Fig 1.
SB225002 induces cell death in ALL cell lines.
Effect of SB225002 [100 to 1.5625 μM] on the survival and proliferation of (A) B-ALL and T-ALL cell lines. (B) Effect of SB225002 [5 and 10 μM] on the survival and proliferation of normal PHA-stimulated human lymphocytes. (C) Cell cycle analysis of B-ALL (REH and RS4;11), T-ALL (Jurkat and TALL-1) and normal human PHA-stimulated lymphocytes treated with DMSO (vehicle; 0.1%) and the following concentrations of SB225002: REH and RS4;11 [10 μM]; Jurkat and TALL-1 [3.125 μM]; PHA-stimulated lymphocytes [10 μM]. Representative PI-staining histograms of cells treated with vehicle (clear area) or SB225002 (shaded area) are shown. (D) Annexin-V and propidium iodide flow cytometry analyses of B-ALL (REH and RS4;11) and T-ALL (Jurkat and TALL-1) treated with DMSO (vehicle; 0.1%) and SB225002 [10 μM]. Cells were treated for 24 h (for cell cycle and Annexin-V analyses) and 48 h (for MTT analysis). ALL = acute lymphoblastic leukemia; PI = propidium iodide; Lym = PHA-stimulated lymphocytes; C or Ctr = DMSO (vehicle control); SB = SB225002 treatment.
Fig 2.
Connectivity Map and Ingenuity Pathway Analysis using the SB225002-derived gene expression signature.
(A) Connectivity Map (C-Map) analysis using the gene expression signature of Jurkat cells treated with SB225002 [IC50] for 9 h. Compounds colored as black bars in each respectively C-Map plot. Compounds are color-coded as follows: blue, PI3K/mTOR inhibitors; green, HSP90 inhibitors; red, tubulin inhibitors. (B) Signaling pathways activated in Jurkat cells in response to 6 h of SB225002 [IC50] treatment. The statistical threshold (line without boxes) represents the cut-off for significance on the log scale (y-axis, left side). The ratio (line with boxes) of the number of significant genes from the data set that mapped to a pathway divided by the total number of genes from the pathway is also shown (y axis, right side). (C) JUN, (D) p53 and (E) TNF pathways are modulated in Jurkat cells after 6 h of SB225002 [IC50] treatment. Analyses were performed using the Ingenuity Pathways Analysis package (Ingenuity Systems).
Fig 3.
Modulation of CX3CR1 and GLIPR1 expression in ALL cells upon SB225002 treatment.
(A) CX3CR1 and (B) GLIPR1 gene expression analysis by quantitative PCR (Q-PCR) and Western blot in Jurkat cells treated with SB225002 [IC50] or DMSO (vehicle control; 0.1%). Treatments were performed for 3h, 6 h, 9 h or 12 h, as indicated. In the Q-PCR analysis, expression values were calculated considering vehicle control (DMSO) as 100%. β-actin was used as loading control in Western blot analysis. Control = DMSO (vehicle control); SB = SB225002 treatment.
Fig 4.
SB225002 induced cell death in ALL is mediated at least in part by the upregulation of GLIPR1.
(A) Relative proliferation of REH, RS4;11, Jurkat and TALL-1 cells upon sh.RNA knockdown of GLIPR1 (sh.GLIPR1) in comparison to control Scramble (sh.Scr). Number of viable cells were counted by flow cytometry and normalized to time-point zero. (B) Annexin-V and propidium iodide flow cytometry analyses of B-ALL (REH and RS4;11) and T-ALL (Jurkat and TALL-1) scramble or GLIPR1-knockdown cells treated with SB225002 [5 μM]. (C) Effect of SB225002 [5 μM or 10 μM] treatment on the proliferation of GLIPR1-knockdown (sh.GLIPR1) versus control (sh.Scramble) B-ALL (REH and RS4;11) and T-ALL (Jurkat and TALL-1) cell lines. B-ALL cells were treated with SB225002 [10 μM] and T-ALL with SB225002 [5 μM]. Treatment control was DMSO 0.1%. Cells were incubated for 48 h. S = scramble transfection control; G-KD = cells infected with GLIPR1-shRNA lentiviral particles (Sigma-Aldrich). P values were calculated using two-tailed Student’s t-test.
Fig 5.
SB225002 and GLIPR1 knockdown effects on ROS generation in ALL cells.
(A) Reactive oxygen species production in B-ALL (REH and RS4;11) and T-ALL (Jurkat and TALL-1) cells treated with DMSO (vehicle; 0.1%) and SB225002 [5 μM and 10 μM]. Cells were treated for 24 h. (B) ROS generation in GLIPR1-knockdown (G-KD) versus control scramble (S) cells treated with DMSO (vehicle; 0.1%) or SB225002 [10 μM] for REH and RS4;11 or SB225002 [5 μM] for Jurkat and TALL-1 for 24 h. (C) Effect of N-Acetyl Cysteine (NAC; a ROS scavenger) pre-treatment on the survival of GLIPR1-knockdown (G-KD) versus control scramble (S) ALL cell lines upon SB225002 treatment for 48 h. Cells were pre-incubated or not with NAC [10 mM] for 3h prior to the SB225002 treatment. SB225002 was used at [10 μM] (REH and RS4;11) or [5 μM] (Jurkat and TALL-1). S = scramble transfection control; G-KD = cells infected with GLIPR1-shRNA lentiviral particles (Sigma-Aldrich). P values were calculated using two-tailed Student’s t-test.