Fig 1.
Effect of CIL-102 on cell viability and morphological characteristics of human colorectal cancer DLD-1 cells, and its role in assessing cell death.
(A) DLD-1, HAT-116 and HCoEpiC cell were treated with either 0.1% DMSO (as control) or CIL-102 (0.1–10 mM) for 24 h and the proportion of surviving cells was measured by the MTT assay. (B) Changes in nuclei by DAPI staining. DLD-1 cells were treated with vehicle or 1 mM CIL-102 for 24 h, and stained with DAPI. Apoptotic cells were measured under fluorescence microscopy. The data were presented as the mean of three repeats from one independent experiment. Other data in this figure is presented as mean ± SD of three independent experiments. * indicate means that are significantly different when compared to the control group of DLD-1 with P < 0.05.
Fig 2.
After an indicated treatment for 24 h, the DLD-1 cells were stained with FITC-conjugated Annexin-V and PI for flow cytometry analysis as described in Materials and methods.
The percentages presented in each frame depicted the apoptotic cells. *P <0.01, compared with the control group (0.2% DMSO).
Fig 3.
Effect of CIL-102 on cell cycle distribution in DLD-1 cells.
(A) After treatment with Erinacine A (1 mM) for 24 h, the cells were fixed and stained with propidium iodide, and the DNA content was analyzed by flow cytometry (FACS). The cell number percentage in each phase (G1, S, and G2/M) of cell cycle was calculated and expressed.
Fig 4.
Effect of CIL-102 on Fas-L, caspase 8, t-Bid, cytochrome c, caspase 3 and caspase 9.
Cells were treated with CIL-102 for 0–24 hr, and were separated by a 15% SDS–PAGE, and, subsequently, immunoblotted with antibodies against Fas-L, cleavage caspase 8, cleavage t-Bid, cytochrome c, cleavage caspase 3 and cleavage caspase 9, or b-actin which served as internal control. CIL-102 induced translocation of cytochrome c. Equal amounts of protein from cytosolic fraction of DLD-1 cells which has been treated with 1 mM of CIL-102.
Fig 5.
Effect of CIL-102 on the p21, GADD45, p50 NFκB, p300/CBP, CREB as well as expression of cell cycle associated proteins.
DLD-1 cells were pretreated with CIL-102 for indicated time points. Whole cell lysate proteins were prepared and analyzed by western blot, with β-actin serving as loading control. CIL-102 for the indicated times were analyzed by 10% SDS-PAGE and subsequently treated with antibodies against p21, GADD45, p50 NFκB, p300/CBP and CREB. The association of cdc2 with Cyclin B were determined by immunoprecipitation followed by western blot with antibody.
Fig 6.
Effect of the kinase inhibitors in blocking CIL-102-induced cell cycle G2/M arrest and apoptosis-related protein.
(A) The expression levels of phosphorylation and protein levels of JNK and ERK, after treatment with CIL-102. DLD-1 cells were treated with CIL-102 for the indicated time points. Total cell lysates were analyzed by SDS-PAGE and subsequently immunoblotted with antisera. (B) DLD-1 were incubated with various concentrations of the specific JNK1/2 inhibitor SP600125, NFκB inhibitor PDTI or the p300/CREB-Binding Protein inhibitor C646 for 1 h. Next, the cells were treated with CIL-102 for 12 h. Total cell lysates of cells treated with or without CIL-102 for the indicated time were extracted, and the expression of p21, GADD45, p50 NFκB, Histone H3 (K4) as well as The association of cdc2 with Cyclin B were determined, as described in “Materials and methods”. Protein levels were quantified by densitometric analysis with the ratio of untreated control being set 1 fold. The quantitative data were presented as the mean of three repeats from one independent experiment. The data were presented as mean ± SD of three independent experiments. *P<0.05, compared to the control group.
Table 1.
Effects of the kinase inhibitor on the CIL-102 treatment associated with apoptosis and cell cycle distribution in DLD-1 cells.
Fig 7.
Schematic presentation of the signaling pathways involved in CIL-102 induced cell cycle arrest G2/M phase and apoptosis in human DLD-1 cancer cells.
The effect of CIL-102 on the activation JNK1/2, and p50 NF-κB/p300 pathways, which leads to p21 and GADD45 expression and cdc2/cyclin B inactivation and increases cell cycle arrest in human DLD-1. CIL-102 triggered the extrinsic apoptosis pathway through activation of Fas-L and induction of Bid cleavage, cytochrome c release and caspase-8, -3, -9 activation.