Synergistic Activity of Deguelin and Fludarabine in Cells from Chronic Lymphocytic Leukemia Patients and in the New Zealand Black Murine Model

Abstract

B-cell chronic lymphocytic leukemia (CLL) remains an incurable disease, and despite the improvement achieved by therapeutic regimes developed over the last years still a subset of patients face a rather poor prognosis and will eventually relapse and become refractory to therapy. The natural rotenoid deguelin has been shown to induce apoptosis in several cancer cells and cell lines, including primary human CLL cells, and to act as a chemopreventive agent in animal models of induced carcinogenesis. In this work, we show that deguelin induces apoptosis in vitro in primary human CLL cells and in CLL-like cells from the New Zealand Black (NZB) mouse strain. In both of them, deguelin dowregulates AKT, NFκB and several downstream antiapoptotic proteins (XIAP, cIAP, BCL2, BCL-XL and survivin), activating the mitochondrial pathway of apoptosis. Moreover, deguelin inhibits stromal cell-mediated c-Myc upregulation and resistance to fludarabine, increasing fludarabine induced DNA damage. We further show that deguelin has activity in vivo against NZB CLL-like cells in an experimental model of CLL in young NZB mice transplanted with spleen cells from aged NZB mice with lymphoproliferation. Moreover, the combination of deguelin and fludarabine in this model prolonged the survival of transplanted mice at doses of both compounds that were ineffective when administered individually. These results suggest deguelin could have potential for the treatment of human CLL.

Fig 1. Deguelin induces cytotoxicity dependent on dose in primary CLL cells.

PBMCs from CLL patients (n = 35) and healthy donors (n = 10) were incubated with or without deguelin for 48 h. Apoptosis was evaluated by flow cytometry after double staining with Annexin V-FITC and PI and it is expressed as a percentage relative to a time-matched untreated control containing diluent (0.1% DMSO). (A) Cells from patients and controls were exposed to increasing concentrations of deguelin (Deg). (B) A dose-response curve was constructed for each sample by non-linear regression fit, and the dose cytotoxic to 50% of the cells (ED50) was calculated. Medians are indicated by horizontal lines. *** P < 0.0005 (Mann-Whitney U test). (C) PBMCs from 5 CLL patients and 5 healthy controls were exposed to 10 μM deguelin (Deg) or diluent (-Deg) for 24 hours and then stained for surface CD3 and CD19 markers for T and B lymphocyte identification. After cell permeabilization, filamentous actin (F-actin) was stained with a fluorescence-conjugated phalloidin and analyzed by flow cytometry (representative samples are shown). Apoptosis was quantified as the loss of F-actin fluorescence in T (dotted line, grey dots) or B lymphocyte subpopulations (solid line, black dots). (D) ED50 values from patients were grouped into previously treated or untreated patients. (E) The same as in (D), in this case stratified by the prognostic markers: percentage of CD38 expression, ZAP70 expression, IgVH mutation status and the presence of cytogenetic alterations different from 13q14 deletions. Horizontal lines represent the median. * P < 0.05 (Mann-Whitney U test).

Fig 2. Deguelin reduces AKT signaling and induces apoptosis via the mitochondrial route in primary CLL cells.

(A) PBMCs from 3 CLL patients were treated with 0, 10, or 100 μM deguelin and viability was evaluated by flow cytometry after Annexin V/IP staining at 24 h (bottom-right bar graphic). Cell lysates were then separated by SDS-PAGE, transferred onto PVDF membranes and analyzed by western blot. Immunoreactive bands for several deguelin target molecules and β-actin from a respresentative cell lysate were visualized with the ECL kit. A representative CLL sample is shown. Numbers indicate the signal intensity of each band from deguelin treated samples (Deg 10, Deg 100) compared to the untreated ones (Deg 0). For caspases (CASP3, CASP9) and PARP, the numbers indicate the proportion of cleaved fragments relative to the total protein (intact plus cleaved). (B) PBMCs from 3 CLL patients and 4 healthy controls were cultured for 24 h in the presence of up to 10 μM deguelin (Deg). The mitochondrial membrane potential was analyzed by flow cytometry after JC-1 staining for 15 min. As depolarization control cells were treated with 50 μM CCCP. Histograms show red fluorescence profiles in samples from a representative control and a CLL patient. (C) Cells from a representative CLL patient were treated with 50μM z-VAD-fmk in the presence or absence of 10 μM deguelin. After 16 h, the percentage of apoptotic cells was determined by Annexin V-FITC/PI staining and flow cytometry (left panels). Caspase 3 activation was assessed in protein lysates of treated cells by western blotting (right panel). The numbers indicate the proportion of cleaved fragments relative to the total protein (intact plus cleaved).

Fig 3. Deguelin overcomes microenvironment mediated pro-survival effects on primary CLL cells.

Primary CLL cells (n = 8) were preincubated for 1 hour with increasing doses of deguelin (Deg; 0–100 μM), and co-cultured in the presence or absence of the murine Ltk^- feeder cell line and fludarabine (Flu; 0–50 μM). After 48h, apoptosis was determined by Annexin V/PI staining and flow cytometry. (A) ED50 was calculated for each sample. Horizontal lines represent the median. * P < 0.05 (Wilcoxon matched paired test). (B) Deguelin and fludarabine were combined at a constant 1:2 molar ratio over the same range of concentrations as in (A), and the combination index (CI) values were computed at several affected fractions (ED50, ED75, ED90). Mean values and SD are shown, CI < 1 indicate synergy.

Fig 4. Deguelin downregulates AKT/NFκB mediated prosurvival signals in CLL cells.

(A) PBMCs from 3 CLL patients were treated for 24h with 0, 10, or 100 μM deguelin. Viability was evaluated by flow cytometry after Annexin V/IP staining. Figure shows immunoreactive bands for several deguelin target molecules and β-actin in western blots from a representative sample. Numbers indicate the signal intensity of β-actin-normalized bands from drug treated samples compared to the untreated ones (Control). (B) PBMCs from two CLL patients were treated with 0, 10, or 100 μM deguelin or 1 μg/ml fludarabine and co-cultured with Ltk^- 24h. Cell viability assays and western blot from a representative sample were done as in (A). (C) PBMCs from 2 CLL patients were co-cultured with Ltk^- or 3T3-CD40L cells and p-AKT expression was evaluated in CLL cells by intracellular staining and flow cytometry at different time points. Histograms show the stains in one of the samples (solid lines from left to right: p-AKT staining at 0, 24, 48 and 120h; dotted lines from left to right: unstained control (samples without primary antibody plus secondary antibody) and isotype control. The lower graph shows the differences in the Mean Fluorescence Intensity (MFI, in channel numbers) of p-AKT compared to the unstained control in both samples (bars show mean and SD). The dotted horizontal line indicates the mean difference in MFI (10 ± 5) between the isotype controls and corresponding unstained controls in the samples with both stains (n = 8), that represents an estimation of the limit for a clearly specific fluorescence signal. (D) PBMCs from 2 CLL patients were treated with 10 μM deguelin, 1 μg/ml fludarabine or the combination of both, and co-cultured with the 3T3-CD40L cell line up to 120h. Upper graphs show viability of CLL cells in both samples (black: control; green: deguelin; red: fludarabine; blue: deguelin plus fludarabine; dotted lines from left to right: unstained control and isotype control). FSC/SSC plots from samples treated with the combination of both drugs are also shown. Histograms show the expression of p-AKT, p-p65 and c-Myc in a representative sample evaluated by intracellular staining and flow cytometry and lower graphs show MFI differences in both samples as in (C).

Fig 5. Potentiation of fludarabine action by deguelin.

PBMCs from 2 CLL patients were treated with 10 μM deguelin, 1 μg/ml fludarabine or the combination of both, and co-cultured with 3T3-CD40L cell line up to 120h (same samples as in Fig 4B, 4C and 4D). Histograms show the expression of γ-H2AX in a representative sample evaluated by intracellular staining and flow cytometry (black: control; green: deguelin; red: fludarabine; blue: deguelin plus fludarabine; dotted lines from left to right: unstained control and isotype control (see Fig 4C)). The lower graphs shows the differences in the Mean Fluorescence Intensity (MFI) of γ-H2AX staining compared to the unstained control in both samples (bars show mean and SD). The dotted horizontal lines indicate the mean difference in MFI between the isotype controls and corresponding unstained controls (see Fig 4C).

Fig 6. Deguelin induces apoptosis in NZB CLL-like cells.

(A) Mononuclear cells isolated from 8 NZB spleens were cultured in the presence of deguelin (Deg; 0–10 μM) and apoptosis was quantified at 24h. (B) Cell lysates obtained from splenic NZB cells treated with 10 μM deguelin for 24h were analyzed by western blot. Immunoreactive bands for several deguelin target molecules from a representative sample are shown. Numbers indicate the signal intensity of β-actin-normalized bands from deguelin treated samples (Deg 10) compared to the untreated ones (Deg 0). For PARP, numbers indicate the proportion of cleaved fragments relative to total protein (intact plus cleaved). (C) Splenic mononuclear cells from 8 NZB mice were treated with increasing doses of deguelin (0–100 μM), fludarabine (0–50 μM) or combinations of both (at a constant 1:2 molar ratio) and co-cultured with or without Ltk^-. After 48h, the combination index (CI) values were computed at several affected fractions (ED50, ED75, ED90). Mean values and SD are shown. CI < 1 indicate synergy.

Fig 7. Effect of deguelin in vivo in transplanted NZB mice.

Five transplanted animals with splenomegaly were selected to study the effect of deguelin in vivo. Two mice received deguelin (4 mg/kg in corn oil) twice a day for three consecutive days by i.g. route and the other three received vehicle. Mice were then sacrificed, spleens were excised and samples for mononuclear cell isolation and histological analysis were obtained. (A) Flow-cytometric analysis of NZB spleen cells (left panels), with neoplasic B220^low CD5^low IgM⁺ cells represented by black dots. Analysis of DNA content from NZB splenic cells are shown in middle panels. Blue peaks represent apoptotic cells, red ones normal diploid cells and yellow ones hyperdiploid neoplastic cells (peaks shown are computer modeled). Histological analysis of spleens stained with hematoxylin and eosin (HE) are shown in right panels (× 40 magnification, scale bar 50 μm). Data from two untreated animals (Control) and two treated animals (Deg) are shown. (B) Paraffin embedded spleen sections from untreated (Control) and deguelin-treated (Deg) animals were processed for immunohistochemical staining of the indicated protein. Brown = specific protein staining, Blue = counterstaining of nuclei with hematoxylin. Representative images were taken at 100 x augments (scale bar 10 μm).

Fig 8. The combination of deguelin and fludarabine prolongs the survival of transplanted NZB mice.

40 transplanted young NZB mice were randomly distributed in four groups and transplanted with leukemic spleen cells from aged NZB. Control group (Control, solid black line) received vehicle i.g. and i.p. Fludarabine (Flu) group received 3 cycles of 35 mg/kg fludarabine i.p., five consecutive days each 28 days. Deguelin group (Deg) received 4 mg/kg deguelin in corn oil i.g. three days per week for 61 days. The last group (Combination) received deguelin plus fludarabine as in groups Deg and Flu. Treatment schedule is shown in the lower diagram (Deg:||||; Flu: ↓). After treatment, mice were monitored and survival was evaluated. Graph shows survival of mice in the four groups (see legend). *P = 0.022 (Gehan-Breslow-Wilcoxon test).