Fludarabine induces apoptosis in chronic lymphocytic leukemia - the role of P53, Bcl-2, Bax, Mcl-1, and Bag-1 proteins

Abstract

The expression of P53, Bcl-2, Bax, Bag-1, and Mcl-1 proteins in CD5/ CD20-positive B-chronic lymphocytic leukemia (B-CLL) cells from 30 typical CLL patients was evaluated before and after 48 h of incubation with 10 -6 M fludarabine using multiparametric flow cytometric analysis. Protein expression was correlated with annexin V expression, Rai modified clinical staging, lymphocyte doubling time, and previous treatment. Our main goal was to determine the predictive value of these proteins in CLL cells in terms of disease evolution. Bcl-2 expression decreased from a median fluorescence index (MFI) of 331.71 ± 42.2 to 245.81 ± 52.2 (P < 0.001) after fludarabine treatment, but there was no difference between viable cells (331.57 ± 44.6 MFI) and apoptotic cells (331.71 ± 42.2 MFI) before incubation (P = 0.859). Bax expression was higher in viable cells (156.24 ± 32.2 MFI) than in apoptotic cells (133.56 ± 35.7 MFI) before incubation, probably reflecting defective apoptosis in CLL (P = 0.001). Mcl-1 expression was increased in fludarabine-resistant cells and seemed to be a remarkable protein for the inhibition of the apoptotic process in CLL (from 233.59 ± 29.8 to 252.04 ± 35.5; P = 0.033). After fludarabine treatment, Bag-1 expression was increased in fludarabine-resistant cells (from 425.55 ± 39.3 to 447.49 ± 34.5 MFI, P = 0.012), and interestingly, this higher expression occurred in patients who had a short lymphocyte doubling time (P = 0.022). Therefore, we could assume that Bag-1 expression in such situation might identify CLL patients who will need treatment earlier.

Effective elimination of fludarabine-resistant CLL cells by PEITC through a redox-mediated mechanism

“…42 Here, we provide evidence that MCL1 may be a redox-sensitive protein and its glutathionylation status could affect its stability. Because MCL1 has a critical role in prolonging the CLL cell survival, especially in a tumor-stroma context, 43,44 and is strongly associated with resistance to fludarabine, 28,29 rituximab, 45 chlorambucil, and prednisone, 46 the thiol-mediated abrogation of MCL1 by PEITC suggests a novel mechanism that may be exploited to overcome drug resistance by combining PEITC with these anti-CLL agents.…”

“…Interestingly, PEITC caused a rapid decrease of MCL1 but not BCL2 in both F-ara-A-sensitive and -resistant CLL cells ( Figure 6A,B). Because a high level of MCL1 was shown to mediate resistance to fludarabine in vivo and in vitro, 28,29 abrogation of MCL1 by PEITC likely facilitated the killing of F-ara-A-refractory CLL cells. As MCL1 may be a better substrate for caspase than BCL2, 30 we used the pan-caspase inhibitor Z-VAD-fmk to test if it could prevent PEITC-induced MCL1 degradation, and showed that the 20 M Z-VAD-fmk largely suppressed MCL1 degradation in PEITC-treated cells ( Figure 6C).…”

Clinical significance of bax/bcl-2 ratio in chronic lymphocytic leukemia

“…7,33,43,44 In particular, significant differences in treatment histories were found between bax/bcl-2 positive and negative patients. In fact, bax/bcl-2 negative patients underwent treatment more frequently and rapidly than bax/bcl-2 positive patients.…”

Utilization of genomic signatures to identify high‐efficacy candidate drugs for chemorefractory endometrial cancers

“…The exact mechanism of apoptosis induction by F‐ara‐A in proliferative and quiescent cells has not been completely clarified although purine nucleoside analogs are reported to activate d‐ATP‐dependent caspase pathways . To investigate the cytotoxic effect of fludarabine, in vitro proliferation assays were done on three endometrial cancer cell lines, which were chosen as representative cell lines according to their probability of sensitivity to conventional chemo agents, as described in the Material and Methods section.…”

“…The exact mechanism of apoptosis induction by F-ara-A in proliferative and quiescent cells has not been completely clarified although purine nucleoside analogs are reported to activate d-ATP-dependent caspase pathways. 26 To investigate the cytotoxic effect of fludarabine, in vitro proliferation assays were done on three endometrial cancer cell lines, which were chosen as representative cell lines according to their probability of sensitivity to conventional chemo agents, as described in the Material and Methods section. As shown in Figure 3, fludarabine has a cytotoxic effect on endometrial cancer, inhibiting tumor growth and inducing apoptosis in a dose-dependent manner in vitro, suggesting that fludarabine may inhibit the proliferation of endometrial cancer cells through induction of apoptosis, consistent with reports using Jurkat cells.…”