Advertisement

Novel di-2-pyridyl–derived iron chelators with marked and selective antitumor activity: in vitro and in vivo assessment

Jun Yuan, David B. Lovejoy and Des R. Richardson

Article Figures & Data

Figures

  • Figure 1.

    Structural formulas of the Fe chelators described in this study. (A) General structure of the DpT analogs showing the numbering scheme used. (B) Structures of DFO, 311, 3-AP, and PKIH. (C) Structures of DpT, Dp2mT, Dp4mT, Dp44mT, Dp4eT, Dp4aT, and Dp4pT.

  • Figure 2.

    The most effective DpT analogs and 311 (internal control) show selective antiproliferative activity against immortal SK-N-MC neuroepithelioma cells (S) compared with mortal MRC-5 fibroblasts (F). Cells were incubated in the presence or absence of the chelators (0-25 μM) for 72 hours at 37° C.23 After this incubation period, cellular density was measured using the MTT assay. Cellular proliferation was expressed as a percentage of that found for the untreated cells. Each data point represents the mean of 2 replicates in a typical experiment of at least 3 to 5 experiments.

  • Figure 3.

    Effect of the DpT analogs compared with DFO and 311 on 59Fe mobilization and cellular 59Fe uptake from 59Fe-Tf in SK-N-MC neuroepithelioma and M109 cells. (A) Effect of chelators on 59Fe mobilization from prelabeled SK-N-MC neuroepithelioma cells. Cells were prelabeled with 59Fe-Tf (0.75 μM) for 3 hours at 37° C, washed, and then reincubated for 3 hours at 37° C in the presence of medium alone (control) or medium containing the chelators (25 μM). (B) Effect of the chelators at preventing 59Fe uptake from 59Fe-Tf by SK-N-MC cells. Cells were incubated for 3 hours at 37° C in media containing either 59Fe-Tf (0.75 μM) alone (control) or 59Fe-Tf (0.75 μM) and the chelators (25 μM). After this incubation, the cells were washed and incubated with pronase (1 mg/mL) for 30 minutes at 4° C to measure internalized 59Fe.13,14 (C) Effect of the chelators on 59Fe mobilization from prelabeled M109 cells as a function of chelator concentration. M109 cells were prelabeled as described for panel A, then reincubated with the chelator (0.2-25 μM) for 3 hours at 37° C. (D) Effect of the chelators at preventing 59Fe uptake from 59Fe-Tf by M109 cells as a function of chelator concentration. M109 cells were incubated with 59Fe-Tf (0.75 μM) in the presence of the chelators (0.2-25 μM) for 3 hours at 37° C. Cells were washed and incubated with pronase as described for panel B. Results are expressed as the mean ± SD of 3 replicates in a typical experiment of 3 performed.

  • Figure 4.

    Dose-dependent inhibition of M109 lung carcinoma growth in mice by Dp44mT and 3-AP. (A) Dp44mT and, to a greater extent, 3-AP markedly decreased the growth of M109 lung carcinoma in mice after a 5-day treatment regimen. (B) Induction of apoptosis in tumors after injection of (i) vehicle control or (ii) Dp44mT, as determined using TUNEL assay. (A) 1 × 105 M109 cells were subcutaneously implanted in CD2F1 mice. The chelators Dp44mT and 3-AP were injected intravenously twice daily for 5 consecutive days starting on the fourth day after tumor implantation. Tumor weight was measured on the 12th day after implantation. n = 8 in each experimental group. Data were analyzed using the Student t test. *P < .05 compared with control. **P < .01 compared with control. ***P < .0001. (B) M109 lung carcinoma specimens from mice treated as for panel A with (i) vehicle control or (ii) Dp44mT were fixed in 10% (vol/vol) buffered formalin and embedded in paraffin. Sections were stained for apoptotic cells in situ using the TUNEL assay. Positive nuclei stained brown, and negative nuclei stained blue. Results in panel A are mean ± SEM for 3 experiments, whereas data in panel B are typical of results found in 3 separate experiments.

  • Figure 5.

    Effect of Dp44mT on M109 cellular apoptosis or necrosis/late-stage apoptosis. (A) Chelator concentration. M109 cells were incubated with Dp44mT at 0 to 250 μM for 24 hours at 37° C. Cellular apoptosis and necrosis/late-stage apoptosis were measured using Annexin V–FITC and PI staining, respectively (see “Materials and methods”). (B) Incubation time. M109 cells were treated with Dp44mT (1 μM) for various incubation times (0-48 hours). Cellular apoptosis and necrosis/late-stage apoptosis were measured as described for panel A. Data plotted are mean ± SEM of 3 separate experiments.

  • Figure 6.

    Effect of Dp44mT (1 μM) on the protein levels of active caspase-3, -8, and -9 and the activity of caspase-3, -8, and -9 in cultured M109 cells in the absence and presence of cell-permeable caspase inhibitors. (A) Caspase-3, -8, and -9 levels after Dp44mT treatment at the indicated times as detected by Western blotting (top blots). Anti–β-actin antibody was used to ensure equal protein loading (bottom blot). (B) Densitometric analysis of the expression of caspase-3, -8, and -9 as a function of time normalized to β-actin. (C) Caspase activity induced by Dp44mT (1 μM) at 0 to 48 hours was expressed as a percentage of the 0-hour time value. (D) Cell-permeable inhibitors of caspase-3, -8, or -9 at 1 μM prevented activation of these enzymes when incubated with Dp44mT (1 μM) for 48 hours. Results are mean ± SEM of 3 separate experiments. Horizontal dashed line indicates 100%.

  • Figure 7.

    Effect of Dp44mT (1 μM) in cultured M109 cells on the holocytochrome c (h-cytc) levels in cytosolic and stromal-mitochondrial membrane (SMM) fractions and mitochondrial protein levels of Bcl-2 and Bax. (A) Cytosolic and SMM fractions of M109 cells were separated and subjected to Western blotting with anti–h-cytc antibody. The blot was reprobed with an anti–β-actin antibody to ensure equal protein loading. Densitometric analyses are shown beneath the blots; expression is normalized to β-actin. (B) Protein levels of Bcl-2 and Bax were determined through Western blotting using the SMM fraction of M109 cells after incubation with Dp44mT for 0 to 48 hours. Anti–β-actin antibody was used to ensure equal protein loading. Densitometric analysis is shown beneath each blot, where expression is normalized to β-actin. Results in panels A and B are representative of 3 experiments.

Tables

  • Table 1.

    IC50 values in neoplastic and normal cells

    Neoplastic cellsNormal cells
    SK-N-MC neuroepitheliomaSK-Mel-28 melanomaMCF-7 breast cancerMRC-5 fibroblasts
    DFO 5 ± 2 15 ± 7 14 ± 9 > 25
    311 0.3 ± 0.2 0.9 ± 0.5 > 25
    DpT > 25 > 25 > 25 > 25
    Dp2mT > 25 > 25 > 25 > 25
    Dp4mT 0.19 ± 0.1 0.6 ± 0.5 0.3 ± 0.2 > 25
    Dp44mT 0.03 ± 0.01 0.06 ± 0.03 0.06 ± 0.01 > 25
    Dp4eT 0.06 ± 0.01 0.09 ± 0.06 0.08 ± 0.01 > 25
    Dp4aT 0.06 ± 0.01 0.10 ± 0.06 0.07 ± 0.01 > 25
    Dp4pT 0.05 ± 0.006 0.09 ± 0.05 0.07 ± 0.01 > 25
    3-AP 0.26 ± 0.01 2.6 ± 0.6 3.0 ± 1.5
    Doxorubicin 0.02 ± 0.01 0.35 ± 0.09 0.6 ± 0.2
    • Each IC50 value (given in μM) is the mean ± SEM of at least 3 experiments performed. Cells were incubated in the presence and absence of DFO, 311, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone, doxorubicin, and the DpT series of chelators (0-25 μM) for 72 hours at 37°C. After this incubation period, cellular proliferation was determined by the MTT assay.23 — indicates not determined.

  • Table 2.

    Weight loss and hematologic indices from mice treated with vehicle alone (control), Dp44mT, or 3-AP

    Dp44mT
    Control0.15 mg/kg0.3 mg/kg0.4 mg/kg3-AP, 6 mg/kg
    Body weight loss, g 0.8 ± 0.3 1.4 ± 0.2 1.3 ± 0.3 1.1 ± 0.2 5.3 ± 0.4
    WBC count, × 109/L 4.4 ± 0.4 4.2 ± 0.8 4.2 ± 0.4 3.9 ± 0.6 1.4 ± 0.3
    RBC count, × 1012/L 9.1 ± 0.2 9.4 ± 0.1 8.9 ± 0.4 8.0 ± 0.6 6.1 ± 0.4
    Hemoglobin level, g/L 137 ± 2 14.0 ± 2 133 ± 5 118 ± 8 86 ± 5
    Hematocrit 0.46 ± 0.01 0.46 ± 0.01 0.40 ± 0.03 0.40 ± 0.01 0.28 ± 0.01
    Platelet count, × 109/L 788 ± 47 1047 ± 42 1019 ± 87 636 ± 88 976 ± 171
    • Female CD2FI mice, aged 8 to 10 weeks, underwent xenografting with M109 tumor cells. Dp44mT or 3-AP was administered intravenously twice daily for 5 consecutive days from day 4 after tumor implantation. Total body weight was measured on the 12th day after implantation (see “Materials and methods”). Mice were killed, and cardiac puncture was performed. Hematologic indices were measured as described in “Materials and methods.” Results are mean ± SEM (3 separate experiments). WBC indicates white blood cell; RBC, red blood cell.