Evaluation of the honey properties and molecular mechanism of cell death induces by acacia honey in MCF-7 cell lines / Muhammad Ashraf Mohd Salleh

Honey’s increasing popularity has prompted medical researchers to study its therapeutic benefits. Honey has been shown to trigger cell death in cancer cells, directly modulate tumour progression. Polyphenols in honey have a significant role in this healing action. Despite its health benefits, the authenticity of honey remains the main challenge for consumers as intake of highly dominant fake honey cause more health issues. The purity and polyphenol content of Malaysian acacia honey were assessed in this study. Methods compiled and harmonised by the International Honey Commission were employed to determine purity per the Codex Alimentarius standard while two spectrophotometric techniques followed with polyphenols identification using HPLC assessed the polyphenol content. The potential of acacia honey to induce cell death was also studied in vitro using a two-pronged approach: detecting biochemical indicators of cell death and examining cell death morphologies. The assessment utilised human breast adenocarcinoma (MCF-7) and mammary gland epithelial (MCF 10A) with tamoxifen as a positive control. In terms of purity, higher moisture content associated with low marketability and shelf-life was observed while enzyme activity, HMF content and sugar profile fulfil the Codex Alimentarius standard. The polyphenol content assessment showed non-correlation between spectrophotometric and HPLC techniques where only two compounds were identified, quercetin and trans-cinnamic acid. Although optimisation may enhance identification, premature harvest which is common in Asian countries is suspected as outlined in a recent study considering higher moisture content was recorded. The inhibition assessment discovered cell death inducement in MCF-7 and MCF 10A cells with IC50 values of 5.5% and 8% respectively. The Annexin V-FITC assay revealed honey induces apoptosis-cell death after 48 hours with the highest apoptosis induction (36.04±9.61%) observed after 72 hours in the IC75 treatment group. The assay also validates the selectivity of acacia honey towards MCF 10A in inhibition assessment. Due to the short treatment period (16 hours), western blotting was unable to produce comprehensive, significant data. The Bcl-2 was found upregulated in MCF 10A treated with IC75 of acacia honey, suggesting the selectivity was achieved by suppressing apoptosis-cell death. However, the upregulation of caspase 8 in MCF-7 treated with IC25 acacia honey did not translate into apoptosis in annexin V-FITC assay. Caspase 8’s capacity to trigger apoptosis via MOMP may be compensated by autophagy’s mitophagy mechanism. Afterwards, CLSM and TEM were used to evaluate morphology. CLSM confirms the selectivity of acacia honey towards MCF 10A, whereas it exhibits apoptotic-like cell blebbing morphology against MCF-7 at IC50. However, the outcome contradicts earlier findings, indicating additional biological reactions produced the single event. TEM investigate ultrastructural changes associated with cell death. Following 24 hours of acacia honey treatment, MCF-7 cells had extensive vacuoles in the cytoplasm, indicating autophagy-cell death. Finally, this study shows the impact of early harvest on honey quality and polyphenol content. The non-correlation between spectrophotometric and HPLC techniques justify the proper method selection for compound identification. The treatment of acacia honey significantly promotes apoptosis-cell death after 48 hours while autophagy-mediated cell death may inhibit the MOMP effect induced through caspase 8 activation post 24 hours treatment.