Assessing N w-hydroxy-l-arginine applicability as a novel ethnic specific estrogen-negative breast cancer marker
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In our prior study we identified N w-hydroxy-l-arginine (NOHA) as a simple, yet sensitive indicator for estrogen negative (ER−) breast cancer early-prognosis, but not estrogen positive (ER+), and to offer ethnic selectivity for ER− detection. However, the ability of NOHA to assess ER− breast tumor based on disease progression, and tumor severity needs further delineation. Also, the overall NOHA storage stability needs to be validated. To assess the NOHA predictive capability based on disease progression, ER−/ER+ 3D-spheroids (from breast tumor cell lines of human origin) were cultured for 10 weeks. We found only ER− 3D-spheroid cultured for 10 weeks to show a gradual reduction in NOHA (both in culture medium and 3D-spheroid lysates) that correlated with a progressive increase in cellular NOS2 expression and NOS2 activity (measured as total nitrites). We additionally identified the NOHA-NOS2 correlation to be ethnically selective between ER− African American versus ER− Caucasian groups. Interestingly, such NOHA reduction was observed earlier in ER− culture medium (viz., after week 1) than from ER− 3D-spheroids lysates (viz., at the end of 3 weeks). When categorized based on 3D-spheroid grade, we found a ≥ 68% NOHA reduction in ER− spheroids that were ≤ 3 weeks old, that was categorized as “low-grade” (based on tumor size ≤ 250 µm, and with cellular characteristics identical to healthy cells). A substantial reduction in NOHA of ≥ 87% occurred with ER− 3D-spheroids grown for 6 weeks, which were categorized as “intermediate-grade” (with tumor size of ≥ 400 µm, and with less characteristic similarity to control spheroids). These in vitro findings thus suggest a distinct correlation between NOHA reduction and ER− tumor grade. Such distinctive correlation between NOHA and ER− tumor grade was additionally observed in de-identified clinical samples where a onefold higher reduction in NOHA occurred in grade-2 than with grade-1 de-identified patient plasma (when compared with control), and such correlation offered ethnic selectivity between ER− African American and ER− Caucasian groups. Of additional interest, when NOHA overall storage stability was assessed by incubating patient plasma and culture medium spiked with 75 pg/ml NOHA at multiple incubation temperatures and time-points, we found NOHA to maintain its stability for up to 6 weeks in culture medium and for 7 days in plasma at 4 °C and below. These results thus provide the first evidence of NOHA as a stable indicator to monitor ER− disease progression and tumor severity in ethnically distinctive populations.
KeywordsNw-hydroxy-l-arginine Estrogen-negative Breast cancer Inflammatory nitric oxide synthase
The University of New England Office of Research and Scholarship, and the College of Pharmacy provided funding support to this study.
Compliance with ethical standards
Conflict of interest
As authors of this manuscript, we declare no conflict of interest, either financial or otherwise.
Research involving human participants and/or animals
No human participants were directly involved with this project. All clinical samples used in this study came from bio-banks.
Only de-identified patient samples from bio-banks were used for this study, thus exempting informed consent requirement.
- Carey LA, Perou CM, Livasy CA, Dressler LG, Cowan D, Conway K, Karaca G, Troester MA, Tse CK, Edmiston S, Deming SL, Geradts J, Cheang MC, Nielsen TO, Moorman PG, Earp HS, Millikan RC (2006) Race, breast cancer subtypes, and survival in the Carolina Breast Cancer Study. JAMA 295(21):2492–2502. https://doi.org/10.1001/jama.295.21.2492 CrossRefPubMedGoogle Scholar
- Dawood S, Merajver SD, Viens P, Vermeulen PB, Swain SM, Buchholz TA, Dirix LY, Levine PH, Lucci A, Krishnamurthy S, Robertson FM, Woodward WA, Yang WT, Ueno NT, Cristofanilli M (2011) International expert panel on inflammatory breast cancer: consensus statement for standardized diagnosis and treatment. Ann Oncol 22(3):515–523. https://doi.org/10.1093/annonc/mdq345 CrossRefPubMedGoogle Scholar
- Dent R, Trudeau M, Pritchard KI, Hanna WM, Kahn HK, Sawka CA, Lickley LA, Rawlinson E, Sun P, Narod SA (2007) Triple-negative breast cancer: clinical features and patterns of recurrence. Clin Cancer Res 13(15 Pt 1):4429–4434. https://doi.org/10.1158/1078-0432.CCR-06-3045 CrossRefPubMedGoogle Scholar
- Ekert JE, Johnson K, Strake B, Pardinas J, Jarantow S, Perkinson R, Colter DC (2014) Three-dimensional lung tumor microenvironment modulates therapeutic compound responsiveness in vitro—implication for drug development. PLoS One 9(3):e92248. https://doi.org/10.1371/journal.pone.0092248 CrossRefPubMedPubMedCentralGoogle Scholar
- Food and Drug Administration (2001) Guidance for industry: bioanalytical method validation. US Department of Health and Human Services, Center for Drug Evaluation and Research, RockvilleGoogle Scholar
- Garlichs CD, Beyer J, Zhang H, Schmeisser A, Plotze K, Mugge A, Schellong S, Daniel WG (2000) Decreased plasma concentrations of l-hydroxy-arginine as a marker of reduced NO formation in patients with combined cardiovascular risk factors. J Lab Clin Med 135(5):419–425. https://doi.org/10.1067/mlc.2000.105975 CrossRefPubMedGoogle Scholar
- Glynn SA, Boersma BJ, Dorsey TH, Yi M, Yfantis HG, Ridnour LA, Martin DN, Switzer CH, Hudson RS, Wink DA, Lee DH, Stephens RM, Ambs S (2010) Increased NOS2 predicts poor survival in estrogen receptor-negative breast cancer patients. J Clin Invest 120(11):3843–3854. https://doi.org/10.1172/JCI42059 CrossRefPubMedPubMedCentralGoogle Scholar
- Haffty BG, Yang Q, Reiss M, Kearney T, Higgins SA, Weidhaas J, Harris L, Hait W, Toppmeyer D (2006) Locoregional relapse and distant metastasis in conservatively managed triple negative early-stage breast cancer. J Clin Oncol Off J Am Soc Clin Oncol 24(36):5652–5657. https://doi.org/10.1200/JCO.2006.06.5664 CrossRefGoogle Scholar
- Huang HL, Stasyk T, Morandell S, Dieplinger H, Falkensammer G, Griesmacher A, Mogg M, Schreiber M, Feuerstein I, Huck CW, Stecher G, Bonn GK, Huber LA (2006) Biomarker discovery in breast cancer serum using 2-D differential gel electrophoresis/MALDI-TOF/TOF and data validation by routine clinical assays. Electrophoresis 27(8):1641–1650. https://doi.org/10.1002/elps.200500857 CrossRefPubMedGoogle Scholar
- Martens-Lobenhoffer J, Bode-Boger SM (2006) Fast and efficient determination of arginine, symmetric dimethylarginine, and asymmetric dimethylarginine in biological fluids by hydrophilic-interaction liquid chromatography-electrospray tandem mass spectrometry. Clin Chem 52(3):488–493. https://doi.org/10.1373/clinchem.2005.060152 CrossRefPubMedGoogle Scholar
- Vinci M, Gowan S, Boxall F, Patterson L, Zimmermann M, Court W, Lomas C, Mendiola M, Hardisson D, Eccles SA (2012) Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation. BMC Biol 10:29. https://doi.org/10.1186/1741-7007-10-29 CrossRefPubMedPubMedCentralGoogle Scholar