T315 Decreases Acute Myeloid Leukemia Cell Viability through a Combination of Apoptosis Induction and Autophagic Cell Death

Abstract

T315, an integrin-linked kinase (ILK) inhibitor, has been shown to suppress the proliferation of breast cancer, stomach cancer and chronic lymphocytic leukemia cells. Here we demonstrate that T315 decreases cell viability of acute myeloid leukemia (AML) cell lines (HL-60 and THP-1) and primary leukemia cells from AML patients in a dose-responsive manner. Normal human bone marrow cells are less sensitive than leukemia cells to T315. T315 down regulates protein kinase B (Akt) and p-Akt and induces caspase activation, poly-ADP-ribose polymerase (PARP) cleavage, apoptosis and autophagy through an ILK-independent manner. Interestingly, pretreatment with autophagy inhibitors rescues cells from apoptosis and concomitant PARP cleavage, which implicates a key role of autophagic cell death in T315-mediated cytotoxicity. T315 also demonstrates efficacy in vivo, suppressing the growth of THP-1 xenograft tumors in athymic nude mice when administered intraperitoneally. This study shows that autophagic cell death and apoptosis cooperatively contribute to the anticancer activity of T315 in AML cells. In conclusion, the complementary roles of apoptotic and autophagic cell death should be considered in the future assessment of the translational value of T315 in AML therapy.

Keywords: T315; acute myeloid leukemia; apoptosis; autophagic cell death; autophagy.

Figure 1

Cell viability inhibition study of T315 in acute myeloid leukemia (AML) cell lines, primary AML cells and normal marrow cells. (A) HL-60 and THP-1 cells (0.25 × 10⁶ cells/mL) were incubated with T315 or dimethyl sulfoxide (DMSO) vehicle for 24 h. The apoptotic cells were analyzed by annexin V-FITC and propidium iodide (PI) staining, as described in Materials and Methods. Upper panel: one example; Lower panel: apoptotic cell percentage (n = 3); (B) HL-60 and THP-1 cells (0.25 × 10⁶ cells/mL) were incubated with T315 or DMSO vehicle for 24 h () or 48 h (□). The cells were analyzed by MTS assay, as described in Materials and Methods; (C) Primary AML cells (0.25 × 10⁶ cells/mL) were incubated with T315 or DMSO for 24 h. The cells were stained with annexin V-FITC and PI to assess apoptotic cells percentage (n = 26); (D) Normal bone marrow nucleated cells (0.25 × 10⁶ cells/mL) were incubated with T315 or DMSO for 24 h. The cells were stained with annexin V-FITC and PI to assess apoptotic cells percentage (n = 16). * denotes p < 0.05; ** denotes p < 0.01 compared to the control group (in panel A) or compared to the primary AML cells at the same concentration of T315 (in panel D).

Figure 1

Cell viability inhibition study of T315 in acute myeloid leukemia (AML) cell lines, primary AML cells and normal marrow cells. (A) HL-60 and THP-1 cells (0.25 × 10⁶ cells/mL) were incubated with T315 or dimethyl sulfoxide (DMSO) vehicle for 24 h. The apoptotic cells were analyzed by annexin V-FITC and propidium iodide (PI) staining, as described in Materials and Methods. Upper panel: one example; Lower panel: apoptotic cell percentage (n = 3); (B) HL-60 and THP-1 cells (0.25 × 10⁶ cells/mL) were incubated with T315 or DMSO vehicle for 24 h () or 48 h (□). The cells were analyzed by MTS assay, as described in Materials and Methods; (C) Primary AML cells (0.25 × 10⁶ cells/mL) were incubated with T315 or DMSO for 24 h. The cells were stained with annexin V-FITC and PI to assess apoptotic cells percentage (n = 26); (D) Normal bone marrow nucleated cells (0.25 × 10⁶ cells/mL) were incubated with T315 or DMSO for 24 h. The cells were stained with annexin V-FITC and PI to assess apoptotic cells percentage (n = 16). * denotes p < 0.05; ** denotes p < 0.01 compared to the control group (in panel A) or compared to the primary AML cells at the same concentration of T315 (in panel D).

Figure 2

T315 induces dephosphorylation of protein kinase B (Akt) without change of integrin-linked kinase (ILK) in AML cell lines. Cells (0.25 × 10⁶ cells/mL) were treated with T315 at the indicated concentration or DMSO for 24 h, and 20 µg protein extract from cell lysates in each condition were used for Western blot analysis. (A) T315 did not change the p^Thr173-ILK and total ILK expression. Histogram of fold change of p^Thr173-ILK/ILK and ILK/glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were shown in lower panels (n = 3); (B) T315 down regulated both p^Thr308-Akt and p^Ser473-Akt, but not Akt, p-ERK and ERK expression. Histogram of fold change of p^Thr308-Akt/Akt, p^Ser473-Akt/Akt, Akt/GAPDH, p-ERK/ERK, and ERK/GAPDH were shown in lower panels (n = 3). * denotes p < 0.05; ** denotes p < 0.01 compared to the control group.

Figure 3

T315-mediated cytotoxicity is dependent on caspase activation and apoptosis. (A) T315 induced poly-ADP-ribose polymerase (PARP) cleavage and activation of caspase-3 and caspase-7 in HL-60 and THP-1 cells at 24 h. Protein extract of 20 µg from cell lysates were used for Western blot analysis; (B) Fold change of cleaved PARP/β-actin, cleaved caspase-3/β-actin, and cleaved caspase-7/β-actin in treatment with T315 of 1, 2 or 3 µM compared with DMSO control (n = 3); (C) Time course change of PARP cleavage and caspase-3 activation induced by T315 of 2 µM or DMSO control; (D) The increased caspase-3 activity in HL-60 cells treated with T315 for 24 h was rescued by pretreatment of 50 µmol/L Z-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD(OMe)-FMK).

Figure 4

T315 induces autophagic cell death but not protective autophagy in AML cells. (A) T315 induced upregulation of LC3B-II in HL-60 and THP-1 cells. Cells (0.25 × 10⁶ cells/mL) were treated with indicated concentrations of T315 for 24 h. 20 µg protein from cell lysates were used for Western blot analysis; (B) Histogram of fold change of LC3B-II/GAPDH protein expression in cells treated with T315 for 24 h (n = 3); (C) T315-induced apoptosis was partially rescued by chloroquine (CQ), an autophagy inhibitor. Cells were treated with DMSO vehicle or T315 for 24 h with or without pretreatment of CQ for 1 h, and then analyzed by a flow cytometer. (n = 3 for HL-60 and n = 4 for THP-1 cells); (D) T315-induced apoptosis was partially rescued by 3-methyladenosine (3-MA), an autophagy inhibitor. Cells were treated with DMSO vehicle or T315 for 24 h with or without pretreatment of 3-MA for 1 h, and then analyzed by a flow cytometer. (n = 4 for HL-60 and n = 5 for THP-1 cells); (E) T315-induced apoptosis was partially rescued by bafilomycin-A1 (Baf), an autophagy inhibitor. Cells were treated with DMSO vehicle or T315 for 24 h with or without pretreatment of Baf for 1 h, and then analyzed by a flow cytometer. (n = 5 for HL-60 and n = 5 for THP-1 cells); (F) T315-induced PARP cleavage was partially rescued by Baf. Cells were treated with DMSO vehicle or T315 for 24 h with or without pretreatment of Baf for 1 h, and then analyzed by Western blotting; (G) Histogram of fold change of cleaved PARP/β-actin protein expression in cells treated with T315 with or without pretreatment of Baf for 1 h (n = 3); (H) T315-induced PARP cleavage in primary AML cells was partially rescued by Baf. Primary AML cells were treated with DMSO vehicle or T315 for 24 h with or without pretreatment of Baf for 1 h, and then analyzed by Western blotting (two patients’ data shown here).

Figure 4

T315 induces autophagic cell death but not protective autophagy in AML cells. (A) T315 induced upregulation of LC3B-II in HL-60 and THP-1 cells. Cells (0.25 × 10⁶ cells/mL) were treated with indicated concentrations of T315 for 24 h. 20 µg protein from cell lysates were used for Western blot analysis; (B) Histogram of fold change of LC3B-II/GAPDH protein expression in cells treated with T315 for 24 h (n = 3); (C) T315-induced apoptosis was partially rescued by chloroquine (CQ), an autophagy inhibitor. Cells were treated with DMSO vehicle or T315 for 24 h with or without pretreatment of CQ for 1 h, and then analyzed by a flow cytometer. (n = 3 for HL-60 and n = 4 for THP-1 cells); (D) T315-induced apoptosis was partially rescued by 3-methyladenosine (3-MA), an autophagy inhibitor. Cells were treated with DMSO vehicle or T315 for 24 h with or without pretreatment of 3-MA for 1 h, and then analyzed by a flow cytometer. (n = 4 for HL-60 and n = 5 for THP-1 cells); (E) T315-induced apoptosis was partially rescued by bafilomycin-A1 (Baf), an autophagy inhibitor. Cells were treated with DMSO vehicle or T315 for 24 h with or without pretreatment of Baf for 1 h, and then analyzed by a flow cytometer. (n = 5 for HL-60 and n = 5 for THP-1 cells); (F) T315-induced PARP cleavage was partially rescued by Baf. Cells were treated with DMSO vehicle or T315 for 24 h with or without pretreatment of Baf for 1 h, and then analyzed by Western blotting; (G) Histogram of fold change of cleaved PARP/β-actin protein expression in cells treated with T315 with or without pretreatment of Baf for 1 h (n = 3); (H) T315-induced PARP cleavage in primary AML cells was partially rescued by Baf. Primary AML cells were treated with DMSO vehicle or T315 for 24 h with or without pretreatment of Baf for 1 h, and then analyzed by Western blotting (two patients’ data shown here).

Figure 5

T315-mediated cytotoxicity is rescued by combination of an apoptosis inhibitor and an autophagy inhibitor. Cells were treated with DMSO vehicle or T315 for 24 h with or without pretreatment of Z-VAD(OMe)-FMK and/or bafilomycin-A1 (Baf) for 1 h, and then analyzed by a flow cytometer. (A) For HL-60 cells (n = 5); (B) For THP-1 cells (n = 5).

Figure 6

T315 mitigates the growth of THP-1 xenografts and prolongs the survival of tumor-bearing athymic nude mice. (A) Mice bearing THP-1 xenografts were treated with DMSO vehicle (······, n = 7) or T315 (――, n = 6) at 37.5 mg/kg/day intraperitoneally. The data represent group means and were plotted until day 10 when one mouse in the control group reached the endpoint tumor size (≥2000 mm³) and was sacrificed; (B) Body weight change of mice. The data were plotted until day 26 when the control group and treated group had 2 and 3 mice remaining, respectively; (C) Overall survival curve plotted by Kaplan-Meier method. * denotes p < 0.05; ** denotes p < 0.01 compared to the control group.