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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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Comparision of the structure and effect of NCX-4040 and NCX-4016 on cell viability. (A) Molecular structure of NCX-4040 (acetyloxy)benzoic acid 4-(nitrooxymethyl)phenyl ester) and NCX-4016 (2-(acetyloxy)benzoic acid 3-(nitrooxymethyl)phenyl ester). The nitroaspirins consist of acetylsalicylate (aspirin) linked to a spacer moiety by an ester bond. An NO-releasing moiety (-ONO2) is attached to the spacer. (B) Effect of NCX-4040 and NCX-4016 (25 μM) on the viability of cisplatin-resistant human ovarian cancer cells (A2780 cDDP). The results show a dose-dependent cytotoxic effect, with NCX-4040 demonstrating a substantially higher effect compared to NCX-4016.
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Figure 1: Comparision of the structure and effect of NCX-4040 and NCX-4016 on cell viability. (A) Molecular structure of NCX-4040 (acetyloxy)benzoic acid 4-(nitrooxymethyl)phenyl ester) and NCX-4016 (2-(acetyloxy)benzoic acid 3-(nitrooxymethyl)phenyl ester). The nitroaspirins consist of acetylsalicylate (aspirin) linked to a spacer moiety by an ester bond. An NO-releasing moiety (-ONO2) is attached to the spacer. (B) Effect of NCX-4040 and NCX-4016 (25 μM) on the viability of cisplatin-resistant human ovarian cancer cells (A2780 cDDP). The results show a dose-dependent cytotoxic effect, with NCX-4040 demonstrating a substantially higher effect compared to NCX-4016.

Mentions: Recently, we showed that NCX-4016, a nitro derivative of aspirin, inhibited the proliferation of cisplatin-sensitive as well as cisplatin-resistant human ovarian cancer cell lines in vitro [18]. We also showed that NCX-4016, on incubation with the cisplatin-resistant cells, generated sustained levels of nitric oxide (NO) and substantially depleted cellular thiols. In a subsequent report, we further showed that NCX-4016, by itself, was capable of inhibiting the growth of cisplatin-resistant human xenograft tumors in mice [19]. We determined that NCX-4016 induced G1 cell-cycle arrest and apoptosis by inhibiting the EGFR/PI3K and STAT3 signaling pathways. Subsequent to the initial reports on NCX-4016, there have been a few reports on NCX-4040 (Figure 1A), a positional isomer of NCX-4016 [20], that demonstrated significant cytotoxic effect on pancreatic [21], bladder [22], and colon cancer [23-25]. It was reported that NCX-4040 was 100 times more potent than NCX-4016 in HT-29 human colon cancer cells [26]. However, its cytotoxic effect on human ovarian cancer has not been investigated. Therefore, the goal of the present work was to study the antitumor efficacy and potential of NCX-4040 to sensitize cisplatin-resistant ovarian cancer cells to cisplatin treatment. The experiments were performed using cisplatin-sensitive (A2780 WT) and cisplatin-resistant (A2780 cDDP) human ovarian cancer cell lines and xenograft tumors. The results showed that administration of NCX-4040 decreased cellular thiol levels, thereby sensitizing the drug-resistant cells to cisplatin. NCX-4040, in combination with cisplatin, inhibited tumor growth by downregulation of EGFR and STAT3 signaling.


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)

Comparision of the structure and effect of NCX-4040 and NCX-4016 on cell viability. (A) Molecular structure of NCX-4040 (acetyloxy)benzoic acid 4-(nitrooxymethyl)phenyl ester) and NCX-4016 (2-(acetyloxy)benzoic acid 3-(nitrooxymethyl)phenyl ester). The nitroaspirins consist of acetylsalicylate (aspirin) linked to a spacer moiety by an ester bond. An NO-releasing moiety (-ONO2) is attached to the spacer. (B) Effect of NCX-4040 and NCX-4016 (25 μM) on the viability of cisplatin-resistant human ovarian cancer cells (A2780 cDDP). The results show a dose-dependent cytotoxic effect, with NCX-4040 demonstrating a substantially higher effect compared to NCX-4016.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Comparision of the structure and effect of NCX-4040 and NCX-4016 on cell viability. (A) Molecular structure of NCX-4040 (acetyloxy)benzoic acid 4-(nitrooxymethyl)phenyl ester) and NCX-4016 (2-(acetyloxy)benzoic acid 3-(nitrooxymethyl)phenyl ester). The nitroaspirins consist of acetylsalicylate (aspirin) linked to a spacer moiety by an ester bond. An NO-releasing moiety (-ONO2) is attached to the spacer. (B) Effect of NCX-4040 and NCX-4016 (25 μM) on the viability of cisplatin-resistant human ovarian cancer cells (A2780 cDDP). The results show a dose-dependent cytotoxic effect, with NCX-4040 demonstrating a substantially higher effect compared to NCX-4016.
Mentions: Recently, we showed that NCX-4016, a nitro derivative of aspirin, inhibited the proliferation of cisplatin-sensitive as well as cisplatin-resistant human ovarian cancer cell lines in vitro [18]. We also showed that NCX-4016, on incubation with the cisplatin-resistant cells, generated sustained levels of nitric oxide (NO) and substantially depleted cellular thiols. In a subsequent report, we further showed that NCX-4016, by itself, was capable of inhibiting the growth of cisplatin-resistant human xenograft tumors in mice [19]. We determined that NCX-4016 induced G1 cell-cycle arrest and apoptosis by inhibiting the EGFR/PI3K and STAT3 signaling pathways. Subsequent to the initial reports on NCX-4016, there have been a few reports on NCX-4040 (Figure 1A), a positional isomer of NCX-4016 [20], that demonstrated significant cytotoxic effect on pancreatic [21], bladder [22], and colon cancer [23-25]. It was reported that NCX-4040 was 100 times more potent than NCX-4016 in HT-29 human colon cancer cells [26]. However, its cytotoxic effect on human ovarian cancer has not been investigated. Therefore, the goal of the present work was to study the antitumor efficacy and potential of NCX-4040 to sensitize cisplatin-resistant ovarian cancer cells to cisplatin treatment. The experiments were performed using cisplatin-sensitive (A2780 WT) and cisplatin-resistant (A2780 cDDP) human ovarian cancer cell lines and xenograft tumors. The results showed that administration of NCX-4040 decreased cellular thiol levels, thereby sensitizing the drug-resistant cells to cisplatin. NCX-4040, in combination with cisplatin, inhibited tumor growth by downregulation of EGFR and STAT3 signaling.

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