Activation of cyclin-dependent kinase activity is frequently observed in many human cancers; therefore, cyclin-dependent kinases that promote cell cycle transition and cell proliferation may be potential targets in the treatment of malignancy. The therapeutic effects of roniciclib, a cyclin-dependent kinase inhibitor for papillary and follicular thyroid cancer (designated as well-differentiated thyroid cancer), were investigated in this study. Roniciclib inhibited cell proliferation in two papillary and two follicular thyroid cancer cell lines in a dose-dependent manner. Roniciclib activated caspase-3 activity and induced apoptosis. Cell cycle progression was arrested in the G2/M phase. Roniciclib treatment in vivo retarded the growth of two well-differentiated thyroid tumors in xenograft models in a dose-dependent fashion. Furthermore, the combination of roniciclib with sorafenib was more effective than either single treatment in a follicular thyroid cancer xenograft model. Acceptable safety profiles appeared in animals treated with either roniciclib alone or roniciclib and sorafenib combination therapy. These findings support roniciclib as a potential drug for the treatment of patients with well-differentiated thyroid cancer.
Supplementary Figure 1. The effects of roniciclib on cell cycle distribution in BHP7-13 cells. Cell cycle analysis using flow cytometry was performed to evaluate DNA content in BHP7-13 cells treated with placebo or roniciclib (25 nmol/L) for 24 h.
Supplementary Figure 2. The effect of roniciclib on mitosis in BHP7-13 cells. Chromosomal appearance was evaluated in BHP7-13 cells treated with roniciclib (25 nmol/L) or placebo for 24 h using immunofluorescence confocal microscopy. DNA was stained with DAPI. Placebo-treated cells in prophase (white arrow), prometaphase (yellow arrowhead) and metaphase (yellow arrow) were identified. A roniciclib-treated cell in metaphase (yellow arrow) was demonstrated. Scale bar, 25 μm.
Supplementary Figure 3. Effects of roniciclib on the expression of cleaved caspase-3, aurora A and cyclin B1 in murine well-differentiated thyroid cancer xenograft tumors. (A) Oral administration of roniciclib (1.3 mg/kg) twice a day increased cleaved caspase-3 level at 2 h and 24 h in K1 tumors. Aurora A level was transiently increased at 2 h and 4 h, decreasing by 10 h. Cyclin B1 level was transiently increased at 2 h and 4h, decreasing by 8 h. (B) Oral administration of roniciclib (1.3 mg/kg) twice a day increased cleaved caspase-3 level at 4 h and 24 h in FTC-133 tumors. Aurora A level was decreased between 2 h and 8 h, elevated at 10 h and subsequently decreased by 24 h. Cyclin B1 level was increased between 2 h and 8 h, slightly decreasing in expression at 10 h and increasing by 24 h.
Supplementary Figure 4. Effects of roniciclib on the expression of survivin in well-differentiated thyroid cancer in vitro and in vivo. (A) The expression of survivin was evaluated by Western blotting in BHP7-13, K1, WRO82-1 and FTC-133 cells treated with roniciclib (25 nmol/L) or placebo for the indicated periods. Roniciclib decreased survivin expression in BHP7-13 (16 h – 24 h), K1 (16 h – 24 h), WRO82-1 (8 h – 24 h) and FTC-133 (24 h) cells. (B) The effects of roniciclib (1.3 mg/kg) administered twice a day on the expression of survivin were evaluated in K1 and FTC-133 tumors. Oral administration of roniciclib decreased survivin expression in K1 (4 h – 24 h) and FTC-133 (10 h) tumors. Arrow, roniciclib treatment.