Article Figures & Data
Figures
- Fig. 1.
Effects of SF-3-026 and analogs on proliferation of cancer cell lines. Data show percent proliferation compared with controls (100%) for A375, RPMI-7951, SK-Mel-28, HL-60, HeLa, or Jurkat cells after treatment with 100 μM of each test compound for 48 hours. The mean and S.D. from three independent experiments are shown.
- Fig. 2.
SF-3-030 interacts with and modifies ERK2 via cysteine adduct formation. (A) Putative mechanisms of SF-3-030 adduct formation with cysteine residues on ERK2. (B) Space-filling model of ERK2 with highlighted cysteine residues and SF-3-030 modification site (adapted from Protein Data Bank Identification (PDB ID): 4GT3). Cysteine modifications determined by high-resolution liquid chromatography-tandem mass spectrometry. Cysteine residues are colored in (green), as well as the TXY motif (magenta), the F-recruitment site (red), and the primary modification site C252 (red font).
- Fig. 3.
Lead compounds differentially regulate IEG levels in A375 cells. (A) Immunoblots for c-Fos, c-Jun, FosB/B2, Fra-1, and c-Myc proteins in cells treated with 10 μM SCH77294 or 25 μM SF-3-030 for 0–24 hours. (B) Phosphorylated (S383) and total Elk-1 levels in cells treated with 10 μM SCH77294 or 25 μM SF-3-030 for 0–24 hours. (C) Cells treated for 4 hours with varying doses of SF-2-110, SF-3-026, or SF-3-030. Controls include untreated or cells treated with 5 μM AZD6244 or SCH772984. Immunoblots show relative c-Fos, c-Jun, FosB/B2, Fra-1, and c-Myc protein levels. Levels of active ERK1/2 (ppERK1/2), total ERK2, and α-tubulin are shown to demonstrate ERK1/2 pathway activity and equal protein loading in (A) and (C). (D) Relative quantification of c-Fos, Fra-1, and c-Myc protein levels after 4 hours of exposure with 25 µM of SF-2-110 or SF-3-030. Mean and S.D. are from three independent experiments, and graph was determined via densitometry as described in the Materials and Methods. * and ** represent P < 0.05 and P < 0.01, respectively. (E) A375 cells were exposed to SF-3-030 for the indicated times and then incubated with fresh media without compound for a total incubation time of 24 hours. Lysates were immunoblotted for cleaved PARP, phosphorylated histone H3 (pH3), and total ERK2 for a loading control. Data are representative of three independent experiments. The numbers in each immunoblot represent the relative levels of protein, normalized to α-tubulin, as determined by densitometry. Molecular mass markers (kDa) are indicated on the right of each immunoblot.
- Fig. 4.
SF-3-030 induces NRF2 levels. (A) Immunoblot analysis of NRF2 in A375 cells treated with 10 μM SCH772984 or 25 μM SF-3-030 for 0–24 hours. (B) NRF2 levels in A375 cells treated with 25 μM SF-3-030 ±5 mM NAC for 0, 1, or 4 hours. Nonspecific bands that crossreact with the NRF2 primary antibody are indicated (ns) for (A) and (B). The graph in (C) shows the densitometry quantitation of NRF2 under the conditions described in (B). Mean and S.D. are from three independent experiments. * indicates statistical significance compared with SF-3-030 treatment only (P < 0.05). The numbers in each immunoblot represent the relative levels of protein, normalized to α-tubulin, as determined by densitometry. Molecular mass markers are indicated on the left of each immunoblot.
- Fig. 5.
SF-3-030 induction of ROS mediates inhibition of A375 cell proliferation. (A) ROS was measured by CellROX Deep Red reagent fluorescence in A375 cells treated with 25 μM SF-3-030 in the absence (white bars) or presence (black bars) of the following ROS inhibitors (ROSi): 10 mM sodium pyruvate (NaPyr), 100 mM mannitol (Mann), or 10 mM NAC for 60 minutes. Graphs represent three independent experiments, and each data point represents the mean ± S.D. from three wells with four fields of view per well and each field containing between 500 and 1000 cells. Statistical significance was determined within each experiment (* and ** represent P < 0.05 and P < 0.01, respectively). (B) A375 cell viability was measured after 48 hours in untreated or SF-3-030–treated cells in the absence (white bars) or presence (black bars) of the ROS inhibitors at the concentrations described in (A). The mean and S.D. for cell proliferation are from three independent experiments. ** indicates statistical significance (P < 0.01) compared with SF-3-030 treatment only. (C) Relative levels of c-Fos after 4 hours of treatment with SF-3-030 alone (white bars) or in combination with the indicated ROS inhibitors (black bars) at the concentrations indicated in (A). Relative c-Fos levels were determined by ProteinSimple immunoanalysis and normalized to total β-actin.
- Fig. 6.
NRF2 inhibitors do not affect SF-3-030 inhibition of A375 cell proliferation. (A) A375 cells were treated for 8 hours with 25 µM SF-3-030 in the absence or presence of ML-385 (50 µM) or brusatol (Bru; 30 nM). Lysates were immunoblotted for relative levels of NRF2, NQO1, HMOX1, and OSGIN1, as shown. (B) A375 lysates from cells treated for 8 hours with 25 µM SF-3-030 (SF) in the absence or presence of Bru (30 nM) were immunoblotted for GCLM and SRXN1. Positive control lysates from HeLa and A549 cells were used for GCLM and SRXN1, respectively. The numbers in each immunoblot represent the relative levels of protein, normalized to β-actin, as determined by densitometry. Molecular mass markers are indicated on the left of each immunoblot. (C) A375 cell viability with varying doses of ML-385 or (D) brusatol. (E) Cell viability with varying doses of SF-3-030 in the absence (white bars) or presence (black bars) of 50 µM ML-385. (F) Combination index with varying doses of SF-3-030 and brusatol. Untreated control cells are indicated with a striped column. Relative cell viability was measured after 48 hours, and data are representative of three independent experiments.
Tables
- TABLE 1
High-resolution liquid chromatography-tandem mass spectrometry analysis of proteins that increase or decrease ≥1.5-fold after treatment with SF-3-030 or SCH772984 for 4 h relative to untreated A375 cells (set at 1)
Protein changes common to both SF-3-030 and SCH772984 are italicized.
Proteins that increase with SF-3-030 Fold increase ZNF774 16.28 MEMO1 10.55 TTC39C 5.18 MYO18A 4.18 TCF4 2.24 HMOX1 1.77 Proteins that decrease with SF-3-030 Fold decrease HCCS 3.35 KIAA1211 2.24 ACTN2 1.66 AHCY 1.53 Proteins that increase with SCH772984 Fold increase TMTC1 5.15 SRGAP1 1.47 Proteins that decrease with SCH772984 Fold decrease TTC29 2.44 AHCY 1.48 ACTN2, α-actinin-2; KIAA1211, Capping protein inhibiting regulator of actin dynamics; MYO18A, unconventional myosin-XVIIIa; SRGAP1, SLIT-ROBO Rho GTPase-activating protein 1; (TMTC1), transmembrane and TPR repeat-containing protein 1; TTC29, tetratricopeptide repeat protein 29; TTC39C, Tetratricopeptide repeat protein 39C
- TABLE 2
High-resolution liquid chromatography-tandem mass spectrometry analysis of proteins that increase or decrease ≥1.5-fold after treatment with SF-3-030 or SCH772984 for 12 h relative to untreated A375 cells (set at 1)
Protein changes common to both SF-3-030 and SCH772984 are italicized.
A. Proteins that increase with SF-3-030 Fold increase OSGIN1 21.04 ZNF774 18.18 TCF4 10.95 PXDN 9.43 Protein MEMO1 8.37 TTC39C 7.31 MYO18A 4.50 SCO2 3.89 Jouberin (AHI1) 3.12 HMOX1 3.02 Protein moonraker (KIAA0753) 2.26 ASB6 2.07 REPS1 1.98 NOC2L 1.73 Sickle tail protein homolog (KIAA1217) 1.71 NCAPD3 1.49 Proteins that decrease with SF-3-030 Fold decrease Myomegalin (PDE4DIP) 2.80 HCCS 2.49 AP1M1 2.24 CNGB1 2.11 FOXR2 1.76 GRIP2 1.72 SUN1 1.68 RSL24D1 1.66 KIAA1211 1.62 INTS2 1.51 B. Proteins that increase with SCH772984 Fold increase FOXD3 32.88 PKP2 4.76 PABPC3 2.85 WDR59 2.76 SCO2 2.69 TCF4 2.64 KIAA1211 1.84 HBS1L 1.71 PDIA3 1.61 GFPT2 1.48 Proteins that decrease with SCH772984 Fold decrease CACNB4 2.65 PDE5A 2.18 RFC4 2.08 RNMT 1.88 ATP11A 1.82 TFAP2A 1.74 RSL24D1 1.70 DCN 1.68 HK2 1.60 ABHD5 1.59 COL1A1 1.48 ABHD5, Abhydrolase domain-containing protein 5; AHI1, Abelson helper integration site 1 protein homolog; AP1M1, AP-1 complex subunit μ-1; ASB6, ankyrin repeat and SOCS box protein 6; ATP11A, ATPase class VI type 11A; CACNB4, Calcium channel voltage-dependent subunit β 4; CNGB1, cyclic nucleotide-gated cation channel β-1; COL1A1, collagen α-1(I) chain; DCN, decorin; FOXR2, forkhead box protein R2; GFPT2, glutamine-fructose-6-phosphate aminotransferase 2; GRIP2, glutamate receptor-interacting protein 2; HBS1L, HBS1-like protein; HK2, hexokinase-2; INTS2, integrator complex subunit 2; KIAA1211, uncharacterized protein KIAA1211; MYO18A, unconventional myosin-XVIIIa; NCAPD3, condensin-2 complex subunit D3; NOC2L, nucleolar complex protein 2 homolog; PABPC3, polyadenylate-binding protein 3; PDE5A, phosphodiesterase 5A cGMP-specific; PDIA3, protein disulfide-isomerase A3; PKP2, plakophilin-2; PXDN, peroxidasin homolog; REPS1, RalBP1-associated Eps domain-containing protein 1; RFC4, replication factor C subunit 4; RNMT, mRNA cap guanine-N7 methyltransferase; SUN1, Sad1/unc-84 protein-like 1; TFAP2A, transcription factor AP-2-α; TTC39C, chromosome 18 open reading frame 17_isoform CRA_c; WDR59, WD repeat-containing protein 59. KIAA0753, moonraker; KIAA1217, Sickle tail protein homolog; PDE4DIP, Phosphodiesterase 4D-interacting protein.
Data Supplement
- Supplemental Data -
Supplemental methods, references, 4 tables, and 7 figures.
- Supplemental Data -
In this issue
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