Fig 1.
Cudraflavone C induces tumor-specific cell death in colorectal cancer cells.
(A) Chemical structure of Cud C. (B) KM12, HT29, Caco-2, HCC2998, HCT116 and SW48 colorectal cancer cells were exposed to various concentrations of Cud C for 72 hours. Cell viability was recorded using CellTitre Glo® luminescence assay. (C) KM12, Caco-2 and CCD 841 CoN were treated with 0.1% DMSO (control) or 10μM Cud C (Cud C) for 72 hours followed by microscopy analysis (×100 magnification). (D) Apoptotic cell death in KM12, Caco-2 and CCD 841 CoN cells was quantified using Annexin V/7-AAD flow cytometry at 72 hours following treatment. (E) Caspase activities in KM12 and Caco-2 cells were assessed by Caspase Glo® assay at 72 hours following treatment. (F) 10μM Cud C induced mitochondrial membrane depolarization. Caco-2 and KM12 cells stained with JC-1 at 72 hours after treatment with Cud C. The green dye represents JC-1 monomers in cytoplasm while the red dye represents JC-1 aggregates in nucleus. Cells were observed under fluorescence microscope (×100 magnification). All data represent the mean ± s.d. from at least three independent experiments. Symbol “*” presents the statistical significance concluded from Student’s independent t-test with p-value ≤0.05.
Table 1.
IC50 of Cud C and 5-fluorouracil in colorectal cancer and non-transformed colon epithelial cells.
Fig 2.
Differential gene expression regulated by cudraflavone C in Caco-2 cells.
(A) Heatmaps generated based on the genes regulated by Cud C. Caco-2 cells were exposed to 10 μM Cud C for 48 hours. GeneChip® Human Transcriptome Array 2.0 (Affymetrix, USA) was applied. Gene expression changes that ≥2-fold were considered significant. Control 1 and Control 2 represent gene expression from cells treated with vehicle control (1% DMSO); Cud C 1 and Cud C 2 were gene expression from cells treated with Cud C (10 μM). (B) qPCR was used to validate the microarray data. Caco-2 cells were exposed to 10 μM Cud C for 12, 24, 48 or 72 hours The left and right panels present genes that are up and down-regulated respectively. All data represent the mean ± s.d. from at least three independent experiments. Symbol “*” indicates the statistical significance concluded from Student’s independent t-test with p-value ≤0.05.
Table 2.
Top 20 pharmaceutical perturbagens exhibiting positive correlation to the gene signature induced by Cud C treatment.
Fig 3.
Cudraflavone C inhibits PI3K activity.
The effect of negative control (1%DMSO), Cud C or LY-294002 (100 μM) on p110α/p85α, p110β/p85α, p110δ/p85α, and p120γ PI3K activity were quantified using the PI3K-Glo™ Class I Profiling Kit. All data represents the mean ± s.d. from at least three independent experiments. Symbol “*” presents the statistical significance concluded from Student’s independent t-test with p-value ≤0.05.
Fig 4.
Cudraflavone C inhibits AKT phosphorylation in Caco-2 and KM12 cells.
Caco-2 and KM12 cells were exposed to either DMSO (1%) or Cud C at 10 μM for 12, 24, 48 and 72hours. Protein lysates were subjected to SDS-PAGE. GAPDH was used as loading control. Numbers displayed below each blot denote the ratio of phosphorylated to total AKT.
Fig 5.
Ectopic expression of myr-AKT confers resistance to cudraflavone C.
(A) Caco2 and KM12 cells were transfected with myristoylated AKT for 24 hours before treatment of cells with test agent. Lysates were collected at 48 hours after transfection for immunoblotting analysis. (B) Cell proliferation was quantified by CellTiterGlo® and (C) Apoptotic cell death was quantified by annexin V/7-AAD flow cytometry. All data represent the mean ± s.d. from at least three independent experiments. Symbol “*” presents the statistical significance concluded from Student’s independent t-test with p-value ≤0.05.