Identifying cellular targets of 3',4',5',5,7-pentamethoxyflavone in colorectal cancer

Despite increasing positive prognosis for those living in high income countries (HICs), the incidence of colorectal cancer (CRC) in low- and middle-income countries (LMICs) such as Malaysia, is rising and increasingly becoming a burden for people in these regions. Natural products with promising bioactive properties have been widely investigated for clinical usage and often show positive properties such as reduced toxicity. Flavonoids are polyphenol phytochemicals that often possess bioactive properties. Flavones, a sub-class of the flavonoid family, have shown promise as potential drug compounds in areas such as anti-inflammatory, anti-obesity, antioxidant, and anticancer medicine. 3',4',5',5,7-Pentamethoxyflavone (PMF) has been shown in previous research to possess anticancer and chemopreventive properties in CRC, this research aims to identify potential cellular targets of PMF to give insight into the mechanisms of action of the compound which are currently unknown. A structure-activity relationship study identified the 3'-position as a suitable modification point on the compound structure that could be changed without compromising biological activity. After synthesis of a photoaffinity labelling probe, protein cellular targets were then investigated using pull down experiments and mass spectrometry proteomics. RNA sequencing was also used to investigate changes in gene expression in CRC cells after treatment with the active compound to further aid in identifying a mechanism of action. Members of the RAB subfamily of small GTPases were isolated using the proteomics analysis. Pathway analysis showed that the key interacting proteins were involved in the cellular response to stress and protein folding. This suggests that the flavone could be involved in the unfolded protein response (UPR). RNA sequencing showed that exposure of the cells to the flavone compound caused changes in expression of genes associated with the cell cycle and the UPR. Future studies of PMF should aim to validate the interaction between the protein targets identified and the compound. Synthetic modification of PMF can improve both the binding properties to the target proteins and aqueous solubility of the compound and enable progression as a potential anticancer treatment.