Cellular and biochemical antileukemic mechanisms of the meroterpenoid Oncocalyxone A

ABSTRACT

Oncocalyxone A, a 1,4-benzoquinone derived from Cordia oncocalyx, exhibits anti-inflammatory, antimicrobial and antidiabetic properties. The aim of this study was to (1) examine the cytotoxic actions of oncocalyxone A on human normal and tumor cell lines and (2) determine mechanistic actions underlying effects upon leukemia cells using cellular and molecular techniques. Antiproliferative studies on cancer cell lines, peripheral blood mononuclear cells, and human erythrocytes were performed using colorimetric assays. To understand cytotoxicity, assessments were performed with HL-60 leukemia cells (8, 16.5, or 33 µM) after 24 hr incubation using light and fluorescence microscopy, trypan blue, flow cytometry, Comet assay, western blot of caspases and poly-ADP-ribose polymerase (PARP), and effects on topoisomerase I and II. Oncocalyxone A exhibited cytotoxic action upon HL-60 cells and dividing leukocytes, but minimal hemolytic action on erythrocytes. Mechanistic investigations demonstrated reduction of cell viability, loss of membrane integrity, cell shrinking, chromatin condensation, blebbings, externalization of phosphatidylserine, caspase activation, PARP cleavage, mitochondrial depolarization, and DNA damage. Pre-treatment with N-acetylcysteine 4 mM significantly reduced DNA damage and prevented membrane integrity loss. Oncocalyxone A displayed free radical dependent antileukemic activity via apoptotic pathways and induced DNA damage in HL-60 cells. Oncocalyxone A possesses structural chemical simplicity enabling it to be a cost-effective alternative. These properties justify further improvements to enhance activity and selectivity and the development of pharmaceutical formulations.

Abbreviations

Acridine orange, AO; ANOVA, analysis of variance; BSA, bovine serum albumin; DI, Damage Index; DMSO, dimethylsulfoxide; EC₅₀, effective concentration 50%; EDTA, ethylenediamine tetraacetic acid; EB, ethidium bromide; HCT-116, colon carcinoma line; HL-60, promyelocytic leukemia line; IC₅₀, inhibitory concentration 50%; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; OVCAR-8, ovarian carcinoma line; NAC, N-acetylcysteine, PBMC, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; PI, propidium iodide; PARP, poly-ADP-ribose polymerase; RPMI-1640, Roswell Park Memorial Institute medium; SF-295, glioblastoma line; ROS, reactive oxygen species; 7-AAD, 7-amino-actinomycin D; H₂-DCF-DA, 7′-dichlorodihydrofluorescein diacetate.

KEYWORDS:

• 1,4-benzoquinone
• Cordia oncocalyx
• apoptosis
• poly-ADP-ribose polymerase
• DNA damage

Acknowledgments

We thank Silvana França dos Santos for her technical assistance and Dr Yves Pommier (Center for Cancer Research, National Cancer Institute, Bethesda, USA) for his help with DNA topoisomerase experiments. Dr Paulo Michel Pinheiro Ferreira [#303247/2019-3] and Dr Cláudia Pessoa [#303102/2013-6] are also grateful to the CNPq for their personal scholarships.

Disclosure statement

There are no conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

Ethics approval

All studies were performed in accordance with Brazilian guidelines (Law 466/2012, National Council of Health), the Declaration of Helsinki and with the Universal Declaration on Bioethics and Human Rights of UNESCO and were approved by the Human Research Ethical Committee of Federal University of Ceará (#0161/2014).

Authorship contributions

ABS: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing - original draft. FWAB-N: Conceptualization, Formal analysis, Investigation, Methodology. BMS: Investigation. BCC: Investigation. RWRS: Visualization and Editing. MPC: Investigation, Methodology, Visualization. ODLP: Conceptualization, Resources. CP: Funding acquisition, Resources, Data curation, Supervision, Visualization. PMPF: Project administration, Conceptualization, Data curation, Supervision, Visualization, Writing - original draft, Writing - review & editing. All authors have read and agreed to the published version of the manuscript.

Submission declaration and verification

The work has not been previously published or submitted for publication elsewhere. The publication is approved by all authors.

Correction Statement

This article has been republished with minor changes. These changes do not impact the academic content of the article.