Pilot study demonstrating metabolic and anti-proliferative effects of in vivo anti-oxidant supplementation with N-Acetylcysteine in Breast Cancer

Abstract

Background

High oxidative stress as defined by hydroxyl and peroxyl activity is often found in the stroma of human breast cancers. Oxidative stress induces stromal catabolism, which promotes cancer aggressiveness. Stromal cells exposed to oxidative stress release catabolites such as lactate, which are up-taken by cancer cells to support mitochondrial oxidative phosphorylation. The transfer of catabolites between stromal and cancer cells leads to metabolic heterogeneity between these cells and increased cancer cell proliferation and reduced apoptosis in preclinical models. N-Acetylcysteine (NAC) is an antioxidant that reduces oxidative stress and reverses stromal catabolism and stromal-carcinoma cell metabolic heterogeneity, resulting in reduced proliferation and increased apoptosis of cancer cells in experimental models of breast cancer. The purpose of this clinical trial was to determine if NAC could reduce markers of stromal-cancer metabolic heterogeneity and markers of cancer cell aggressiveness in human breast cancer.

Methods

Subjects with newly diagnosed stage 0 and I breast cancer who were not going to receive neoadjuvant therapy prior to surgical resection were treated with NAC before definitive surgery to assess intra-tumoral metabolic markers. NAC was administered once a week intravenously at a dose of 150 mg/kg and 600 mg twice daily orally on the days not receiving intravenous NAC. Histochemistry for the stromal metabolic markers monocarboxylate transporter 4 (MCT4) and caveolin-1 (CAV1) and the Ki67 proliferation assay and TUNEL apoptosis assay in carcinoma cells were performed in pre- and post-NAC specimens.

Results

The range of days on NAC was 14–27 and the mean was 19 days. Post-treatment biopsies showed significant decrease in stromal MCT4 and reduced Ki67 in carcinoma cells. NAC did not significantly change stromal CAV1 and carcinoma TUNEL staining. NAC was well tolerated.

Conclusions

NAC as a single agent reduces MCT4 stromal expression, which is a marker of glycolysis in breast cancer with reduced carcinoma cell proliferation. This study suggests that modulating metabolism in the tumor microenvironment has the potential to impact breast cancer proliferation.

Introduction

Breast cancer is the most common type of cancer in women [1] and is the fourth leading cause of cancer death in women in the United States [1]. Moreover, current breast treatment strategies have significant side effects. There is a need for novel treatments in breast cancer, especially those with low morbidity. Epidemiological studies suggest that antioxidants such as N-Acetylcysteine (NAC) may reduce breast cancer mortality by reducing progression [2]. Antioxidants, which are well tolerated, offer the potential to fill this need.

NAC is one of the best-characterized antioxidants and it is approved by the US Food and Drug Administration (FDA) for intravenous or oral treatment of acetaminophen overdose and is also FDA-approved by inhalation as a mucolytic agent [3]. Because of its antioxidant properties, NAC is also commonly administered prophylactically for contrast-induced nephropathy and is being investigated in many diseases, including chemotherapy-induced toxicity such as doxorubicin cardiotoxicity, ischemia–reperfusion cardiac injury, acute respiratory distress syndrome, bronchitis, heavy metal toxicity, neurologic and psychiatric disorders, interstitial lung diseases, hepatitis, influenza, and HIV [4].

NAC is a precursor for reduced glutathione (GSH), which is the main intracellular antioxidant [5]. GSH is a water-soluble molecule found in millimolar concentration in cells. It is a low-molecular-weight peptide containing a thiol group, which provides its antioxidant activity [5]. The physiologic roles of GSH include being a scavenger of free radical and reactive oxygen species, forming conjugates with metabolites and xenobiotics, being a thiol buffer for many cellular proteins such as metallothioneins and thioredoxins, altering protein structures by reducing disulfide bonds, being an essential cofactor for many enzymes, and allowing the regeneration of other antioxidants such as tocopherols and ascorbate [4], [6]. GSH is composed of three amino acids: cysteine, glycine, and glutamate [7]. Cysteine is the rate-limiting amino acid in the generation of GSH, and NAC has been developed as a drug instead of cysteine because of its greater stability [4]. NAC was investigated in the current breast cancer clinical trial because of its ability to reduce oxidative stress by increasing GSH levels.

Dysfunctional GSH homeostasis causes a reduced antioxidant response, activation of redox-regulated signal transduction with increased glycolysis, immune impairment, reduced ability to detoxify electrophilic xenobiotics, and increased cellular proliferation [8]. GSH is implicated in the etiology of several diseases, including cancer, aging, neurodegenerative diseases, pulmonary diseases, liver diseases, immune disorders, and cardiovascular diseases [6]. GSH is rapidly degraded extracellularly by γ-glutamyl transpeptidase (GGT) [4]. NAC, orally and intravenously in human subjects, enhances GSH intracellular production [9]. NAC is also an antioxidant directly without needing to be metabolized to GSH [4]. Hence, most studies investigating targeting dysfunctional GSH and redox homeostasis have used NAC instead of GSH itself [4]. No clinical trials have been conducted with NAC in breast cancer; NAC was investigated in the current trial because redox and GSH homeostasis is altered in this disease.

Oxidative stress induces a glycolytic and catabolic state in tumor stromal cells with the release of catabolites such as lactate [10]. These catabolites drive metabolic heterogeneity with transfer of catabolites from stromal cells to carcinoma cells to support mitochondrial metabolism [11], [12]. Metabolic heterogeneity increases cancer cell proliferation, reduces apoptosis, and induces larger tumors with more frequent metastasis and shorter overall survival [11], [12], [13], [14], [15], [16], [17], [18].

Metabolic heterogeneity exists in breast cancer [10]. High stromal monocarboxylate transporter 4 (MCT4) and low caveolin-1 (CAV1) expression are markers of glycolytic stromal cells in metabolically heterogeneous tumors [10], [12], [19], [20], [21]. NAC preferentially targets cells with altered glycolysis and, hence, stromal cells with high MCT4 and low CAV1 would presumably be more susceptible to NAC.

High stromal staining for MCT4 and low CAV1 occurs in the majority of breast cancers, suggesting transport of catabolites from cancer-associated stroma to highly proliferative cancer cells [22]. MCT4 is an exporter of glycolytic byproducts such as pyruvate and lactate [19]. Oxidative stress induces the expression of MCT4 in stromal cells and NAC can reduce MCT4 expression in preclinical models [10]. Also, loss of CAV1 in cancer-associated stroma induces glycolysis and the upregulation of MCT4 and stromal CAV1 expression can be rescued with NAC [18]. The purpose of this clinical trial was to determine if NAC could reduce markers of stromal-cancer metabolic heterogeneity and markers of cancer cell proliferation and apoptosis in human breast cancer.

NAC, which reduces oxidative stress, has been extensively studied as an anticancer agent in vitro and in vivo and has been shown to reduce cancer aggressiveness with reduced proliferation and increased apoptosis of cancer cells [10], [11], [23], [24], [25], [26], [27]. NAC’s ability to limit tumor growth in some in vivo models is dependent on its antioxidant properties [28]. NAC also reduces catabolism, glycolysis, mitochondrial dysfunction, and inflammatory mediators by reducing oxidative stress [5], [7], [24], [25]. However, NAC has not been investigated systematically in breast cancer. Also, no clinical trials have been performed to assess the effect of drugs on markers of the metabolic profile of human tumors as a primary end-point.

In sum, oxidative stress drives metabolic heterogeneity between tumor stromal cells and cancer cells and metabolic heterogeneity induces aggressive behavior in cancer. NAC preferentially targets tumors with increased stromal glycolysis, such as breast cancer [29]. Because of its antioxidant effect, NAC can reverse stromal-cancer metabolic heterogeneity, which drives cancer aggressiveness [10]. Hence NAC may be a drug with anticancer activity in human breast cancer. We hypothesized that, because of the metabolic effects of NAC in the tumor microenvironment, it can reduce cancer cell proliferation and increase apoptosis rates in subjects with breast cancer.