α-Tomatine inhibits growth and induces apoptosis in HL-60 human myeloid leukemia cells

Abstract

α‑Tomatine is a glycoalkaloid that occurs naturally in tomatoes (Lycopersicon esculentum). In the present study, the effects of α‑tomatine on human myeloid leukemia HL‑60 cells were investigated. Treatment of HL‑60 cells with α‑tomatine resulted in growth inhibition and apoptosis in a concentration‑dependent manner. Tomatidine, the aglycone of tomatine had little effect on the growth and apoptosis of HL‑60 cells. Growth inhibition and apoptosis induced by α‑tomatine in HL‑60 cells was partially abrogated by addition of cholesterol indicating that interactions between α‑tomatine and cell membrane‑associated cholesterol may be important in mediating the effect of α‑tomatine. Activation of nuclear factor‑κB by the phorbol ester, 12‑O‑tetradecanoylphorbol‑13‑acetate failed to prevent apoptosis in HL‑60 cells treated with α‑tomatine. In animal experiments, it was found that treatment of mice with α‑tomatine inhibited the growth of HL‑60 xenografts in vivo. Results from the present study indicated that α‑tomatine may have useful anti‑leukemia activities.

Figure 1

Structures of α-tomatine and tomatidine.

Figure 2

Effects of α-tomatine, tomatidine and Ara-C on the growth of HL-60 cells. HL-60 cells were seeded at a density of 1×10⁵ cells/ml in 35 mm tissue culture dishes and treated with various concentrations of (A) α-tomatine, (B) Ara-C and (C) tomatidine for 24, 48 and 72 h. The number of viable cells was determined using the trypan blue exclusion assay. Each value represents the mean ± standard error of the mean from three separate experiments. Ara-c, cytosine arabinoside.

Figure 3

Effect of α-tomatine and tomatidine on the apoptosis of HL-60 cells. HL-60 cells were seeded at a density of 1×10⁵ cells/ml in 35 mm tissue culture dishes and treated with various concentrations of α-tomatine and tomatidine for 48 h. Apoptosis was determined by propidium iodide staining and morphological assessment. Representative micrographs of propidium iodide-stained cells from the (A) control group and cells treated with (B) 2 μM α-tomatine (arrows indicate apoptotic cells). (C) Percentage of apoptotic cells as determined by morphological assessment in HL-60 cells treated with different concentrations of α-tomatine (grey bar) and tomatidine (open bar). Each value represents the mean ± standard error of the mean from three separate experiments.

Figure 4

Effects of CH on TM-induced growth inhibition and apoptosis. HL-60 cells were seeded at a density of 1×10⁵ cells/ml in 35 mm tissue culture dishes and treated with TM in the presence or absence of CH for 48 h. (A) The number of viable cells was determined by the trypan blue exclusion assay. (B) Apoptosis was determined by propidium iodide staining and morphological assessment. Each value represents the mean ± standard error of the mean from three separate experiments. Differences in the number of viable and apoptotic cells between the TM-treated group and TM+CH-treated group were analyzed by analysis of variance with the Tukey-Kramer multiple comparison test. ^*P<0.01 vs. TM-treated group. CH, cholesterol; TM, α-tomatine.

Figure 5

Effects of pre-treatment with TPA on TM-induced apoptosis in HL-60 cells. (A) HL-60 cells were seeded in 100 mm culture dishes and treated with 0.32 nM TPA for different time intervals. Activation of NF-κB in HL-60 cells was determined by electrophoretic mobility shift assay. P=probe only. C=control. Treatment of HL-60 cells with TPA for 1 h resulted in a marked activation of NF-κB. (B and C) HL-60 cells were seeded at a density of 1×10⁵ cells/ml in 35 mm culture dishes. The cells were treated with TM for 48 h with or without pre-treatment with 0.32 nM TPA for 1 h. (B) The number of viable cells was determined by the trypan blue exclusion assay and expressed as a percentage of the control. (C) Apoptosis was determined by propidium iodide staining and morphological assessment. TPA, 12-O-tetradecanoylphorbol-13-acetate; TM, α-tomatine; CH, cholesterol; DMSO, dimethyl sulfoxide.

Figure 6

Effects of α-tomatine on (A) the growth of HL-60 xenografts and (B) body weight of SCID mice. Female SCID mice were injected subcutaneously with HL-60 cells in 50% Matrigel (1.0×10⁶ cells/0.1 ml) suspended in RPMI medium. After 3 weeks, mice with HL-60 xenograft tumors (0.6–1.0 cm wide and 0.6–1.0 cm long) were intraperitoneally injected with vehicle (5 μl/g body weight; n=12) or with α-tomatine (5 mg/kg body weight; n=12) three times a week for 3 weeks. (A) Tumor size (length x width; cm²) was measured and expressed as a percentage of initial tumor size. (B) Body weight (g). SCID, severe combined immunodeficient.