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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

We previously reported that many ingenol compounds derived from Euphorbia kansui exhibit topoisomerase inhibitory activity and/or inhibitory activity of cell proliferation. The inhibitory effects of 20-O-(2′E,4′Z-decadienoyl) ingenol and 3-O-(2′E,4′Z-decadienoyl)-ingenol among these compounds on topoisomerase II activity and on the cell proliferative activity and arrest phase of the cell cycle were studied using a mouse breast cancer (MMT) cell line. Although 20-O-ingenolEZ exerted inhibitory effects on both topoisomerase II activity and cell proliferative activity, 3-O-ingenolEZ exerted inhibitory activity on neither. The 20-O-ingenolEZ-induced cell arrest of MMT-cell proliferation led to a cell cycle arrest in the G2/M phase. Topoisomerase II inhibition can be divided into the poison and catalytic inhibitor types. A checkpoint mechanism is activated when cells are treated with these topoisomerase II inhibitors. Poison-type inhibition occurs via induction of the DNA damage checkpoint and the catalytic-type inhibition occurs via induction of the DNA-decatenation checkpoint, suggestive of distinct checkpoint reactions. 20-O-ingenolEZ inhibited topoisomerase IIα activity through inhibition of ATPase, and induced DNA-decatenation checkpoint without signaling for phosphorylation of H2AX. (Cancer Sci 2010; 101: 374–378)

E uphorbia kansui has been used in herbal remedies employed for treating ascites, leukemia, and some tumors,(1,2,3) and extracts from kansui have been demonstrated to show antitumor activity.(4–6) Many ingenol derivatives from Euphoria have been reported to exhibit antiproliferative and antitumor properties(7–9) and biologically active ingenol derivatives have also been synthesized.(10) We showed in a previous study that ingenol compounds inhibited topoisomerase II activity and/or the proliferative activity of cancer cells.(1,11) In cancer chemotherapy, topoisomerase II is a major target for a variety of anticancer drugs. According to their mode of action, these drugs have been divided into two classes: anticancer topoisomerase II poisons, such as adriamycin and etoposide, which stabilize an intermediate in the topoisomerase II reaction in which two topoisomerase II subunits are covalently linked to DNA via a phosphotyrosine linkage.(12,13) This covalent intermediate, termed the cleavable complexes, induces the DNA damage checkpoint with signaling for phosphorylation of H2AX(14) and plays a critical role in the cell killing by anti-topoisomerase II agents. The agents of the second class of topoisomerase II inhibitor do not stabilize the covalent intermediate of the topoisomerase II reaction described above, but inhibit the enzyme at other points of the reaction cycle.(12,15) Since blocking of the enzyme at other points of the reaction cycle would not result in DNA damage, it is thought that this second class of agents arrests cell growth by depriving them of the essential enzymatic activity of topoisomerase II. This second class of inhibitors has been termed catalytic inhibitors to distinguish them from agents that act by stabilizing the covalent complex. Examples of the latter class of inhibitors include aclarubicin, which intercalates with DNA and prevents the binding of the enzyme to DNA,(16) and merbarone, which inhibits DNA cleavage by the enzyme.(17) Furthermore, bisdioxopiperazine (ICRF 193) inhibits re-opening of the closed clamp by inhibition of ATPase activity of the enzyme and prevents decatenation of replicated chromosomes by topoisomerase II and arrests cell proliferation or induces apoptosis.(14,18–21)

We studied the inhibition mechanisms of topoisomerase II activity and effects on the cell proliferation through DNA damage or blockade of topoisomerase II by ingenol compounds.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

Cell culture.  Growing mouse mammary tumor (MMT) cells suspended in MEM supplemented with 10% fetal calf serum were transplanted in microculture plates (1–2 × 10 000 cells/0.1 mL) and incubated for 1 day at 37°C in an atmosphere of 95% air and 5% CO2. The diterpene compounds, 20-O-(2′E,4′Z-decadienoyl)-ingenol (20-O-ingenolEZ) (Fig. 1a ) and 3-O-(2′E,4′Z-decadienoyl)-ingenol (3-O-ingenolEZ) (Fig. 1b) were dissolved in dimethylsulfoxide. After appropriate dilution, 1 μL of the drug was added to 100 μL of medium and the cells were incubated at 37°C for 3 days.

image

Figure 1.  Structures of the diterpene compounds. (a) 20-O-(2′E,4′Z-decadienoyl)ingenol; (b) 3-O-(2′E,4′Z-decadienoyl)-ingenol.

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Topoisomerase II mediated supercoiled pBR322 relaxation.  The activity of the compounds on the relaxation by DNA topoisomerase IIα (human recombinant in E.coli; USB Corporation, Cleveland, OH, USA) was determined by measuring the conversion of supercoiled pBR322 plasmid DNA to its relaxed form. The reaction mixture contained 10 mm Tris-HCl (pH 7.9), 50 mm NaCl, 50 mm KCl, 5 mm MgCl2, 0.1 mm EDTA, 15 μg/mL BSA, 1 mm ATP, 30 ng pBR322, 0.2 U of enzyme, and different concentrations of the drugs in a total volume of 20 μL. After incubation for 1 h at 37°C, the mixtures were subjected to electrophoresis on 1% agarose gel. After the electrophoresis, the gels were stained with ethidium bromide and photographed under UV light.

Kinetoplast DNA (kDNA) decatenation assay.  Topoisomerase II activity was measured by the amount of ATP-dependent decatenation of kDNA. The standard reaction mixture consisted of 50 mm Tris-HCl (pH 7.9), 120 mm KCl, 10 mm MgCl2, 0.5 mm ATP, 0.4 μg of kDNA, and nuclear protein of MMT cells in a total volume of 20 μL. Nuclear protein was prepared from the MMT cells by the method of Miller et al. (1981).(22) After incubation at 37°C for 30 min, the reaction was stopped by the addition of 2 μL of the stop solution. The DNA samples were subjected to electrophoresis under the same conditions as described above.

ATPase assay.  The hydrolysis catalyzed by human topoisomerase IIα was studied with measure of liberated phosphate as described by Cogan et al. (1999).(23) The free phosphate measure carried out with a malachite green phosphate assay kit as described in the instruction manual (BioAssay Systems, Hayward, CA, USA). The reaction mixture contained 20 mm Tris, 40 mm NaCl, 4 mm MgAc2, 0.5 mm EDTA, 5 mm ATP, 200 ng pBR322, and two units of human topoisomerase IIα (pH 7.5). After 30 min, 200 μL of reagent was added and the mixture was incubated for 30 min at room temperature for color development and was measured absorbance at 620 nm on the micro plate reader.

Cell cycle analysis.  MMT cells and 3T3 cells were treated or not treated with 20 μm 20-O-ingenolEZ for 12, 24, and 48 h. The cells were then harvested and fixed with 70% ethanol for 24 h. After centrifugation at 1800g for 5 min, the cell pellets were washed with PBS and resuspended in PBS containing propidium iodide (50 μg/mL), Triton X-100 (0.1%), and DNase-free RNase (50 μg/mL). The cells were then incubated at 37°C for 20 min, and the DNA content was determined by flow cytometry.

Western blot analysis.  MMT cells were cultured for 24 h in the presence of 20 μm 20-O-ingenolEZ or 0.9 μm adriamycin, washed in phosphate-buffered saline (PBS), suspended in a lysis buffer (20 mm HEPES [pH7.8], 10 mm NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 0.1% Triton-X100, 20% glycerol, and 1 mm DTT, supplemented with protease inhibitors) and lysed. After centrifugation at 320g for 4 min, the precipitate was resuspended in an lysis buffer containing 500 mm NaCl instead of 10 mm NaCl and placed on ice for 30 min. The suspension was centrifuged at 15 300g for 15 min at 4°C. Twenty μg of nuclear protein was resolved on 15% SDS–polyacrylamide gels, and transferred to a polyvinylidene difluoride membrane. Mouse anti-γ-H2AX antibody (Millipore, Billerica, MA, USA) was used. The blots were developed using an enhanced chemiluminescence system.

Immunofluorescence assay.  MMT cells were cultured on coverslips for 24 or 48 h in the presence of 20 μm 20-O-ingenolEZ or 0.9 μm adriamycin, and the coverslips were washed three times with PBS for 5 min. Apoptotic cells and nuclei were stained for 10 min at room temperature with 4,6-diamino-2-phenyl indole (DAPI, at 1 μg/mL PBS) and detected by fluorescence microscopy.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

Topoisomerase II mediated supercoiled pBR322 relaxation.  The effect of 20-O-ingenolEZ (Fig. 1a) and 3-O-ingenolEZ (Fig. 1b) on the strand passage activity of topoisomerase II was determined by the substance effect on enzyme-mediated supercoiled pBR322 relaxation. As shown in Figure 2, although 20-O-ingenolEZ exerted slight inhibition of this reaction at the concentration of 4 μm and significant inhibition at the increased concentration of 40 μm (lane 3), 3-O-ingenolEZ did not exhibit any inhibitory activity, even up to the concentration of 200 μm (lane 4).

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Figure 2.  Topoisomerase II mediated supercoiled pBR322 relaxation. To determine inhibition of relaxation of the supercoils, pBR322 DNA was incubated with topoisomerase IIα. Each reaction mixture (20 μL) contained 30 ng pBR322 plasmid DNA, 0.2 U of human topoisomerase IIα, and 4, 40, or 200 μm 20-O-ingenolEZ or 3-O-ingenolEZ. After incubation for 1 h at 37°C, the mixture was loaded onto a gel well for agarose gel electrophoresis. Lanes: 1, no enzyme; 2, plus enzyme, 3, plus 20-O-ingenolEZ; 4, plus 3-O-ingenolEZ; 4 μm of lane 4, no sample. R, relaxed DNA; S, supercoiled DNA.

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Kinetoplast DNA (kDNA) decatenation assay.  To further substantiate the effect of 20-O-ingenolEZ on topoisomerase II activity, the effect on kDNA decatenation was assessed. kDNA is a massive network consisting of thousands of interlocked circular DNA molecules called ‘minicircles’. Since a transient double-strand break is necessary to release a minicircle from the network, assay of decatenation of the kDNA is believed to be one of the highly specific assays for topoisomerase II, the enzyme that catalyzes the double-strand passing reaction. As shown in Figure 3, the strand passage activity of topoisomerase II was inhibited with the addition of 20-O-ingenolEZ (lane 3), and obvious inhibition was observed at the concentrations of 40 μm and 100 μm (lanes 3 and 4).

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Figure 3.  Kinetoplast DNA (kDNA) decatenation assay. To demonstrate inhibition of decatenation, kDNA was incubated with nuclear protein in mouse breast cancer (MMT) cells. Each mixture (20 μL) contained 0.4 μg of kDNA, nuclear protein, and 40, 100, or 200 μm 20-O-ingenolEZ. After incubation for 1 h at 37°C, the mixture was loaded onto a gel well for agarose gel electrophoresis. Lanes: 1, no enzyme; 2, plus enzyme; 3, plus 40 μm 20-O-ingenolEZ; 4, plus 100 μm 20-O-ingenolEZ; 5, plus 200 μm 20-O-ingenolEZ. C, catenated kDNA; D, decatenated kDNA.

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ATPase assay.  To further investigate the inhibition of eukaryotic topoisomerase II by 20-O-ingenolEZ, we studied the effects on the DNA-stimulated ATPase activities of human topoisomerase II. The inhibition of the ATPase domains by 20-O-ingenolEZ could be detected (Fig. 4). There was a 50% reduction in ATP hydrolysis activity with the addition of 30–40 μm 20-O-ingenolEZ, when DNA-stimulated activity was reduced to approximately 20% of the control value.(24) The function of 20-O-ingenolEZ appeared to be similar to ICRF 193.

image

Figure 4.  Effects on ATPase activity of topoisomerase II by 20-O-ingenolEZ. DNA-dependent ATPase activity was measured in a reaction mixture containing 20 mm Tris, 40 mm NaCl, 4 mm MgAc2, 0.5 mm EDTA, 5 mm ATP, 200 ng pBR322, and two units of human topoisomerase IIα (pH 7.5) at various 20-O-ingenolEZ concentrations. The free phosphate measure was carried out with a malachite green reagent. Relative ATPase activity is shown by normalizing against activity under conditions without inhibitors.

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Effects on the proliferative activity of tumor cells.  To investigate the inhibitory effects of 20-O-ingenolEZ and 3-O-ingenolEZ on the growth of cancer cell lines, a concentration–response range (20 and 100 μm) was established using an exposure time of 3 days. The effects of the ingenol compounds on the MMT cells were compared. Although the structure of 20-O-ingenolEZ is very similar to that of 3-O-ingenolEZ, 3-O-ingenolEZ did not trigger supercoiled pBR322 relaxation (Fig. 2, lane 4). Furthermore, the compound exhibited no inhibitory activity on the cellular proliferative activity of the MMT over the concentration range of 20–100 μm, whereas 20-O-ingenolEZ exerted marked inhibition on MMT cell proliferative activity over the entire concentration range examined (Fig. 5). Thus, inhibition of the proliferative activity of MMT cells by 20-O-ingenolEZ may depend on its topoisomerase II inhibitory activity. The 50% inhibitory concentration of 20-O-ingenolEZ in relation to the inhibitory activity against MMT cell proliferation was about 20 μm. When MMT cells were cultured for 24 h in the presence of 60 μm 20-O-ingenolEZ, washed in PBS, and subsequently cultured in the absence of ingenolEZ for 48 h, their cell proliferative activity was almost completely restored to the control level (data not shown).

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Figure 5.  Effects of ingenolEZ on cell proliferative activity. Mouse breast cancer (MMT) cells were cultured in microplates at 37°C for 3 days in the absence or presence of 20, 40, 60, and 100 μm 20-O-ingenolEZ or 3-O-ingenolEZ. Relative cell growth was determined by MTT assay. The cell growth in untreated MMT cells was set as 100%, and the cell growth of the MMT cells treated with 20, 40, 60, and 100 μm 20-O-ingenolEZ or 3-O-ingenolEZ was expressed relative to the level in untreated MMT cells (100%). The expressions were assessed in triplicate and the data are shown as means ± SD.

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Flow cytometric analysis.  Furthermore, we investigated the mechanisms underlying the inhibition of MMT cell growth by 20-O-ingenolEZ. Because MMT cell growth was effectively inhibited by 20-O-ingenolEZ, we reasoned that this inhibitory activity might be attributable to its ability to interfere with the specific site of cell cycle. To resolve this question, we treated MMT cells with 20 μm 20-O-ingenolEZ for 12, 24, and 48 h, followed by cell cycle analysis using flow cytometry (Fig. 6a). The results showed that the MMT cells treated with 20-O-ingenolEZ started to show weak arrest in the G2/M phase at 12 h, and accumulation of cells in the G2/M phase continued until 48 h. At 12 h, 16.5% of the cell population was in the G2/M phase, the percentages being 30% and 27%, respectively, after 24 and 48 h of treatment (Fig. 6b), as compared to 12% of untreated cells. On the other hand, the G1 population decreased to 19% and 25% at 24 and 48 h, respectively, as compared to 36% of untreated cells. 20-O-ingenol EZ induced arrest in the G2/M phase in the MMT cells, whereas the G1 phase was depleted. In contrast to the observation in the MMT cells, the results of flow-cytometric analysis of the 3T3 cells treated with 20 μm 20-O-ingenolEZ at 24 h were the same as observations of the untreated control 3T3 cells (Fig. 6a).

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Figure 6.  Cell cycle analysis. 3T3-fibrobrast cells were cultured at 37°C for 24 h with 20 μm 20-O-ingenolEZ. Mouse breast cancer (MMT) cells were cultured at 37°C for 12, 24, and 48 h with 20-O-ingenolEZ at 10 μg/mL. The cells stained with propidium iodide were subjected to flow-cytometric analysis (a). (b) Shows the results of (a) with % the cell distributions in each G1, S, and G2/M phase of the cell cycle in untreated cells and at 12, 24, and 48 h after 20-O-ingenolEZ treatment.

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Influence of 20-O-ingenolEZ on the phosphorylation of H2AX.  We also observed the appearance of phosphorylated H2AX (γ-H2AX) which serves as a marker for DNA double strand breaks (DSBs).(25) Although γ-H2AX was not induced in MMT cells treated with 20-O-ingenolEZ, it could be visualized as a band stained with anti-γ-H2AX treatment in nuclei of adriamycin-treated MMT cells (Fig. 7). 20-O-ingenolEZ effectively induced G2/M phase arrest in MMT cells, with little evidence of DNA damage.

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Figure 7.  Influence of 20-O-ingenolEZ on phosphorylation of H2AX. For immunoblotting of γ-H2AX in the mouse breast cancer (MMT) cells, cells were cultured in the presence of 20 μm 20-O-ingenolEZ or 0.9 μm adriamycin for 24 h. The nuclear protein fraction (20 μg) was resolved by SDS-PAGE, followed by Western blot analysis and chemiluminescence detection. γ-H2AX was detected using the specific antibody against γH2AX. Lane 1, control; lane 2, 20-O-ingenolEZ; lane 3, adriamycin.

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Influence of 20-O-ingenolEZ on apoptosis.  The morphological characteristics of apoptotic cells in each sample were determined based on staining with DAPI. After 24-h treatment with 0.9 μm adriamycin, MMT cells were stained with DAPI (Fig. 8a). Apoptosis was induced in the cells after 24 and 48 h of continuous treatment with adriamycin (Fig. 8b). Apoptosis of more than 60% of the cells was induced. However, only 13% of the 20-O-ingenolEZ-treated MMT cells showed the morphological characteristics of apoptosis (Fig. 8b) and the results were the almost same as those of controls.

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Figure 8.  Effects of 20-O-ingenolEZ on mouse breast cancer (MMT) cell apoptosis. MMT cells were treated at 37°C with 0.9 μm adriamycin, 20 μm 20-O-ingenol EZ, or control for 24 and 48 h. Apoptosis of the cells was detected by DAPI staining and the arrows in the photograph indicate examples of apoptotic cells after 24 h treatment with adriamycin (a). The percentage of apoptotic MMT cells at different time-points after adriamycin and 20-O-ingenolEZ treatments are shown (b).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

Topoisomerase II inhibitors are widely used as anticancer drugs. There are two well-characterized checkpoint signals that are activated by topoisomerase II inhibitors which are classified as poisons or catalytic inhibitors. We previously reported that some diterpenes exhibited inhibitory activity on topoisomerase II and/or the cell growth. In this study, we showed that 20-O-ingenolEZ is a catalytic inhibitor which inhibits the ATPase activity of topoisomerase IIα and induces the DNA-decatenation checkpoint.

Topoisomerase II poisons induce topisomerase II-mediated DNA breaks in mammalian cells.(13) Following DNA damage, the cells can be arrested in a specific phase of the cell cycle or eliminated by apoptosis. Adriamycin is known to stabilize the topoisomerase II DNA-cleavable complex, and the DNA damage checkpoint is induced via many signalings. For example, H2AX is also rapidly phosphorylated in response to agents that introduce DSBs.(14,25) Adriamycin-treated MMT cells exhibited the strongest γ-H2AX signals by DNA damage, and these cells showed apoptosis.

In contrast to the changes observed in the cells treated with adriamycin, many studies have suggested that the decatenation checkpoint, as distinct from the DNA damage checkpoint, is activated when cells are treated with catalytic topoisomarase II inhibitors.(15) ICRF 193 is a typical catalytic inhibitor that stabilizes the closed clamp form of the enzyme, an intermediate in which the N-terminal domains of the protein have dimerized. In addition to stabilizing the closed clamp form of topoisomerase II, ICRF 193 inhibits the ATPase hydrolysis activity of the yeast and human enzyme(24,26) and induces G2 arrest which occurs with little evidence of DNA damage, as determined by the lack of appearance of γ-H2AX signaling using anti-γ-H2AX antibody.(14)

In this study, 20-O-ingenolEZ inhibited relaxation of pBR322, decatenation of kDNA and the ATPase activity of human DNA topoisomerase II, and produced growth arrest of MMT cells in the G2/M phase of the cell cycle, without inducing γ-H2AX by DNA breaks. After topoisomerase II inhibitor treatment, many cells appeared to select sustained arrest in the G2 phase of the cell cycle or apoptosis with characteristics of cell type. The MMT cells treated with 20-O-ingenolEZ did not exhibit apoptosis, while the MMT cells treated with adriamycin showed significant apoptosis. In conclusion, the decatenation inhibitor 20-O-ingenolEZ induced cell arrest in the G2/M phase of the cell cycle without the DSBs. Therefore, 20-O-ingenolEZ was concluded to have shown the characteristics of a catalytic inhibitor, similar to the topoisomerase II inhibitor ICRF 193.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

This investigation was supported in part by a grant from Nihon University to S. Miyata.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References
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