Figure 1.
Chemical structures of 4,3′,4′,5′- tetramethoxychalcone (TMOC) (A), and TMOC did not interrupt tubulin polymerization (B).
Figure 2.
The effect of TMOC on cell proliferation and colony formation.
(A) TMOC inhibited cell proliferation in dose- and time-dependent manner. Cell viability was determined by MTT assays. (B) Representative images of cell colonies after treatment with various concentrations of TMOC for 24 h. (C) Colony formation rate after treatment with TMOC for 24 h.
Table 1.
The IC50 values and water solubility of TMOC and chalcone.
Figure 3.
The effect of TOMC on cell cycle progression.
(A) Cell cycle distribution after treatment with different concentrations of TMOC for 24 h. (B) Quantitative analysis of TMOC-treated cells. (C) Regulation of cell cycle associated proteins in A2780 cells. The experiments were repeated three times, and a representative experiment is shown. * p<0.05, ** p<0.01 compared to control.
Figure 4.
(A) DAPI nuclei staining was used to visualize the apoptosis induced by TMOC. Arrows represent the apoptotic cells. Scale bar = 10 µm. (B) PI uptake was analyzed under a fluorescent microscope. Scale bar = 50 µm. (C) Representative flow cytometry profiles of apoptosis and (D) quantitative results obtained using Annexin V/PI staining. (E) Western blots of apoptotic related proteins. The assay was repeated three times, and a representative result is shown. * p<0.05, ** p<0.01 compared to control.
Figure 5.
The inhibitory effect of TMOC on cell migration and invasion.
(A) TMOC inhibited cell migration. Representative photographs of the cells were taken at time 0, 36 or 72 h. (B) Quantification of wound healing. The experiments were repeated three times. (C) Representative photographs of invasive cells in transwell invasive assays. Scale bar = 20 µm. (C) Quantification of invasive A2780 cells. The experiments were repeated three times. * p<0.05, ** p<0.01 compared to control.
Figure 6.
The molecular mechanism of in vitro anti-tumor effects of TMOC.
(A) TMOC treatment inhibited the STAT3 phosphorylation and down-regulated the level of c-myc in a dose dependent manner. (B) A2780 and A2780/CDDP cells were transfected with STAT3 constitutively active (S3-CA) plasmid, STAT3 dominant negative (S3 DN) plasmid or control vector. (C) Introduction of S3-CA significant blocked the anti-proliferative activity of TMOC. In contrast, introduction of S3-DN sensitized the cancer cells to TMOC treatment. (D) TMOC inhibited the c-Src kinase phosphorylation and up-regulated the tumor surpressor PTEN in all of the three cell lines, but only up-regulated p53 in A2780 cells. β-actin was used as an equal loading control. The experiments were repeated three times and a representative experiment is shown. * p<0.05, ** p<0.01 compared to control.