Royal jelly with ellagic acid inhibits the glycolytic pathway and triggers apoptosis through multiple pathways in colorectal cancer

Background: Developing novel chemotherapeutics with high anticancer efficacy and low toxicity remains a critical challenge in oncology. Natural products, with their multi-target activities and favorable safety profiles, offer promising candidates.This study investigated the combined anticancer effects of royal jelly (RJ) and ellagic acid (EA), two potent antioxidants of animal and plant origin.

Materials and methods: Royal jelly (RJ) and Ellagic Acid (EA) were applied to HT29 (ATCC^® HTB-38^™, Human colorectal adenocarcinoma), HCT116 (ATCC^® CCL-247^™, Human colorectal carcinoma), and BEAS-2B (ATCC^®CRL-3588 ™, human bronchial epithelium), their antiproliferative effects were investigated by xCELLigence Real-Time Cell Analyzer (RTCA MP). To investigate the mechanisms underlying the antiproliferative activity, the effect of the combination of RJ and EA on the glycolytic pathway was determined by SeaHorse XFe24. The apoptotic process was evaluated by DNA laddering and the expression of Bcl-2 and Bax genes in the apoptotic pathway by Real-Time quantitative PCR  (RT-qPCR). The transcriptome profiling of RJ and EA on colorectal cancer cells when used together was performed by Total RNA Sequencing analysis.

Results: The combination of RJ with EA significantly reduced the extracellular acidification rate (ECAR), effectively inhibiting aerobic glycolysis, especially in HCT116. The combination of RJ with EA in HCT116 and HT29 cells induced apoptosis by increasing the Bax/Bcl-2 ratio compared to cases with EA or RJ (p<0.05). In the GSEA analyses, the increase in apoptosis and p53 pathway-related genes and the suppression of genes involved in E2F target, G2M checkpoint, oxidative phosphorylation, and MYC target mechanism with the treatment applied in both cell lines clearly show the directly proportional relationship with antiproliferative effect on cancer cells and increased apoptosis.

Conclusion: The combination of RJ with EA combination demonstrates potent anticancer effects in colorectal cancer by suppressing glycolysis and activating apoptosis, highlighting its therapeutic potential as a novel combinatorial strategy for cancer treatment.

Key words: xCELLigence, SeaHorse XFe24, Apoptosis, Transcriptome, Colorectal Cancer