Synergistic Apoptosis-Inducing Antileukemic Effects of Arsenic Trioxide and Mucuna macrocarpa Stem Extract in Human Leukemic Cells via a Reactive Oxygen Species-Dependent Mechanism

Abstract

The objective of this study was to examine the potential of enhancing the antileukemic activity of arsenic trioxide (ATO) by combining it with a folk remedy, crude methanolic extract of Mucuna macrocarpa (CMEMM). Human leukemia cells HL-60, Jurkat, and Molt-3 were treated with various doses of ATO, CMEMM, and combinations thereof for 24 and 48 h. Results indicated that the combination of 2.5 μM ATO and 50 μg/mL CMEMM synergistically inhibited cell proliferation in HL-60 and Jurkat cell lines. Apoptosis triggered by ATO/CMEMM treatment was confirmed by accumulation of cells in the sub-G(1) phase in cell cycle analyses, characteristic apoptotic nuclear fragmentation, and increased percentage of annexin V-positive apoptotic cells. Such combination treatments also led to elevation of reactive oxygen species (ROS). The antioxidants N-acetyl cysteine (NAC), butylated hydroxytoluene, and α-tocopherol prevented cells from ATO/CMEMM-induced apoptosis. The ATO/CMEMM-induced activation of caspase-3 and caspase-9 can be blocked by NAC. In summary, these results suggest that ATO/CMEMM combination treatment exerts synergistic apoptosis-inducing effects in human leukemic cells through a ROS-dependent mechanism and may provide a promising antileukemic approach in the future.

Figure 1

Antiproliferative effects of combined application of arsenic trioxide (ATO) and crude methanolic extract of Mucuna macrocarpa (CMEMM) on human leukemia cells. HL-60, Jurkat, or Molt-3 cells (1 × 10⁵ cells/mL) were seeded into 6-well plates and exposed to 0, 2.5, or 5 μM ATO alone or together with 0, 25, 50, or 75 μg/mL CMEMM for 24 and 48 h. Vehicle control cells were treated with 0.1% DMSO in medium. The percentages of cell growth were measured by the trypan blue exclusion assay and calculated by comparing the cells number with that of the vehicle controls. Each value represents the mean ± S.E. of duplicate cultures from three independent experiments. *P < 0.05, **P < 0.01 indicate significant difference from the respective control value.

Figure 2

Cell cycle progression in leukemia cells exposed to arsenic trioxide (ATO) and/or crude methanolic extract of Mucuna macrocarpa (CMEMM). HL-60 (a) or Jurkat (b) cells (1 × 10⁵ cells/mL) were first treated with 5 mM N-acetyl cysteine (NAC) or untreated, followed by treatment with 0.1% DMSO (CTL), 2.5 μM ATO, and/or 50 μg/mL CMEMM as indicated. After 24 or 48 h of treatment, cells were collected and stained with propidium iodide and determined for DNA content using flow cytometry. The percentages of sub-G₁ or hypodiploid cells were analyzed by ModFit LT software. The representative cell cycle progressions in ATO- and/or CMEMM-treated or control cells were from one of three independent experiments.

Figure 3

Nuclear morphological changes induced by arsenic trioxide (ATO) and/or crude methanolic extract of Mucuna macrocarpa (CMEMM). HL-60, Jurkat, or Molt-3 cells (1 × 10⁵ cells/mL) were treated with 0.1% DMSO (control), 2.5 μM ATO, 50 μg/mL CMEMM, or 2.5 μM ATO plus 50 μg/mL CMEMM. After 24 or 48 h of incubation, cells were washed with PBS and collected on microscope slides by cytospin. The nuclei were stained with 2.5 μg/mL DAPI. Arrows indicate apoptotic bodies of nuclear fragmentation. Magnification × 200.

Figure 4

Changes in the level of intracellular reactive oxygen species (ROS) in leukemia cells exposed to arsenic trioxide (ATO) and/or crude methanolic extract of Mucuna macrocarpa (CMEMM). HL-60 (a) or Jurkat (b) cells (1 × 10⁵ cells/mL) were treated with 0.1% DMSO (control), 2.5 μM ATO, 50 μg/mL CMEMM, or 2.5 μM ATO plus 50 μg/mL CMEMM for 4 to 48 h. Then, cells were washed with PBS, incubated with dihydroethidium for 30 min, and analyzed for red fluorescence by flow cytometry. The mean fluorescence intensity was used as read-out for intracellular ROS levels.

Figure 5

Annexin V-FITC/propidium iodide (PI) analyses of arsenic trioxide (ATO)- and/or crude methanolic extract of Mucuna macrocarpa (CMEMM)-treated cells. HL-60 (a) and Jurkat (b) cells (1 × 10⁵ cells/mL) were first treated with 5 mM N-acetyl cysteine (NAC), 50 μM butylated hydroxytoluene (BHT), or 40 μM α-tocopherol (VitE) or untreated, followed by treatment with 0.1% DMSO (CTL), 2.5 μM ATO, and/or 50 μg/mL CMEMM as indicated for 24 h. Quantitative percentages of apoptotic cells of ATO/CMEMM-treated cells were measured by flow cytometry. Data represent the result from one of three independent experiments.

Figure 6

Expressions of apoptosis-related proteins in leukemia cells treated with arsenic trioxide (ATO), crude methanolic extract of Mucuna macrocarpa (CMEMM), and/or N-acetyl cysteine (NAC). Whole cell lysates were prepared from HL-60 or Jurkat cells treated with 2.5 μM ATO, 50 μg/mL CMEMM, 5 mM NAC, or indicated combinations for 24 h. Proteins as indicated were analyzed by Western blotting with β-actin as loading control. Representative blots shown were from one of three independent experiments.