Fig 1.
SMA suppressed colony formation and PI3K/Akt signaling in colon cancer cells.
(A) Single-cell suspensions of CT26 and HT29 cells in agarose medium in the absence or presence of different concentrations of SMA were incubated for three weeks. Colonies that formed on five separate dishes were counted, and the mean colony numbers are shown in the bar graphs. *P<0.05 vs. the vehicle-treated control. (B) SMA potently inhibited cancer cell proliferation induced by serum. (C) Western blot analyses of the phosphorylation of PI3K/p85, Akt, and mTOR in SMA-treated HT29 colon cancer cells. The bar graph represents the relative band intensities of PI3K and its downstream signals. The measurements were performed in three independent experiments. *P<0.05 vs. the vehicle-treated control. #P<0.05 vs. serum-stimulated cells. D-E, GSK3β inhibitors (LiCl, BIO) induced cancer cell proliferation, which was inhibited by SMA.
Table 1.
The half maximal inhibitory concentration (IC50) of SMA in cancer cell proliferation.
Fig 2.
SMA inhibited nuclear translocation of β-catenin and TCF/LEF1 transcriptional activity in HT29 human colon cancer cells.
(A-B) Immunofluorescence confocal images of β-catenin in HT29 cells treated with serum (A) or Wnt3α (B). β-Catenin and cell nuclei are stained green and blue, respectively. The images are representative of three independent experiments. (C) Western blots showing the levels of β-catenin in the cytosolic, nuclear, and total fractions of HT29 cells. The bar graph shows the relative band intensities of β-catenin. (D) HT29 cells were transfected with TCF/LEF-1-Luc reporter gene and pretreated with vehicle or SMA 1 hour prior to treatment with serum or Wnt3. *P<0.05 vs. vehicle-treated control cells. #P<0.05 vs. serum- or Wnt3α-treated cells. (E) The nuclear protein expressions of cyclin D1 and c-myc were inhibited in HT29 cells by SMA in a concentration-dependent manner. *P<0.05 vs. vehicle-treated control cells. #P<0.05 vs. serum-treated cells (n = 3).
Fig 3.
SMA facilitated GSK3β activity by inhibiting PI3K/Akt and tankyrase in HT29 colon cancer cells.
(A) Western blot analyses of phosphorylation of PI3K, Akt, GSK3β, and β-catenin and the expression level of axin in SMA- or XAV939-treated HT29 cells. *P<0.05 vs. vehicle-treated controls. (B) Tankyrase activity was measured using the Tankyrase 1 Colorimetric Activity Assay Kit and TNKS2 Histone Ribosylation Colorimetric Assay Kit. XAV939 was used as a positive control. *P<0.05 vs. vehicle-treated control cells. (C) HT29 cells transfected with TCF/LEF-1-Luc reporter gene were pretreated with vehicle or SMA 1 hour prior to being treated with serum.
Table 2.
The IC50 of SMA with respect to tankyrase and TCF/LEF transcription activities.
Fig 4.
Suppression of colon tumor growth by SMA in murine syngenic and xenograft tumor models.
CT26 murine or HT29 human colon cancer cells were subcutaneously injected into BALB/c mice or BALB/c nude mice, respectively. When tumors reached 120 mm3, SMA were administered intraperitoneally, and 5-FU was used as a positive control. (A) Schematic representation of drug treatment in CT26 allograft and HT29 xenograft tumor models. (B-D) Tumor growth in CT26 syngenic model as measured by tumor size (B) and excised tumor weights (C) along with body weight reductions (D). (E-F) In the HT29 xenograft tumor model, SMA dose-dependently inhibited tumor growth (E) without reducing body weight (F). *P<0.05 vs. the vehicle-treated controls. *P<0.05 vs. vehicle-treated controls. #P<0.05 vs. 5-FU alone-treated animals. $P<0.05 vs. SMA alone-treated animals. In all experiments, six mice per group were used.
Fig 5.
Anti-tumor effect of SMA alone and in combination with cisplatin in the A549 human non-small cell lung cancer xenograft model.
(A) Tumor-bearing BALB/c nude mice were treated intraperitoneally with SMA, cisplatin, or both, for 7 consecutive days following a 3-day intermission as one cycle. Six mice per group were used. (B) Tumor growth was monitored by measuring tumor size. (C) Twenty eight days after commencing three cycles of treatment, tumor tissues were isolated and the tumor weights were measured. (D-F) Western blotting of tumor tissues on signaling molecule activation (D), nuclear localization of β-catenin (E), and protein expression of target genes (F). C and N in (E) represent cytosol and nucleus, respectively. *P<0.05 vs. vehicle-treated controls. #P<0.05 vs. cisplatin alone-treated animals. $P<0.05 vs. SMA alone-treated animals.