PMC

Zyflamend induces expression of death receptors. HCT-116 cells were treated with the indicated concentrations of Zyflamend for 24 h (A) or with 250 μg/mL Zyflamend for the indicated times (B). Whole-cell extracts were prepared and analyzed for DR5 and DR4 by Western blotting. β-Actin was used as an internal control to verify equal protein loading. (C) HCT-116 cells were treated with 100 μg/mL Zyflamend for 24 h and then harvested for analysis of cell surface expression of DR5 and DR4 by FACS. (D) Zyflamend up-regulated DR5 in various types of cancer cells. Cells were treated with the indicated concentrations of Zyflamend for 24 h, and whole-cell extracts were prepared and analyzed by Western blotting using antibodies against DR5 and DR4. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. (Zy, Zyflamend) (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).

Zyflamend activates PKA signalling pathway in differentiating 3T3-MBX adipocytes. (a-b) Total cell lysates from control and Zyflamend treated cells for the entire duration of differentiation were immunoblotted for phosphorylated P38, JNK, c-JUN, their respective unphosphorylated proteins, c-FOS, and β-Actin to control for loading. (a) Representative immunoblots from three independent experiments are shown. (b) Bar graphs represent pP38^T180/Y182/P38, pJNK^T183/Y185/JNK, pc-JUN^S63/c-JUN, and c-FOS/β-Actin as means + SEM. p < 0.05, **p < 0.01 indicate significant difference between indicated time points and day 1 for each treatment. †p < 0.05, ††p < 0.01 indicate significant difference between Zyflamend and control (DMSO) treated cells. (c) Immunoblots of phosphorylated PKA substrate in 3T3-MBX cells treated or non-treated with Zyflamend (200 μg/ml) for the entire duration of differentiation. Representative immunoblots from three independent experiments are shown. (d) Bar graphs represent phosphorylated PKA substrate/β-Actin as means + SEM. *p < 0.05, **p < 0.01 indicate significant difference between indicated time points and day 1 for each treatment. †p < 0.05, ††p < 0.01 indicate significant difference between Zyflamend and control (DMSO) treated cells. (e) Immunoblots of pHSL^S660, and HSL in 3T3-MBX cells treated or non-treated with Zyflamend (200 μg/ml) for the entire duration of differentiation. Representative Immunoblots from three independent experiments are shown. (f) Bar graphs represent pHSL^S660/HSL, and HSL/β-Actin as means + SEM. *p < 0.05, **p < 0.01 indicate significant difference between the indicated time points and day 1 for each treatment. †p < 0.05, ††p < 0.01 indicate significant difference between Zyflamend and control (DMSO) treated cells

Up-regulation of death receptors by Zyflamend is CHOP dependent. (A, B) HCT-116 cells were treated with the indicated concentrations of Zyflamend for 24 h (A) or with 250 μg/mL Zyflamend for the indicated times (B). Whole-cell extracts were prepared and analyzed by Western blotting using the relevant antibodies. β-Actin was used as an internal control to verify equal protein loading. (C) Zyflamend up-regulated CHOP in various types of cancer cells. Cells were treated with the indicated concentrations of Zyflamend for 24 h, and whole-cell extracts were prepared and analyzed by Western blotting using antibodies against CHOP. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. (D) Silencing of CHOP reversed the effect of Zyflamend on DR5 expression. Cells were transfected with either CHOP siRNA or control siRNA. After 48 h, cells were treated with 250 μg/mL Zyflamend for 24 h, and whole-cell extracts were subjected to Western blotting for CHOP and DR5. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. (E) Mutation of CHOP binding site on DR5 promoter abolishes Zyflamend-induced DR5 transactivation. A293 cells were transfected with DR5/-552 luciferase plasmid containing wild type or mutated CHOP binding site. After 24 h, cells were treated with Zyflamend for 24 h and luciferase activity was determined as described in materials and methods. Each bar represents means of luciferase activity±SD of three independent experiments (F) Gene silencing of CHOP abolishes the effect of Zyflamend on TRAIL-induced apoptosis in colon cancer cells. HCT-116 cells were transfected with CHOP siRNA for 48 h. Cells were then treated with 100 μg/mL Zyflamend for 12 h, washed with PBS, and then incubated with 25 ng/mL TRAIL for 24 h. Cell death was determined by the live/dead assay. (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).

(A, left), MIA PaCa-2 (1 × 10⁶) cells were treated with Zyflamend (25 and 100 μg/mL) for 4 h, nuclear extracts were prepared and then assayed for NF-κB activation by EMSA; (Left) MIA PaCa-2 cells were incubated with Zyflamend (10 μg/mL) for indicated time, nuclear extracts were prepared and then assayed for NF-κB activation by EMSA. (B–D), Western blot analysis showed that Zyflamend inhibited constitutive expression of NF-κB-regulated gene products that regulate apoptosis (B), proliferation (C), and metastasis and angiogenesis (D) in pancreatic cancer cells. The MIA PaCa-2 (1 × 10⁶) cells were treated with Zyflamend (25 and 100 μg/mL) for 24 h. Whole-cell lysates were prepared and assayed for NF-κB-regulated gene products by Western blotting. Data represent two independent experiments.