Article Figures & Data
Figures
- Fig. 1.
Modulation of thiols by Bar-Me, CDODA-Me, and CF3DODA-Me in leukemia cells. (A) Structures of oleanolic and glycyrrhetinic acid–derived triterpenoids CDDO-Me (Bar-Me), CDODA-Me, and CF3DODA-Me. (B) Spectral changes in Bar-Me, CDODA-Me, and CF3DODA-Me after incubation with GSH. (C) Comparative A-, B-, and C-ring structures of Bar-Me and CDODA-Me/CF3DODA-Me. (D) Cells were treated with dimethylsulfoxide (DMSO), Bar-Me, and CF3DODA-Me for 6 hours, and TNFα was then added for 15 minutes. Whole cell lysates were analyzed by western blots as outlined in Materials and Methods.
- Fig. 2.
Bar-Me and CF3DODA-Me inhibit leukemia cell growth and induce apoptosis and differentiation of leukemia cells. HL-60 and Jurkat cells were treated with Bar-Me (A) or CF3DODA-Me (B) and growth inhibition was determined using the XTT assay. HL-60 and Jurkat cells were treated with Bar-Me or CF3DODA-Me for 24 hours and the effects of cleaved PARP and caspases (C), annexin V staining (D), and apoptosis were determined by western blots and FACS analysis for annexin V and propidium iodide staining, respectively, as outlined in Materials and Methods. Cells were treated with dimethylsulfoxide (DMSO), Bar-Me, and CF3DODA-Me for 72 hours and differentiation markers (CD11b) (E) in HL-60 and CD4 in Jurkat (F) cells were determined by FACS analysis as outlined in Materials and Methods. Results are expressed as mean ± S.E.M. for at least three replicate determinations and significant (P < 0.05) changes (compared with DMSO) are indicated (#).
- Fig. 3.
Bar-Me and CF3DODA-Me induce ROS in leukemia cells. HL-60 (A) and Jurkat (B) cells were pretreated with GSH for 1 hour, and then treated with dimethylsulfoxide (DMSO), Bar-Me, or CF3DODA-Me for 3 hours. ROS was determined by FACS analysis as outlined in Materials and Methods. (C) Cells were treated with DMSO, Bar-Me, or CF3DODA-Me alone or in combination with GSH for 24 hours and cell proliferation was determined using the XTT assay as outlined in Materials and Methods. (D) Cells were treated with DMSO, Bar-Me, or CF3DODA-Me alone or in combination with GSH for 24 hours, and cleaved PARP and caspases were determined by western blots of whole cell lysates as outlined in Materials and Methods. Results are expressed as mean ± S.E.M. for at least three replicate determinations and significant (P < 0.05) effects of the compounds (#) and reversal of specific effects by GSH (*) are indicated.
- Fig. 4.
Bar-Me and CF3DODA-Me induce markers of differentiation and maturation. HL-60 cells were treated with Bar-Me or CF3DODA-Me alone or in combination with GSH and the effects of CD11b mRNA (A) and protein levels (B) were determined after 24 and 72 hours, respectively, as outlined in Materials and Methods. Jurkat cells were treated with Bar-Me or CF3DODA-Me alone or in combination with GSH and CD4 (C) and were determined by FACS analysis after 72 hours and IL-2, IL-4, and IL-13 mRNA after 24-hour levels (D) were determined by real-time PCR as outlined in Materials and Methods. Results are expressed as mean ± S.E.M. (at least three replicate determinations), and significant (P < 0.05) induction by the compound (#) and inhibition by GSH (*) are indicated.
- Fig. 5.
Bar-Me and CF3DODA-Me decrease Sp proteins and cMyc in leukemia cells. (A) Cells were treated with dimethylsulfoxide (DMSO), Bar-Me, or CF3DODA-Me for 24 hours and whole cell lysates were analyzed by western blots as outlined in Materials and Methods. (B) Cells were treated with DMSO, Bar-Me, or CF3DODA-Me alone or in combination with GSH for 24 hours and whole cell lysates were analyzed by western blots as outlined in Materials and Methods. (C) Cells were treated with DMSO, Bar-Me, or CF3DODA-Me for 3, 6, or 12 hours and whole cell lysates were analyzed by western blots as outlined in Materials and Methods. (D) Cells were treated with DMSO, Bar-Me, or CF3DODA-Me alone or in combination with GSH for 9 hours and analyzed by western blots as outlined in Materials and Methods, and results are expressed as mean ± S.E.M. for at least three replicate determinations. Significant (P < 0.05) effects of the compounds (#) and reversal by GSH (*) are indicated.
- Fig. 6.
Role of cMyc and Sp TFs in leukemia cell growth, differentiation, and maturation. Knockdown of Sp1 (siSp1), Sp3 (siSp3), Sp4 (siSp4), all three Sp TFs (siSp1/3/4), and cMyc (sicMyc) in HL-60 cells was carried out by RNA interference, and the effects on protein expression (A) and cell proliferation (B) were determined by western blots and the XTT assay. Effects of knockdown on CD11b mRNA and cellular expression of CD11b protein (C) were determined by real-time PCR and FACS analysis, respectively. Jurkat cells were transfected with siSp1, siSp3, siSp4, siSp1/3/4, and sicMyc, and effects on protein levels (D), cell growth (E), CD4-positive cells (F), and IL-2, IL-4, and IL-3 mRNA levels (G) were determined by western blots, XTT assay, FACS analysis, and real-time PCR, respectively, as outlined in Materials and Methods. Results are expressed as mean ± S.E.M. (three replicate determinations) and significant (P < 0.05) effects compared with dimethylsulfoxide control are indicated (#).
- Fig. 7.
Inactivation of mTOR and Akt by Bar-Me and CF3DODA-Me. (A) Cells were treated with Bar-Me or CF3DODA-Me alone or in combination with 5 mM GSH for 12 hours and analyzed by western blots. Cells were treated with LY294002 (PI3K) and MK2208 (Akt) kinase inhibitors for 24 hours, and whole cell lysates were analyzed by western blots (B) by determining the effects on cell growth in HL-60 and Jurkat cells (C) and on CD11b (HL-60 cells) (D) and CD4 (Jurkat cells) (E) differentiation markers as outlined in Materials and Methods. Results (C–E) are mean ± S.E.M. for at least three replicate determinations and significant (P < 0.05) differences compared with dimethylsulfoxide (DMSO) (control) are indicated (#).
Additional Files
Data Supplement
Files in this Data Supplement:
- Supplemental Figures -
Supplemental Figure 1 - GSH inhibits Bar-Me- and CF3DODA-Meinduced apoptosis
Supplemental Figure 2 - Bar-Me and CF3DODA-Me decrease mitochondrial membrane potential and Sp proteins in leukemia cells
Supplementary Figure 3 - Effects of Tiron. HL-60 and Jurkat cells were treated with DMSO, Bar-Me, or CF3DODA-Me alone or in combination with 100 mu-M Tiron for 24 hrs and effects on induction of ROS in HL60 (A) and Jurkat (B) cells and cell growth inhibition (C) were determined
Supplementary Figure 4 - Effects of Tiron. HL-60 and Jurkat cells were treated with DMSO, Bar-Me, or CF3DODA-Me alone or in combination with 100 mu-M Tiron for 72 hr (FACS analysis) or 9 hr (Western blot) and effects on CD11b (A), CD4 (B), and protein levels (C) were determined
Supplemental Figure 5 - Bar-Me and CF3DODA-Me do not induce ZBTB proteins
Supplemental Figure 6 - HL-60 and Jurkat cells were treated with DMSO (control), 0.5 mu-M Bar- Me, and 2 mu-M CF3DODA-Me for 3, 6 or 12 hr
- Supplemental Figures -
In this issue
Triterpenoids Induce ROS-Dependent cMyc Downregulation
