Novel dichlorophenyl urea compounds inhibit proliferation of human leukemia HL-60 cells by inducing cell cycle arrest, differentiation and apoptosis

Abstract

Two novel dichlorophenyl urea compounds, SR4 and SR9, were synthesized in our laboratory and evaluated for anti-cancer activities. Specifically, we investigated the antiproliferative properties of these new compounds on promyelocytic HL-60 leukemia cells by analyzing their effects on cell differentiation, cell cycle progression and apoptosis. SR4 and SR9 were both cytotoxic to HL-60 cells in a dose-and time-dependent manner, with IC(50) of 1.2 μM and 2.2 μM, respectively, after 72 h treatment. Both compounds strongly suppressed growth of HL-60 cells by promoting cell cycle arrest at the G0/G1 transition, with concomitant decrease in protein levels of cyclins D1 and E2 and cyclin-dependent kinases (CDK 2 and CDK 4), and increased protein expression of CDK inhibitors p21(WAF1/Cip1) and p27(Kip1). In addition, either compounds induce cell differentiation as detected by increased NBT staining and expression of CD11b and CD14. Treatment with SR compounds also promoted mitochondrial-dependent apoptosis as confirmed by Annexin V-FITC double staining, DNA fragmentation, increased expression of caspase 3, 7 and 9, cytochrome c release, PARP degradation, and collapse in mitochondrial membrane potential (ΔΨ(MT)). Collectively, these results provide evidence that SR4 and SR9 have the potential for the treatment of human leukemia and merit further investigation as therapeutic agents against other types of cancer.

Small-molecule COH-SR4 inhibits adipocyte differentiation via AMPK activation

“…Similarly, an AMPK activator, AICAR, has been recently reported to induce G1/S cell cycle arrest and inhibit proliferation in cancer cells (35). We also observed cell cycle arrest in human leukemia melanoma and lung and breast cancer cells (19,20, unpublished data). Overall, these data indicate that COH-SR4 induction of AMPK in both adipocytes and cancer cells results in cell cycle arrest, and thus, imply a common molecular target.…”

“…A recent study showed that chemicals that can cause depolarization of mitochondrial membrane potential (Δψm) can also activate AMPK (32). Interestingly, we previously found that COH-SR4 decreased Δψm in HL-60 leukemia cells (19). We are currently investigating the effects of COH-SR4 on the mitochondria to identify the exact molecular target of the compound.…”

“…Cells were harvested and fixed with 70% ethanol at 4˚C for 24 h. After removal of ethanol by washing with ice cold PBS, cells were resuspended in 1 ml propidium iodide (PI) staining solution (4% PI, 2 µg/ml RNase in PBS) and incubated for 30 min at 37˚C. Fluorescenceactivated cell sorting (FACS) analysis was performed using a CyAn ADP cytometer (Beckman Coulter, Inc.) as previously described (19).…”

“…Total cytosolic proteins were extracted with cell lysis buffer (Cell Signaling Technology), and the protein concentration was determined using the DC Protein Assay Kit (Bio-Rad, Hercules, CA, USA). Protein electrophoresis and western blotting were performed as described previously (19). Equal loading of proteins was confirmed by stripping and restaining the membranes with β-actin antibodies.…”

SR4 Uncouples Mitochondrial Oxidative Phosphorylation, Modulates AMP-dependent Kinase (AMPK)-Mammalian Target of Rapamycin (mTOR) Signaling, and Inhibits Proliferation of HepG2 Hepatocarcinoma Cells

“…SR4 was synthesized according to a previously validated protocol at the Chemical GMP Synthesis Facility, Translational Medicinal Chemistry Laboratory, Beckman Research Institute of the City of Hope (30). FCCP, antimycin A, rotenone, 6-ketocholestanol (6-KCH), cyclosporin A (CSA), bongkrekic acid, N,N,NЈ,NЈ-tetramethyl-p-phenylenediamine (TMPD), valinomycin, ascorbic acid, malic acid, succinate, MitoTempo, guanosine diphosphate (GDP), N-acetyl-L-cysteine (NAC), fatty acid/endotoxin-free bovine serum albumin (BSA), tetramethylrhodamine ester (TMRE), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and crystal violet (CV) were purchased from Sigma.…”