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Comparative Study
, 13 (11), 1043-57

A Novel Topoisomerase Inhibitor, Daurinol, Suppresses Growth of HCT116 Cells With Low Hematological Toxicity Compared to Etoposide

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Comparative Study

A Novel Topoisomerase Inhibitor, Daurinol, Suppresses Growth of HCT116 Cells With Low Hematological Toxicity Compared to Etoposide

Kyungsu Kang et al. Neoplasia.

Abstract

We report that daurinol, a novel arylnaphthalene lignan, is a promising potential anticancer agent with adverse effects that are less severe than those of etoposide, a clinical anticancer agent. Despite its potent antitumor activity, clinical use of etoposide is limited because of its adverse effects, including myelosuppression and the development of secondary leukemia. Here, we comprehensively compared the mechanistic differences between daurinol and etoposide because they have similar chemical structures. Etoposide, a topoisomerase II poison, is known to attenuate cancer cell proliferation through the inhibition of DNA synthesis. Etoposide treatment induces G(2)/M arrest, severe DNA damage, and the formation of giant nuclei in HCT116 cells. We hypothesized that the induction of DNA damage and nuclear enlargement due to abnormal chromosomal conditions could give rise to genomic instability in both tumor cells and in actively dividing normal cells, resulting in the toxic adverse effects of etoposide. We found that daurinol is a catalytic inhibitor of human topoisomerase IIa, and it induces S-phase arrest through the enhanced expression of cyclins E and A and by activation of the ATM/Chk/Cdc25A pathway in HCT116 cells. However, daurinol treatment did not cause DNA damage or nuclear enlargement in vitro. Finally, we confirmed the in vivo antitumor effects and adverse effects of daurinol and etoposide in nude mice xenograft models. Daurinol displayed potent antitumor effects without any significant loss of body weight or changes in hematological parameters, whereas etoposide treatment led to decreased body weight and white blood cell, red blood cell, and hemoglobin concentration.

Figures

Figure 1
Figure 1
Daurinol inhibits cell proliferation and DNA synthesis. (A) Chemical structures of daurinol and etoposide. (B) Antiproliferative activity of daurinol was determined by cell viability assays in various human cancer cell lines. Cells were treated with various concentrations of daurinol for 24 and 48 hours. IC50, the concentration that inhibits 50% of cell proliferation. Means ± SD from three independent experiments are shown. (C) Inhibition of cell viability and DNA synthesis by treatment with daurinol or etoposide for 24 and 48 hours was determined by CCK-8 assay (mitochondrial dehydrogenase activity) and BrdU incorporation ELISA in HCT116 cells. Cell viability (%) and BrdU incorporation (%) were calculated as percents of the vehicle control. Data are expressed as mean ± SD from triplicate experiments. **P < .01 and ***P < .001, for significant differences from the vehicle control. Graphs are representative of three independent experiments. (D) Cell viability versus BrdU incorporation (left). Relative DNA synthesis was calculated by BrdU incorporation (%) over cell viability (%) (right). Columns and error bars indicate mean ± SD. *P < .05, **P < .01, and ***P < .001, for significant differences from the vehicle control. Graphs are representative of three independent experiments.
Figure 2
Figure 2
Daurinol induces S-phase arrest in HCT116 cells. Cell cycle distribution was evaluated by using flow cytometric DNA content analysis. (A) HCT116 cells were treated with 5 µM daurinol for 24, 48, and 72 hours. Columns and error bars indicate mean ± SD from triplicate experiments. *P < .05 and ***P < .001, for significant differences from the vehicle control at each treatment time. The graph is representative of three independent experiments. (B) HCT116 cells were treated with various concentrations of daurinol (0, 2.5, 5, and 10 µM) for 48 hours. Columns and error bars indicate mean ± SD from quadruplicate experiments. ***P < .001, for significant differences from the vehicle control. (C) Daurinol-induced S-phase arrest of HCT116 cells was confirmed in hydroxyurea-synchronized cells. Cells were pretreated with 2 mM hydroxyurea (HU) for 12 hours to synchronize in the G1/S-phase. Then, cells were treated with daurinol (0, 2.5, 5, and 10 µM) for 12, 24, and 48 hours. C indicates vehicle control. The graph is representative of three independent experiments.
Figure 3
Figure 3
Daurinol is a catalytic inhibitor of human topoisomerase IIα. (A) Inhibitory activity of daurinol and etoposide on human topoisomerase IIα was evaluated by in vitro biochemical assays. The supercoiled DNA (pHOT1) substrate was incubated with human topoisomerase IIα in the presence of daurinol (1 and 2 mM) or etoposide (0.5 and 2 mM). DNA relaxation was evaluated by 1% agarose gel electrophoresis in the presence of ethidium bromide. (B and C) Effects of daurinol and etoposide on DNA damage were determined by comet assays. HCT116 cells were treated with daurinol (5, 20, and 50 µM) or 10 µM etoposide for 6 hours. (B) Images of cellular DNA damage were detected by fluorescence microscopy. Pictures are representative of three independent experiments. (C) The DNA damage index (% DNA in tail) was determined using comet score software. Columns and error bars indicate mean ± SD (n = 50). ***P < .001, for significant differences from the vehicle control.
Figure 4
Figure 4
Effect of daurinol on the expression and phosphorylation of cell cycle regulatory proteins. HCT116 cells were treated with 5 µM daurinol for 2, 12, 24, and 48 hours. (A) Representative immunoblots are shown from three independent experiments. The relative expression of p-ATM (Ser1981)/ATM (B), p-Chk1 (Ser317)/Chk1 (C), p-Chk1 (Ser345)/Chk1 (D), p-Chk2 (Thr68)/Chk2 (E), p-Cdk2 (Tyr15)/Cdk2 (F), p-Cdc2 (Tyr15)/Cdc2 (G), E2F-1/β-actin (H), Cyc E/β-actin (I), Cyc A/β-actin (J), Cdc25A/β-actin (K), Cdk4/β-actin (L), and Cyc D1/β-actin (M) were determined by densitometry. Columns and error bars indicate mean ± SEM from three independent experiments. *P <.05, **P <.01, and ***P < .001 for significant differences between the vehicle control and daurinol-treated cells at each time point.
Figure 5
Figure 5
Effects of daurinol and etoposide on the nucleus size of HCT116 cells were evaluated by fluorescence microscopy (A–C) and flow cytometry (D and E). HCT116 cells were treated with daurinol (2.5, 5, and 10 µM) or 10 µM etoposide for 48 hours. Cellular DNA was labeled with propidium iodide for visualization and flow cytometric DNA content analysis. (A) Fluorescence microscopic images of HCT116 cells treated with 5 µM daurinol or 10 µM etoposide for 48 hours. Pictures are representative of three independent experiments (bar, 10 µm). Size of nucleus after treatment with daurinol or etoposide was determined using fluorescence microscopy and the circle measurement algorithm of the microscope software. (B) Distribution of nucleus size of HCT116 cells treated with 5 µM daurinol or 10 µM etoposide for 48 hours. (C) Mean value of nucleus diameter. Columns and error bars indicate mean ± SD (n = 200). ***P < .001, for significant differences from the vehicle control. (D) Flow cytometric analysis of HCT116 cells treated with 5 µM daurinol or 10 µM etoposide. The FSC-H, FL2-A, and FL2-W histogram plots represent cell size, cell cycle, and nucleus size, respectively. Histograms are representative of quadruplicate experiments. (E) Differences in the distributions of the FL2-W value (nucleus size) between vehicle control and chemical-treated cells were quantitatively determined using Kolmogorov-Smirnov statistics. Index of similarity is the D/s(n) value of Kolmogorov-Smirnov statistics. Columns and error bars indicate mean ± SD from quadruplicate experiments. ***P < .001, for significant differences from the etoposide treatment.
Figure 6
Figure 6
Antitumor activity of daurinol and etoposide in nude mice xenograft models of HCT116 cells. Daurinol (1, 5, 10, and 20 mg/kg) or etoposide (20 mg/kg) were administered intraperitoneally three times weekly for 2 weeks. Body weights (A) and tumor volumes (B) were monitored for 4 weeks after the initial injection of chemicals to evaluate toxicity, antitumor effects, and their persistence of daurinol and etoposide treatment. (C) On day 29, tumors were removed and weighed. Data are expressed as mean ± SD (n = 10). *P < .05 and **P < .01, for significant differences from the vehicle-treated group. (D) Photographs of tumors removed from mice treated with vehicle control, daurinol (20 mg/kg), or etoposide (20 mg/kg).
Figure 7
Figure 7
Evaluation of antitumor activities and molecular effects of daurinol and etoposide in nude mice xenograft models. Daurinol (5, 10, and 20 mg/kg) or etoposide (20 mg/kg) were administered intraperitoneally twice weekly for 3 weeks. Body weights (A) and tumor volumes (B) were monitored for 3 weeks. On day 18, tumors were removed for immunohistochemistry and Western blot analysis. (C) Tumor weights. (D) Inhibition of DNA synthesis in xenograft tumors was measured by BrdU incorporation immunostaining. Data are expressed as mean ± SD (n = 10). *P < .05 and **P < .01, for significant differences from the vehicle-treated group. (E) Western blot analysis of expression and phosphorylation of cell cycle regulatory proteins from tumors of nude mice treated with the vehicle control or daurinol (20 mg/kg). Columns and error bars indicate mean ± SEM (n = 5). *P < .05 for significant differences from the vehicle-treated group.
Figure 8
Figure 8
(A) Comparison of daurinol and etoposide. (B) Schematic representation of the possible molecular mechanism of S-phase arrest induced by daurinol treatment in human colon cancer cells.

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