Small molecule inhibition of arylamine N-acetyltransferase Type I inhibits proliferation and invasiveness of MDA-MB-231 breast cancer cells

doi:10.1016/j.bbrc.2010.01.087

Biochemical and Biophysical Research Communications

Volume 393, Issue 1, 26 February 2010, Pages 95-100

https://doi.org/10.1016/j.bbrc.2010.01.087 Get rights and content

Abstract

Arylamine N-acetyltransferase 1 is a phase II metabolizing enzyme that has been associated with certain breast cancer subtypes. While it has been linked to breast cancer risk because of its role in the metabolic activation and detoxification of carcinogens, recent studies have suggested it may be important in cell growth and survival. To address the possible importance of NAT1 in breast cancer, we have used a novel small molecule inhibitor (Rhod-o-hp) of the enzyme to examine growth and invasion of the breast adenocarcinoma line MDA-MB-231. The inhibitor significantly reduced cell growth by increasing the percent of cells in G2/M phase of the cell cycle. Rhod-o-hp also reduced the ability of the MDA-MB-231 cells to grow in soft agar. Using an in vitro invasion assay, the inhibitor significantly reduced the invasiveness of the cells. To test whether this effect was due to inhibition of NAT1, the enzyme was knocked down using a lentivirus-based shRNA approach and invasion potential was significantly reduced. Taken together, the results of this study demonstrate that NAT1 activity may be important in breast cancer growth and metastasis. The study suggests that NAT1 is a novel target for breast cancer treatment.

Introduction

The arylamine N-acetyltransferases (NATs) are a family of conjugating enzymes that were originally linked to cancer risk because of their ability to metabolically activate many arylamine and heterocyclic amine carcinogens [1]. In humans, the NATs are encoded by 2 closely related genes located on chromosome 8. Arylamine N-acetyltransferase II (NAT2) is primarily found in the liver and gastrointestinal tract while arylamine N-acetyltransferase I (NAT1) is more widely distributed [2]. Both the NAT1 and NAT2 genes are genetically polymorphic and many studies have suggested that they are low penetrance genes that affect cancer risk [3]. In breast cancer, NAT2 ‘rapid’ genotype has been linked with increased cancer risk, and the link is strongest in combination with smoking history [4], [5], [6], [7], [8].

In 2000, Perou and associates reported on the gene expression profiles for a number of different breast cancer subtypes [9]. They showed that NAT1 clustered with a number of genes that included the estrogen receptor (ER), GATA binding protein and X-box binding protein. Interestingly, NAT1 expression is 17-fold greater in vessels microdissected from breast tumors compared to vessels from normal tissue [10].

Despite several associations with cancer, NAT1 has not been shown to influence growth or survival other than in a non-transformed breast cell-line where elevated NAT1 appeared to provide a growth advantage [11]. Until recently, it was difficult to address the importance of NAT1 in cancer as there were no useful small molecule inhibitors available. A screen of a library of 5000 compounds against five different recombinant NATs from prokaryote and eukaryote sources identified several specific inhibitors for NAT1 that were based on the structure of rhodanine [12]. One of these, (Z)-5-(2′-hydroxybenzylidene)-2-thioxothiazolidin-4-one, demonstrated potent inhibition of human NAT1 (IC₅₀ = 1.1 μM). Docking studies suggested that this compound interacted with the hydrophobic pocket of the active site of the enzyme. The crystal structure of this compound (Rhod-o-hp) has suggested that it may interact with adjacent molecules forming several weak bonds such as phi bonds and hydrogen bonds [13].

In the present study, we have used the small molecule inhibitor Rhod-o-hp to decrease NAT1 activity in the breast carcinoma cell-line MDA-MB-231 in order to study what effect, if any, loss of NAT1 activity has on cell function. In particular, we examined the effect of NAT1 inhibition on MDA-MB-231 cell growth and invasion.

Section snippets

Materials and methods

Reagents. Rhod-o-hp was a kind gift from Prof. Jose Sordo, Universidade de Santiago de Compostela. The synthesis and purification of the compound has been reported previously [13]. All other chemicals were obtained from Sigma–Aldrich (Sigma–Aldrich, St. Louis, MO, USA) and were of the highest purity available.

Cell culture. Human breast cancer cell-line, MDA-MB-231, was obtained from Ms. Damara McAndrew, University of Queensland, Australia. Cells were cultured in DMEM (Invitrogen, Melbourne,

NAT1 activity is inhibited by Rhod-o-hp

Rhod-o-hp is a 5-benzylidenerhodanine derivative that contains a hydroxyl group in the ortho position of the benzene ring (Fig. 1A). Using recombinant NAT1 and PABA as substrate, Rhod-o-hp inhibited NAT1 enzyme activity in a concentration-dependent manner (Fig. 1B). The estimated IC₅₀ was 12.1 ± 2.0 μM, which is slightly higher than reported values [12]. This may be due to different assay conditions. At 100 μM, Rhod-o-hp inhibited NAT1 activity by more than 95%. To test whether Rhod-o-hp could

Discussion

Rhod-o-hp is a small molecule inhibitor of NAT1 first identified in a library screen of compounds against several recombinant NATs [12]. In solution, it inhibits NAT1 with an IC₅₀ in the low micromolar range. However, it is somewhat less effective in whole cells suggesting it is degraded or poorly taken up. Nevertheless, it inhibited NAT1 activity in a dose-dependent manner that correlated closely with its ability to inhibit cell proliferation and to inhibit growth in soft agar. Cell toxicity

Acknowledgement

This work was supported by the National Health and Medical Research Council, Australia (NHMRC 569695).

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Accumulating epidemiological evidence supports the mis-regulation of hNAT1 in cancers of the breast, prostate, skin, lung, brain, colon and renal system (for a detailed review see (Butcher & Minchin, 2012)). hNAT1 overexpression alone in breast cancer cells resulted in increased cell growth and conversely, hNAT1 inhibition decreased cell growth and invasiveness both in breast and colon cancer cells, potentially through a mechanism where hNAT1 stabilizes the gain-of-function form of p53 (Adam et al., 2003; Tiang, Butcher, Cullinane, Humbert, & Minchin, 2011; Tiang, Butcher, & Minchin, 2010; Tiang, Butcher, & Minchin, 2015; Wang, Minchin, & Butcher, 2018). These studies suggest that hNAT1 may play other carcinogenic roles apart from its classical role as a metabolizing enzyme for aromatic and heterocyclic amines (Fig. 3).
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Small molecule inhibition of arylamine N-acetyltransferase Type I inhibits proliferation and invasiveness of MDA-MB-231 breast cancer cells

Abstract

Introduction

Section snippets

Materials and methods

NAT1 activity is inhibited by Rhod-o-hp

Discussion

Acknowledgement

Mutat. Res.

Am. J. Pathol.

Bioorg. Med. Chem.

Anal. Biochem.

Lab. Invest.

Biochem. Biophys. Res. Commun.

Pharmacogenetics of the arylamine N-acetyltransferases

Pharmacogenomics J.

Molecular genetics and epidemiology of the NAT1 and NAT2 acetylation polymorphisms

Cancer Epidemiol. Biomarkers Prev.

Cigarette smoking, N-acetyltransferase 2 genetic polymorphisms, and breast cancer risk

Jama

N-acetyltransferase 1 genetic polymorphism, cigarette smoking, well-done meat intake, and breast cancer risk

Cancer Epidemiol. Biomarkers Prev.

Is the association between cigarette smoking and breast cancer modified by genotype? A review of epidemiologic studies and meta-analysis

Cancer Epidemiol. Biomarkers Prev.

A meta-analysis of the association of N-acetyltransferase 2 gene (NAT2) variants with breast cancer

Am. J. Epidemiol.

Cigarette smoking, N-acetyltransferase 2 genotypes, and breast cancer risk: pooled analysis and meta-analysis

Cancer Epidemiol. Biomarkers Prev.