Distinct effects of etoposide on glutamine-addicted neuroblastoma

Cellular and Molecular Life Sciences https://doi.org/10.1007/s00018-019-03232-z Cellular and Molecular Life Sciences ORIGINAL ARTICLE Distinct effects of etoposide on glutamine‑addicted neuroblastoma Kadri Valter1 · Polina Maximchik2 · Alibek Abdrakhmanov2 · Viacheslav Senichkin2 · Boris Zhivotovsky1,2 · Vladimir Gogvadze1,2 Received: 27 October 2018 / Revised: 9 July 2019 / Accepted: 15 July 2019 © The Author(s) 2019 Abstract The majority of anticancer drugs are DNA-damaging agents, and whether or not they may directly target mitochondria remains unclear. In addition, tumors such as neuroblastoma exhibit addiction to glutamine in spite of it being a nonessential amino acid. Our aim was to evaluate the direct effect of widely used anticancer drugs on mitochondrial activity in combination with glutamine withdrawal, and possible apoptotic effects of such interaction. Our results revealed that etoposide inhibits mitochondrial respiratory chain Complex I causing the leakage of electrons and the superoxide radical formation. However, it was not sufficient to induce apoptosis, and apoptotic manifestation was detectable only alongside the withdrawal of glutamine, a precursor for antioxidant glutathione. Thus, the simultaneous depletion of glutathione and destabilization of mitochondria by ROS can compromise the barrier properties of the mitochondrial membrane, leading to cytochrome c release and the activation of the mitochondrial apoptotic pathway. Thus, the depletion of antioxidants or the inhibition of the pathways responsible for cellular antioxidant response can enhance mitochondrial targeting and strengthen antitumor therapy. Keywords Apoptosis · Neuroblastoma · Mitochondria · Oxidative stress · Respiratory chain Introduction Tumor development can be counteracted by the stimulation of apoptosis or other forms of programmed cell death [1, 2]. In the majority of tumor cells, apoptotic pathways are dormant owing to the low expression of pro-apoptotic factors, such as pro-apoptotic members of Bcl-2 family proteins [3, 4], or glycolytic shift, described by Otto Warburg, which stimulates glycolysis in tumor cells and silences mitochondria [5, 6]. Mitochondria are paramount in the stimulation/execution of apoptosis. Targeting mitochondria and the stimulation of mitochondrial apoptotic pathway has proven to be a Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00018-019-03232-z) contains supplementary material, which is available to authorized users. * Vladimir Gogvadze 1 Division of Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Box 210, 171 77 Stockholm, Sweden 2 Faculty of Medicine, MV Lomonosov Moscow State University, 119991 Moscow, Russia promising strategy in tumor cell elimination [7, 8]. Mitochondria can be targeted by compounds that affect mitochondrial activity and/or contribute to the outer mitochondrial membrane (OMM) permeabilization, which may lead to the activation of programmed cell death. Various conventionally used anticancer therapeutic drugs are DNA-damaging agents, but their ability to target mitochondria remains unclear. Targeting the mitochondrial electron transport chain can influence proton gradient formation and instigate the dissipation of the mitochondrial membrane potential (∆ψ). Furthermore, obstructions in respiratory chain electron flow can induce electron leakage and the formation of reactive oxygen species (ROS) [9–12]. These events can cause OMM permeabilization, leading to cytochrome c release and the activation of apoptosis. Although mitochondria are regarded as a source of ROS, they also possess a powerful detoxifying system responsible for ROS elimination [13]. Moreover, in some studies, mitochondria are regarded as “a sink for ROS generated in other cellular compartments”, because they can also rapidly detoxify ROS [14]. Thus, the ability of mitochondria to diminish ROS constitutes an important factor regulating apoptosis induction. 13 Vol.:(0123456789) K. Valter et al. In addition to glycolytic shift, some tumors display addiction to glutamine, even though this is a nonessential amino acid [15]. Attained glutamine is converted by glutaminase into glutamate, which is used as an important pre-substrate for the Krebs cycle, synthesis of glutathione, and amino and fatty acids to support intensive proliferation of cancer cells [16, 17]. One such glutamine-dependent tumor is neuroblastoma (NB), the most common solid cancer in childhood, which is often driven by oncogene MYCN amplification [18, 19]. MYCN is a transcription factor that belongs to the family of MYC oncoproteins. MYC and MYCN are involved in glutaminolysis. Thus, suppression of MYC in human glioma SF188 cells using shRNA caused the reduction in glutamine consumption and ammonia production [20]. Knockdown of MYCN inhibited glutaminolysis in NB cells, while overexpression of MYCN in neural crest progenitor cells enhanced glutaminolysis [21]. Therefore, targeting glutaminolysis might be beneficial for tumor cell elimination. Our aim was to evaluate the direct effect of widely used anticancer drugs on mitochondrial activity in combination with glutamine withdrawal, and the possible apoptotic effects of such interaction. Materials and methods Cell lines TET21N derived from SHEP cells possess an inducible expression system on the basis of the tetracycline repressor of E. coli to reversibly express MYCN in a human NB cell line [22]. TET21N cell line was cultured in 37 °C humidified air/CO2 (5%) atmosphere with RPMI 1640 (Sigma) complete medium including heat-inactivated fetal calf serum 10% (w/v), penicillin/streptomycin (100 U/ml), 100 µg/ml hygromycin B, and 200 µg/ml geneticin. Glutamine deprivation was achieved by changing the regular medium to glutamine-free RPMI 1640 (Sigma) 24 h prior to treatment. Switching off the oncogene MYCN was done by incubation of the cells with 0.1 μg/ml doxycycline 24 h before seeding the sample plates. HCT116 cell line was cultured in 37 °C humidified air/CO2 (5%) atmosphere with DMEM (Gibco®) complete medium including heat-inactivated fetal calf serum 10% (w/v) and penicillin/streptomycin (100 U/ml). Mitochondrial oxygen consumption 15,000 cells/well were seeded on 96-well plates (Seahorse Bioscience, Billerica, MA) and allowed to adhere overnight. The attached cells were washed with Seahorse assay medium and incubated in the same medium (0.175 ml) for 1 h at 37 °C. Respiration analysis began with the assessment of basal oxygen consumption rate (OCR), followed by the 13 injection of various drugs into the wells. The compounds of interest were injected from port A, and subsequent additions from ports B to D were oligomycin, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), and rotenone plus antimycin A. Injections from B to D were conducted to respectively assess phosphorylating respiration, spare respiratory capacity, and non-mitochondrial respiration. The data w (...truncated)