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

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.

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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

References

  1. Akhtar W, Shaquiquzzaman M, Akhter M, Verma G, Khan MF, Alam MM. The therapeutic journey of pyridazinone. Eur J Med Chem. 2016;123:256–81.

    CAS  PubMed  Google Scholar 

  2. Al-Lazikani B, Banerji U, Workman P. Combinatorial drug therapy for cancer in the post-genomic era. Nat Biotechnol. 2012;30:679–92.

    CAS  PubMed  Google Scholar 

  3. Almond JB, Cohen GM. The proteasome: a novel target for cancer chemotherapy. Leukemia. 2002;16:433–43.

    CAS  PubMed  Google Scholar 

  4. American Cancer Society. 2017. Cancer Facts & Figures 2017.

  5. Asif M. A mini review on biological activities of pyridazinone derivatives as antiulcer, antisecretory, antihistamine and particularly against histamine H3R. Mini-Rev Med Chem. 2015;14:1093–103.

    PubMed  Google Scholar 

  6. Bates DJ, Lewis LD. Manipulating the apoptotic pathway: potential therapeutics for cancer patients. Br J Clin Pharmacol. 2006;76:381–95.

    Google Scholar 

  7. Brnjic S, Mazurkiewicz M, Fryknas M, Sun C, Zhang X, Larsson R, et al. Induction of tumor cell apoptosis by a proteasome deubiquitinase inhibitor is associated with oxidative stress. Antioxid Redox Signal. 2014;21:2271–85.

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Chen L, Brewer MD, Guo L, Wang R, Jiang P, Yang X. Enhanced degradation of misfolded proteins promotes tumorigenesis. Cell Rep. 2017;18:3143–54.

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Contreras L, Calderon RI, Varela-Ramirez A, Zhang HY, Quan Y, Das U, et al. Induction of apoptosis via proteasome inhibition in leukemia/lymphoma cells by two potent piperidones. Cell Oncol. 2018;41(6):623–36.

    CAS  Google Scholar 

  10. Costas T, Costas-Lago MC, Vila N, Besada P, Cano E, Teran C. New platelet aggregation inhibitors based on pyridazinone moiety. Eur J Med Chem. 2015;94:113–22.

    CAS  PubMed  Google Scholar 

  11. D’Arcy P, Brnjic S, Olofsson MH, Fryknas M, Lindsten K, De Cesare M, et al. Inhibition of proteasome deubiquitinating activity as a new cancer therapy. Nat Med. 2011;17:1636–40.

    PubMed  Google Scholar 

  12. Dorsch D, Schadt O, Stieber F, Meyring M, Gradler U, Bladt F, et al. Identification and optimization of pyridazinones as potent and selective c-met kinase inhibitors. Bioorg Med Chem Lett. 2015;25:1597–602.

    CAS  PubMed  Google Scholar 

  13. Dou QP, Zonder JA. Overview of proteasome inhibitor-based anti-cancer therapies: perspective on Bortezomib and second generation proteasome inhibitors versus future generation inhibitors of ubiquitin proteasome system. Curr Cancer Drug Targets. 2014;14:517–36.

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Elagawany M, Ibrahim MA, Ali Ahmed HE, El-Etrawy AS, Ghiaty A, Abdel-Samii ZK, et al. Design, synthesis, and molecular modelling of pyridazinone and phthalazinone derivatives as protein kinases inhibitors. Bioorg Med Chem Lett. 2013;23:2007–13.

    CAS  PubMed  Google Scholar 

  15. Elmore S. Apoptosis: a review of programmed cell death. Toxicol Pathol. 2007;35:495–516.

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Eruslanov E, Kusmartsev S. Identification of ROS using oxidized DCFDA and flow-cytometry. Methods Mol Biol. 2010;594:57–72.

    CAS  PubMed  Google Scholar 

  17. Faidallah HM, Rostom SAF, Basaif SA, Makki MST, Khan KA. Synthesis and biological evaluation of some novel urea and thiourea derivatives of isoxazolo[4,5-d]pyridazine and structurally related thiazolo[4,5-d]pyridazine as anti-microbial agents. Arch Pharm Res. 2013;36:1354–68.

    CAS  PubMed  Google Scholar 

  18. Fribley A, Zeng Q, Wang CY. Proteasome inhibitor PS-341 induces apoptosis through induction of endoplasmic reticulum stress-reactive oxygen species in head and neck squamous cell carcinoma cells. Mol Cell Biol. 2004;24:9695–704.

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Fulda S, Debatin KM. Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene. 2006;25:4798–811.

    CAS  PubMed  Google Scholar 

  20. Gong J, Zheng Y, Wang Y, Sheng W, Li Y, Liu X, et al. A new compound of thiophenylated pyridazinone IMB5043 showing potent antitumor efficacy through ATM-Chk2 pathway. PLoS One. 2018;13:e0191984.

    PubMed  PubMed Central  Google Scholar 

  21. Guo N, Peng Z. MG132, a proteasome inhibitor, induces apoptosis in tumor cells. Asia Pac J Clin Oncol. 2013;9:6–11.

    PubMed  Google Scholar 

  22. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. 2000;100:57–70.

    CAS  PubMed  Google Scholar 

  23. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144:646–74.

    CAS  PubMed  Google Scholar 

  24. Huang Z, Pinto JT, Deng H, Richie JP Jr. Inhibition of caspase-3 activity and activation by protein glutathionylation. Biochem Pharmacol. 2008;75:2234–44.

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Invitrogen. Reactive oxygen species (ROS) detection reagents. In: Eugene, OR. Invitrogen: Molecular Probes; 2006.

    Google Scholar 

  26. Jacomini AP, Silva MJV, Silva RGM, Goncalves DS, Volpato H, Basso EA, et al. Synthesis and evaluation against Leishmania amazonensis of novel pyrazolo[3,4-d]pyridazinone-N-acylhydrazone-(bi)thiophene hybrids. Eur J Med Chem. 2016;124:340–9.

    CAS  PubMed  Google Scholar 

  27. Jin X, Wang Y, Tan L, He Y, Peng J, Hai L, et al. An efficient injectable formulation with block copolymer micelles for hydrophobic antitumor candidate-pyridazinone derivatives. Nanomedicine (London). 2015;10:2153–65.

    CAS  Google Scholar 

  28. King KL, Cidlowski JA. Cell cycle regulation and apoptosis. Annu Rev Physiol. 1998;60:601–14.

    CAS  PubMed  Google Scholar 

  29. Lee YJ, Shacter E. Hydrogen peroxide inhibits activation, not activity, of cellular caspase-3 in vivo. Free Radic Biol Med. 2000;29:684–92.

    CAS  PubMed  Google Scholar 

  30. Lema C, Varela-Ramirez A, Aguilera RJ. Differential nuclear staining assay for high-throughput screening to identify cytotoxic compounds. Curr Cell Biochem. 2011;1:1–14.

    PubMed  PubMed Central  Google Scholar 

  31. Leonard JP, Furman RR, Coleman M. Proteasome inhibition with bortezomib: a new therapeutic strategy for non-Hodgkin’s lymphoma. Int J Cancer. 2006;119:971–9.

    CAS  PubMed  Google Scholar 

  32. Leventis PA, Grinstein S. The distribution and function of phosphatidylserine in cellular membranes. Annu Rev Biophys. 2010;39:407–27.

    CAS  PubMed  Google Scholar 

  33. Li B, Dou QP. Bax degradation by the ubiquitin/proteasome-dependent pathway: involvement in tumor survival and progression. Proc Natl Acad Sci. 2000;97:3850–5.

    CAS  PubMed  Google Scholar 

  34. Li K, Schurig-Briccio LA, Feng X, Upadhyay A, Pujari V, Lechartier B, et al. Multitarget drug discovery for tuberculosis and other infectious diseases. J Med Chem. 2014;57:3126–39.

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Ling HE, Liebes L, Zou Y, Perez-Soler R. Reactive oxygen species generation and mitochondrial dysfunction in the apoptotic response to Bortezomib, a novel proteasome inhibitor, in human H460 non-small cell lung Cancer cells. J Biol Chem. 2003a;278:33714–23.

    CAS  PubMed  Google Scholar 

  36. Ling YH, Liebes L, Zou Y, Perez-Soler R. Reactive oxygen species generation and mitochondrial dysfunction in the apoptotic response to Bortezomib, a novel proteasome inhibitor, in human H460 non-small cell lung cancer cells. J Biol Chem. 2003b;278:33714–23.

    CAS  PubMed  Google Scholar 

  37. Liu Y, Jin S, Peng X, Lu D, Zeng L, Sun Y, et al. Pyridazinone derivatives displaying highly potent and selective inhibitory activities against c-met tyrosine kinase. Eur J Med Chem. 2016;108:322–33.

    CAS  PubMed  Google Scholar 

  38. Loda M, Cukor B, Tam SW, Lavin P, Fiorentino M, Draetta GF, et al. Increased proteasome-dependent degradation of the cyclin-dependent kinase inhibitor p27 in aggressive colorectal carcinomas. Nat Med. 1997;3:231–4.

    CAS  PubMed  Google Scholar 

  39. Matsura T, Serinkan BF, Jiang J, Kagan VE. Phosphatidylserine peroxidation/externalization during staurosporine-induced apoptosis in HL-60 cells. FEBS Lett. 2002;524:25–30.

    CAS  PubMed  Google Scholar 

  40. Mogilski S, Kubacka M, Redzicka A, Kazek G, Dudek M, Malinka W, et al. Antinociceptive, anti-inflammatory and smooth muscle relaxant activities of the pyrrolo[3,4-d]pyridazinone derivatives: possible mechanisms of action. Pharmacol Biochem Behav. 2015;133:99–110.

    CAS  PubMed  Google Scholar 

  41. Munoz R, Man S, Shaked Y, Lee CR, Wong J, Francia G, et al. Highly efficacious nontoxic preclinical treatment for advanced metastatic breast cancer using combination oral UFTCyclophosphamide metronomic chemotherapy. Cancer Res. 2006;66:3386–91.

    CAS  PubMed  Google Scholar 

  42. Nastiuk KL, Krolewski JJ. Opportunities and challenges in combination gene cancer therapy. Adv Drug Deliv Rev. 2016;98:35–40.

    CAS  PubMed  Google Scholar 

  43. Nath KA, Balla G, Vercellotti G. Induction of heme oxygenase is a rapid, protective response in rhabdomyolysis in the rat. J Clin Invest. 1992;90:267–70.

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Naujokat C, Hoffmann S. Role and function of the 26S proteasome in proliferation and apoptosis. Lab Investig. 2002;82:965–80.

    CAS  PubMed  Google Scholar 

  45. Ovais S, Javed K, Yaseen S, Bashir R, Rathore P, Yaseen R, et al. Synthesis, antiproliferative and anti-inflammatory activities of some novel 6-aryl-2-(p-(methanesulfonyl)phenyl)-4,5-dihydropyridazi3(2H)-ones. Eur J Med Chem. 2013;67:352–8.

    CAS  PubMed  Google Scholar 

  46. Perez-Galan P, Roue G, Villamor N, Montserrat E, Campo E, Colomer D. The proteasome inhibitor bortezomib induces apoptosis in mantle-cell lymphoma through generation of ROS and Noxa activation independent of p53 status. Blood. 2006;107:257–64.

    CAS  PubMed  Google Scholar 

  47. Qin JZ, Ziffra J, Stennett L, Bodner B, Bonish BK, Chaturvedi V, et al. Proteasome inhibitors trigger NOXA-mediated apoptosis in melanoma and myeloma cells. Cancer Res. 2005;65:6282–93.

    CAS  PubMed  Google Scholar 

  48. Ramaswami R, Harding V, Newsom-Davis T. Novel cancer therapies: treatments driven by tumour biology. Postgrad Med J. 2013;89:652–8.

    CAS  PubMed  Google Scholar 

  49. Rathish IG, Javed K, Bano S, Ahmad S, Alam MS, Pillai KK. Synthesis and blood glucose lowering effect of novel pyridazinone substituted benzenesulfonylurea derivatives. Eur J Med Chem. 2009;44:2673–8.

    CAS  PubMed  Google Scholar 

  50. Reece DE, Hegenbart U, Sanchorawala V, Merlini G, Palladini G, Bladé J, et al. Efficacy and safety of once-weekly and twice-weekly bortezomib in patients with relapsed systemic AL amyloidosis: results of a phase 1/2 study. Blood. 2011;118:865–73.

    CAS  PubMed  Google Scholar 

  51. Robles-Escajeda E, Das U, Ortega NM, Parra K, Francia G, Dimmock JR, et al. A novel curcumin-like dienone induces apoptosis in triple-negative breast cancer cells. Cell Oncol. 2016;39:265–77.

    CAS  Google Scholar 

  52. Robles-Escajeda E, Lerma C, Nyakeriga AM, Ross JA, Kirken RA, Aguilera RJ, et al. Searching in Mother Nature for Anti-Cancer Activity: AntiProliferative and Pro-Apoptotic Effect Elicited by Green Barley on Leukemia/Lymphoma Cells. PLoS One. 2013;8:e73508.

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Rossig L, Fichtlscherer B, Breitschopf K, Haendeler J, Zeiher AM, Mulsch A, et al. Nitric oxide inhibits caspase- 3 by S-nitrosation in vivo. J Biol Chem. 1999;274:6823–6.

    CAS  PubMed  Google Scholar 

  54. Saini M, Mehta DK, Das R, Saini G. Recent advances in anti-inflammatory potential of Pyridazinone derivatives. Mini-Rev Med Chem. 2016;16:996–1012.

    CAS  PubMed  Google Scholar 

  55. Santiago-Vazquez Y, Das U, Varela-Ramirez A, Baca ST, Ayala-Marin Y, Lema C, et al. Tumor-selective cytotoxicity of a novel pentadiene analogue on human leukemia/ lymphoma cells. Clin Cancer Drugs. 2016;3:138–46.

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative CT method. Nat Protoc. 2008;3:1101–8.

    CAS  PubMed  Google Scholar 

  57. Shunguang Z, Huimin L, Chao H, Yanan D, Mingyan J, Lixiang R, et al. Design, synthesis and structureeactivity relationships of novel 4-phenoxyquinoline derivatives containing pyridazinone moiety as potential antitumor agents. Eur J Med Chem. 2014;83:581–93.

    Google Scholar 

  58. Suski J, Lebiedzinska M, Bonora M, Pinton P, Duszynski J, Wieckowski MR. Relation between mitochondrial membrane potential and ROS formation. Methods Mol Biol. 2018;1782:357–81.

    CAS  PubMed  Google Scholar 

  59. Suski JM, Lebiedzinska M, Bonora M, Pinton P, Duszynski J, Wieckowski MR. Relation between mitochondrial membrane potential and ROS formation. Methods Mol Biol. 2012;810:183–205.

    CAS  PubMed  Google Scholar 

  60. Tewari KM, Dhaneshwar SS. Inhibitors of apoptosis proteins (IAPs): clinical significance in Cancer treatment research. J Can Res Updates. 2012;1:212–20.

    Google Scholar 

  61. Torres-Roca JF, Lecoeur H, Amatore C, Gougeon ML. The early intracellular production of a reactive oxygen intermediate mediates apoptosis in dexamethasone-treated thymocytes. Cell Death Differ. 1995;2:309–19.

    CAS  PubMed  Google Scholar 

  62. Varela-Ramirez A, Costanzo M, Carrasco YP, Pannell KH, Aguilera RJ. Cytotoxic effects of two organotin compounds and their mode of inflicting cell death on four mammalian cancer cells. Cell Biol Toxicol. 2011;27:159–68.

    CAS  PubMed  Google Scholar 

  63. Wada N, Kawano Y, Fujiwara S, Kikukawa Y, Okuno Y, Tasaki M, et al. Shikonin, dually functions as a proteasome inhibitor and a necroptosis inducer in multiple myeloma cells. Int J Oncol. 2015;46:963–72.

    CAS  PubMed  Google Scholar 

  64. Wang YJ, Lu D, Xu YB, Xing WQ, Tong XK, Wang GF, et al. A novel pyridazinone derivative inhibits hepatitis B virus replication by inducing genome-free capsid formation. Antimicrob Agents Chemother. 2015;59:7061–72.

    CAS  PubMed  PubMed Central  Google Scholar 

  65. WHO. 2018. Cancer. WHO [Online] Available: http://www.who.int/news-room/fact-sheets/detail/cancer [Accessed 7–31-18].

  66. Wu CC, Bratton SB. Regulation of the intrinsic apoptosis pathway by reactive oxygen species. Antioxid Redox Signal. 2013;19:546–58.

    CAS  PubMed  PubMed Central  Google Scholar 

  67. Xing W, Ai J, Jin S, Shi Z, Peng X, Wang L, et al. Enhancing the cellular anti-proliferation activity of pyridazinones as cmet inhibitors using docking analysis. Eur J Med Chem. 2015;95:302–12.

    CAS  PubMed  Google Scholar 

  68. Zhang J, Wu P, Hu Y. Clinical and marketed proteasome inhibitors for Cancer treatment. Curr Med Chem. 2013;20:2537–51.

    CAS  PubMed  Google Scholar 

  69. Zhou S, Liao H, He C, Dou Y, Jiang M, Ren L, et al. Design, synthesis and structure-activity relationships of novel 4-phenoxyquinoline derivatives containing pyridazinone moiety as potential antitumor agents. Eur J Med Chem. 2014;83:581–93.

    CAS  PubMed  Google Scholar 

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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

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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Correspondence to Renato J. Aguilera.

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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

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Keywords

  • Anti-cancer
  • Apoptosis
  • hmox-1
  • Proteasome inhibition
  • Pyridazinone
  • ROS