Long-term adaptation of breast tumor cell lines to high concentrations of nitric oxide

Abstract

Nitric oxide (NO), a free radical, has been implicated in the biology of human cancers, including breast cancer, yet it is still unclear how NO affects tumor development and propagation. We herein gradually adapted four human breast adenocarcinoma cell lines (BT-20, Hs578T, T-47D, and MCF-7) to increasing concentrations of the NO donor DETA-NONOate up to 600 muM. The resulting model system consisted of a set of fully adapted high nitric oxide ("HNO") cell lines that are biologically different from the "parent" cell lines from which they originated. Although each of the four parent and HNO cell lines had identical morphologic appearance, the HNO cells grew faster than their corresponding parent cells and were resistant to both nitrogen- and oxygen-based free radicals. These cell lines serve as a novel tool to study the role of NO in breast cancer progression and potentially can be used to predict the therapeutic response leading to more efficient therapeutic regimens.

Part I. Molecular and cellular characterization of high nitric oxide-adapted human breast adenocarcinoma cell lines

“…As indicated in our prior works, a number of tumor cell lines have been adapted to HNO concentration levels including those from lung [11,[46][47][48], head and neck [3,[49][50][51][52], and breast [7], suggesting that NO adaptation is also a universal property. In an attempt to further elucidate the impact of extended exposure from NO on underlying biological systems, we herein characterize the four newly The ten most up-regulated appear first and the ten most down-regulated appear below within each cell line type NA not available a Gene symbols used vary according to the supplier of the gene chip created breast adenocarcinoma HNO cell lines [7], along with each respective parent cell line.…”

“…However, a dichotomy exists between enhanced radiosensitization and conditions where elevated levels of NO and up-regulated NOS are contributors to tumor progression. The relationship between NOS expression and tumor growth is evident in the literature from studies with a variety of cancer types including: (1) human oral squamous cell carcinoma [10,40], (2) gastric carcinoma [41], (3) lymph node metastases, and most significantly for this current work, (4) breast cancer, to a point that it is a universal property for human solid tumors [7,[15][16][17][42][43][44][45].…”

“…These four cell lines have previously been reported on by our group as to how they were adapted to grow in comparatively high levels of NO [7]. To summarize, the cell adaptation process was conducted over a period of time by exposing parent cell lines to an NO donor, (Z)-1-[2-(2-aminoethyl)-N-(2-amminoethyl) amino] diazen-1-ium-1,2-diolate (DETA-NONOate) (Sigma Life Sciences, St. Louis, MO, USA), at an initial 50 μM concentration.…”

“…At lower concentrations, it is beneficial being antiinflammatory and having a tumor suppressive function. At high levels, however, it can become mutagenic and lead to tumor formation [6,7]. NO, when produced by nitric oxide synthesis (NOS) in low concentrations, can be a highly effective hypoxic radiosensitizer; it has been shown to inhibit damage to DNA caused by radiation [8].…”

“…To further clarify NO behavior patterns we created a model cell line system within our laboratory using a variety of breast cancer cell lines that were adapted to NO. These cells were gradually adapted to high concentrations of NO over a prolonged period of months [7]. Two significant results were observed: (1) the original "parent" cell lines adapted to the high nitric oxide (HNO), and (2) the resultant HNO adapted cell lines grew faster and more aggressively than did each respective "parent" cell line.…”