Table 1.
Effect of SC-1 on the Tumor Take Rate of the Seven Colon Tumor Lines of the NCI60 Panel*.
Table 2.
Calculated Time to 1000 mg Tumor Volume in 5 Colon Lines Forming Tumors*.
Figure 1.
Effect of SC-1 on Tumor Formation in HCT-15 Colon Tumor Line with a 10 Cell Inoculum and Matrigel Co-injection.
Limiting dilution tumorigenicity assay was performed with simultaneous injection of Matrigel. A. Control treated HCT-15 tumor line. B. SC-1 treated HCT-15 tumor line. Tumor weight for each mouse is depicted. Tumor formation was increased in SC-1 treated cells when 10 cells/mouse were injected (SC-1 treated: 5 of 5 tumors formed, Control treated: 2 of 5 tumors formed). At higher cell inocula, all mice formed tumors independent of treatment. Each line in both graphs represents growth of one tumor per mouse (A. A–E, B. F–J).
Figure 2.
Tumor Lines Treated with SC-1 Possess Characteristics Consistent with the CSC Phenotype.
The colon tumor lines were treated for five days with 0.1 µM SC-1, harvested and then analyzed for their ability to form colonies in soft agar, to alter expression of putative CSC surface markers, and to form spheres. A. The cloning efficiency of the colon tumor lines was determined following treatment with SC-1. In 5/7 tumor lines, SC-1 treated cells had statistically significant increases in cumulative colony forming unit (CFU) mass compared to control treated cells (***p<0.001, **p<0.1 *p<0.05, n = 3). In one instance (#p<0.05), SC-1 treatment reduced cloning efficiency. B. The CD133 positive subpopulation was increased 2.5-fold in SC-1 treated HT29 colon tumor line (p = 0.03, n = 3). The CD44+ CD24- subpopulation was increased after SC-1 treatment in the HCT-116 colon tumor line (*p<0.05, n = 3). The CD44+ subpopulation was increased significantly following SC-1 treatments (*p<0.05, n = 3) in SW-620 colon tumor line. C. Three tumor lines (COLO 205, HCT-116, HT-29) formed nonadherent spheres in the presence of 0.1 µM SC-1 cultured in standard media containing 5% FBS. These observations were also apparent at day 5 in culture. All images were prepared at 400× magnification.
Figure 3.
Effect of SC-1 on Protein Expression Levels of phospho-ERK1/2, and OCT4 in Colon Tumor Lines.
Colon tumor lines were treated with SC-1 (0.1 µM), harvested, lysed, and probed for the proteins of interest following electrophoresis in SDS-PAGE gels at the indicated time points. A. In the SC-1 treated HT29 tumor line, phospho-ERK1/2/total ERK1/2 protein levels were decreased to 67±0.06% of control value at 5 min, 56±0.06% of control value at 30 min, and 46±0.06% of control value at 1 hr (n = 3, p<0.05). B. Increased OCT4 protein expression due to SC-1 was dependent on the tumor line. Representative experiments are shown for Figure S4A–B (n = 2).
Figure 4.
SC-1 Inhibited RSK2 Kinase Activity in vitro and an RSK2 Inhibitor Increased Colony Formation.
COLO 205 tumor cell line was treated with SL0101 (1.25 µM) and SC-1 (0.1 µM) for 24 hrs prior to soft agar cloning, lysate preparation, and immunoblotting. A. SC-1 inhibited RSK2 N-terminal domain in vitro kinase activity with an EC50 of 2.5±1.8 µM (mean±SD, n = 5) but not in other like assays for Aurora kinase B, CHK1, or CHK2 kinases. B. Decreased RSK2 protein levels (33%) were found at 5 days following SC-1 treatment in the COLO 205 tumor line (n = 2). C. Treatment (24 hrs) of COLO 205 tumor line with SL0101, a kaempferol glycoside RSK2 inhibitor, increased colony formation (≥60 µm) in soft agar (n = 2, representative experiment shown).
Table 3.
Effect of SC-1 on In Vitro Kinase Inhibition in Selected Kinases*.
Table 4.
Summary of SC-1 Effects in the NCI60 Colon Tumor Lines.