Triterpenoids display single agent anti-tumor activity in a transgenic mouse model of chronic lymphocytic leukemia and small B cell lymphoma

Abstract

Background: The synthetic triterpenoid 2-Cyano-3,12-Dioxooleana-1,9-Dien-28-Oic Acid (CDDO) and derivatives display anti-tumor activity against a variety of cultured tumor cell lines and in mouse xenografts. In this report, we have studied the effects of CDDO and its imidazolide derivative (CDDO-Im) on chronic lymphocytic leukemia (CLL), using patients' CLL cells and a mouse model of CLL and small B cell lymphoma (SBL).

Principal findings: CDDO and CDDO-Im efficiently induced apoptosis of malignant human and mouse B-cells ex vivo, although CDDO-Im was over 10-fold more potent than CDDO. Treating mice with CLL/SBL with liposome-formulated CDDO or CDDO-Im resulted in significant reductions of B cells in blood, spleen and lung. CDDO-Im was shown to be more potent than CDDO, while treatment with empty liposomes had no impact on disease. CDDO-Im treatment initially resulted in an increase of circulating B cells, which correlates with a reduction in resident lymphocytes in spleen, and lungs, suggesting that CDDO-Im induces mobilization of tumor cells from lymphoid organs and infiltrated tissues into the circulation. Analysis of blood cells recovered from treated mice also showed that CDDO-Im is a potent inducer of tumor cells death in vivo. Furthermore, CDDO-Im efficiently eradicated mouse CLL/SBL cells but had little effect on the viability of normal B and T cells in vivo.

Significance: The presented data demonstrate that triterpenoids CDDO and CDDO-Im reduce leukemia and lymphoma burden in vivo in a transgenic mouse model of CLL/SBL, and support the clinical testing of CDDO-based synthetic triterpenoids in patients with CLL.

Figure 1. Chemical structures of CDDO and CDDO-imidazolide.

The synthesis of CDDO and CDDO-Im was previously described , .

Figure 2. Effects of CDDO and CDDO-Im on apoptosis of human CLL and TRAF2DN/Bcl-2 cells.

Human CLL cells (A) and splenocytes isolated from TRAF2DN/Bcl-2 transgenic mice (B) were cultured with or without CDDO and CDDO-Im at the indicated concentrations. Human CLL cells (n = 4) were obtained from 2 patients previously untreated with standard therapies (⋄ and X), one patient refractory to chlorambucil (▪) and one patient refractory to F-ara-A (Δ). Cells were harvested after 24 h, washed and incubated with Annexin-V-FITC and PI. Mouse splenocytes (n = 6) were also harvested after 24 h, but first they were incubated with APC-anti-B220 mAb for 30 min, then washed and incubated with Annexin-V-FITC and PI. Lymphocytes (A) or B lymphocytes (B220⁺ cells) (B) were gated and the percentage of apoptotic cells (Annexin V⁺) was determined by flow cytometry. The percentage of non-apoptotic viable cells is shown. Data were corrected for differences in spontaneous cell death.

Figure 3. In vivo effect of CDDO and CDDO-Im on CLL/SBL cells and normal lymphocytes (A).

Mice with leukemia were treated as indicated. Total numbers of circulating B cells (B220⁺) in blood were quantified 1–2 days before the beginning of the treatment (pre) and 7–10 days after the end of treatment (post). Statistical significance was determined by Student's two-tailed paired t-test. (B) Total numbers of circulating B (B220⁺) and T (CD4⁺) cells were quantified in blood before and after treatment with empty liposomes (n = 5), CDDO (20 mg/Kg; n = 5), or CDDO-Im at 5 (n = 6) or 10 mg/Kg (n = 9). The ratio of the concentration of cells before and after the treatment was calculated. Figure shows the average percentages ±SEM. (C). Wild-type mice were treated with 5 or 10 mg/Kg CDDO-Im and the concentration of B (B220+) or T (CD4+) lymphocytes in blood before and after the treatment was determined. Figure shows the average percentages ±SEM as described in B.

Figure 4. CDDO-Im induces massive cell death in vivo.

A. The percentage of viable (black columns) and apoptotic (dotted columns) cells in the blood of representative mice after 3 inoculations with empty liposomes or liposomes containing CDDO-Im is shown. Viability was assessed using an assay based on the alterations in the permeability of cells at different viability stages using a combination of DNA-binding dyes (ViaCount, Guava Technologies) and a Guava PCA96 fluorometer and analyzed with Guava's multi-caspase software. B. Representative flow cytometry profile of the viability of blood cells from mice treated with empty liposomes or liposomes containing CDDO-Im using Viacount (Guava Technologies). Analysis was performed with Flowjo software (Tree Star, Inc, Ashland, OR). PM2 (red channel) shows nuclear staining of cells using a cell permeable dye. PM1 (yellow-orange channel) shows staining of non-viable cells with a non-cell permeable dye.

Figure 5. Amelioration of splenomegaly by CDDO and CDDO-Im treatment.

A. TRAF2DN/Bcl-2 mice with leukemia were treated with empty liposomes or with liposomes containing either CDDO (20 mg/Kg/day) or CDDO-Im (5 or 10 mg/Kg/day). Mice were euthanized 7–10 days after the final drug administration, and the spleens were weighted. The average weight ±SEM of the spleens after the different treatments was: empty liposomes: 1405±133 mg, n = 7; CDDO: 937±117, n = 4; CDDO-Im (5 mg/Kg): 821±83, n = 4; and CDDO-Im (10 mg/Kg): 635±71 mg, n = 6. Statistical significance of liposomes vs CDDO (*, p = 0.04) and liposomes vs CDDO-Im (5 mg/Kg**, p = 0.013; 10 mg/Kg*** p = 0.0005) was determined using unpaired t-test. B. Total number of lymphocytes isolated from spleens of mice treated with empty liposomes (324±44.7×10⁶; n = 3) or with liposomes containing CDDO-Im (10 mg/Kg) (12.7±6.7×10⁶; n = 3) is shown.

Figure 6. Histochemical analysis of the spleen and lung of leukemic mice treated with empty liposomes or liposomes containing CDDO or CDDO-Im.

Tissues and organs from TRAF2DN/Bcl2 leukemic mice were treated as indicated in the figure. After treatment, tissues were fixed in Z-fix solution (Anatech Ltd.), embedded in paraffin, and tissue sections (5 µm) were stained with hematoxylin and eosin (H&E). One representative example for each treatment is shown. Arrows indicate the presence of infiltrating lymphocytes.

Figure 7. Analysis of B cell populations in spleen and blood of leukemic mice treated with empty liposomes or liposomes containing CDDO (20 mg/Kg) or CDDO-Im (10 mg/Kg).

Three-color flow-cytometry analysis was performed to determine the phenotype of B lymphocytes. Two different gates were used to identify normal B cells and CLL/SBL. First, lymphocytes were selected by gating the lymphocyte population in a forward scattered (FSC) and side scattered (SSC) plot (not shown). Then, B cell populations were identified by plotting B220 expression and FCS. Gate R1 contains cells with high expression of B220 (B220^H) and small size (FSC^L). Red dots within gate R1 were also contained in the lymphocyte gate and represent normal B cells. Gate R2 includes cells with medium expression of B220 (B220^M) and larger in size (FSC^M). Purple dots within gate R2 were also contained in the lymphocyte gate and represent CLL/SBL cells. The analysis of the lymphocyte populations expressing B220 and CD5 is also shown. Representative results are provided for mice that completed the treatment with each drug.

Figure 8. Determination of the total number of normal B lymphocytes and CLL/SBL cells in spleen of mice treated with empty liposomes or with CDDO-Im containing liposomes.

The total number of B lymphocytes (CD45⁺/B220⁺) in spleen was quantified using a cell analysis and microfluorocytometer (Guava technologies). The percentages of the B220^H FSC^L normal B cells and of the B220^M FSC^M CLL/SBL cells were assessed by FACS (see Figure 6). The total number of CLL/SBL cells (white) and normal B lymphocytes (grey) in 3 mice treated with 10 mg/Kg CDDO-Im and 3 mice treated with empty liposomes is shown.