The Antioxidant N-Acetylcysteine Prevents HIF-1 Stabilization under Hypoxia In Vitro but Does Not Affect Tumorigenesis in Multiple Breast Cancer Models In Vivo

Jaclyn Sceneay,Christina S F Wong,Anna Chen,Andreas Möller,Mira C P Liu,David D L Bowtell,Victoria Lawson

doi:10.1371/journal.pone.0066388

Abstract

Intratumoral hypoxia is a poor prognostic factor associated with reduced disease-free survival in many cancer types, including breast cancer. Hypoxia encourages tumor cell proliferation, stimulates angiogenesis and lymphangiogenesis, and promotes epithelial-mesenchymal transition and metastasis. Tumor cells respond to a hypoxic state by stabilizing the Hif-1α subunit of the Hypoxia-Inducible Factor (HIF) transcription factor to promote expression of various tumor- and metastasis-promoting hypoxic response genes. The antioxidant N-acetylcysteine (NAC) was recently shown to prevent Hif-1α stabilization under hypoxia, and has been identified as a potential alternative method to target the hypoxic response in tumors. We utilized three orthotopic syngeneic murine models of breast cancer, the PyMT, EO771 and 4T1.2 models, to investigate the ability of NAC to modulate the hypoxic response in vitro and in vivo. While NAC prevented Hif-1α stabilization under hypoxia in vitro and increased levels of glutathione in the blood of mice in vivo, this did not translate to a difference in tumor growth or the hypoxic state of the tumor compared to untreated control mice. In addition, NAC treatment actually increased metastatic burden in an experimental metastasis model. This work raises questions regarding the validity of NAC as an anti-tumorigenic agent in breast cancer, and highlights the need to further investigate its properties in vivo in different cancer models.

Highlights

Hypoxic conditions in tumors correlate with a poor prognosis, reduced disease-free survival and overall survival in cancer patients [1,2]
In vitro Hif-1a Staining For cellular Hif-1a staining, PyMT and EO771 tumor cells were plated on glass slides and stained with anti-Hif-1a antibody (Novus Biologicals), co-stained with diaminobenzidine-stained (DAB) and images taken on a BX-51 microscope (Olympus)
NAC Prevents Hif-1a Stabilization in vitro To examine the effect of NAC on the hypoxic response in vitro, PyMT and EO771 breast tumor cell lines were exposed to normoxic (20% O2) or hypoxic (2% O2) conditions

Summary

Introduction

Hypoxic conditions in tumors correlate with a poor prognosis, reduced disease-free survival and overall survival in cancer patients [1,2]. Hypoxia refers to conditions in solid tumors where the oxygen pressure is less than 5–10 mm Hg, which initially occurs when the vasculature of the local environment is unable to meet the needs of rapidly proliferating tumor cells, and persists due to aberrant tumor vasculature [2,3,4,5]. The cellular response to hypoxia is largely mediated by formation of the hypoxia-inducible factor (HIF) transcription factor, a basic helix-loop-helix heterodimeric protein consisting of the oxygen-dependent a and constitutively expressed b subunits [9,10,11,12,13,14]. Subsequent polyubiquitination by pVHL marks Hif-1a for degradation by the 26S proteasome [19,20,21,22,23,24,25]

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Results

Conclusion