Implications of Altered Glutathione Metabolism in Aspirin-Induced Oxidative Stress and Mitochondrial Dysfunction in HepG2 Cells

Haider Raza,Annie John,Ashraf B Abdel-Naim

doi:10.1371/journal.pone.0036325

Abstract

We have previously reported that acetylsalicylic acid (aspirin, ASA) induces cell cycle arrest, oxidative stress and mitochondrial dysfunction in HepG2 cells. In the present study, we have further elucidated that altered glutathione (GSH)-redox metabolism in HepG2 cells play a critical role in ASA-induced cytotoxicity. Using selected doses and time point for ASA toxicity, we have demonstrated that when GSH synthesis is inhibited in HepG2 cells by buthionine sulfoximine (BSO), prior to ASA treatment, cytotoxicity of the drug is augmented. On the other hand, when GSH-depleted cells were treated with N-acetyl cysteine (NAC), cytotoxicity/apoptosis caused by ASA was attenuated with a significant recovery in oxidative stress, GSH homeostasis, DNA fragmentation and some of the mitochondrial functions. NAC treatment, however, had no significant effects on the drug-induced inhibition of mitochondrial aconitase activity and ATP synthesis in GSH-depleted cells. Our results have confirmed that aspirin increases apoptosis by increased reactive oxygen species production, loss of mitochondrial membrane potential and inhibition of mitochondrial respiratory functions. These effects were further amplified when GSH-depleted cells were treated with ASA. We have also shown that some of the effects of aspirin might be associated with reduced GSH homeostasis, as treatment of cells with NAC attenuated the effects of BSO and aspirin. Our results strongly suggest that GSH dependent redox homeostasis in HepG2 cells is critical in preserving mitochondrial functions and preventing oxidative stress associated complications caused by aspirin treatment.

Highlights

Inflammation induced response have been implicated in the pathogenesis of numerous diseases including cancer
buthionine sulfoximine (BSO) treatment, which inhibits the rate limiting enzyme for GSH synthesis, resulted in almost 80% reduction in extra mitochondrial GSH concentration compared to about 50% reduction in the mitochondrial GSH pool suggesting that cytosolic GSH is more sensitive to BSO compared to mitochondrial GSH
Our results in the present study show that ASA-induced mitochondrial dysfunction and oxidative stress were further augmented in GSH-depleted cells, while N-acetyl cysteine (NAC) treatment attenuated the effects of aspirin in GSH-depleted cells

Summary

Introduction

Inflammation induced response have been implicated in the pathogenesis of numerous diseases including cancer. Non-steroidal anti-inflammatory drugs (NSAIDs), including aspirin (ASA) reduce inflammation by inhibiting the synthesis of prostaglandins (PGs) and induce apoptosis in a variety of cancer cells [5], [6], [7].Tumor cells are known to develop resistance towards therapeutic drugs and irradiation due to inhibition of apoptotic stimuli in these cells. There are multiple reports suggesting several additional mechanisms of action, independent of their ability to inhibit COX activity, that may contribute to its anticancer and anti-inflammatory effects [9],[10],[11].There is little information on the selectivity and specificity of NSAID-mediated effects and a better understanding of the molecular and biochemical mechanisms for aspirin and other NSAIDs is essential for therapeutic use of drugs in multiple disorders associated with inflammation. Recent epidemiological studies on humans and experimental models in diabetes, cancer and cardiovascular diseases have demonstrated that regular use of ASA alone or as an adjuvant may improve the outcome of disease prevention/protection in favor of benefit: risk ratio [11]

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Conclusion