoptosis, co-immunoprecipitation of complexes and western blot analysis, caspase luminescent activity assay and apoptotic DNA fragmentation assay were used to demonstrate the effect of TW-37 on different B-cell lines, patient derived samples, as well as in animal xenograft models.TW-37 was able to block Bim-Bcl-XL and Bim-Mcl-1 heterodimerization and induced apoptosis via activation of caspases -9, -3, PARP and DNA fragmentation.TW-37 failed to induce changes in the Bcl-2 proteins levels suggesting that assessment of baseline Bcl-2 family proteins can be used to predict response to the drug.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Internal Medicine, Division of Hematology/Oncology, Wayne State University School of Medicine, Detroit, Michigan, USA. alkatib@med.wayne.edu

ABSTRACT
The Bcl-2 family of proteins is critical to the life and death of malignant B-lymphocytes. Interfering with their activity using small-molecule inhibitors (SMI) is being explored as a new therapeutic strategy for treating B-cell tumors. We evaluated the efficacy of TW-37, a non-peptidic SMI of Bcl-2 against a range spectrum of human B-cell lines, fresh patient samples and animal xenograft models. Multiple cytochemical and molecular approaches such as acridine orange/ethidium bromide assay for apoptosis, co-immunoprecipitation of complexes and western blot analysis, caspase luminescent activity assay and apoptotic DNA fragmentation assay were used to demonstrate the effect of TW-37 on different B-cell lines, patient derived samples, as well as in animal xenograft models. Nanomolar concentrations of TW-37 were able to induce apoptosis in both fresh samples and established cell lines with IC50 in most cases of 165-320 nM. Apoptosis was independent of proliferative status or pathological classification of B-cell tumor. TW-37 was able to block Bim-Bcl-XL and Bim-Mcl-1 heterodimerization and induced apoptosis via activation of caspases -9, -3, PARP and DNA fragmentation. TW-37 administered to tumor-bearing SCID mice led to significant tumor growth inhibition (T/C), tumor growth delay (T-C) and Log10kill, when used at its maximum tolerated dose (40 mg/kg x 3 days) via tail vein. TW-37 failed to induce changes in the Bcl-2 proteins levels suggesting that assessment of baseline Bcl-2 family proteins can be used to predict response to the drug. These findings indicate activity of TW-37 across the spectrum of human B-cell tumors and support the concept of targeting the Bcl-2 system as a therapeutic strategy regardless of the stage of B-cell differentiation.

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Immunoprecipitation and western-blot analysis of heterodimerization interaction by TW-37 between anti-apoptosis and pro-apoptosis Bcl-2 family proteins. WSU-FSCCL cells were treated with 1 or 2 μM of TW-37 for 24 hr, lysed and 300 μg of whole cell lysate was immunoprecipitated with anti-Bim followed by Western-Blot with anti-Mcl-1, anti-Bcl-XL, anti-Bim and anti β-actin.
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Figure 6: Immunoprecipitation and western-blot analysis of heterodimerization interaction by TW-37 between anti-apoptosis and pro-apoptosis Bcl-2 family proteins. WSU-FSCCL cells were treated with 1 or 2 μM of TW-37 for 24 hr, lysed and 300 μg of whole cell lysate was immunoprecipitated with anti-Bim followed by Western-Blot with anti-Mcl-1, anti-Bcl-XL, anti-Bim and anti β-actin.

Mentions: Protein lysates of TW-37-treated WSU-FSCCL cells were immunoprecipitated with antibody to Bim BH3-only proapoptotic protein. Immunoprecipitates were separated by SDS-PAGE and electroblotted to a membrane. Subsequent immunoblotting with Mcl-1 and Bcl-XL revealed a decrease in Bim-Mcl-1 and Bim-Bcl-XL complexes in the WSU-FSCCL-treated cells compared with untreated (control) cell lysates (Fig. 6). The blocking of Bim-Mcl-1 heterodimerization is evident at 1 μM TW-37 and increased at 2 μM; the blocking of Bim-Bcl-XL heterodimerization is evident only at the highest drug concentration. This finding confirms the ability of TW-37 to block Bim-Mcl-1 and Bim-Bcl-XL heterodimerization. Using similar technique, previously we have shown that TW-37 blocks Bid-Bcl-2 and Bid-Mcl-1 but not Bid-Bcl-XL in WSU-DLCL2 cell lysate [27].


SMI of Bcl-2 TW-37 is active across a spectrum of B-cell tumors irrespective of their proliferative and differentiation status.

Al-Katib AM, Sun Y, Goustin AS, Azmi AS, Chen B, Aboukameel A, Mohammad RM - J Hematol Oncol (2009)

Immunoprecipitation and western-blot analysis of heterodimerization interaction by TW-37 between anti-apoptosis and pro-apoptosis Bcl-2 family proteins. WSU-FSCCL cells were treated with 1 or 2 μM of TW-37 for 24 hr, lysed and 300 μg of whole cell lysate was immunoprecipitated with anti-Bim followed by Western-Blot with anti-Mcl-1, anti-Bcl-XL, anti-Bim and anti β-actin.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651908&req=5

Figure 6: Immunoprecipitation and western-blot analysis of heterodimerization interaction by TW-37 between anti-apoptosis and pro-apoptosis Bcl-2 family proteins. WSU-FSCCL cells were treated with 1 or 2 μM of TW-37 for 24 hr, lysed and 300 μg of whole cell lysate was immunoprecipitated with anti-Bim followed by Western-Blot with anti-Mcl-1, anti-Bcl-XL, anti-Bim and anti β-actin.
Mentions: Protein lysates of TW-37-treated WSU-FSCCL cells were immunoprecipitated with antibody to Bim BH3-only proapoptotic protein. Immunoprecipitates were separated by SDS-PAGE and electroblotted to a membrane. Subsequent immunoblotting with Mcl-1 and Bcl-XL revealed a decrease in Bim-Mcl-1 and Bim-Bcl-XL complexes in the WSU-FSCCL-treated cells compared with untreated (control) cell lysates (Fig. 6). The blocking of Bim-Mcl-1 heterodimerization is evident at 1 μM TW-37 and increased at 2 μM; the blocking of Bim-Bcl-XL heterodimerization is evident only at the highest drug concentration. This finding confirms the ability of TW-37 to block Bim-Mcl-1 and Bim-Bcl-XL heterodimerization. Using similar technique, previously we have shown that TW-37 blocks Bid-Bcl-2 and Bid-Mcl-1 but not Bid-Bcl-XL in WSU-DLCL2 cell lysate [27].

Bottom Line: Multiple cytochemical and molecular approaches such as acridine orange/ethidium bromide assay for apoptosis, co-immunoprecipitation of complexes and western blot analysis, caspase luminescent activity assay and apoptotic DNA fragmentation assay were used to demonstrate the effect of TW-37 on different B-cell lines, patient derived samples, as well as in animal xenograft models.TW-37 was able to block Bim-Bcl-XL and Bim-Mcl-1 heterodimerization and induced apoptosis via activation of caspases -9, -3, PARP and DNA fragmentation.TW-37 failed to induce changes in the Bcl-2 proteins levels suggesting that assessment of baseline Bcl-2 family proteins can be used to predict response to the drug.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Internal Medicine, Division of Hematology/Oncology, Wayne State University School of Medicine, Detroit, Michigan, USA. alkatib@med.wayne.edu

ABSTRACT
The Bcl-2 family of proteins is critical to the life and death of malignant B-lymphocytes. Interfering with their activity using small-molecule inhibitors (SMI) is being explored as a new therapeutic strategy for treating B-cell tumors. We evaluated the efficacy of TW-37, a non-peptidic SMI of Bcl-2 against a range spectrum of human B-cell lines, fresh patient samples and animal xenograft models. Multiple cytochemical and molecular approaches such as acridine orange/ethidium bromide assay for apoptosis, co-immunoprecipitation of complexes and western blot analysis, caspase luminescent activity assay and apoptotic DNA fragmentation assay were used to demonstrate the effect of TW-37 on different B-cell lines, patient derived samples, as well as in animal xenograft models. Nanomolar concentrations of TW-37 were able to induce apoptosis in both fresh samples and established cell lines with IC50 in most cases of 165-320 nM. Apoptosis was independent of proliferative status or pathological classification of B-cell tumor. TW-37 was able to block Bim-Bcl-XL and Bim-Mcl-1 heterodimerization and induced apoptosis via activation of caspases -9, -3, PARP and DNA fragmentation. TW-37 administered to tumor-bearing SCID mice led to significant tumor growth inhibition (T/C), tumor growth delay (T-C) and Log10kill, when used at its maximum tolerated dose (40 mg/kg x 3 days) via tail vein. TW-37 failed to induce changes in the Bcl-2 proteins levels suggesting that assessment of baseline Bcl-2 family proteins can be used to predict response to the drug. These findings indicate activity of TW-37 across the spectrum of human B-cell tumors and support the concept of targeting the Bcl-2 system as a therapeutic strategy regardless of the stage of B-cell differentiation.

Show MeSH
Related in: MedlinePlus