In the last 15 years, pyridazinone derivatives have acquired extensive attention due to their widespread biological activities and pharmacological applications. Pyridazinones are well known for their anti-microbial, anti-viral, anti-inflammatory, anti-cancer, and cardiovascular activities, among others. In this study, we evaluated the anti-cancer activity of a new pyridazinone derivative and propose it as a potential anti-neoplastic agent in acute promyelocytic leukemia cells. Pyr-1 cytotoxicity was assessed on several human cancer and two non-cancerous cell lines by the DNS assay. Pyr-1 demonstrated potent cytotoxicity against 22 human cancer cell lines, exhibiting the most favorable selective cytotoxicity on leukemia (CEM and HL-60), breast (MDA-MB-231 and MDA-MB-468), and lung (A-549) cancer cell lines, when compared with non-cancerous breast epithelial MCF-10A cells. Analyses of apoptosis/necrosis pathways, reactive oxygen species (ROS) production, mitochondria health, caspase-3 activation, and cell cycle profile were performed via flow cytometry. Both hmox-1 RNA and protein expression levels were evaluated by quantitative real-time PCR and Western blotting assays, respectively. Pyr-1 induced apoptosis in acute promyelocytic leukemia cells as confirmed by phosphatidylserine externalization, mitochondrial depolarization, caspase-3 activation, DNA fragmentation, and disrupted cell cycle progression. Additionally, it was determined that Pyr-1 generates oxidative and proteotoxic stress by provoking the accumulation of ROS, resulting in the overexpression of the stress-related hmox-1 mRNA transcripts and protein and a marked increase in poly-ubiquitinated proteins. Our data demonstrate that Pyr-1 induces cell death via the intrinsic apoptosis pathway by accumulating ROS and by impairing proteasome activity.
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amyloidosis: immunoglobulin light chain amyloidosis
- CC25 :
Concentration that results in 25% cytotoxicity
- CC50 :
Concentration that results in 50% cytotoxicity
Differential nuclear staining
- GI50 :
Concentration that results in 50% of cell growth inhibition
- H2O2 :
Heme oxygenase 1
Inhibitor of apoptosis proteins
- IC50 :
concentration that results in 50% of cell growth inhibition
- JC-1 reagent:
- LC50 :
Lethal concentration that results in 50% of cell death
Nuclear isolation medium
protein form of the PMAIP1 gene
Pro-apoptotic receptor agonists
Poly [ADP-ribose] polymerase 1
Phosphate buffer saline
Phorbol-12-myristate-13-acetate-induced protein 1
Reactive oxygen species
Selective cytotoxicity index
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The authors thank the personnel of the Genomic Analysis and the Cytometry, Screening and Imaging Core Facilities at the University of Texas at El Paso (UTEP), which are supported by a Research Centers in Minority Institutions (RCMI) program grant 5G12MD007592 to the Border Biomedical Research Center (BBRC) at UTEP from the National Institute on Minority Health and Health Disparities, a component of NIH. The authors thank Ms. Gladys Almodovar (with UTEP) for cell culture expertise assistance.
Funding for this work was provided by the National Institute of General Medical Sciences-Support of Competitive Research (SCORE) grant 1SC3GM103713 to RJA. LC, LM, ML, RDJ, and PV were supported by NIGMS RISE training grant R25 GM069621-15. KSB was supported by UTEP BUILDing Scholars grants RL5GM118969, TL4GM118971, and UL1GM118970.
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Gutierrez, D.A., DeJesus, R.E., Contreras, L. et al. A new pyridazinone exhibits potent cytotoxicity on human cancer cells via apoptosis and poly-ubiquitinated protein accumulation. Cell Biol Toxicol 35, 503–519 (2019). https://doi.org/10.1007/s10565-019-09466-8
- Proteasome inhibition