Bryostatin-1, Fenretinide and 1α,25 (OH)(2)D(3) Induce Growth Inhibition, Apoptosis and Differentiation in T and B Cell-Derived Acute Lymphoblastic Leukemia Cell Lines (CCRF-CEM and Nalm-6)

Abstract

In many acute leukemias, normal differentiation does not occur. However, in many cell lines derived from hematologic malignancies, differentiation or apoptosis can be induced by variety of agents. Despite advances in the treatment of Acute Lymphoblastic Leukemia (ALL), in most patients long-term survival rates remain unsatisfactory, especially in T-cell derived ALL. Thus we studied the anti-cancer effects of fenretinide, 1α,25(OH)(2)D(3), and bryostatin-1 in CCRF-CEM (T-cell derived) and Nalm-6 (B-cell derived) ALL cell lines. Using MTT assays, both cell lines were shown to exhibit increased inhibition of proliferation at micro (fenretinide) and nanomolar (1α,25(OH)(2)D(3), bryostatin-1) concentrations. These anti-cancer agents were shown to induce apoptosis and activate caspase-3 pathway in both ALL cell lines. Furthermore, for the first time we are reporting consistent anti-proliferative and apoptotic effects of Bryostatin-1 in ALL T-cell derived cell line with the lowest ED(50) (ranging 4.6-7.4 nM). To evaluate the differentiation induction by fenretinide, 1α,25(OH)(2)D(3), and bryostatin-1 in ALL cell lines, we assayed for the expressions of CD19, CD38 markers on Nalm-6 and CD7 marker on CCRF-CEM cell line. The flow cytometric analysis showed a significant increase in expression of CD markers in response to anti-cancer drug treatments. To assay the effects of anti-cancer drugs on cell cycle distribution, cell cycle analysis using flow cytometry was employed. These anti-cancer drugs appear to affect the CCRF-CEM and Nalm-6 cell cycles differently (G0/G1 and G2/M arrest, respectively). Overall results demonstrate that the anti-cancer agents used in this study are strong inhibitors of ALL cell proliferation and inducers of apoptosis and differentiation in vitro. These findings may be quite helpful if these drugs are to be used for differentiation therapy of ALL patients in clinics in the future. Further studies are warranted to establish the in vivo effect of these drugs particularly in patients with T-cell derived ALL.

Keywords: Acute lymphoblastic leukemia; Apoptosis; Cell differentiation; Flow cytometry.

Figure 1

Effect of (A) 4-HPR, (B) 1α,25(OH)₂D₃, and (C) Bryostatin-1 on proliferation of two ALL cell lines (CCRF-CEM, Nalm-6). (D and E) Synergistic effects of 4-HPR and 1α,25(OH)₂D₃ (VD₃) on proliferation of CCRF-CEM and Nalm-6 cells. Results show the mean±S.E.M. of three independently performed experiments. Each concentration was tested in eight wells per experiment. P-values are for individual treatment groups compared to control (*p<0.001, **p<0.01, # p<0.05)

Figure 2

Effects of 4-HPR, 1α,25(OH)₂D₃, and Bryostatin-1on cell cycle distribution of CCRF-CEM and Nalm-6 cells. ALL cells were cultured with anti-cancer drugs. After 24 hr, cell distribution was analyzed by flow cytometry

Figure 3

Flow cytometric analysis of cell surface molecules on Nalm-6 and CCRF-CEM cells treated with 4-HPR, 1α,25(OH)₂D₃, and Bryostatin-1. Nalm-6 cells were assessed via flow cytometric analysis using PE-conjugated anti-CD19 mAb (A) or anti-CD38 mAb (B), 24, 48 and 72 hr post treatment. (C) Expression of CD7 by CCRF-CEM cells using FITC-conjugated anti-CD7 mAb. Results represent the mean±S.E.M. of three separate experiments. P-values are for individual treatment groups compared to control (*p < 0.001, **p < 0.01, # p < 0.05).

Figure 4

Flow cytometric analysis of cell surface molecules on Nalm-6 and CCRF-CEM cells treated with 4-HPR, 1α,25(OH)₂ D₃, and Bryostatin-1. Nalm-6 cells were assessed via flow cytometric analysis using PE-conjugated anti-CD19 mAb (A) or anti-CD38 mAb (B), 24, 48 and 72 hr post treatment. (C) Expression of CD7 by CCRF-CEM cells using FITC-conjugated anti-CD7 mAb. Results represent the mean±S.E.M. of three separate experiments. MIF is the mean fluorescence intensity (expressed as the ratio of sample mean channel:control mean channel). P-values are for individual treatment groups compared to control (*p<0.001, **p<0.01, # p<0.05)

Figure 5

Effects of 1α,25(OH)2D3 (VD3) on CD19(A), bryostatin-1 (Bryo1) on CD38(B) and fenretinide (4-HPR) on CD7(C) expressions in ALL cell lines after 72 hr treatment. The cells stained with PE-or FITC- conjugated isotype mAbs were used as a control

Figure 6

Effects of bryostatin-1 (Lane 2), 1α,25(OH)2D3 (Lane 3), and 4-HPR (Lane 4) on activation of caspase-3 (cleaved form) in CCRF-CEM and Nalm-6 cell lines 24 hr post treatment. C: negative control; Lane 1: positive control representing ALL cells treated with Staurosporine. β-actin: loading control