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TIMP-2 mediates the anti-invasive effects of the nitric oxide-releasing prodrug JS-K in breast cancer cells.

Simeone AM, McMurtry V, Nieves-Alicea R, Saavedra JE, Keefer LK, Johnson MM, Tari AM - Breast Cancer Res. (2008)

Bottom Line: Under conditions by which JS-K was not cytotoxic, JS-K significantly decreased (P < 0.05) the invasiveness of breast cancer cells across the Matrigel basement membrane, which was directly correlated with NO production.JS-K decreased p38 activity, whereas the activity and the expression of extracellular signal-regulated kinase 1/2 and c-Jun N-terminal kinase were unaffected.Upregulation of TIMP-2 production is one mechanism by which JS-K mediates its anti-invasive effects.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.

ABSTRACT

Introduction: Tumor invasion and metastasis remain a major cause of mortality in breast cancer patients. High concentrations of nitric oxide (NO) suppress tumor invasion and metastasis in vivo. NO prodrugs generate large amounts of NO upon metabolism by appropriate intracellular enzymes, and therefore could have potential in the prevention and therapy of metastatic breast cancer.

Methods: The present study was designed to determine the effects of the NO-releasing prodrug O2-(2,4-dinitrophenyl) 1- [(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) on breast cancer invasion and the mechanisms involved. MDA-MB-231, MDA-MB-231/F10, and MCF-7/COX-2 were the three breast cancer cell lines tested. NO levels were determined spectrophotometrically using a NO assay kit. Invasion and the expression of matrix metalloproteinases (MMPs) and tissue inhibitor of MMPs were determined using Matrigel invasion assays, an MMP array kit and ELISAs. The activity and expression of extracellular signal-regulated kinase 1/2, p38, and c-Jun N-terminal kinase mitogen-activated protein kinases were determined using western blot analyses.

Results: Under conditions by which JS-K was not cytotoxic, JS-K significantly decreased (P < 0.05) the invasiveness of breast cancer cells across the Matrigel basement membrane, which was directly correlated with NO production. JS-43-126, a non-NO-releasing analog of JS-K, had no effect on NO levels or invasion. JS-K increased (P < 0.05) TIMP-2 production, and blocking TIMP-2 activity with a neutralizing antibody significantly increased (P < 0.05) the invasive activity of JS-K-treated cells across Matrigel. JS-K decreased p38 activity, whereas the activity and the expression of extracellular signal-regulated kinase 1/2 and c-Jun N-terminal kinase were unaffected.

Conclusion: We report the novel findings that JS-K inhibits breast cancer invasion across the Matrigel basement membrane, and NO production is vital for this activity. Upregulation of TIMP-2 production is one mechanism by which JS-K mediates its anti-invasive effects. JS-K and other NO prodrugs may represent an innovative biological approach in the prevention and treatment of metastatic breast cancer.

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JS-K, but not JS-43-126, increases nitric oxide production in breast cancer cells. Conditioned medium supernatant was collected from MDA-MB-231, F10, and MCF-7/COX-2 cells treated in the absence or presence of JS-K or JS-43-126 for 72 hours. Total nitric oxide (NO) was determined by quantifying nitrite, the stable end product of NO oxidation, spectrophotometrically using a colorimetric nonenzymatic nitric oxide assay kit. Nitrite values were normalized for total cell counts and expressed as picomoles per 106 cells. Columns indicate the mean of triplicate wells ± standard deviation. *Significant increase in NO levels relative to untreated cells, P < 0.05.
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Figure 3: JS-K, but not JS-43-126, increases nitric oxide production in breast cancer cells. Conditioned medium supernatant was collected from MDA-MB-231, F10, and MCF-7/COX-2 cells treated in the absence or presence of JS-K or JS-43-126 for 72 hours. Total nitric oxide (NO) was determined by quantifying nitrite, the stable end product of NO oxidation, spectrophotometrically using a colorimetric nonenzymatic nitric oxide assay kit. Nitrite values were normalized for total cell counts and expressed as picomoles per 106 cells. Columns indicate the mean of triplicate wells ± standard deviation. *Significant increase in NO levels relative to untreated cells, P < 0.05.

Mentions: NO levels were determined in untreated and JS-K-treated MDA-MB-231, F10, and MCF-7/COX-2 cells to confirm drug activation. The NO production was significantly increased (P < 0.05) in the three cell lines as a result of JS-K treatment (Figure 3).


TIMP-2 mediates the anti-invasive effects of the nitric oxide-releasing prodrug JS-K in breast cancer cells.

Simeone AM, McMurtry V, Nieves-Alicea R, Saavedra JE, Keefer LK, Johnson MM, Tari AM - Breast Cancer Res. (2008)

JS-K, but not JS-43-126, increases nitric oxide production in breast cancer cells. Conditioned medium supernatant was collected from MDA-MB-231, F10, and MCF-7/COX-2 cells treated in the absence or presence of JS-K or JS-43-126 for 72 hours. Total nitric oxide (NO) was determined by quantifying nitrite, the stable end product of NO oxidation, spectrophotometrically using a colorimetric nonenzymatic nitric oxide assay kit. Nitrite values were normalized for total cell counts and expressed as picomoles per 106 cells. Columns indicate the mean of triplicate wells ± standard deviation. *Significant increase in NO levels relative to untreated cells, P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2481491&req=5

Figure 3: JS-K, but not JS-43-126, increases nitric oxide production in breast cancer cells. Conditioned medium supernatant was collected from MDA-MB-231, F10, and MCF-7/COX-2 cells treated in the absence or presence of JS-K or JS-43-126 for 72 hours. Total nitric oxide (NO) was determined by quantifying nitrite, the stable end product of NO oxidation, spectrophotometrically using a colorimetric nonenzymatic nitric oxide assay kit. Nitrite values were normalized for total cell counts and expressed as picomoles per 106 cells. Columns indicate the mean of triplicate wells ± standard deviation. *Significant increase in NO levels relative to untreated cells, P < 0.05.
Mentions: NO levels were determined in untreated and JS-K-treated MDA-MB-231, F10, and MCF-7/COX-2 cells to confirm drug activation. The NO production was significantly increased (P < 0.05) in the three cell lines as a result of JS-K treatment (Figure 3).

Bottom Line: Under conditions by which JS-K was not cytotoxic, JS-K significantly decreased (P < 0.05) the invasiveness of breast cancer cells across the Matrigel basement membrane, which was directly correlated with NO production.JS-K decreased p38 activity, whereas the activity and the expression of extracellular signal-regulated kinase 1/2 and c-Jun N-terminal kinase were unaffected.Upregulation of TIMP-2 production is one mechanism by which JS-K mediates its anti-invasive effects.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.

ABSTRACT

Introduction: Tumor invasion and metastasis remain a major cause of mortality in breast cancer patients. High concentrations of nitric oxide (NO) suppress tumor invasion and metastasis in vivo. NO prodrugs generate large amounts of NO upon metabolism by appropriate intracellular enzymes, and therefore could have potential in the prevention and therapy of metastatic breast cancer.

Methods: The present study was designed to determine the effects of the NO-releasing prodrug O2-(2,4-dinitrophenyl) 1- [(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) on breast cancer invasion and the mechanisms involved. MDA-MB-231, MDA-MB-231/F10, and MCF-7/COX-2 were the three breast cancer cell lines tested. NO levels were determined spectrophotometrically using a NO assay kit. Invasion and the expression of matrix metalloproteinases (MMPs) and tissue inhibitor of MMPs were determined using Matrigel invasion assays, an MMP array kit and ELISAs. The activity and expression of extracellular signal-regulated kinase 1/2, p38, and c-Jun N-terminal kinase mitogen-activated protein kinases were determined using western blot analyses.

Results: Under conditions by which JS-K was not cytotoxic, JS-K significantly decreased (P < 0.05) the invasiveness of breast cancer cells across the Matrigel basement membrane, which was directly correlated with NO production. JS-43-126, a non-NO-releasing analog of JS-K, had no effect on NO levels or invasion. JS-K increased (P < 0.05) TIMP-2 production, and blocking TIMP-2 activity with a neutralizing antibody significantly increased (P < 0.05) the invasive activity of JS-K-treated cells across Matrigel. JS-K decreased p38 activity, whereas the activity and the expression of extracellular signal-regulated kinase 1/2 and c-Jun N-terminal kinase were unaffected.

Conclusion: We report the novel findings that JS-K inhibits breast cancer invasion across the Matrigel basement membrane, and NO production is vital for this activity. Upregulation of TIMP-2 production is one mechanism by which JS-K mediates its anti-invasive effects. JS-K and other NO prodrugs may represent an innovative biological approach in the prevention and treatment of metastatic breast cancer.

Show MeSH
Related in: MedlinePlus