Cytotoxic effects of tetracycline analogues (doxycycline, minocycline and COL-3) in acute myeloid leukemia HL-60 cells

Abstract

Tetracycline analogues (TCNAs) have been shown to inhibit matrix metalloproteinases and to induce apoptosis in several cancer cell types. In the present study, the cytotoxic effects of TCNAs doxycycline (DOXY), minocycline (MINO) and chemically modified tetracycline-3 (COL-3) were investigated in the human acute myeloid leukemia HL-60 cell line. Cells were incubated with TCNAs in final concentrations of 0.5-100 µg/ml for 24 h. Viability of the leukemic cells was inhibited in a concentration-dependent manner using resazurin assay. The estimated IC50s were 9.2 µg/ml for DOXY, 9.9 µg/ml for MINO and 1.3 µg/ml for COL-3. All three TCNAs induced potent cytotoxic effects and cell death. Apoptosis, which was assessed by morphological changes and annexin V positivity, was concentration- and time-dependent following incubation with any one of the drugs. TCNAs induced DNA double strand breaks soon after treatment commenced as detected by γH2AX and western blot. The loss of mitochondrial membrane potential (Δψm), caspase activation and cleavage of PARP and Bcl-2 were observed; however, the sequence of events differed among the drugs. Pancaspase inhibitor Z-VAD-FMK improved survival of TCNAs-treated cells and decreased TCNAs-induced apoptosis. In summary, we demonstrated that TCNAs had a cytotoxic effect on the HL-60 leukemic cell line. Apoptosis was induced via mitochondria-mediated and caspase-dependent pathways in HL-60 cells by all three TCNAs. COL-3 exerted the strongest anti-proliferative and pro-apoptotic effects in concentrations that have been achieved in human plasma in reported clinical trials. These results indicate that there is a therapeutic potential of TCNAs in leukemia.

Figure 1. The effect of TCNAs on the viability of human leukemic HL-60 cell line.

HL-60 cells were incubated with DOXY, MINO or COL-3 in concentrations within the range 0.5–100 µg/ml for 24 h. Cells incubated in DMSO in a final concentration of 0.2% were used as controls for solvent toxicity and cells incubated in complete medium served as controls. Viability was studied using resazurin fluorescence assay and expressed as percent of the control. Results are presented as a mean ± SD of three independent experiments.

Figure 2. TCNAs-induced apoptosis.

HL-60 cells were incubated with DOXY (A) and MINO (B) in concentrations of 5, 10, 25 and 50 µg/ml or COL-3 (C) in concentrations of 0.75, 1, and 5 µg/ml until 48 h. Cells incubated in DMSO in a final concentration of 0.2% were used as controls for solvent toxicity and cells incubated in complete medium served as controls. Cells were stained with annexin V and PI for 15 min at room temperature in the dark. Twenty thousand events were acquired and analyzed using flow cytometry. Subpopulations are expressed as percentages of total events. Results are expressed as a mean ± SD of three independent experiments. (D), (E): Examples on flow cytometric analysis of TCNAs-induced apoptosis using annexin V/PI assay. D: DOXY, M: MINO COL: COL-3 and PI: propidium iodide.

Figure 3. The loss of mitochondrial membrane potential (Δψm) in TCNAs-induced apoptosis.

Mitochondrial membrane potential was assessed using tetramethylrhodamine methyl ester (TMRM) and flow cytometry. (A) HL-60 cells were incubated with DOXY or MINO in final concentrations of 25 and 50 µg/ml or (B) COL-3 in final concentrations of 0.5, 1, 2.5 and 5 µg/ml for 2, 4, 6 and 24 h. Cells incubated in DMSO in a final concentration of 0.2% were used as controls for solvent toxicity and cells incubated in complete medium served as controls. Results are expressed as mean ± SD of three independent experiments. (C), (D): Example on flow cytometric analysis of loss of Δψm using TMRM assay. D: DOXY, M: MINO, COL: COL-3.

Figure 4. The role of mitochondria in TCNAs-induced apoptosis.

Cytosolic translocation of cytochrome c (cyt c) and Bcl-2 cleavage were assessed with Western blot. Cells were incubated with DOXY or MINO in final concentrations of 2.5, 10 and 25 µg/ml or COL-3 in final concentrations of 0.75, 1 and 5 µg/ml until 24 h. DMSO (0.2%) treated cells served as controls for solvent toxicity, etoposide (6 µg/ml) treated cells as positive controls for apoptosis and cells incubated in complete medium as controls. Co: control, D: DOXY, M: MINO, C: COL-3 and E: etoposide.

Figure 5. The effect of TCNAs on DNA.

HL-60 cells were incubated with DOXY or MINO in final concentrations of 25 µg/ml or COL-3 in a final concentration of 5 µg/ml for 10 min and 1, 2 and 4 h. Cells incubated in DMSO in a final concentration of 0.2% were used as controls for solvent toxicity and cells incubated in complete medium served as controls. Cells incubated with etoposide in a final concentration of 6 µg/ml served as positive controls for apoptosis. The TCNAs-induced DNA damage was assessed using SDS-PAGE and immunostaining for the DNA double strand breaks marker γH2AX. Co: control, E: etoposide, D: DOXY, M: MINO and C: COL-3.

Figure 6. TCNAs-mediated poly (ADP-ribose) polymerase-1(PARP-1) cleavage.

HL-60 cells were treated with DOXY or MINO in final concentrations of 2.5, 10 and 25 µg/ml or COL-3 in final concentrations of 0.75, 1 and 5 µg/ml for 6 and 24 h. Cells incubated with DMSO in a final concentration of 0.2% served as controls for solvent toxicity, cells incubated with complete medium served as controls. The pattern of PARP-1 cleavage was assessed using WB. Actin was used as a control for equal protein loading. Co: control, D: DOXY, M: MINO and C: COL-3.

Figure 7. The effect of Z-VAD-FMK on TCNAs-induced cytotoxicity.

HL-60 cells were treated with Z-VAD-FMK in a final concentration of 100 µM for 1 h before treatment with TCNAs for 6 h and 24 h. Cells incubated with DMSO in a final concentration of 0.2% served as controls for solvent toxicity, cells incubated in complete medium served as controls. (A) Total cell death was assessed using resazurin viability assay and calculated as 100% - viability %. (B) Apoptosis was assessed by morphological criteria in Giemsa staining. DMSO and Z-VAD-FMK exerted no cytotoxic effects on HL-60 cells compared to controls incubated with complete medium (data not shown on the graph). Results are presented as mean ± SD of three independent experiments. D: DOXY 25 µg/ml, M: MINO 25 µg/ml, Z: Z-VAD-FMK 100 µM; C: COL-3 5 µg/ml in total cell death experiments (A) and 2.5 µg/ml apoptosis experiments (B).