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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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Inventory of Bcl-2 family protein by Western-blot quantification of anti-, pro-apoptotic Bcl-2 family protein of 4 NHL cell lines (4.A) and 5 fresh patient derived samples (4.B). Cells were harvested and lysed for Western-blot analysis. Forty μg of total lysate was subjected to detect multi-domain anti-apoptotic proteins Bcl-2, Bcl-XL and Mcl-1 proteins in NHL cell lines and patient derived samples. 80 μg of total cell lysate was loaded to detect multi-domain pro-apoptotic and BH3-only Bax, Bak, Bok, Bad, Bim and Puma profiles in WSU cell lines and patient-derived fresh samples.
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Figure 4: Inventory of Bcl-2 family protein by Western-blot quantification of anti-, pro-apoptotic Bcl-2 family protein of 4 NHL cell lines (4.A) and 5 fresh patient derived samples (4.B). Cells were harvested and lysed for Western-blot analysis. Forty μg of total lysate was subjected to detect multi-domain anti-apoptotic proteins Bcl-2, Bcl-XL and Mcl-1 proteins in NHL cell lines and patient derived samples. 80 μg of total cell lysate was loaded to detect multi-domain pro-apoptotic and BH3-only Bax, Bak, Bok, Bad, Bim and Puma profiles in WSU cell lines and patient-derived fresh samples.

Mentions: To determine if certain Bcl-2 family protein expression profiles are associated with increased susceptibility to TW-37, we determined the expression of major proteins in this family in all 4 cell lines and 5 of the fresh cases using Western Blotting analysis (Fig. 4). In all cases, fresh and cell lines, cells expressed at least 2 of the 3 anti-apoptotic proteins examined (Bcl-2, Bcl-XL, and Mcl-1). Bcl-2 was over-expressed in all fresh cases, and cell lines except the WSU-WM (expressed low levels), Bcl-XL was expressed in all patient cells and cell lines (except WSU-ALL cell line) and Mcl-1 was low only in WSU-ALL, WSU-DLCL2 and pt4. There was variation in the expression of the pro-apoptotic proteins examined. In every case there was at least 3 pro-apoptotic proteins expressed.


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)

Inventory of Bcl-2 family protein by Western-blot quantification of anti-, pro-apoptotic Bcl-2 family protein of 4 NHL cell lines (4.A) and 5 fresh patient derived samples (4.B). Cells were harvested and lysed for Western-blot analysis. Forty μg of total lysate was subjected to detect multi-domain anti-apoptotic proteins Bcl-2, Bcl-XL and Mcl-1 proteins in NHL cell lines and patient derived samples. 80 μg of total cell lysate was loaded to detect multi-domain pro-apoptotic and BH3-only Bax, Bak, Bok, Bad, Bim and Puma profiles in WSU cell lines and patient-derived fresh samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Inventory of Bcl-2 family protein by Western-blot quantification of anti-, pro-apoptotic Bcl-2 family protein of 4 NHL cell lines (4.A) and 5 fresh patient derived samples (4.B). Cells were harvested and lysed for Western-blot analysis. Forty μg of total lysate was subjected to detect multi-domain anti-apoptotic proteins Bcl-2, Bcl-XL and Mcl-1 proteins in NHL cell lines and patient derived samples. 80 μg of total cell lysate was loaded to detect multi-domain pro-apoptotic and BH3-only Bax, Bak, Bok, Bad, Bim and Puma profiles in WSU cell lines and patient-derived fresh samples.
Mentions: To determine if certain Bcl-2 family protein expression profiles are associated with increased susceptibility to TW-37, we determined the expression of major proteins in this family in all 4 cell lines and 5 of the fresh cases using Western Blotting analysis (Fig. 4). In all cases, fresh and cell lines, cells expressed at least 2 of the 3 anti-apoptotic proteins examined (Bcl-2, Bcl-XL, and Mcl-1). Bcl-2 was over-expressed in all fresh cases, and cell lines except the WSU-WM (expressed low levels), Bcl-XL was expressed in all patient cells and cell lines (except WSU-ALL cell line) and Mcl-1 was low only in WSU-ALL, WSU-DLCL2 and pt4. There was variation in the expression of the pro-apoptotic proteins examined. In every case there was at least 3 pro-apoptotic proteins expressed.

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