## 3 Results
### Torsol Induces Apoptosis in HI-60 Associated with a Reduction in Cells in the G1 and S Phases  
There are numerous reports of the inhibition of cell proliferation and even cancer chemo-preventive effects by EVOO phenols [24; 31; 32]. To further explore the observed reduction in cell numbers (MTT assay--Supplementary Figure S2), the cell cycle status of tyrosol-exposed cells was analysed. The exposure of HL-60 cells to tyrosol resulted in a marked increase in cells in the Sub G\({}_{0}\)/G\({}_{1}\) section (Figure 6). This suggests a significant difference in mechanism from the positive controls, with tyrosol appearing to induce cell death following induced differentiation. This appears to coincide with a reduction in the G\({}_{0}\)/G\({}_{1}\) and S phase, with cells accumulating in the G\({}_{2}\)M phase of the cycle. This may suggest that tyrosol-exposed cells cannot progress beyond G\({}_{2}\)M, and when attempting to do so, they proceed to cellular demise. Figure 6: Percentage gated HL-60 cells in the Sub G\({}_{0}\)/G\({}_{1}\), G\({}_{0}\)/G\({}_{1}\), S, and G\({}_{2}\)/M phases of the cell cycle on day 1, day 3, and day 5 and representative histograms for each condition on day 5. Histograms from left to right = Untreated, 1.6% DMSO, 10 nM PMA, and 9.23 ppm tyrosol. Untreated = negative (medium only) control, 1.6% DMSO, 10 nM PMA are positive controls for granulocyte and monocytic differentiation, respectively. Each bar represents the median value where \(n=4\). Error bars represent differences between the median and upper and lower quartiles. Statistically significant differences from the negative control are denoted by (*) \(p<0.05\) and (**) \(p<0.01\). To assess the mechanism of tyrosol-induced cell death, Annexin V/PI staining was performed and analysed by flow cytometry (Figure 7). Tyrosine exposure causes cells to primarily undergo apoptosis, with cells on day 5 showing late apoptosis (35.52%), as well as necrosis (9.35%). This contrasts with the results obtained for both differentiation controls, which do not appear to induce significant amounts of cell death by day 5, with cells (21.28% and 19.25% for DMSO and PMA, respectively) appearing to be in early apoptosis. These results indicate that the polyphenol tested here arrests the leukaemia cell line, subsequently inducing apoptosis. Figure 7: Percentage gated live, early apoptotic, late apoptotic and necrotic HL-60 cells on day 1, day 3, and day 5 and representative quadrants for each condition on day 5. Days 3 and 5 and representative histogram overlays and subtractions  
for each condition on day 5. From left to right = Untreated, 10 nM PMA, 1.6% DMSO, and 9.23 ppm tyrosol. Untreated = negative (medium only) control, 1.6% DMSO, 10 nM PMA = positive controls for granulocyte and monocytic differentiation respectively. Each bar represents the median value where \(n=4\). Error bars represent differences between the median and upper and lower quartiles. Statistically significant differences from the negative control are denoted by (*) \(p<0.05\), (**) \(p<0.01\), and (***) \(p<0.001\). Tyrosol Downregulates Neutrophil and Cholesterol Biosynthesis Genes and Upregulates Monocytic Differentiation Genes  
In order to determine the Tyrosol-mediated global gene expression changes in the ATRA-resistant HL-60 cell line, an RNA-seq analysis was performed after stimulation with Tyrosol for 1, 6, and 12 h. This defined a total of 199 differentially expressed (DE) genes (DE-seq2 \(p<0.01\) and Intensity Difference filter \(p<0.05\), with multiple testing correction), with 142 genes resulting in increased expression, whilst 57 genes were decreased in expression (Supplementary Table S1). Figure 8A,B show the hierarchical clustering of the negative and positively regulated genes respectively. In order to explore the biological significance of negatively regulated genes we used Reactome pathway analysis (Reactome.org).