Teriflunomide (Leflunomide) Promotes Cytostatic, Antioxidant, and Apoptotic Effects in Transformed Prostate Epithelial Cells: Evidence Supporting a Role for Teriflunomide in Prostate Cancer Chemoprevention

Teriflunomide (TFN) is an inhibitor of de novo pyrimidine synthesis and the active metabolite of leflunomide. Leflunomide is prescribed to patients worldwide as an immunomodulatory and anti-inflammatory disease-modifying prodrug. Leflunomide inhibited the growth of human prostate cancer xenographs in mice, and leflunomide or TFN promoted cytostasis and/or apoptosis in cultured cells. These findings suggest that TFN could be useful in prostate cancer chemoprevention. We investigated the possible mechanistic aspects of this tenet by characterizing the effects of TFN using premalignant PWR-1E and malignant DU-145 human prostate epithelial cells. TFN promoted a dose- and time-dependent cytostasis or apoptosis induction in these cells. The cytostatic effects of TFN, which were reversible but not by the presence of excess uridine in the culture medium, included diminished cellular uridine levels, an inhibition in oxygen consumption, a suppression of reactive oxygen species (ROS) generation, S-phase cell cycle arrest, and a conspicuous reduction in the size and number of the nucleoli in the nuclei of these cells. Conversely, TFN's apoptogenic effects were characteristic of catastrophic mitochondrial disruption (i.e., a dissipation of mitochondrial inner transmembrane potential, enhanced ROS production, mitochondrial cytochrome c release, and cytoplasmic vacuolization) and followed by DNA fragmentation. The respiration-deficient derivatives of the DU-145 cells, which are also uridine auxotrophs, were markedly resistant to the cytostatic and apoptotic effects of TFN, implicating de novo pyrimidine synthesis and mitochondrial bioenergetics as the primary targets for TFN in the respiration competent cells. These mechanistic findings advocate a role for TFN and mitochondrial bioenergetics in prostate cancer chemoprevention

Phosphorylation of GSK3α/β correlates with activation of AKT and is prognostic for poor overall survival in acute myeloid leukemia patients

Background: Acute myeloid leukemia (AML) patients with highly active AKT tend to do poorly. Cell cycle arrest and apoptosis are tightly regulated by AKT via phosphorylation of GSK3α and β isoforms which inactivates these kinases. In the current study we examine the prognostic role of AKT mediated GSK3 phosphorylation in AML. Methods: We analyzed GSK3α/β phosphorylation by reverse phase protein analysis (RPPA) in a cohort of 511 acute myeloid leukemia (AML) patients. Levels of phosphorylated GSK3 were correlated with patient characteristics including survival and with expression of other proteins important in AML cell survival. Results: High levels of p-GSK3α/β correlated with adverse overall survival and a lower incidence of complete remission duration in patients with intermediate cytogenetics, but not in those with unfavorable cytogenetics. Intermediate cytogenetic patients with FLT3 mutation also fared better respectively when p-GSK3α/β levels were lower. Phosphorylated GSK3α/β expression was compared and contrasted with that of 229 related cell cycle arrest and/or apoptosis proteins. Consistent with p-GSK3α/β as an indicator of AKT activation, RPPA revealed that p-GSK3α/β positively correlated with phosphorylation of AKT, BAD, and P70S6K, and negatively correlated with β-catenin and FOXO3A. PKCδ also positively correlated with p-GSK3α/β expression, suggesting crosstalk between the AKT and PKC signaling pathways in AML cells. Conclusions: These findings suggest that AKT-mediated phosphorylation of GSK3α/β may be beneficial to AML cell survival, and hence detrimental to the overall survival of AML patients. Intrinsically, p-GSK3α/β may serve as an important adverse prognostic factor for a subset of AML patients

AMD3465 reduces CD11b positive cells within metastatic lesions

<p>. <b>A,</b> A quantitative representations of CD11b positive cells in the lungs, liver, and spleen of immunocompetent syngeneic mouse model with the indicated cell line were calculated and the results are shown as bar graphs. The reductions in CD11b positive cells after a 14-d AMD3465 treatment in lung, spleen, and liver was observed in all three different breast cancer cell lines in an immunocompetent syngeneic mouse model. The percent positive cells were calculated based on total number of cells counted per image in triplicate samples, and expressed as the mean value ± SD (error bars) (**p<0.01 and ***P<0.001). <b>B,</b> A qualitative immunohistochemical depiction of the CD11b positive cells in representative lung tissues is shown for the 4T1, 4T07, and 168Farn cells in the immunocompetent syngeneic mouse model. <b>C,</b> Co-staining of metastatic nodules with CXCR4 (green fluorescence) and CD11b (red fluorescence) revealed ∼66% of the cells in the field are positive both for CD11b and CXCR4 (resulting yellow fluorescence) based on quantitative analysis of 10 images of the spleen tissue harvested from PBS-treated mice.</p

AMD3465 affects the <i>in vitro</i> invasiveness of 4T1 cells.

<p>4T1 cells in serum-free medium were seeded in matrigel transwells and allowed to migrate 48 h towards compartments with medium containing 10% FBS without (PBS) and with AMD3465 (2.5, 5, and 10 µM) present. All of the samples were conducted in triplicate and expressed as the mean value ± SD (error bars, **p<0.001).</p

AMD3465 reduces tumor metastases in a syngenic breast cancer model.

<p>The size/number (indicated by arrows) of metastatic nodules in the 4T1 tumor bearing mice treated with PBS (control) or AMD3465 in both the lung and liver as determined by H&E staining. We also confirmed the metastatic nodules were GFP positive as were the primary tumors. A detailed treatment procedure for the metastasis assay is described in the Method section.</p

AMD3465 inactivates CXCR4 in 4T1 tumors and slows tumor progression.

<p><b>A,</b> Tumor-bearing mice were injected with AMD3465 a single subcutaneous dose of 2.5 mg/kg. The tumor tissue was collected 1 h after treatment and sectioning was carried out. Immunohistochemical staining of pCXCR4, pAKT and pERK1/2 positive tumor cells can be seen in PBS controls compared to AMD3465 treated tumor sections. The slides were analyzed with an Olympus BX 41 microscope equipped with a digital camera (Olympus DP70). <b>B,</b> The top panel illustrates the BLI of 5 representative mice 1 d after injection of the 4T1 cells. The middle panel shows representative BLI in 5 mice treated with PBS 20 d after tumor injection, and the lower panel displays imaging of the 4T1 tumor masses following a similar exposure to AMD3465 (please see the Methods section for details). <b>C,</b> A bar graph representation of the end-point integrated photon (photons/cm²/sec) data collected in the experiment described in <b>B</b>. The tumor size was measured by BLI between control mice (n = 5) and AMD3465-treated mice (n = 10) and are expressed as the mean value ± SD (*p<0.05).</p

AMD3465 modulates intracellular oncogenic signaling mediators in mouse breast cancer cell lines.

<p><b>A,</b> A western blot analysis showing a 24-h knockdown of STAT3 expression using shRNA in the 4T1 cells and the concomitant abrogation of CXCR4 expression. <b>B,</b> Western blot analysis of oncogenic intermediates following a 24-h treatment of the 4T1 cells with 5 µM AMD3465. The band intensities ware normalized relative to β-actin expression and presented as % of Control in <b>C</b>. <b>D</b> and <b>E,</b> Western blot analyses of oncogenic intermediates following a 24-h treatment of the 4T1, 4T07, and 168Fran cells with 5 µM AMD3465.</p

AMD3465 Inhibits Breast Cancer Metastases.

<p>Site and number of metastases (Met.),</p>*<p>p<0.001 and **p<0.01 statically significant differences.</p