Figure 1.
EF24 inhibited liver cancer cell proliferation.
(A). The topological structures of curcumin (diferuloylmethane) and EF24 (diphenyl difluoroketone). (B). EF24 treatment on proliferation of PLC/PRF/5, SK-HEP-1, Hep3B, HepG2, Huh 7 and LO2 cells. These cells were incubated with increasing doses of EF24 (0.5–8 µmol/L) for 48-h and 72-h periods and analyzed for cell proliferation using cell counting kit-8 assay.
Figure 2.
EF24 induced cell cycle arrest and inhibited the NF-κB pathway.
(A). DNA content (propidium iodide) and cell cycle analysis of EF24-treated cells. The five HCC cells were incubated with 0, 2 µmol/L EF24 for 48 h. The numbers of cells in G0/G1 phase, S phase and G2/M phase was determined via flow cytometry. (B). Representative histograms from cytometrically analyzed the five cell lines treated with control and EF24. Assay was done in triplicate and p<0.05 is denoted by “*”. (C). Expression of G2/M cell cycle relative proteins Tyr15 and Thr161 phosphorylation of cdc2 and Cyclin B1 were determined via western blot after treatment with 2 and 4 µmol/L EF24 for 24 h. β-actin was used as the internal control. (D). Nuclear extracts were prepared and subjected to EMSA to measure NF-κB DNA-binding activity. (E). Expression of p65, Bcl-2, Bax and COX-2 via western blot.
Figure 3.
EF24 induced cancer cell apoptosis.
(A). Five liver cancer cells were treated with 0, 2 and 4 µmol/L EF24 for 48 h and harvested. Flow cytometry was performed to observe apoptosis rates. (B). Representative histograms from cytometrically analyzed the five cell lines treated with control (DMSO) and EF24. Assay was done in triplicate. *p<0.05; **p<0.01. (C).The target proteins were detected by Western blot analyses. (D). Histograms from three cell lines treated with EF24 and EF24 in combination with pan-caspase inhibitor (z-VAD-fmk). (E). Electron microscopic findings in PLC/PRF/5 cancer cells treated with EF24. Cancer cells without treatment exhibited innumerable microvilli and well developed filopodia on the cell surface with intact nuclei (a). In contrast, EF24 induced distinct changes on the cell surface with decreased filopodias and microvilli, and changes in nuclei (b). Original magnification: ×6,000 in a; ×8,000 in b.
Figure 4.
EF24 inhibits liver cancer tumor xenograft growth in vivo.
(A) and (B). PLC/PRF/5 cells were injected to the flanks of nude mice and palpable tumors were allowed to develop for 7 d. Subsequently, 20 mg/kg/d of EF24 was injected daily i.p. for up to 21 d. On day 22, tumors were excised and subjected to further analyses. Tumor volumes in EF24 given mice were smaller than that of control mice. (C). Tumor size was measured every two days. There was a significant reduction in relative tumor volume from EF24-treated animals when compared with untreated controls. (D). Liver and spleen weight of the nude mice in EF24 treated and control group. (E) Tumor sections were stained with an anti-Ki-67 Ab to detect proliferating cells, or TUNEL agent to visualize apoptotic cells. (F). Cells expressing Ki-67 were counted to calculate the proliferation index, or TUNEL-positive cells. Assay was done in triplicate and p<0.05 is denoted by “*”. (G). Western blot analysis on the expressions of p65, Cyclin B1, p-cdc2 (Thr161), p-cdc2 (Tyr15), Bcl-2, Bax, PCNA, pro-caspase-3 and pro-caspase-9 from respective tumoral homogenate, with β-actin as protein loading control.