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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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Related in: MedlinePlus

NO is involved in GTN/H89-induced apoptosis(A) Exponentially growing SW480 cells (3 × 105/mL) were treated with 10 μM GTN and/or 10 μM H89 for 48 h at 37°C. The concentration of nitrite in the medium was quantified with the Griess method. (B) Exponentially growing SW480 cells (3 × 105/mL) were treated with NO scavenger carboxy-PTIO (200 μM) for 1 h before exposure to 10 μM GTN and 10 μM H89 for 48 h at 37°C. The nitrite concentration in the medium was then measured and (C) apoptotic cells counted after Hoechst 33342 staining. Results are the means of 3 independent experiments. (D) cGMP content analysis using the colorimetric cGMP Direct immunoassay kit using lysates from SW480 cells treated with 10 μM GTN, 500 μM ISDN, 500 μM SNAP and/or 10 μM H89 for 16 h at 37°C. Data are from 1 experiment made in triplicate representative of three independent experiments (E) SW480 cells were treated with 500 μM Rp-8-Br-PET-cGMPS, a competitive inhibitor of PKG (inhib PKG), 10 μM GTN and 10 μM H89 for 48 h at 37°C, and apoptotic cells were counted. Results are the means of 3 independent experiments. *P < .05. (F) SW480 cells were treated with 200 μM zaprinast a phosphodiesterase 5 inhibitor, 10 μM GTN and 10 μM H89 for 48 h at 37°C. Results are the means of 3 independent experiments. *P < .05.
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Figure 4: NO is involved in GTN/H89-induced apoptosis(A) Exponentially growing SW480 cells (3 × 105/mL) were treated with 10 μM GTN and/or 10 μM H89 for 48 h at 37°C. The concentration of nitrite in the medium was quantified with the Griess method. (B) Exponentially growing SW480 cells (3 × 105/mL) were treated with NO scavenger carboxy-PTIO (200 μM) for 1 h before exposure to 10 μM GTN and 10 μM H89 for 48 h at 37°C. The nitrite concentration in the medium was then measured and (C) apoptotic cells counted after Hoechst 33342 staining. Results are the means of 3 independent experiments. (D) cGMP content analysis using the colorimetric cGMP Direct immunoassay kit using lysates from SW480 cells treated with 10 μM GTN, 500 μM ISDN, 500 μM SNAP and/or 10 μM H89 for 16 h at 37°C. Data are from 1 experiment made in triplicate representative of three independent experiments (E) SW480 cells were treated with 500 μM Rp-8-Br-PET-cGMPS, a competitive inhibitor of PKG (inhib PKG), 10 μM GTN and 10 μM H89 for 48 h at 37°C, and apoptotic cells were counted. Results are the means of 3 independent experiments. *P < .05. (F) SW480 cells were treated with 200 μM zaprinast a phosphodiesterase 5 inhibitor, 10 μM GTN and 10 μM H89 for 48 h at 37°C. Results are the means of 3 independent experiments. *P < .05.

Mentions: As a NO donor, the contribution of GTN to the synergistic effect of the GTN/H89 combination is likely due to the release of NO. Exposure of SW480 cells to 10 μM GTN induces the release of nitrite in the culture medium (> 4 μM at 48 h compared to < 1 μM in controls; Figure 4A). H89 did not significantly increase the GTN-induced NO production (Figure 4A). Interestingly, the NO scavenger (4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) significantly lowered GTN and GTN/H89-induced nitrite levels (Figure 4B), and abrogated GTN/H89-mediated apoptosis (Figure 4C). This suggests that NO production was required for the combination to induce apoptosis.


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)

NO is involved in GTN/H89-induced apoptosis(A) Exponentially growing SW480 cells (3 × 105/mL) were treated with 10 μM GTN and/or 10 μM H89 for 48 h at 37°C. The concentration of nitrite in the medium was quantified with the Griess method. (B) Exponentially growing SW480 cells (3 × 105/mL) were treated with NO scavenger carboxy-PTIO (200 μM) for 1 h before exposure to 10 μM GTN and 10 μM H89 for 48 h at 37°C. The nitrite concentration in the medium was then measured and (C) apoptotic cells counted after Hoechst 33342 staining. Results are the means of 3 independent experiments. (D) cGMP content analysis using the colorimetric cGMP Direct immunoassay kit using lysates from SW480 cells treated with 10 μM GTN, 500 μM ISDN, 500 μM SNAP and/or 10 μM H89 for 16 h at 37°C. Data are from 1 experiment made in triplicate representative of three independent experiments (E) SW480 cells were treated with 500 μM Rp-8-Br-PET-cGMPS, a competitive inhibitor of PKG (inhib PKG), 10 μM GTN and 10 μM H89 for 48 h at 37°C, and apoptotic cells were counted. Results are the means of 3 independent experiments. *P < .05. (F) SW480 cells were treated with 200 μM zaprinast a phosphodiesterase 5 inhibitor, 10 μM GTN and 10 μM H89 for 48 h at 37°C. Results are the means of 3 independent experiments. *P < .05.
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Related In: Results  -  Collection

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Figure 4: NO is involved in GTN/H89-induced apoptosis(A) Exponentially growing SW480 cells (3 × 105/mL) were treated with 10 μM GTN and/or 10 μM H89 for 48 h at 37°C. The concentration of nitrite in the medium was quantified with the Griess method. (B) Exponentially growing SW480 cells (3 × 105/mL) were treated with NO scavenger carboxy-PTIO (200 μM) for 1 h before exposure to 10 μM GTN and 10 μM H89 for 48 h at 37°C. The nitrite concentration in the medium was then measured and (C) apoptotic cells counted after Hoechst 33342 staining. Results are the means of 3 independent experiments. (D) cGMP content analysis using the colorimetric cGMP Direct immunoassay kit using lysates from SW480 cells treated with 10 μM GTN, 500 μM ISDN, 500 μM SNAP and/or 10 μM H89 for 16 h at 37°C. Data are from 1 experiment made in triplicate representative of three independent experiments (E) SW480 cells were treated with 500 μM Rp-8-Br-PET-cGMPS, a competitive inhibitor of PKG (inhib PKG), 10 μM GTN and 10 μM H89 for 48 h at 37°C, and apoptotic cells were counted. Results are the means of 3 independent experiments. *P < .05. (F) SW480 cells were treated with 200 μM zaprinast a phosphodiesterase 5 inhibitor, 10 μM GTN and 10 μM H89 for 48 h at 37°C. Results are the means of 3 independent experiments. *P < .05.
Mentions: As a NO donor, the contribution of GTN to the synergistic effect of the GTN/H89 combination is likely due to the release of NO. Exposure of SW480 cells to 10 μM GTN induces the release of nitrite in the culture medium (> 4 μM at 48 h compared to < 1 μM in controls; Figure 4A). H89 did not significantly increase the GTN-induced NO production (Figure 4A). Interestingly, the NO scavenger (4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) significantly lowered GTN and GTN/H89-induced nitrite levels (Figure 4B), and abrogated GTN/H89-mediated apoptosis (Figure 4C). This suggests that NO production was required for the combination to induce apoptosis.

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