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H89 enhances the sensitivity of cancer cells to glyceryl trinitrate through a purinergic receptor-dependent pathway.

Cortier M, Boina-Ali R, Racoeur C, Paul C, Solary E, Jeannin JF, Bettaieb A - Oncotarget (2015)

Bottom Line: This synergistic effect requires the generation of reactive oxygen species (ROS) from H89 and NO from GTN treatment that causes cGMP production and PKG activation.Furthermore, the GTN/H89 synergy was attenuated by inhibition of P2-purinergic receptors with suramin and competition with ATP/UDP.Thus, H89 likely acts as an ATP mimetic synergizing with GTN to trigger apoptosis in aggressive cancer cells.

View Article: PubMed Central - PubMed

Affiliation: EPHE, Tumor Immunology and Immunotherapy Laboratory, Dijon, F-21000, France.

ABSTRACT
High doses of the organic nitrate glyceryl trinitrate (GTN), a nitric oxide (NO) donor, are known to trigger apoptosis in human cancer cells. Here, we show that such a cytotoxic effect can be obtained with subtoxic concentrations of GTN when combined with H89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulphonamide.2HCl. This synergistic effect requires the generation of reactive oxygen species (ROS) from H89 and NO from GTN treatment that causes cGMP production and PKG activation. Furthermore, the GTN/H89 synergy was attenuated by inhibition of P2-purinergic receptors with suramin and competition with ATP/UDP. By down-regulating genes with antisense oligonucleotides, P2-purinergic receptors P2X3, P2Y1, and P2Y6 were found to have a role in creating this cytotoxic effect. Thus, H89 likely acts as an ATP mimetic synergizing with GTN to trigger apoptosis in aggressive cancer cells.

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ROS is involved in GTN/H89-induced apoptosis(A) SW480 cells were treated with 10 μM GTN and 10 μM H89 for 48 h at 37°C. Then the amounts of ROS in cells were monitored. Left, the relative amount of superoxide anion per cell was monitored by flow cytometry using the DHE dye. Right, the relative amount of hydrogen peroxide per cell was monitored by flow cytometry using the DHR123 dye. One readout representative of six experiments is shown for each dye. The horizontal axis shows the geometric green fluorescence intensity and the vertical axis shows the percentage of cells. Before exposure to 10 μM GTN and 10 μM H89 for 48 h at 37°C, exponentially growing SW480 cells (3 × 105/mL) were treated for 1 h (B) with ROS production inhibitors, DPI (5 μM) and apocynin (500 μM), (C) with 10 mM N-acetylcysteine (NAC), or (D) with ONOO− scavenger FeTPPS at the indicated concentrations. Apoptotic cells were counted after Hoechst 33342 staining. Results are the means of 3 independent experiments. (E) SW480 cells were treated with 500 μM ISDN, 500 μM SNAP and/or 10 μM H89 for 48 h at 37°C. The relative amount of superoxide anion per cell was monitored by flow cytometry using the DHE dye. Results are representative of 2 independent experiments.
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Figure 5: ROS is involved in GTN/H89-induced apoptosis(A) SW480 cells were treated with 10 μM GTN and 10 μM H89 for 48 h at 37°C. Then the amounts of ROS in cells were monitored. Left, the relative amount of superoxide anion per cell was monitored by flow cytometry using the DHE dye. Right, the relative amount of hydrogen peroxide per cell was monitored by flow cytometry using the DHR123 dye. One readout representative of six experiments is shown for each dye. The horizontal axis shows the geometric green fluorescence intensity and the vertical axis shows the percentage of cells. Before exposure to 10 μM GTN and 10 μM H89 for 48 h at 37°C, exponentially growing SW480 cells (3 × 105/mL) were treated for 1 h (B) with ROS production inhibitors, DPI (5 μM) and apocynin (500 μM), (C) with 10 mM N-acetylcysteine (NAC), or (D) with ONOO− scavenger FeTPPS at the indicated concentrations. Apoptotic cells were counted after Hoechst 33342 staining. Results are the means of 3 independent experiments. (E) SW480 cells were treated with 500 μM ISDN, 500 μM SNAP and/or 10 μM H89 for 48 h at 37°C. The relative amount of superoxide anion per cell was monitored by flow cytometry using the DHE dye. Results are representative of 2 independent experiments.

Mentions: As NO utilization is obligatory linked to the mitochondrial production of reactive oxygen species (ROS) [19], the possible involvement of ROS in apoptosis induced by the GTN/H89 combination was investigated. Exposure of SW480 cells to 10 μM H89 for 48 h induced the production of ROS including superoxide anions (O2−) and H2O2, as evaluated by flow cytometry using cell-permeable dihydroethamine (DHE) and dihydrorhodamine 123 (DHR123), respectively (Figure 5A). GTN alone, at 10 μM for 48 h, did not induce the production of ROS or did not increase ROS production when combined with H89 (Figure 5A). In order to determine the origin of ROS produced in H89-treated cells, we investigated the involvement of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, a well known enzyme that catalyzes ROS production. The combination's ability to trigger apoptosis was attenuated by approximately 60 and 40% by two NADPH oxidase inhibitors [20], namely diphenylene iodonium (DPI) and apocynin, respectively (Figure 5B), suggesting that this enzyme is required for the superoxide production resulting from exposure to H89. ROS production may be required for the H89/GTN combination to induce apoptosis as the latter was prevented by the antioxidant N-acetyl-L-cysteine (NAC) (Figure 5C). Because NO and superoxide anions together generate peroxynitrite, a high toxic compound, we wondered whether this oxidative agent was responsible for GTN/H89-induced apoptosis. Treatment of cells with the peroxynitrite scavenger FeTPPS [21] (at concentrations up to 100 μM) did not significantly affect the sensitivity of SW480 cells to the H89/GTN combination (Figure 5D), suggesting that peroxynitrite generation was not involved in apoptosis induction. Of note, one can speculate that the failure of the other NO donors to synergize with H89 could be due to their capacity to inhibit H89 activity. To address this question, we evaluated their impact on H89-induced ROS production. Exposure of SW480 cells to SNAP or ISDN and H89 did not abolish the ability of H89 to induce ROS production (Figure 5E).


H89 enhances the sensitivity of cancer cells to glyceryl trinitrate through a purinergic receptor-dependent pathway.

Cortier M, Boina-Ali R, Racoeur C, Paul C, Solary E, Jeannin JF, Bettaieb A - Oncotarget (2015)

ROS is involved in GTN/H89-induced apoptosis(A) SW480 cells were treated with 10 μM GTN and 10 μM H89 for 48 h at 37°C. Then the amounts of ROS in cells were monitored. Left, the relative amount of superoxide anion per cell was monitored by flow cytometry using the DHE dye. Right, the relative amount of hydrogen peroxide per cell was monitored by flow cytometry using the DHR123 dye. One readout representative of six experiments is shown for each dye. The horizontal axis shows the geometric green fluorescence intensity and the vertical axis shows the percentage of cells. Before exposure to 10 μM GTN and 10 μM H89 for 48 h at 37°C, exponentially growing SW480 cells (3 × 105/mL) were treated for 1 h (B) with ROS production inhibitors, DPI (5 μM) and apocynin (500 μM), (C) with 10 mM N-acetylcysteine (NAC), or (D) with ONOO− scavenger FeTPPS at the indicated concentrations. Apoptotic cells were counted after Hoechst 33342 staining. Results are the means of 3 independent experiments. (E) SW480 cells were treated with 500 μM ISDN, 500 μM SNAP and/or 10 μM H89 for 48 h at 37°C. The relative amount of superoxide anion per cell was monitored by flow cytometry using the DHE dye. Results are representative of 2 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 5: ROS is involved in GTN/H89-induced apoptosis(A) SW480 cells were treated with 10 μM GTN and 10 μM H89 for 48 h at 37°C. Then the amounts of ROS in cells were monitored. Left, the relative amount of superoxide anion per cell was monitored by flow cytometry using the DHE dye. Right, the relative amount of hydrogen peroxide per cell was monitored by flow cytometry using the DHR123 dye. One readout representative of six experiments is shown for each dye. The horizontal axis shows the geometric green fluorescence intensity and the vertical axis shows the percentage of cells. Before exposure to 10 μM GTN and 10 μM H89 for 48 h at 37°C, exponentially growing SW480 cells (3 × 105/mL) were treated for 1 h (B) with ROS production inhibitors, DPI (5 μM) and apocynin (500 μM), (C) with 10 mM N-acetylcysteine (NAC), or (D) with ONOO− scavenger FeTPPS at the indicated concentrations. Apoptotic cells were counted after Hoechst 33342 staining. Results are the means of 3 independent experiments. (E) SW480 cells were treated with 500 μM ISDN, 500 μM SNAP and/or 10 μM H89 for 48 h at 37°C. The relative amount of superoxide anion per cell was monitored by flow cytometry using the DHE dye. Results are representative of 2 independent experiments.
Mentions: As NO utilization is obligatory linked to the mitochondrial production of reactive oxygen species (ROS) [19], the possible involvement of ROS in apoptosis induced by the GTN/H89 combination was investigated. Exposure of SW480 cells to 10 μM H89 for 48 h induced the production of ROS including superoxide anions (O2−) and H2O2, as evaluated by flow cytometry using cell-permeable dihydroethamine (DHE) and dihydrorhodamine 123 (DHR123), respectively (Figure 5A). GTN alone, at 10 μM for 48 h, did not induce the production of ROS or did not increase ROS production when combined with H89 (Figure 5A). In order to determine the origin of ROS produced in H89-treated cells, we investigated the involvement of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, a well known enzyme that catalyzes ROS production. The combination's ability to trigger apoptosis was attenuated by approximately 60 and 40% by two NADPH oxidase inhibitors [20], namely diphenylene iodonium (DPI) and apocynin, respectively (Figure 5B), suggesting that this enzyme is required for the superoxide production resulting from exposure to H89. ROS production may be required for the H89/GTN combination to induce apoptosis as the latter was prevented by the antioxidant N-acetyl-L-cysteine (NAC) (Figure 5C). Because NO and superoxide anions together generate peroxynitrite, a high toxic compound, we wondered whether this oxidative agent was responsible for GTN/H89-induced apoptosis. Treatment of cells with the peroxynitrite scavenger FeTPPS [21] (at concentrations up to 100 μM) did not significantly affect the sensitivity of SW480 cells to the H89/GTN combination (Figure 5D), suggesting that peroxynitrite generation was not involved in apoptosis induction. Of note, one can speculate that the failure of the other NO donors to synergize with H89 could be due to their capacity to inhibit H89 activity. To address this question, we evaluated their impact on H89-induced ROS production. Exposure of SW480 cells to SNAP or ISDN and H89 did not abolish the ability of H89 to induce ROS production (Figure 5E).

Bottom Line: This synergistic effect requires the generation of reactive oxygen species (ROS) from H89 and NO from GTN treatment that causes cGMP production and PKG activation.Furthermore, the GTN/H89 synergy was attenuated by inhibition of P2-purinergic receptors with suramin and competition with ATP/UDP.Thus, H89 likely acts as an ATP mimetic synergizing with GTN to trigger apoptosis in aggressive cancer cells.

View Article: PubMed Central - PubMed

Affiliation: EPHE, Tumor Immunology and Immunotherapy Laboratory, Dijon, F-21000, France.

ABSTRACT
High doses of the organic nitrate glyceryl trinitrate (GTN), a nitric oxide (NO) donor, are known to trigger apoptosis in human cancer cells. Here, we show that such a cytotoxic effect can be obtained with subtoxic concentrations of GTN when combined with H89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulphonamide.2HCl. This synergistic effect requires the generation of reactive oxygen species (ROS) from H89 and NO from GTN treatment that causes cGMP production and PKG activation. Furthermore, the GTN/H89 synergy was attenuated by inhibition of P2-purinergic receptors with suramin and competition with ATP/UDP. By down-regulating genes with antisense oligonucleotides, P2-purinergic receptors P2X3, P2Y1, and P2Y6 were found to have a role in creating this cytotoxic effect. Thus, H89 likely acts as an ATP mimetic synergizing with GTN to trigger apoptosis in aggressive cancer cells.

Show MeSH
Related in: MedlinePlus