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NCX-4040, a nitric oxide-releasing aspirin, sensitizes drug-resistant human ovarian xenograft tumors to cisplatin by depletion of cellular thiols.

Bratasz A, Selvendiran K, Wasowicz T, Bobko A, Khramtsov VV, Ignarro LJ, Kuppusamy P - J Transl Med (2008)

Bottom Line: Cells treated with NCX-4040 (25 microM) showed a significant reduction of cell viability (A2780 WT, 34.9 +/- 8.7%; A2780 cDDP, 41.7 +/- 7.6%; p < 0.05).EPR imaging of tissue redox and thiol measurements showed a 5.5-fold reduction (p < 0.01) of glutathione in NCX-4040-treated A2780 cDDP tumors when compared to untreated controls.The results suggested that NCX-4040 could resensitize drug-resistant ovarian cancer cells to cisplatin possibly by depletion of cellular thiols.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Biomedical EPR Spectroscopy and Imaging, Davis Heart and Lung Research Institute, Department of Internal Medicine, The Ohio State University, Columbus, OH 43210, USA. kuppusamy.1@osu.edu

ABSTRACT

Background: Ovarian carcinoma is the leading cause of mortality among gynecological cancers in the world. The high mortality rate is associated with lack of early diagnosis and development of drug resistance. The antitumor efficacy and mechanism of NCX-4040, a nitric oxide-releasing aspirin derivative, against ovarian cancer is studied.

Methods: NCX-4040, alone or in combination with cisplatin (cis-diamminedichloroplatinum, cDDP), was studied in cisplatin-sensitive (A2780 WT) and cisplatin-resistant (A2780 cDDP) cell lines as well as xenograft tumors grown in nude mice. Electron paramagnetic resonance (EPR) was used for measurements of nitric oxide and redox state. Immunoblotting analysis of A2780 cDDP tumor xenografts from mice was used for mechanistic studies.

Results: Cells treated with NCX-4040 (25 microM) showed a significant reduction of cell viability (A2780 WT, 34.9 +/- 8.7%; A2780 cDDP, 41.7 +/- 7.6%; p < 0.05). Further, NCX-4040 significantly enhanced the sensitivity of A2780 cDDP cells (cisplatin alone, 80.6 +/- 11.8% versus NCX-4040+cisplatin, 26.4 +/- 7.6%; p < 0.01) and xenograft tumors (cisplatin alone, 74.0 +/- 4.4% versus NCX-4040+cisplatin, 56.4 +/- 7.8%; p < 0.05), to cisplatin treatment. EPR imaging of tissue redox and thiol measurements showed a 5.5-fold reduction (p < 0.01) of glutathione in NCX-4040-treated A2780 cDDP tumors when compared to untreated controls. Immunoblotting analysis of A2780 cDDP tumor xenografts from mice treated with NCX-4040 and cisplatin revealed significant downregulation of pEGFR (Tyr845 and Tyr992) and pSTAT3 (Tyr705 and Ser727) expression.

Conclusion: The results suggested that NCX-4040 could resensitize drug-resistant ovarian cancer cells to cisplatin possibly by depletion of cellular thiols. Thus NCX-4040 appears to be a potential therapeutic agent for the treatment of human ovarian carcinoma and cisplatin-resistant malignancies.

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Generation of nitric oxide (NO) by NCX-4040 in A2780 WT and A2780 cDDP cells. (A) Representative EPR spectra obtained from control (NCX-4040, A2780 cDDP cells) and NCX-4040 treatment (100 μM for both A2780 WT and A2780 cDDP) groups. The cells were incubated with NCX-4040 in the presence of spin trap (MGD)2Fe. Also shown is an EPR spectrum of an authentic complex of NO with Fe(MGD)2 obtained by adding SNAP (an NO donor) to spin trap. (B) Time-course of nitric oxide release by NCX-4040 in A2780 WT, A2780 cDDP, and A2780 cDDP cells treated with 1 mM buthionine sulfoximine (BSO) for 24 h. (C) Representative DAF-FM DA fluorescence images of untreated (control) and treated (1, 2 or 4 hours) with NCX-4040 (100 μM). (D) Quantitative results on the DAF-FM DA fluorescence data. *p < 0.01 versus Control group.
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Figure 3: Generation of nitric oxide (NO) by NCX-4040 in A2780 WT and A2780 cDDP cells. (A) Representative EPR spectra obtained from control (NCX-4040, A2780 cDDP cells) and NCX-4040 treatment (100 μM for both A2780 WT and A2780 cDDP) groups. The cells were incubated with NCX-4040 in the presence of spin trap (MGD)2Fe. Also shown is an EPR spectrum of an authentic complex of NO with Fe(MGD)2 obtained by adding SNAP (an NO donor) to spin trap. (B) Time-course of nitric oxide release by NCX-4040 in A2780 WT, A2780 cDDP, and A2780 cDDP cells treated with 1 mM buthionine sulfoximine (BSO) for 24 h. (C) Representative DAF-FM DA fluorescence images of untreated (control) and treated (1, 2 or 4 hours) with NCX-4040 (100 μM). (D) Quantitative results on the DAF-FM DA fluorescence data. *p < 0.01 versus Control group.

Mentions: To determine whether NCX-4040 can generate NO in cells, we performed EPR spectroscopic measurements on A2780 WT and A2780 cDDP cells incubated with 100 μM of NCX-4040. Fe(MGD)2 was used as a spin-trap for real-time detection of NO. Fe(MGD)2 reacts with NO and forms a stable paramagnetic adduct, Fe(MGD)2-NO, which can be detected by EPR spectroscopy [34]. NCX-4040 or cells alone in media did not generate any signal (Figure 3A). However, a prominent triplet signal with a hyperfine coupling constant of 12.5 G, characteristic of Fe(MGD)2-NO, was observed upon incubation of NCX-4040 with the cells. The authenticity of the spectra was verified using S-nitroso-N-acetylpenicillamine (SNAP, 100 μM), a known NO-releasing compound. Both cell lines demonstrated a time-dependent increase in the intensity of Fe(MGD)2-NO signal (Figure 3B) starting at 40–50 min of co-incubation time. While the rate of NO generation was steady in the case of A2780 WT cells, a rapid increase followed by slow attenuation was observed in the case of A2780 cDDP cells. In order to determine whether or not the increased rate of NO generation in A2780 cDDP cells was due to thiols, we pre-incubated A2780 cDDP cells with 1 mM buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, for 24 h and then treated with NCX-4040. As seen in Figure 3B, the NO generation was significantly decreased upon BSO treatment, suggesting that the NO generation was mediated by cellular thiols. In contrast, NO generation by SNAP was instant and lasted only for a few minutes (data not shown). We further imaged the intracellular NO generation using DAF-FM DA (Figure 3C). These results shown in Figure 3D clearly demonstrated a sustained generation of NO by NCX-4040 in the cancer cells. Thus, the NO generation by NCX-4040 in cancer cells was low, but persisted for longer periods.


NCX-4040, a nitric oxide-releasing aspirin, sensitizes drug-resistant human ovarian xenograft tumors to cisplatin by depletion of cellular thiols.

Bratasz A, Selvendiran K, Wasowicz T, Bobko A, Khramtsov VV, Ignarro LJ, Kuppusamy P - J Transl Med (2008)

Generation of nitric oxide (NO) by NCX-4040 in A2780 WT and A2780 cDDP cells. (A) Representative EPR spectra obtained from control (NCX-4040, A2780 cDDP cells) and NCX-4040 treatment (100 μM for both A2780 WT and A2780 cDDP) groups. The cells were incubated with NCX-4040 in the presence of spin trap (MGD)2Fe. Also shown is an EPR spectrum of an authentic complex of NO with Fe(MGD)2 obtained by adding SNAP (an NO donor) to spin trap. (B) Time-course of nitric oxide release by NCX-4040 in A2780 WT, A2780 cDDP, and A2780 cDDP cells treated with 1 mM buthionine sulfoximine (BSO) for 24 h. (C) Representative DAF-FM DA fluorescence images of untreated (control) and treated (1, 2 or 4 hours) with NCX-4040 (100 μM). (D) Quantitative results on the DAF-FM DA fluorescence data. *p < 0.01 versus Control group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 3: Generation of nitric oxide (NO) by NCX-4040 in A2780 WT and A2780 cDDP cells. (A) Representative EPR spectra obtained from control (NCX-4040, A2780 cDDP cells) and NCX-4040 treatment (100 μM for both A2780 WT and A2780 cDDP) groups. The cells were incubated with NCX-4040 in the presence of spin trap (MGD)2Fe. Also shown is an EPR spectrum of an authentic complex of NO with Fe(MGD)2 obtained by adding SNAP (an NO donor) to spin trap. (B) Time-course of nitric oxide release by NCX-4040 in A2780 WT, A2780 cDDP, and A2780 cDDP cells treated with 1 mM buthionine sulfoximine (BSO) for 24 h. (C) Representative DAF-FM DA fluorescence images of untreated (control) and treated (1, 2 or 4 hours) with NCX-4040 (100 μM). (D) Quantitative results on the DAF-FM DA fluorescence data. *p < 0.01 versus Control group.
Mentions: To determine whether NCX-4040 can generate NO in cells, we performed EPR spectroscopic measurements on A2780 WT and A2780 cDDP cells incubated with 100 μM of NCX-4040. Fe(MGD)2 was used as a spin-trap for real-time detection of NO. Fe(MGD)2 reacts with NO and forms a stable paramagnetic adduct, Fe(MGD)2-NO, which can be detected by EPR spectroscopy [34]. NCX-4040 or cells alone in media did not generate any signal (Figure 3A). However, a prominent triplet signal with a hyperfine coupling constant of 12.5 G, characteristic of Fe(MGD)2-NO, was observed upon incubation of NCX-4040 with the cells. The authenticity of the spectra was verified using S-nitroso-N-acetylpenicillamine (SNAP, 100 μM), a known NO-releasing compound. Both cell lines demonstrated a time-dependent increase in the intensity of Fe(MGD)2-NO signal (Figure 3B) starting at 40–50 min of co-incubation time. While the rate of NO generation was steady in the case of A2780 WT cells, a rapid increase followed by slow attenuation was observed in the case of A2780 cDDP cells. In order to determine whether or not the increased rate of NO generation in A2780 cDDP cells was due to thiols, we pre-incubated A2780 cDDP cells with 1 mM buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, for 24 h and then treated with NCX-4040. As seen in Figure 3B, the NO generation was significantly decreased upon BSO treatment, suggesting that the NO generation was mediated by cellular thiols. In contrast, NO generation by SNAP was instant and lasted only for a few minutes (data not shown). We further imaged the intracellular NO generation using DAF-FM DA (Figure 3C). These results shown in Figure 3D clearly demonstrated a sustained generation of NO by NCX-4040 in the cancer cells. Thus, the NO generation by NCX-4040 in cancer cells was low, but persisted for longer periods.

Bottom Line: Cells treated with NCX-4040 (25 microM) showed a significant reduction of cell viability (A2780 WT, 34.9 +/- 8.7%; A2780 cDDP, 41.7 +/- 7.6%; p < 0.05).EPR imaging of tissue redox and thiol measurements showed a 5.5-fold reduction (p < 0.01) of glutathione in NCX-4040-treated A2780 cDDP tumors when compared to untreated controls.The results suggested that NCX-4040 could resensitize drug-resistant ovarian cancer cells to cisplatin possibly by depletion of cellular thiols.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Biomedical EPR Spectroscopy and Imaging, Davis Heart and Lung Research Institute, Department of Internal Medicine, The Ohio State University, Columbus, OH 43210, USA. kuppusamy.1@osu.edu

ABSTRACT

Background: Ovarian carcinoma is the leading cause of mortality among gynecological cancers in the world. The high mortality rate is associated with lack of early diagnosis and development of drug resistance. The antitumor efficacy and mechanism of NCX-4040, a nitric oxide-releasing aspirin derivative, against ovarian cancer is studied.

Methods: NCX-4040, alone or in combination with cisplatin (cis-diamminedichloroplatinum, cDDP), was studied in cisplatin-sensitive (A2780 WT) and cisplatin-resistant (A2780 cDDP) cell lines as well as xenograft tumors grown in nude mice. Electron paramagnetic resonance (EPR) was used for measurements of nitric oxide and redox state. Immunoblotting analysis of A2780 cDDP tumor xenografts from mice was used for mechanistic studies.

Results: Cells treated with NCX-4040 (25 microM) showed a significant reduction of cell viability (A2780 WT, 34.9 +/- 8.7%; A2780 cDDP, 41.7 +/- 7.6%; p < 0.05). Further, NCX-4040 significantly enhanced the sensitivity of A2780 cDDP cells (cisplatin alone, 80.6 +/- 11.8% versus NCX-4040+cisplatin, 26.4 +/- 7.6%; p < 0.01) and xenograft tumors (cisplatin alone, 74.0 +/- 4.4% versus NCX-4040+cisplatin, 56.4 +/- 7.8%; p < 0.05), to cisplatin treatment. EPR imaging of tissue redox and thiol measurements showed a 5.5-fold reduction (p < 0.01) of glutathione in NCX-4040-treated A2780 cDDP tumors when compared to untreated controls. Immunoblotting analysis of A2780 cDDP tumor xenografts from mice treated with NCX-4040 and cisplatin revealed significant downregulation of pEGFR (Tyr845 and Tyr992) and pSTAT3 (Tyr705 and Ser727) expression.

Conclusion: The results suggested that NCX-4040 could resensitize drug-resistant ovarian cancer cells to cisplatin possibly by depletion of cellular thiols. Thus NCX-4040 appears to be a potential therapeutic agent for the treatment of human ovarian carcinoma and cisplatin-resistant malignancies.

Show MeSH
Related in: MedlinePlus