br Acknowledgements This work is
Acknowledgements This work is sponsored by the National Natural Science Foundation of China (grant no. 31201963) and Youth Backbone Teachers Project in Henan Province Department of Education, China (grant no. 2016GGJS-061).
Introduction Acute lymphoblastic leukemia (ALL) is a malignant disorder of the bone marrow characterized by excessive and rapid proliferation of lymphoblasts that originate from T or B cells. These PHA-848125 australia invade the blood and affect mostly children and adults younger than 20 years old (www.seer.cancer.gov). Generally, T-cell ALL in children has proven to be more sinister than B-cell ALL (Raetz and Teachey, 2016). Although cure rates for ALL have improved, 15%–20% of pediatric patients relapse (Desjonqueres et al., 2016). Unfortunately, the mortality rate among children who relapse is between 50% and 70%, despite aggressive treatment (Cooper and Brown, 2015). Therapeutic failure might be caused by either primary (intrinsic) resistance resulting from genetic alterations existing before treatment or acquired (secondary) resistance induced by drug treatment (Ebinger et al., 2016). Consequently, ALL cells evade apoptosis (Rebucci and Michiels, 2013). Apoptosis is a type of regulated cell death characterized by specific morphological and biochemical features, such as cell shrinkage, nuclear condensation/fragmentation, loss of plasma and mitochondria membrane potential (ΔΨm), and activation of a family of caspases (Kroemer et al., 2009; Galluzzi et al., 2012). Evasion of apoptosis is the chief hallmark of oncogenic cells (Hanahan and Weinberg, 2011). Therefore, discovering new agents that act against pediatric ALL has become a research priority. However, drug discovery is a complex process associated with high costs, a lengthy development process, high failure rates, and an interplay between academia and pharmaceutical companies (Chung, 2014). Therefore, drug repurposing, i.e., finding new therapeutic indications for already established drugs, offers an attractive alternative to treat ALL patients (McCabe et al., 2015). Minocycline (Mino, 7-dimethylamino-6-desoxytertracycline, PubMed CID: 54675783) is a second-generation, semi-synthetic tetracycline derivative that has well-established clinical pharmacokinetic and pharmacodynamic properties (Agwuh and MacGowan, 2006). Interestingly, this tetracycline not only has a proven safe clinical track record as an antibiotic but also has non-antibiotic properties (Garrido-Mesa et al., 2013). Effectively, Mino exerts both antioxidant and anti-apoptotic activity. However, there is compelling pre-clinical evidence that Mino induces apoptosis in an acute myeloid leukemia HL-60 cell line through mitochondria-mediated and caspase-dependent pathways (Song et al., 2014) and a chronic myeloid leukemia K562 cell line via DNA damage, caspase 3 activation, Bcl-xL deamination, lysosomal degradation and activation of the mitochondrial pathway of apoptosis (Fares et al., 2015). However, whether Mino induces apoptosis in Jurkat cells has not yet been determined. Mino presents several aldehydes (=keto) and hydroxyl (=enol) groups in the lower peripheral region of the DCBA naphthacene ring structure (Nelson and Levy, 2011). Therefore, Mino has the potential to engage in redox reactions resulting in the generation of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) (e.g., http://nzic.org.nz/ChemProcesses/production/). Since tumor cells might have a reduced capacity to remove H2O2 (Doskey et al., 2016), we theorize that Mino can induce apoptosis in leukemia cells through H2O2-mediated cell death signaling pathways (Jimenez-Del-Rio and Velez-Pardo, 2012). To test this hypothesis, we used the Jurkat cell line, a well-established ALL tumor line from the peripheral blood of a 14-year-old boy (Schneider et al., 1977), and examined the mechanism of action of Mino on this cell line. Moreover, to assess the selective effect of Mino against T-cell ALL cells, human peripheral blood lymphocyte cells (hPBLCs) were included in the assay. Several morphological and biochemical parameters of apoptosis were assessed such as cell viability, nuclear and ΔΨm integrity, DNA fragmentation and ROS generation. Activation of transcription factors (e.g., nuclear factor kappaB (NF-κB), P53, c-JUN) and expression of proteins involved in the dismantling of the nuclei (e.g., apoptosis-inducing factor (AIF), CASPASE-3), proteins involved in the maintenance of mitochondrial function (e.g., PTEN-induced putative kinase 1 (PINK1) and PARKIN), pro-apoptotic Bcl-2-associated X (BAX) and p53-up-regulated modulator of apoptosis (PUMA) proteins, and redox sensor proteins (e.g., DJ-1) were evaluated by Western blotting. Furthermore, the effects of the antioxidant N-acetyl-cysteine (NAC) and specific pharmacological signaling inhibitors in cells exposed to Mino were evaluated by flow cytometry. Taken together, our results suggest that Mino selectively induces apoptosis in Jurkat cells via an oxidative stress-mediated mechanism. The mechanism involves the generation of H2O2, which serves as a signaling molecule to trigger the oxidation of the sensor protein DJ-1, activation of transcription factors, up-regulation of pro-apoptotic proteins and proteins involved in the maintenance of mitochondrial function and mitochondrial damage, and activation of CASPASE-3/AIF, which are responsible for nuclear fragmentation. Therefore, Mino is proposed to be a promising drug for the treatment of ALL.