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A new pyridazinone exhibits potent cytotoxicity on human cancer cells via apoptosis and poly-ubiquitinated protein accumulation

  • Denisse A. Gutierrez
  • Rebecca E. DeJesus
  • Lisett Contreras
  • Isela A. Rodriguez-Palomares
  • Paulina J. Villanueva
  • Karol S. Balderrama
  • Lenore Monterroza
  • Manuel Larragoity
  • Armando Varela-Ramirez
  • Renato J. AguileraEmail author
Original Article

Abstract

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.

Keywords

Anti-cancer Apoptosis hmox-1 Proteasome inhibition Pyridazinone ROS 

Abbreviations

AL

amyloidosis: immunoglobulin light chain amyloidosis

CC25

Concentration that results in 25% cytotoxicity

CC50

Concentration that results in 50% cytotoxicity

Carboxy-H2DCFDA

6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate

CAD

Caspase-activated DNase

DAPI

4′,6-diamidino-2-phenylindole

DNS

Differential nuclear staining

DMSO

Dimethyl sulfoxide

FITC

Fluorescein isothiocyanate

GI50

Concentration that results in 50% of cell growth inhibition

H2O2

Hydrogen peroxide

Hmox-1

Heme oxygenase 1

IAP

Inhibitor of apoptosis proteins

IC50

concentration that results in 50% of cell growth inhibition

JC-1 reagent

5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcar-bocyanine iodide

LC50

Lethal concentration that results in 50% of cell death

MM

Multiple myeloma

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NIM

Nuclear isolation medium

Noxa

protein form of the PMAIP1 gene

PARAs

Pro-apoptotic receptor agonists

PARP-1

Poly [ADP-ribose] polymerase 1

PBS

Phosphate buffer saline

PEG

Polyethylene glycol

PI

Propidium Iodide

PMAIP1

Phorbol-12-myristate-13-acetate-induced protein 1

PS

Phosphatidylserine

Pyr-1

4,5-dichloro-2-[4-chloro-3-(trifluoromethyl)phenyl]-3(2H)-pyridazinone

ROS

Reactive oxygen species

SCI

Selective cytotoxicity index

SRB

Sulforhodamine B

Notes

Acknowledgments

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 information

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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Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Denisse A. Gutierrez
    • 1
  • Rebecca E. DeJesus
    • 1
  • Lisett Contreras
    • 1
  • Isela A. Rodriguez-Palomares
    • 1
  • Paulina J. Villanueva
    • 1
  • Karol S. Balderrama
    • 1
  • Lenore Monterroza
    • 1
  • Manuel Larragoity
    • 1
  • Armando Varela-Ramirez
    • 1
  • Renato J. Aguilera
    • 1
    Email author
  1. 1.The Cytometry, Screening, and Imaging Core Facility, Border Biomedical Research Center, Department of Biological SciencesThe University of Texas at El PasoEl PasoUSA

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