Arylamine N-acetyltransferase 1 protects against reactive oxygen species during glucose starvation: Role in the regulation of p53 stability.

Lili Wang,Rodney F Minchin,Neville J Butcher,Fernando Rodrigues-Lima

doi:10.1371/journal.pone.0193560

Abstract

Human arylamine N-acetyltransferase 1 (NAT1) has been associated with cancer cell growth and invasion, but the underlying molecular mechanisms remain unknown. NAT1 is located on the short arm of chromosome 8 (8p21), a region that is commonly deleted in colon cancer. Previously, it was reported that HT-29 colon cancer cells, which have a large deletion at 8p21-22, show marked morphological changes, increased E-cadherin expression and altered cell-cell contact inhibition following down-regulation of NAT1 with shRNA. By contrast, no effects on growth were observed in HeLa cells. In the present study, cellular changes following knockout of NAT1 with CRISPR/Cas9 in HT-29 and HeLa cells were compared in the presence and absence of glucose. Cell growth decreased in both cell-lines during glucose starvation, but it was enhanced in HT-29 cells following NAT1 deletion. This was due to an increase in ROS production that induced cell apoptosis. Both ROS production and cell death were prevented by the glutathione precursor N-acetylcysteine. NAT1 knockout also resulted in a loss of the gain-of-function p53 protein in HT-29 cells. When p53 expression was inhibited with siRNA in parental HT-29 cells, ROS production and apoptosis increased to levels seen in the NAT1 knockout cells. The loss of p53 may explain the decreased colony formation and increased contact inhibition previously reported following NAT1 down-regulation in these cells. In conclusion, NAT1 is important in maintaining intracellular ROS, especially during glucose starvation, by stabilizing gain-of-function p53 in HT-29 cells. These results suggest that NAT1 may be a novel target to decrease intracellular gain-of -function p53.

Highlights

The arylamine N-acetyltransferases are a family of Phase II drug metabolizing enzymes that utilise acetyl coenzyme A to acetylate hydrazines, aromatic amines and heterocyclic amines [1]
The growth rate of HT-29 cells was significantly decreased following N-acetyltransferase 1 (NAT1) knockout and the effect was more pronounced in low glucose (Fig 1A)
NAT1 deletion further enhanced apoptosis in the HT-29 cells but had no effect in the HeLa cells. These results suggest that the differences in growth over time seen in Fig 1 are due to an increase in apoptosis and that NAT1 enhances apoptosis in a celldependent manner

Summary

Introduction

The arylamine N-acetyltransferases are a family of Phase II drug metabolizing enzymes that utilise acetyl coenzyme A to acetylate hydrazines, aromatic amines and heterocyclic amines [1]. Several recent studies suggest that NAT1 can protect cells during nutrient deprivation. This was seen when HT-29 cells were grown continuously for 6 days without a change in medium during which inhibition of NAT1 significantly reduced cell survival [3]. Over-expression of NAT1 in HB4a cells protected them from growth inhibition in low serum conditions [5]. Tumors from NAT1 knockdown HT-29 cells developed significantly more slowly than those from the parental cell-line [6] while NAT1 inhibition in MDA-MB-231 cells slowed growth in vitro and inhibited in vivo metastasis to the lungs [7]. Knockdown of NAT1 promotes a more epithelial phenotype with up-regulation of E-cadherin, cell-cell contact inhibition and loss of filopodia [3, 6, 7]

Methods

Results

Conclusion