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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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Effect of NCX-4040 and/or cisplatin on the viability of A2780 WT and A2780 cDDP cell lines. Cells were incubated with 25 μM of NCX-4040 for 6 h, then with cisplatin for 1 h (IC67 dose for A2780 WT) with a 24 h follow up time. Cell viability was evaluated using MTT assay. Data represent mean ± SD obtained from 4 independent experiments and expressed as a percentage of the control cells treated with vehicle (1% DMSO). *p < 0.01 versus Control group. **p < 0.05 versus cDDP or NCX-4040 group.
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Figure 2: Effect of NCX-4040 and/or cisplatin on the viability of A2780 WT and A2780 cDDP cell lines. Cells were incubated with 25 μM of NCX-4040 for 6 h, then with cisplatin for 1 h (IC67 dose for A2780 WT) with a 24 h follow up time. Cell viability was evaluated using MTT assay. Data represent mean ± SD obtained from 4 independent experiments and expressed as a percentage of the control cells treated with vehicle (1% DMSO). *p < 0.01 versus Control group. **p < 0.05 versus cDDP or NCX-4040 group.

Mentions: NCX-4040 is a positional isomer of NCX-4016 (Figure 1A), which we have previously reported to be cytotoxic to human ovarian cancer cell lines [18]. In order to evaluate the anticancer efficacy of NCX-4040 and to compare with that of NCX-4016, we performed a dose-response study using cisplatin-resistant cell line (A2780 cDDP). As seen in Figure 1B, a dose-dependent decrease of cell viability was observed in both cases. However, NCX-4040 showed a substantial decrease in cell viability when compared to NCX-4016. The cytotoxic effects of NCX-4040, either alone or in combination with cisplatin, on A2780 WT and A2780 cDDP cell lines were determined. Cells were treated with NCX-4040 (25 μM) for 6 h followed by cisplatin (IC67 dose for A2780 WT cells) for 1 h with a 24-h follow up time. Cytotoxicity was assessed using an MTT viability assay. The results (Figure 2) showed that NCX-4040 alone significantly decreased the viability of both A2780 WT (34.9 ± 8.7%) and A2780 cDDP (41.7 ± 7.6%) cell lines. Cisplatin (cDDP) alone significantly decreased the viability of A2780 WT (31.5 ± 3.4%), but not A2780 cDDP (80.6 ± 11.8%) cell lines. On the other hand, combination of cisplatin and NCX-4040 was more effective than cisplatin or NCX-4040 alone in decreasing the viability of both A2780 WT (9.4 ± 6.0%) and A2780 cDDP (26.4 ± 7.6%) cell lines. A weak synergism was observed (R = 1.27, see Methods for calculation) between NCX-4040 and cisplatin in the killing of A2780 cDDP cells. The results clearly demonstrated that NCX-4040 was not only cytotoxic, but also capable of sensitizing cisplatin-resistant ovarian cancer cells to cisplatin.


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)

Effect of NCX-4040 and/or cisplatin on the viability of A2780 WT and A2780 cDDP cell lines. Cells were incubated with 25 μM of NCX-4040 for 6 h, then with cisplatin for 1 h (IC67 dose for A2780 WT) with a 24 h follow up time. Cell viability was evaluated using MTT assay. Data represent mean ± SD obtained from 4 independent experiments and expressed as a percentage of the control cells treated with vehicle (1% DMSO). *p < 0.01 versus Control group. **p < 0.05 versus cDDP or NCX-4040 group.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2267444&req=5

Figure 2: Effect of NCX-4040 and/or cisplatin on the viability of A2780 WT and A2780 cDDP cell lines. Cells were incubated with 25 μM of NCX-4040 for 6 h, then with cisplatin for 1 h (IC67 dose for A2780 WT) with a 24 h follow up time. Cell viability was evaluated using MTT assay. Data represent mean ± SD obtained from 4 independent experiments and expressed as a percentage of the control cells treated with vehicle (1% DMSO). *p < 0.01 versus Control group. **p < 0.05 versus cDDP or NCX-4040 group.
Mentions: NCX-4040 is a positional isomer of NCX-4016 (Figure 1A), which we have previously reported to be cytotoxic to human ovarian cancer cell lines [18]. In order to evaluate the anticancer efficacy of NCX-4040 and to compare with that of NCX-4016, we performed a dose-response study using cisplatin-resistant cell line (A2780 cDDP). As seen in Figure 1B, a dose-dependent decrease of cell viability was observed in both cases. However, NCX-4040 showed a substantial decrease in cell viability when compared to NCX-4016. The cytotoxic effects of NCX-4040, either alone or in combination with cisplatin, on A2780 WT and A2780 cDDP cell lines were determined. Cells were treated with NCX-4040 (25 μM) for 6 h followed by cisplatin (IC67 dose for A2780 WT cells) for 1 h with a 24-h follow up time. Cytotoxicity was assessed using an MTT viability assay. The results (Figure 2) showed that NCX-4040 alone significantly decreased the viability of both A2780 WT (34.9 ± 8.7%) and A2780 cDDP (41.7 ± 7.6%) cell lines. Cisplatin (cDDP) alone significantly decreased the viability of A2780 WT (31.5 ± 3.4%), but not A2780 cDDP (80.6 ± 11.8%) cell lines. On the other hand, combination of cisplatin and NCX-4040 was more effective than cisplatin or NCX-4040 alone in decreasing the viability of both A2780 WT (9.4 ± 6.0%) and A2780 cDDP (26.4 ± 7.6%) cell lines. A weak synergism was observed (R = 1.27, see Methods for calculation) between NCX-4040 and cisplatin in the killing of A2780 cDDP cells. The results clearly demonstrated that NCX-4040 was not only cytotoxic, but also capable of sensitizing cisplatin-resistant ovarian cancer cells to cisplatin.

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