Potent effects of roniciclib alone and with sorafenib against well-differentiated thyroid cancer

in Endocrine-Related Cancer
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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.

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  • 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.

 

      Society for Endocrinology

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    Roniciclib induces cytotoxicity in well-differentiated thyroid cancer cells. Cytotoxicity was evaluated in cells treated with a series of six two-fold dilutions of roniciclib starting from 100 nmol/L. Dose–response curves were obtained on day 4 using LDH assays. The median-effect dose (IC50) of roniciclib on day 4 was calculated for each cell line using CompuSyn software.

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    Roniciclib stimulates caspase-3 activity and induces apoptosis in well-differentiated thyroid cancer cells. (A) Caspase-3 activity was evaluated using a fluorometric assay kit in BHP7-13, K1, WRO82-1 and FTC-133 cells treated with roniciclib (25 nmol/L) or vehicle for 24 h. (B) BHP7-13 cells were treated with roniciclib (25 nmol/L) or placebo for 24 h and stained with DAPI (blue) and fluorescent antibodies against cleaved caspase-3 (red) and α-tubulin (green). A cell with cleaved caspase-3 expression is shown (arrow). (C) The percentages of cells with cleaved caspase-3 expression were assessed after treatment with placebo or roniciclib (25 nmol/L) for 24 h. Cells were stained with fluorescent antibodies against cleaved caspase-3, and its expression was evaluated using immunofluorescence confocal microscopy. A minimum of 861 cells from at least 12 different fields was counted for each condition. Roniciclib significantly increased the proportion of BHP7-13, K1, WRO82-1 and FTC-133 cells with cleaved caspase-3 expression. (D) Sub-G1 apoptotic cells were detected by measuring the DNA content using flow cytometry in cells treated with roniciclib (25 nmol/L) or vehicle for 24 h. Roniciclib increased the proportion of sub-G1 cells in four well-differentiated thyroid cancer cell lines. Scale bar, 20 μm.

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    Roniciclib decreases the protein levels of aurora A and cyclin B1 and induces G2/M phase accumulation in well-differentiated thyroid cancer cells. (A) Cell cycle distribution was analyzed by evaluating the DNA content in BHP7-13 cells treated with placebo or roniciclib (25 nmol/L) for 24 h using flow cytometry. (B) Statistical analyses revealed that roniciclib treatment (25 nmol/L) significantly arrested BHP7-13, K1, WRO82-1 and FTC-133 cells in the G2/M phase at 24 h. (C) The proportion of well-differentiated thyroid cancer cells in mitosis was assessed after treatment with roniciclib (25 nmol/L) or placebo for 24 h. Cells were stained with DAPI, and chromosome characteristics were evaluated using immunofluorescence confocal microscopy. The mitotic index was assessed with a minimum of 420 cells counted from at least 10 different fields for each condition. Roniciclib significantly decreased the proportion of BHP7-13, K1, WRO82-1 and FTC-133 cells in mitosis. (D) The expression of aurora A and cyclin B1 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.

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    Roniciclib inhibits subcutaneous xenograft growth of two well-differentiated thyroid cancer models. (A) The therapeutic efficacy of roniciclib for papillary thyroid cancer was evaluated in mice bearing K1 flank tumors. Serial oral gavage of low-dose (1.0 mg/kg) and high-dose (1.3 mg/kg) roniciclib significantly repressed K1 tumor growth by day 3 when compared with control mice, and the effect persisted through day 14. (B) Serial treatment of low-dose (1.0 mg/kg) and high-dose (1.3 mg/kg) roniciclib did not significantly decrease body weight when compared with control mice. (C) The therapeutic efficacy of roniciclib for follicular thyroid cancer was evaluated in mice bearing FTC-133 flank tumors. Serial oral gavage of low-dose (1.0 mg/kg) and high-dose (1.3 mg/kg) roniciclib significantly repressed FTC-133 tumor growth by day 3 when compared with control mice, and the effect persisted through day 21. (D) Serial treatment of high-dose roniciclib (1.3 mg/kg) slightly, but significantly induced body weight loss on day 10 when compared with control mice. This effect was not observed in mice treated with low-dose (1.0 mg/kg) roniciclib. K1 (E) and FTC-133 (F) xenograft tumors (arrowhead) were photographed on day 15. The molecular effects of roniciclib (1.3 mg/kg) treatment were evaluated in K1 (G) and FTC-133 tumors (H) using Western blot analysis. Arrow, roniciclib or placebo treatment.

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    Combination therapy of roniciclib and sorafenib in well-differentiated thyroid cancer cells. (A) Cytotoxicity was evaluated in BHP7-13, K1, WRO82-1 and FTC-133 cells treated with a series of six two-fold dilutions of sorafenib starting at 10 µmol/L. Dose–response curves were obtained on day 4 using LDH assays. (B) The median-effect dose (IC50) of sorafenib on day 4 was calculated for each cell line using CompuSyn software. (C) The cytotoxic effects of roniciclib and sorafenib alone and in combination after a 4-day treatment in BHP7-13, K1, WRO82-1 and FTC-133 cells were evaluated using LDH assays. (D) The combination index (CI) of roniciclib and sorafenib was calculated using CompuSyn software. The combination therapy of roniciclib and sorafenib had synergistic effects in WRO82-1 and FTC-133 cells, synergistic to additive effects in BHP7-13 cells and synergistic to antagonistic effects in K1 cells. Synergistic effects appeared when the affected fractions of BHP7-13 and K1 cells was lower (affected fraction <0.9 and <0.7, respectively). The horizontal line at CI = 1 was drawn for discrimination of synergism (CI < 1) and antagonism (CI > 1).

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    Daily therapy of roniciclib and sorafenib in murine well-differentiated thyroid cancer xenograft tumor models. (A) Daily treatment of roniciclib (1.4 mg/kg) for 4 days significantly inhibited subcutaneous xenograft growth of K1 tumors compared with control mice on day 7. (B) Serial daily administration of roniciclib did not result in significant changes in body weight during the study period. (C) After FTC-133 flank tumors were established in nude mice, they were treated with oral gavage of placebo, roniciclib (1.4 mg/kg), sorafenib (25 mg/kg) or combination therapy once a day for three cycles of 4-day on and 3-day off therapy. Compared with control therapy, roniciclib, sorafenib and combination treatment all significantly inhibited tumor growth by day 3, and the inhibitory effects persisted through day 13. Combination therapy significantly retarded xenograft growth when compared with either single modality treatment on days 17 and 20. (D) No significant decreases in body weight were attributable to placebo, roniciclib, sorafenib or combination therapy compared with the baseline weight. Arrow, placebo, roniciclib, sorafenib and combination treatment.

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