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Amino Acids (2018) 50:373–382
https://doi.org/10.1007/s00726-017-2523-1
ORIGINAL ARTICLE
Assessing N
w
‑hydroxy‑
l
‑arginine applicability as a novel ethnic specic
estrogen‑negative breast cancer marker
Srinidi Mohan
1
· Ian Greenstein
1
· Cathy Ng
1
· Kelly Frazier
1
· Giang Nguyen
1
· Lisa Harding
1
· David Barlow
1
Received: 14 March 2017 / Accepted: 5 December 2017 / Published online: 19 December 2017
© Springer-Verlag GmbH Austria, part of Springer Nature 2017
Abstract
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.
Keywords N
w
-hydroxy-
l
-arginine · Estrogen-negative · Breast cancer · Inflammatory nitric oxide synthase
Introduction
As a heterogeneous disease, breast cancer has been recog-
nized as the most common form of cancer among women,
and second amongst cancer-related deaths in women in the
United States (Chavez et al. 2010; Zhou et al. 2013). 15–20%
of all breast cancer cases in the United States are diagnosed
to belong to an aggressive subtype of estrogen-receptor neg-
ative (ER
−
) breast cancer (Chavez et al. 2010; Garcia et al.
2007), which is predominantly caused due to mutation in the
human tumor suppressor gene (viz., BRCA-1 gene muta-
tion) (Haffty et al. 2006). Based on gene expression profile
Handling Editor: G. J. Peters.
* Srinidi Mohan
smohan@une.edu
1
Department of Pharmaceutical Sciences, College
of Pharmacy, University of New England, Portland,
Maine 04103, USA
@Phil_Robichaud
@deepthiw
@JoseServera