Naphthalimides induce G(2) arrest through the ATM-activated Chk2-executed pathway in HCT116 cells

Abstract

Naphthalimides, particularly amonafide and 2-(2-dimethylamino)-6-thia-2-aza-benzo[def]chrysene-1,3-diones (R16), have been identified to possess anticancer activities and to induce G(2)-M arrest through inhibiting topoisomerase II accompanied by Chk1 degradation. The current study was designed to precisely dissect the signaling pathway(s) responsible for the naphthalimide-induced cell cycle arrest in human colon carcinoma HCT116 cells. Using phosphorylated histone H3 and mitotic protein monoclonal 2 as mitosis markers, we first specified the G(2) arrest elicited by the R16 and amonafide. Then, R16 and amonafide were revealed to induce phosphorylation of the DNA damage sensor ataxia telangiectasia-mutated (ATM) responding to DNA double-strand breaks (DSBs). Inhibition of ATM by both the pharmacological inhibitor caffeine and the specific small interference RNA (siRNA) rescued the G(2) arrest elicited by R16, indicating its ATM-dependent characteristic. Furthermore, depletion of Chk2, but not Chk1 with their corresponding siRNA, statistically significantly reversed the R16- and amonafide-triggered G(2) arrest. Moreover, the naphthalimides phosphorylated Chk2 in an ATM-dependent manner but induced Chk1 degradation. These data indicate that R16 and amonafide preferentially used Chk2 as evidenced by the differential ATM-executed phosphorylation of Chk1 and Chk2. Thus, a clear signaling pathway can be established, in which ATM relays the DNA DSBs signaling triggered by the naphthalimides to the checkpoint kinases, predominantly to Chk2,which finally elicits G(2) arrest. The mechanistic elucidation not only favors the development of the naphthalimides as anticancer agents but also provides an alternative strategy of Chk2 inhibition to potentiate the anticancer activities of these agents.

Figure 1

Chemical structures of amonafide and R16.

Figure 2

R16 arrests HCT116 cells at G₂ phase. (A and B) R16 and amonafide increased the G₂/M population of HCT116 cells in a concentration-dependent manner. HCT116 cells were treated with different compounds at indicated concentrations for 24 hours and then subjected to flow cytometry. The data were expressed as mean ± SD from three independent experiments (A) and the typical histograms were shown (B). (C) R16 and amonafide increased the G₂/M population in a time-dependent fashion. HCT116 cells were treated with R16 (2.5 µM) or amonafide (2.5 µM) for the different periods and were then subjected to flow cytometry. The data were expressed as mean ± SD from three independent experiments. (D and E) Treatments with R16 or amonafide did not increase the molecular markers of mitosis arrest phosphorylated histone H3 (p-Histone H3) and MPM-2. HCT116 cells were treated with indicated concentrations of the tested compounds for 24 hours and were then subjected to Western blot analysis. All the Top2 inhibitors (R16, amonafide, VP16, and ADR) did not increase p-Histone H3 (D) and MPM-2 (E) as the mitosis inhibitor vincristine did (D and E). The images were representative of three separate experiments.

Figure 3

DNA DSBs contribute to R16-mediated G₂ arrest. (A and B) R16 and amonafide induced DNA DSBs. HCT116 cells were treated with different compounds at indicated concentrations for 2 hours and were then subjected to Western blot analysis to detect the level of γ-H2Ax (A) or to NSCGE assays to detect the broken DNA (comet tails; B). (C) R16 (10 µM for 2 hours) induced the formation of p-ATM foci in HCT116 cells detected by immunofluorescence assays. (D) R16 and amonafide (at indicated concentrations for 2 hours) increased the phosphorylation of ATM detected by Western blot analysis. (E and F) The ATM/ATR inhibitor caffeine (2 mM, 30 minutes) abrogated R16- and amonafide-induced (24 hours) G₂ arrest in HCT116 cells detected by flow cytometry. The data were expressed as the typical histograms (E) or as mean ± SD from three independent experiments (F), *P < .05; **P < .01.

Figure 4

Depletion of ATM but not ATR abates the G₂ arrest elicited by R16 and amonafide. (A and B) ATM knockdown rescued HCT116 cells from the G₂ arrest triggered by R16 and amonafide. Cells were transfected with 100 nM ATM siRNA1 (A) or 100 nM ATM siRNA2 (B) for 24 hours before being exposed to R16 or amonafide for the following 24 hours. Then the cell cycle distribution was analyzed by flow cytometry. Left panel indicates the efficiency of ATM depletion; middle panel, typical histograms; right panel, mean ± SD from three separate experiments. *P < .05; **P < .01. (C) ATR knockdown with ATR siRNA (100 nM) did not prevent the G₂ arrest induced by R16 and amonafide although it effectively abated the S arrest elicited by HU. HCT116 cells were transfected with 100 nM ATR siRNA for 24 hours before the treatment with different compounds at the indicated concentrations for the following 24 hours. Left panel indicates the efficiency of ATR depletion; middle panel, S arrest and HU; right panel, G₂/M arrest and R16, amonafide, VP16, and ADR. The data were expressed as mean ± SD from three independent experiments; *P < .05.

Figure 5

Depletion of Chk2 but not Chk1 prevents the G₂ arrest induced by R16 and amonafide. (A) Chk1 knockdown with its siRNA (100 nM) did not decrease the G₂ arrest triggered by R16 and amonafide statistically significantly although reducing the G₂ arrest triggered by the other two Top2 inhibitors VP16 and ADR. Left panel indicates the efficiency of Chk1 depletion; right panel, mean ± SD from three separate experiments. *P < .05. (B and C) Silencing of Chk2 diminished G₂ arrest but potentiated apoptosis induction by R16 significantly. HCT116 ells were treated with the compounds at the indicated concentrations for 24 hours after Chk2 silencing with its siRNA (100 nM, 24 hours). Then the cells were subjected to flow cytometry. The percentage of G₂ population (B) or apoptotic cells (C) were expressed as mean ± SD from three separate experiments. *P < .05. B: Left panel indicates the efficiency of Chk2 depletion; right panel, mean ± SD of G₂ population (%). *P < .05. C: Left panel indicates typical histograms to show the sub-G₁ population (apoptotic cells); right panel, mean ± SD of apoptotic cells (%). *P < .05.

Figure 6

R16 induces differential phosphorylation of Chk2 and Chk1. (A) Chk2 phosphorylation triggered by R16 was blocked by ATM siRNA1 (100 nM). HCT116 cells were treated with R16, amonafide, or VP16 at the indicated concentrations for 24 hours after being transfected with 100 nM ATM siRNA1 for 24 hours. Then the cells were subjected to Western blot analysis for Chk1, Chk2, p-Chk1, and p-Chk2. The images were representative of three independent experiments. (B) Schematic presentation of the possible mechanistic link between the inhibition of Top2 and G₂ arrest elicited by the naphthalimides.