Journal: Frontiers in pharmacology

Article Title: Bax/Tubulin/Epithelial-Mesenchymal Pathways Determine the Efficacy of Silybin Analog HM015k in Colorectal Cancer Cell Growth and Metastasis.

doi: 10.3389/fphar.2018.00520

Figure Lengend Snippet: FIGURE 1 | The selectivity and cytotoxicity of 15k, 15j on colon cancer cell lines; (A) The chemical structures of silybin A and the two potential lead silybin derivatives 15k, 15j; (B) Survival of colon cancer cells (HCT116, S1, LOVO) compared to that of normal colon cells (CRL1459); IC50 Values of 15k, 15j respectively on colon cancer cells (HCT116, S1, LOVO) compared to that of normal colon cells (CRL1459); Cell survival was determined by the MTT assay. IC50 values are represented as means ± SD of three independent experiments performed in triplicate. Statistically, ***P < 0.001; (C,D) Colony formation assay with quantification of colony number represented as colony formation rate. HCT116 CRC cancer cells were incubated with different concentrations (0, 2, 4 µM) of 15k and15j. The pictures show the effect of 15k (C), and 15j (D) on colony formation in whole well, colonies density, and colony size; a bar graph summarizing the results for 15k and 15j, respectively. The results are represented as means ± SD of three independent experiments with *p < 0.05, **p < 0.01, ***p < 0.001. (E) Green cytotox (green fluorescence) to quantify cell proliferation and death; Representative pictures of the fluorescence level green cytotox at the 0 and 36 h time points; time line curve quantitatively summarizing the results is also shown. The data are presented as the means ± SEM of three independent studies.

Article Snippet: The cells were then incubated in a IncuCyte Zoom live cell imaging apparatus and pictures were taken every 2 h for up to 50 h.The IncuCyte R© integrated analysis software was used to quantify the fluorescent objects and minimize the background fluorescence (IncuCyte ZOOM version 2016A, Essen BioScience, Ann Arbor, MI 48108, USA).

Techniques: MTT Assay, Colony Assay, Incubation

Journal: Frontiers in pharmacology

Article Title: Bax/Tubulin/Epithelial-Mesenchymal Pathways Determine the Efficacy of Silybin Analog HM015k in Colorectal Cancer Cell Growth and Metastasis.

doi: 10.3389/fphar.2018.00520

Figure Lengend Snippet: FIGURE 4 | The effect of 15k on apoptosis induction in fluorescence time dependent studies at different concentrations in HCT116 cells. Apoptosis induction in fluorescence time dependent studies (Annexin V red, and caspase 3/7 reagent) on HCT116 cells incubated with different concentrations of 15k (0, 2, 4, and 8 µM) for 48 h. The fluorescent reagents were added along with vehicle or 15k for 48 h. Annexin V (red fluorescence) and caspases 3/7 reagent (green fluorescence). (A,B) are representative pictures of the fluorescence level of the two reagents at the 0 and 36 h time points. Time line curves quantitatively summarizing the results are also shown. (C) Western blots for the proteins BCL-2, Bak, Bax (p21 and p18), caspase-3, PARP, and cleaved PARP following overnight incubation with 15k (4 µM). The values of the cytosolic proteins were normalized to β-actin levels where nuclear proteins (PARP) are normalized to histone levels. A histogram summarizing the levels of each protein is also shown. All the data are presented as the means ± SEM of three independent studies with *p < 0.05, **p < 0.01, ***p < 0.001 vs. control group.

Article Snippet: The cells were then incubated in a IncuCyte Zoom live cell imaging apparatus and pictures were taken every 2 h for up to 50 h.The IncuCyte R© integrated analysis software was used to quantify the fluorescent objects and minimize the background fluorescence (IncuCyte ZOOM version 2016A, Essen BioScience, Ann Arbor, MI 48108, USA).

Techniques: Incubation, Western Blot, Control

Journal: Frontiers in pharmacology

Article Title: Bax/Tubulin/Epithelial-Mesenchymal Pathways Determine the Efficacy of Silybin Analog HM015k in Colorectal Cancer Cell Growth and Metastasis.

doi: 10.3389/fphar.2018.00520

Figure Lengend Snippet: FIGURE 6 | The effect of 15k on the expression of α- tubulin protein level in HCT116 cells; (A) Western blot showing the protein level of α-tubulin following overnight incubation with 15k (0, 4 uM). A histogram quantitatively summarizing the results of expression is also shown where Expression R stands for relative expression; (B) The effect of 15k on spindle microtubule formation in HCT116 cells. Representative pictures of α-tubulin fluorescence from cells incubated overnight with 15k at different concentrations (0, 2, 4 µM). Cells were then fixed and labeled with a monoclonal α-tubulin (green) antibody conjugated to FITC and nuclei stained with DAPI. A histogram quantitatively summarizing the results is also shown. The data are presented as the means ± SEM of three independent studies with **p < 0.01, ***p < 0.001; (C) The docking studies with the structure of DAMA Colchicine (Col) and 15k, respectively, flowed by XP-Glide predicted binding mode of 15k in the colchicine binding site of tubulin (PDB ID: 1SA0). Important amino acids are depicted as sticks with the atoms colored as carbon—green, hydrogen—white, nitrogen—blue, oxygen—red, sulfur—yellow, whereas the ligand 15k is shown with the same color scheme as above except for carbon atoms which are represented in orange. The red dotted lines represent hydrogen bonding. An overlay of the ligand 15k on DAMA-colchicine (carbon atoms presented in yellow) in the colchicine binding site of tubulin is also shown.

Article Snippet: The cells were then incubated in a IncuCyte Zoom live cell imaging apparatus and pictures were taken every 2 h for up to 50 h.The IncuCyte R© integrated analysis software was used to quantify the fluorescent objects and minimize the background fluorescence (IncuCyte ZOOM version 2016A, Essen BioScience, Ann Arbor, MI 48108, USA).

Techniques: Expressing, Western Blot, Incubation, Labeling, Staining, Binding Assay

Journal: Frontiers in pharmacology

Article Title: Bax/Tubulin/Epithelial-Mesenchymal Pathways Determine the Efficacy of Silybin Analog HM015k in Colorectal Cancer Cell Growth and Metastasis.

doi: 10.3389/fphar.2018.00520

Figure Lengend Snippet: FIGURE 8 | The effect of 15k on embryonic signaling pathways: (A) Representative image of HCT116 cell morphology at 40X. The morphological changes are consistent with EMT inhibition as the cells where converted from a mesenchymal shape to epithelial shape. Images were taken from EVOS microscope; (B) Western blots illustrating the levels of E-cadherin, N-cadherin, DVL3, c-Myc, Cyclin B1, and β-catenin fragments following overnight incubation with 15k (4 µM). The values of the cytosolic proteins were normalized to β-actin and histone levels. A histogram quantitatively summarizing the results for each protein where expression (R) stands for the relative expression level of each protein and a histogram quantitatively summarizing the effects of 15k on the levels of the β-catenin protein fragments are both shown where P1, P2, and P3 are the fragments of β-catenin; (C,D) representative pictures of E-cadherin and β- catenin fluorescence in HCT116 cells incubated with 15k at 0, 2 or 4 µM for 24 h, respectively. Histograms quantitatively summarizing the results are shown (Fluorescence R is for relative fluorescence levels). All data are presented as the means ± SEM of three independent experiments with *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The cells were then incubated in a IncuCyte Zoom live cell imaging apparatus and pictures were taken every 2 h for up to 50 h.The IncuCyte R© integrated analysis software was used to quantify the fluorescent objects and minimize the background fluorescence (IncuCyte ZOOM version 2016A, Essen BioScience, Ann Arbor, MI 48108, USA).

Techniques: Protein-Protein interactions, Inhibition, Microscopy, Western Blot, Incubation, Expressing, Fluorescence