Ammonium chloride enhances cisplatin cytotoxicity through DNA double-strand breaks in human cervical cancer cells

Abstract

Abstract. Cisplatin (cis-diamminedichloroplatinum II, CDDP) acts as a therapeutic agent by initiating cellular apoptosis. However, side-effects and drug resistance limit the clinical use of cisplatin. Numerous studies have focused on the drug-target interactions, cellular pharmacology and pharmacokinetics of cisplatin. Newly developed treatment strategies are needed in order to be used in combination with cisplatin, with the aim to minimize toxicity and to circumvent cisplatin resistance. Ammonium chloride (NH 4 Cl) is widely used in various areas, but its use as a combination agent with cisplatin for the treatment of cancer cells has not been previously reported. In the present study, we showed that NH 4 Cl could be potentially used as an effective agent in cisplatin combination treatment of HeLa human cervical cancer (HCC) cells. Cisplatin was found to inhibit cell growth, as well as to induce cell apoptosis and DNA double-strand breaks. In addition, treatment with NH 4 Cl increased the rate of cell apoptosis and the activation of caspase-3. Particularly, we found that NH 4 Cl treatment increased cisplatin-induced phosphorylation of H 2 AX. In conclusion, our data indicate that NH 4 Cl enhances cisplatin cytotoxicity through increased DNA damage in HeLa HCC cells. IntroductionCisplatin (cis-diamminedichloroplatinum II, CDDP) is one of the most effective chemotherapeutic agents widely used for the treatment of solid tumors. However, the side-effects of cisplatin chemotherapy and resistance during the course of the treatment limit its clinical use (1-3). Cisplatin is generally considered as a cytotoxic drug which kills cancer cells by damaging DNA and inhibiting DNA synthesis. Cisplatin-induced DNA damage activates various signaling pathways to prevent or promote cell death predominantly by inducing apoptosis (4). Based on the mechanism of action of cisplatin-induced cell death, modifications to the combination methods used in chemotherapy are required to reduce the side-effects and increase the therapeutic effects of cisplatin. Autophagy inhibitors, such as 3-methyladenine and chloroquine, have been shown to effectively enhance cisplatin cytotoxity (5-8). Additional agents have been recently assessed such as bortezomib (PS-341, Velcade), a proteasome inhibitor (9,10), and histone acetyltransferase inhibitor, suberoylanilide hydroxamic acid (SAHA) (11,12). Some of these agents have been shown to confer sensitizing effects to cisplatin combination therapy.Ammonium chloride (NH 4 Cl) is an agent used for the treatment of metabolic alkalosis in clinical practice (13). NH 4 Cl was recently used as an autophagy inhibitor in in vitro studies, where it was found to affect the pH of lysosomes and to disturb the activity of autolysosomes (14-16). In the present study, we used NH 4 Cl combined with cisplatin to investigate the potentially effective use of NH 4 Cl as an agent in cisplatin combination chemotherapy.In the present study, we tested the hypothesis that the use of NH 4 Cl increases the apoptosis induced by cispl...

Molecular mechanisms of cisplatin resistance in cervical cancer

“…NH 4 Cl has been found to increase DNA damage and consequently enhance cisplatin cytotoxicity in HeLa cells. 77 Similarly, pretreatment with the autophagy inhibitor bafilomycin sensitizes both cervical adenocarcinoma HeLa cells and squamous cell carcinoma CaSki cells effectively to cisplatin. 78 …”

Ammonia-based enrichment and long-term propagation of zone I hepatocyte-like cells

“…This competition of ammonia with potassium ions lead to predictable intracellular and/or extracellular pH change and subsequent cell death. Neural toxicity of ammonia is associated with intracellular pH change through inhibition of potassium channel and depolarization of GABA neuron [17,18].…”

Ammonia-based enrichment and long-term propagation of zone I hepatocyte-like cells

“…This competition of ammonia with potassium ions lead to predictable intracellular and/or extracellular pH changes and subsequent cell death. Neural toxicity of ammonia is associated with intracellular pH change through inhibition of potassium channel and depolarization of GABA neurons 18 , 19 . Hepatocytes predictably have high metabolic activity towards ammonia and can evade its cytotoxic effects 12 .…”