Arenobufagin intercalates with DNA leading to G2 cell cycle arrest via ATM/ATR pathway

Abstract

Arenobufagin, a representative bufadienolide, is the major active component in the traditional Chinese medicine Chan'su. It possesses significant antineoplastic activity in vitro. Although bufadienolide has been found to disrupt the cell cycle, the underlying mechanisms of this disruption are not defined. Here, we reported that arenobufagin blocked the transition from G2 to M phase of cell cycle through inhibiting the activation of CDK1-Cyclin B1 complex; The tumor suppressor p53 contributed to sustaining arrest at the G2 phase of the cell cycle in hepatocellular carcinoma (HCC) cells. Moreover, arenobufagin caused double-strand DNA breaks (DSBs) and triggered the DNA damage response (DDR), partly via the ATM/ATR-Chk1/Chk2-Cdc25C signaling pathway. Importantly, we used a synthetic biotinylated arenobufagin-conjugated chemical probe in live cells to show that arenobufagin accumulated mainly in the nucleus. The microscopic thermodynamic parameters measured using isothermal titration calorimetry (ITC) also demonstrated that arenobufagin directly bound to DNA in vitro. The hypochromicity in the UV-visible absorption spectrum, the significant changes in the circular dichroism (CD) spectrum of DNA, and the distinct quenching in the fluorescence intensity of the ethidium bromide (EB)-DNA system before and after arenobufagin treatment indicated that arenobufagin bound to DNA in vitro by intercalation. Molecular modeling suggested arenobufagin intercalated with DNA via hydrogen bonds between arenobufagin and GT base pairs. Collectively, these data provide novel insights into arenobufagin-induced cell cycle disruption that are valuable for the further discussion and investigation of the use of arenobufagin in clinical anticancer chemotherapy.

Keywords: DNA damage response; DNA intercalator; G2 cell cycle arrest; arenobufagin.

Figure 1. Arenobufagin induces G₂ cell cycle arrest in HCC cells

A. After treatment with 10 nmol/L (Hep3B cells) or 20 nmol/L (HepG2 and HepG2/ADM cells) of arenobufagin for 0, 24, 36, and 48 h, the cell cycle distributions were measured using flow cytometry. Representative pictures (left panel) and a quantification of the cell population in the G₂/M phase (right panel) are shown. Each column represents the mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 versus the DMSO control. B. Effect of arenobufagin on the mitotic index in HCC cells. Cells were treated with arenobufagin for 0, 24 and 48 h and Taxel for 12 h (25 nmol/L for HepG2 and Hep3B cells, 5 μmol/L for HepG2/ADM cells) as a positive control. Representative pictures are shown (left panel). Original magnification: 100×; Scale bar: 200 μm. The mitotic indexes were calculated using the number of p-Histone H3-positive cells per total number of cells (DAPI-positive cells). Each column represents the mean ± SD of triplicates. **P < 0.01, ***P < 0. 001 versus the DMSO control (right panel).

Figure 2. The role of p53 in arenobufagin-induced G₂ arrest

A. After treatment with arenobufagin for 48 h, the apoptotic cells were measured using flow cytometry. At least 10,000 cells were analyzed per sample. Representative pictures (left panel) and a quantification of the apoptotic cells (right panel) are shown. Each column represents the mean ± SD of triplicates. *P < 0.05, ***P < 0.001 versus the DMSO control. B. HepG2 and HepG2/ADM cells were incubated with arenobufagin for 0, 6, 12, 24, 36 and 48 h. The total protein cell lysates were harvested and evaluated by Western blotting with the indicated antibodies. C. The knockdown efficiency of p53 by siRNA in HepG2 and HepG2/ADM cells was evaluated by Western blotting. D. The effect of combined p53 siRNA and arenobufagin on the DNA content of HepG2 and HepG2/ADM cells. Cell cycle distributions of cells were assessed by flow cytometry. Each column represents the mean ± SD of at least three independent experiments. **P < 0.01, ***P < 0.001 versus the DMSO control; ^##P < 0.01, ^###P < 0.001 versus the arenobufagin alone.

Figure 3. Arenobufagin inhibits the activation of CDK1-Cyclin B1 complex

A. Total cell lysates from HepG2, HepG2/ADM and Hep3B cells treated with arenobufagin for 0, 24, 36 and 48 h. The lysates were evaluated by Western blotting with the indicated antibodies. B. Co-immunoprecipitation of the CDK1-Cyclin B1 complex. Protein extracts (1 mg) were incubated with CDK1 primary antibody. Immunoprecipitated complex were subjected to SDS electrophoresis. Total cell lysates were used as an input control. C. Arenobufagin degraded the expression of Cdc25C protein. Total cell lysates were evaluated by Western blotting with the indicated antibodies.

Figure 4. Arenobufagin activates the ATM/ATR-Chk1/Chk2 signaling pathway

HepG2, HepG2/ADM and Hep3B cells treated with arenobufagin for 0, 24, 36 and 48 h. 293 cell extracts were exposed to UV for 4 h. Cells were collected and lysed. The lysates were assayed by Western blotting with the indicated antibodies. The representative pictures from 3 independent experiments are shown.

Figure 5. Arenobufagin induces DSBs in cells

A. Cells treated with arenobufagin for 0, 24 and 48 h and then harvested and evaluated with a comet assay. The DNA was stained with Vista Green DNA dye. Representative images of arenobufagin-induced DNA damage are shown. Original magnification: 200×; Scale bar: 50 μm. The percentage of Tail DNA, Tail Length, and Olive Tail Moment were evaluated using MetaXpress software. Each sample includes at least 20 cells. Each column represents the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 versus the DMSO control. B. Cells were incubated with arenobufagin for the indicated times and then stained with γ-H2AX and DAPI. Representative images are shown (left panel). Original magnification: 400×; Scale bar: 50 μm. The granules were calculated using the number of γ-H2AX-positive cells per the total number of cells (DAPI-positive cells). Each column represents the mean ± SD of triplicates. **P < 0.01, ***P < 0.001 versus the DMSO control (right panel).

Figure 6. Arenobufagin-induced G₂ arrest results from DNA damage

A. Caffeine antagonized the arenobufagin-induced phosphorylation of ATM and ATR. HepG2 cells were pretreated with 2 mmol/L of caffeine for 2 h and then incubated with arenobufagin (20 nmol/L) for 24 h. Cell lysates were harvested and evaluated by Western blotting with the indicated antibodies. B. HCC cells were treated with caffeine combined with arenobufagin (20 nmol/L) and then stained with an antibody to γ-H2AX and the DNA dye DAPI. The granules were calculated using the number of γ-H2AX-positive cells per the total number of cells (DAPI-positive cells). Each column represents the mean ± SD of triplicates. ***P < 0.001 versus the DMSO control; ^### P < 0.001 versus the arenobufagin alone. C. Effect of caffeine on arenobufagin-induced G₂ arrest. The cell cycle distributions were measured by flow cytometry. Representative images from 3 independent experiments are shown.

Figure 7. The cellular localization of arenobufagin in live cells

A. The chemical structure of biotinylated arenobufagin. B. HepG2 cells were incubated with biotinylated arenobufagin for various times and then probed with SP. Nuclear DNA was stained with DAPI. Arrows indicated the granules of accumulation of biotinylated-arenobufagin. Original magnification: 630×; Scale bar: 10 μm.

Figure 8. Arenobufagin directly binds with DNA via intercalation

A. Arenobufagin binding to DNA was measured by ITC. A total of 30 μmol/L of DNA was titrated with 0.4 mmol/L of arenobufagin. The resulting thermograms were analyzed based on the one set of binding sites model using Microcal Origin 7.0 (Microcal. Inc.). B. The effect of arenobufagin on the UV absorption spectrum of DNA. 1 mmol/L DNA solution was mixed with 20 nmol/L arenobufagin. After the solution was mixed and equilibrated for approximately 5 min, the absorption spectra were measured at wavelengths ranging from 200 nm to 400 nm. C. The effect of arenobufagin on the CD spectra of DNA. The CD spectra of DNA (1 mmol/L) in 50 mmol/L Tris-HCl (pH = 8.0) with 20 nmol/L of arenobufagin. Each spectrum was analyzed from 200 nm to 370 nm at 25°C with a 10 mm path length cell. D. Fluorescence titration of EB-DNA complex with arenobufagin. EB-DNA complex was excited at 524 nm, and emission spectra was recorded from 530 to 700 nm at 25°C. E. The docked conformations suggested the intercalation between arenobufagin and d(CCGGCGGT)₂. The green dotted lines represent the hydrogen bonds formed between arenobufagin and the DNA duplex.

Figure 9. Proposed model for the mechanisms of arenobufagin-induced G₂ arrest in HCC cells

Arenobufagin directly binds with DNA via intercalation, leading to DSBs and triggering DDR via the ATM/ATR signaling pathway, which subsequently results in G₂ phase arrest in HCC cells.