Limits...
Irreversible dual inhibitory mode: the novel Btk inhibitor PLS-123 demonstrates promising anti-tumor activity in human B-cell lymphoma.

Ding N, Li X, Shi Y, Ping L, Wu L, Fu K, Feng L, Zheng X, Song Y, Pan Z, Zhu J - Oncotarget (2015)

Bottom Line: Here we present a novel covalent irreversible Btk inhibitor PLS-123 with more potent anti-proliferative activity compared with ibrutinib in multiple cellular and in vivo models through effective apoptosis induction and dual-action inhibitory mode of Btk activation.The phosphorylation of BCR downstream activating AKT/mTOR and MAPK signal pathways was also more significantly reduced after treatment with PLS-123 than ibrutinib.In addition, PLS-123 dose-dependently attenuated BCR- and chemokine-mediated lymphoma cell adhesion and migration.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Lymphoma, Peking University Cancer Hospital and Institute, Beijing, China.

ABSTRACT
The B-cell receptor (BCR) signaling pathway has gained significant attention as a therapeutic target in B-cell malignancies. Recently, several drugs that target the BCR signaling pathway, especially the Btk inhibitor ibrutinib, have demonstrated notable therapeutic effects in relapsed/refractory patients, which indicates that pharmacological inhibition of BCR pathway holds promise in B-cell lymphoma treatment. Here we present a novel covalent irreversible Btk inhibitor PLS-123 with more potent anti-proliferative activity compared with ibrutinib in multiple cellular and in vivo models through effective apoptosis induction and dual-action inhibitory mode of Btk activation. The phosphorylation of BCR downstream activating AKT/mTOR and MAPK signal pathways was also more significantly reduced after treatment with PLS-123 than ibrutinib. Gene expression profile analysis further suggested that the different selectivity profile of PLS-123 led to significant downregulation of oncogenic gene PTPN11 expression, which might also offer new opportunities beyond what ibrutinib has achieved. In addition, PLS-123 dose-dependently attenuated BCR- and chemokine-mediated lymphoma cell adhesion and migration. Taken together, Btk inhibitor PLS-123 suggested a new direction to pharmacologically modulate Btk function and develop novel therapeutic drug for B-cell lymphoma treatment.

No MeSH data available.


Related in: MedlinePlus

PLS-123-mediated B-cell lymphoma cytotoxicity is caspase-dependentA. OCI-Ly7 cells (1 × 106) were cultured with 1 μM ibrutinib, 1 μM PLS-123 or vehicle for the indicated periods, and apoptotic cells were analyzed by flow cytometry. B & D. OCI-Ly7 and SU-DHL-2 cells (1 × 106) were treated with 1 μM ibrutinib, 1 μM PLS-123 or vehicle for the indicated periods. Whole cell extracts were probed by Western blot for Caspase-3, Caspase-8, Caspase-9, PARP, XIAP, BCL-xL, BCL-2, MCL-1 and MAX. β-actin is shown as a loading control. C. OCI-Ly7 cells (1 × 106) were pre-treated in presence or absence of the pan-caspase inhibitor z-VAD-fmk for 1 hour and then incubated with 1 μM ibrutinib, 1 μM PLS-123 or vehicle for 24 hours. Caspase-3 enzymatic activity was measured in tumor cells as described in the Materials and Methods. The results were expressed as the mean of the relative activity and S.D. from triplicate cultures. *Significantly changed compared with the control (p < 0.05). Results are representative of at least three similar experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4558140&req=5

Figure 2: PLS-123-mediated B-cell lymphoma cytotoxicity is caspase-dependentA. OCI-Ly7 cells (1 × 106) were cultured with 1 μM ibrutinib, 1 μM PLS-123 or vehicle for the indicated periods, and apoptotic cells were analyzed by flow cytometry. B & D. OCI-Ly7 and SU-DHL-2 cells (1 × 106) were treated with 1 μM ibrutinib, 1 μM PLS-123 or vehicle for the indicated periods. Whole cell extracts were probed by Western blot for Caspase-3, Caspase-8, Caspase-9, PARP, XIAP, BCL-xL, BCL-2, MCL-1 and MAX. β-actin is shown as a loading control. C. OCI-Ly7 cells (1 × 106) were pre-treated in presence or absence of the pan-caspase inhibitor z-VAD-fmk for 1 hour and then incubated with 1 μM ibrutinib, 1 μM PLS-123 or vehicle for 24 hours. Caspase-3 enzymatic activity was measured in tumor cells as described in the Materials and Methods. The results were expressed as the mean of the relative activity and S.D. from triplicate cultures. *Significantly changed compared with the control (p < 0.05). Results are representative of at least three similar experiments.

Mentions: To investigate the mechanisms by which PLS-123 induced stronger cytotoxic effects than ibrutinib in malignant B cells, annexin V- and PI-stained apoptotic cells were analyzed by flow cytometry in the PLS-123-sensitive OCI-Ly7 cells. The percentage of annexin V-positive and PI-negative apoptotic cells peaked 24 hours after PLS-123 treatment (23.5%), whereas ibrutinib resulted in apoptosis in only 9.9% of the cells (Figure 2A). These findings correlated with the cell viability results shown in Figure 1. Furthermore, Western blotting analysis suggested that PLS-123 more strongly activated cleaved caspase-3, -8 and -9 as well as PARP compared with ibrutinib during apoptosis induction in OCI-Ly7 and SU-DHL-2 cells (Figure 2B). To confirm caspase-3 involvement in this apoptosis process, caspase-3 enzymatic activity was determined after treatment with PLS-123 and ibrutinib for indicated period. As shown in Figure 2C, the active form of caspase-3 was more significantly increased upon treatment with PLS-123 compared with ibrutinib. Meanwhile, PLS-123-induced caspase-3 cleavage was completely inhibited by the pan-caspase inhibitor z-VAD-fmk, thereby demonstrating that PLS-123 induced cytotoxicity against B-cell lymphoma cells is caspase-dependent.


Irreversible dual inhibitory mode: the novel Btk inhibitor PLS-123 demonstrates promising anti-tumor activity in human B-cell lymphoma.

Ding N, Li X, Shi Y, Ping L, Wu L, Fu K, Feng L, Zheng X, Song Y, Pan Z, Zhu J - Oncotarget (2015)

PLS-123-mediated B-cell lymphoma cytotoxicity is caspase-dependentA. OCI-Ly7 cells (1 × 106) were cultured with 1 μM ibrutinib, 1 μM PLS-123 or vehicle for the indicated periods, and apoptotic cells were analyzed by flow cytometry. B & D. OCI-Ly7 and SU-DHL-2 cells (1 × 106) were treated with 1 μM ibrutinib, 1 μM PLS-123 or vehicle for the indicated periods. Whole cell extracts were probed by Western blot for Caspase-3, Caspase-8, Caspase-9, PARP, XIAP, BCL-xL, BCL-2, MCL-1 and MAX. β-actin is shown as a loading control. C. OCI-Ly7 cells (1 × 106) were pre-treated in presence or absence of the pan-caspase inhibitor z-VAD-fmk for 1 hour and then incubated with 1 μM ibrutinib, 1 μM PLS-123 or vehicle for 24 hours. Caspase-3 enzymatic activity was measured in tumor cells as described in the Materials and Methods. The results were expressed as the mean of the relative activity and S.D. from triplicate cultures. *Significantly changed compared with the control (p < 0.05). Results are representative of at least three similar experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4558140&req=5

Figure 2: PLS-123-mediated B-cell lymphoma cytotoxicity is caspase-dependentA. OCI-Ly7 cells (1 × 106) were cultured with 1 μM ibrutinib, 1 μM PLS-123 or vehicle for the indicated periods, and apoptotic cells were analyzed by flow cytometry. B & D. OCI-Ly7 and SU-DHL-2 cells (1 × 106) were treated with 1 μM ibrutinib, 1 μM PLS-123 or vehicle for the indicated periods. Whole cell extracts were probed by Western blot for Caspase-3, Caspase-8, Caspase-9, PARP, XIAP, BCL-xL, BCL-2, MCL-1 and MAX. β-actin is shown as a loading control. C. OCI-Ly7 cells (1 × 106) were pre-treated in presence or absence of the pan-caspase inhibitor z-VAD-fmk for 1 hour and then incubated with 1 μM ibrutinib, 1 μM PLS-123 or vehicle for 24 hours. Caspase-3 enzymatic activity was measured in tumor cells as described in the Materials and Methods. The results were expressed as the mean of the relative activity and S.D. from triplicate cultures. *Significantly changed compared with the control (p < 0.05). Results are representative of at least three similar experiments.
Mentions: To investigate the mechanisms by which PLS-123 induced stronger cytotoxic effects than ibrutinib in malignant B cells, annexin V- and PI-stained apoptotic cells were analyzed by flow cytometry in the PLS-123-sensitive OCI-Ly7 cells. The percentage of annexin V-positive and PI-negative apoptotic cells peaked 24 hours after PLS-123 treatment (23.5%), whereas ibrutinib resulted in apoptosis in only 9.9% of the cells (Figure 2A). These findings correlated with the cell viability results shown in Figure 1. Furthermore, Western blotting analysis suggested that PLS-123 more strongly activated cleaved caspase-3, -8 and -9 as well as PARP compared with ibrutinib during apoptosis induction in OCI-Ly7 and SU-DHL-2 cells (Figure 2B). To confirm caspase-3 involvement in this apoptosis process, caspase-3 enzymatic activity was determined after treatment with PLS-123 and ibrutinib for indicated period. As shown in Figure 2C, the active form of caspase-3 was more significantly increased upon treatment with PLS-123 compared with ibrutinib. Meanwhile, PLS-123-induced caspase-3 cleavage was completely inhibited by the pan-caspase inhibitor z-VAD-fmk, thereby demonstrating that PLS-123 induced cytotoxicity against B-cell lymphoma cells is caspase-dependent.

Bottom Line: Here we present a novel covalent irreversible Btk inhibitor PLS-123 with more potent anti-proliferative activity compared with ibrutinib in multiple cellular and in vivo models through effective apoptosis induction and dual-action inhibitory mode of Btk activation.The phosphorylation of BCR downstream activating AKT/mTOR and MAPK signal pathways was also more significantly reduced after treatment with PLS-123 than ibrutinib.In addition, PLS-123 dose-dependently attenuated BCR- and chemokine-mediated lymphoma cell adhesion and migration.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Lymphoma, Peking University Cancer Hospital and Institute, Beijing, China.

ABSTRACT
The B-cell receptor (BCR) signaling pathway has gained significant attention as a therapeutic target in B-cell malignancies. Recently, several drugs that target the BCR signaling pathway, especially the Btk inhibitor ibrutinib, have demonstrated notable therapeutic effects in relapsed/refractory patients, which indicates that pharmacological inhibition of BCR pathway holds promise in B-cell lymphoma treatment. Here we present a novel covalent irreversible Btk inhibitor PLS-123 with more potent anti-proliferative activity compared with ibrutinib in multiple cellular and in vivo models through effective apoptosis induction and dual-action inhibitory mode of Btk activation. The phosphorylation of BCR downstream activating AKT/mTOR and MAPK signal pathways was also more significantly reduced after treatment with PLS-123 than ibrutinib. Gene expression profile analysis further suggested that the different selectivity profile of PLS-123 led to significant downregulation of oncogenic gene PTPN11 expression, which might also offer new opportunities beyond what ibrutinib has achieved. In addition, PLS-123 dose-dependently attenuated BCR- and chemokine-mediated lymphoma cell adhesion and migration. Taken together, Btk inhibitor PLS-123 suggested a new direction to pharmacologically modulate Btk function and develop novel therapeutic drug for B-cell lymphoma treatment.

No MeSH data available.


Related in: MedlinePlus