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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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Acridine orange/ethidium bromide (AO/EB) staining showing apoptosis induction by TW-37. A, Apoptosis induction of TW-37 on 4 WSU-cell lines was assayed after 72-h exposure. WSU cell lines were seeded and treated with 500 nM of TW-37 and with inactive congener TW-37a [designated as "(-)"] in triplicate and apoptosis was determined by AO/EB staining after 72 h. B, Apoptosis induction of TW-37 on patient-derived NHL cells was determined on 3 selected samples. Apoptotic cells were assayed by AO/EB staining after exposure of TW-37 with concentrations ranging from 0 to 750 nM (0 is the same as TW-37a). C, Bax-to-Mcl-1 ratio positively correlates with induction of apoptosis by TW-37. The Bax/Mcl-1 ratio was plotted on the abscissa against this AO/EB metric on the ordinate for four cell lines (the filled diamonds represent 48 h and the empty squares represent 72 h treatments). Each line is calculated by linear regression using equal weighting of the four points; the lines described closely emanate from the origin (x-intercept = 0.046 to 0.084). Patient data (Patients-1 and 3, empty triangles) lie close to the lines fitted to the data for the four established NHL cell lines.
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Figure 2: Acridine orange/ethidium bromide (AO/EB) staining showing apoptosis induction by TW-37. A, Apoptosis induction of TW-37 on 4 WSU-cell lines was assayed after 72-h exposure. WSU cell lines were seeded and treated with 500 nM of TW-37 and with inactive congener TW-37a [designated as "(-)"] in triplicate and apoptosis was determined by AO/EB staining after 72 h. B, Apoptosis induction of TW-37 on patient-derived NHL cells was determined on 3 selected samples. Apoptotic cells were assayed by AO/EB staining after exposure of TW-37 with concentrations ranging from 0 to 750 nM (0 is the same as TW-37a). C, Bax-to-Mcl-1 ratio positively correlates with induction of apoptosis by TW-37. The Bax/Mcl-1 ratio was plotted on the abscissa against this AO/EB metric on the ordinate for four cell lines (the filled diamonds represent 48 h and the empty squares represent 72 h treatments). Each line is calculated by linear regression using equal weighting of the four points; the lines described closely emanate from the origin (x-intercept = 0.046 to 0.084). Patient data (Patients-1 and 3, empty triangles) lie close to the lines fitted to the data for the four established NHL cell lines.

Mentions: TW-37 induced significant apoptosis in the cell lines and fresh patient samples (Fig. 2). This effect was specific since there was significant difference between TW-37 and TW-37a used under the same conditions. The highest proportion of cells in apoptosis was observed in WSU-FSCCL indicating higher sensitivity to TW-37 whereas the lowest was in WSU-WM (Fig. 2A). Similarly, TW-37 induced apoptosis on each of the three patient samples examined (Fig. 2B) with lower values in pt.2 that also showed less growth inhibition (Fig. 1B). Interestingly, the Bax-to-Mcl-1 ratio positively correlated with induction of apoptosis in the cell lines and in the 2 fresh cases studied (R2 = 0.9682 and 0.9653 after 48 and 72 h of exposure to TW-37, respectively, Figure 2C).


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)

Acridine orange/ethidium bromide (AO/EB) staining showing apoptosis induction by TW-37. A, Apoptosis induction of TW-37 on 4 WSU-cell lines was assayed after 72-h exposure. WSU cell lines were seeded and treated with 500 nM of TW-37 and with inactive congener TW-37a [designated as "(-)"] in triplicate and apoptosis was determined by AO/EB staining after 72 h. B, Apoptosis induction of TW-37 on patient-derived NHL cells was determined on 3 selected samples. Apoptotic cells were assayed by AO/EB staining after exposure of TW-37 with concentrations ranging from 0 to 750 nM (0 is the same as TW-37a). C, Bax-to-Mcl-1 ratio positively correlates with induction of apoptosis by TW-37. The Bax/Mcl-1 ratio was plotted on the abscissa against this AO/EB metric on the ordinate for four cell lines (the filled diamonds represent 48 h and the empty squares represent 72 h treatments). Each line is calculated by linear regression using equal weighting of the four points; the lines described closely emanate from the origin (x-intercept = 0.046 to 0.084). Patient data (Patients-1 and 3, empty triangles) lie close to the lines fitted to the data for the four established NHL cell lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Acridine orange/ethidium bromide (AO/EB) staining showing apoptosis induction by TW-37. A, Apoptosis induction of TW-37 on 4 WSU-cell lines was assayed after 72-h exposure. WSU cell lines were seeded and treated with 500 nM of TW-37 and with inactive congener TW-37a [designated as "(-)"] in triplicate and apoptosis was determined by AO/EB staining after 72 h. B, Apoptosis induction of TW-37 on patient-derived NHL cells was determined on 3 selected samples. Apoptotic cells were assayed by AO/EB staining after exposure of TW-37 with concentrations ranging from 0 to 750 nM (0 is the same as TW-37a). C, Bax-to-Mcl-1 ratio positively correlates with induction of apoptosis by TW-37. The Bax/Mcl-1 ratio was plotted on the abscissa against this AO/EB metric on the ordinate for four cell lines (the filled diamonds represent 48 h and the empty squares represent 72 h treatments). Each line is calculated by linear regression using equal weighting of the four points; the lines described closely emanate from the origin (x-intercept = 0.046 to 0.084). Patient data (Patients-1 and 3, empty triangles) lie close to the lines fitted to the data for the four established NHL cell lines.
Mentions: TW-37 induced significant apoptosis in the cell lines and fresh patient samples (Fig. 2). This effect was specific since there was significant difference between TW-37 and TW-37a used under the same conditions. The highest proportion of cells in apoptosis was observed in WSU-FSCCL indicating higher sensitivity to TW-37 whereas the lowest was in WSU-WM (Fig. 2A). Similarly, TW-37 induced apoptosis on each of the three patient samples examined (Fig. 2B) with lower values in pt.2 that also showed less growth inhibition (Fig. 1B). Interestingly, the Bax-to-Mcl-1 ratio positively correlated with induction of apoptosis in the cell lines and in the 2 fresh cases studied (R2 = 0.9682 and 0.9653 after 48 and 72 h of exposure to TW-37, respectively, Figure 2C).

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