Basic Study
Copyright ©The Author(s) 2021. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Clin Oncol. May 24, 2021; 12(5): 342-354
Published online May 24, 2021. doi: 10.5306/wjco.v12.i5.342
Thymoquinone anticancer activity is enhanced when combined with royal jelly in human breast cancer
Maya M Moubarak, Nour Chanouha, Najwa Abou Ibrahim, Hala Khalife, Hala Gali-Muhtasib
Maya M Moubarak, Nour Chanouha, Department of Biology, American University of Beirut, Beirut 1107-2020, Lebanon
Najwa Abou Ibrahim, Hala Khalife, Rammal Rammal Laboratory (ATAC group), Faculty of Sciences I, Hadath 1003, Lebanon
Hala Gali-Muhtasib, Department of Biology and Center for Drug Discovery, American University of Beirut, Beirut 1107-2020, Lebanon
Author contributions: Moubarak MM carried out lab work as part of her MSc thesis, performed analysis and interpretation of data (e.g., biostatistics, statistical analysis and editing), and wrote the first draft of the manuscript; Chanouha N performed initial lab work and determined dose responses and performed data analysis; Abou Ibrahim N brought the importance of royal jelly to the attention of the corresponding author, provided it, and contributed intellectually to the study; Khalife H reviewed the manuscript and contributed in the critical appraisal of data; Gali-Muhtasib H conceived the project, supervised the work, and edited the manuscript draft; All authors have read and approved the final manuscript.
Supported by The Lebanese National Council for Scientific Research and the American University of Beirut, No.103482; and the Undergraduate Research Experience of the Faculty of Arts and Sciences, American University of Beirut.
Conflict-of-interest statement: Authors declare no conflict of interest for this manuscript.
Data sharing statement: No additional data are available.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
Corresponding author: Hala Gali-Muhtasib, PhD, Professor, Department of Biology and Center for Drug Discovery, American University of Beirut, Bliss Street, Biology Bldg, Room 207, PO Box 11-0236, Riad El Solh, Lebanon, Beirut 1107-2020, Lebanon.
Received: January 28, 2021
Peer-review started: January 28, 2021
First decision: March 1, 2021
Revised: March 13, 2021
Accepted: April 26, 2021
Article in press: April 26, 2021
Published online: May 24, 2021

Breast cancer is the most common cause of the majority of cancer-related deaths in women, among which triple-negative breast cancer is the most aggressive type of breast cancer diagnosed with limited treatment options. Thymoquinone (TQ), the main bioactive constituent of Nigella sativa, has been extensively studied as a potent anticancer molecule against various types of cancers. Honeybee products such as the royal jelly (RJ), the nutritive secretion fed to honeybee queens, exhibit a variety of biological activities besides its anticancer effect. However, the anticancer activity of the combination of TQ and RJ against breast cancer is still unknown.


To investigate cytotoxicity of RJ in FHs 74 Int cells and the anticancer effects of TQ, RJ, and their combinations in the MDA-MB-231 cell line.


Cells were treated with TQ, RJ, and their combinations for 24 h. Using 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, we determined the half-maximal inhibitory concentration of TQ. Trypan blue and 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays were then performed to assess the cell viability in response to different treatment conditions. Cell death and cycle regulation were investigated using propidium iodide deoxyribonucleic acid staining followed by flow cytometry in response to a single dose of TQ, RJ, and their combination. Immunostaining for cleaved caspase 3 and Ki67 expression was used to determine apoptosis induction and changes in cell proliferation.


TQ alone inhibited cell viability in a dose-dependent manner at concentrations below and above the half-maximal inhibitory concentration. RJ exhibited relatively nontoxic effects against MDA-MB-231 cells and FHs 74 Int small intestinal cells at concentrations below 5 µg/mL. High doses of RJ (200 µg/mL) had greater toxicity against MDA-MB-231 cells. Interestingly, the inhibition of cell viability was most pronounced in response to 15 µmol/L TQ and 5 µg/mL RJ. A dose of 15 µmol/L TQ caused a significant increase in the PreG1 population, while a more pronounced effect on cell viability inhibition and PreG1 increase was observed in response to TQ and RJ combinations. TQ was the main inducer of caspase 3-dependent apoptosis when applied alone and in combination with RJ. In contrast, no significant regulation of Ki67 expression was observed, indicating that the decrease in cell viability was due to apoptosis induction rather than to inhibition of cell proliferation.


This study is the first to report enhanced anticancer effects of TQ and RJ combination against MDA-MB-231 breast cancer cells, which could confer an advantage for cancer therapy.

Keywords: Anticancer activity, Breast cancer cells, Drug combination, Natural products, Royal jelly, Thymoquinone

Core Tip: Royal jelly enhances thymoquinone (TQ) anticancer activity against breast cancer. TQ induces the apoptotic response in breast cancer cells while royal jelly when combined with TQ potentiates the reduction in cell viability more than each drug alone.