Suppression of autophagy enhances the cytotoxicity of the DNA-damaging aromatic amine p-anilinoaniline

Abstract

p-Anilinoaniline (pAA) is an aromatic amine that is widely used in hair dying applications. It is also a metabolite of metanil yellow, an azo dye that is commonly used as a food coloring agent. Concentrations of pAA between 10–25 μM were cytostatic to cultures of the normal human mammary epithelia cell line MCF10A. Concentrations ≥ 50 μM were cytotoxic. Cytostatic concentrations induced transient G1 and S cell cycle phase arrests; whereas cytotoxic concentrations induced protracted arrests. Cytotoxic concentrations of pAA caused DNA damage, as monitored by the alkaline single-cell gel electrophoresis (Comet) assay, and morphological changes consistent with cells undergoing apoptosis and/or autophagy. Enzymatic and western blot analyses, and binding analyses of fluorescent labeled VAD-FMK, suggested that caspase family members were activated by pAA. Western blot analyses documented the conversion of LC3-I to LC3-II, a post-translational modification involved in the development of the autophagosome. Suppression of autophagosome formation, via knockdown of ATG7 with shRNA, prevented pAA-induced vacuolization, enhanced the activation of pro-caspase-3, and increased susceptibility of ATG7-deficient cells to the cytostatic and cytotoxic activities of markedly lower concentrations of pAA. Cells stably transfected with a nonsense shRNA behaved like parental MCF10A cells. Collectively, these data suggest that MCF10A cultures undergo autophagy as a pro-survival response to concentrations of pAA sufficient to induce DNA damage.

Autophagy-Dependent Regulation of the DNA Damage Response Protein Ribonucleotide Reductase 1

“…It is possible that MMS-induced protein damage, aggregation, or a posttranslational modification provides the signal, but this has yet to be determined and is the focus of future work. Previous studies in human cell systems report that defects in autophagy promote genome instability, arising from increased metabolic stress that generates increased levels of endogenous damaging agents (56). In addition, mTOR inhibition and the induction of autophagy have been linked to p53 and Ataxia-telangiectasia mutated (ATM) signaling after DNA damage, with ATM activation also occurring by reactive oxygen species in the cytoplasm (57,58).…”

DNA damage and autophagy

“…Recent reports using the DNA-damaging agents camptothecin [108], etoposide and tomozolomide [109], p-Anilioaniline [110], and ionizing radiation (IR) [107] demonstrate that cells, in addition to initiate cell cycle arrest, also initiate autophagy. Autophagy delays apoptotic cell death induced by the DNA-damaging agent camptothecin and this is associated with mitophagy [108].…”

Autophagy Inhibition Enhances Apoptosis Induced by Ginsenoside Rk1 in Hepatocellular Carcinoma Cells

“…Recent investigations indicate that chemotherapeutic drugs, or any of several other anticancer stimuli, such as aromatic amine p-anilinoaniline, induce autophagy but not apoptosis in various cancer cells. 3) Furthermore, the cytotoxicity of many anticancer agents is mediated by autophagy activation, which is associated with G 1 arrest. 8,24,25) In order to determine whether Rk1 causes autophagy in HepG2 cells, we measured the incorporation of MDC, a marker for the acidic compartment of autolysosomes.…”