Citrus Peel in Maintaining Cellular Quiescence of Prostate Cancer

Abstract

Cancer recurrence remains a major concern for cancer patients despite the significant advancement in cancer treatment. Cell cycle re-entry of quiescent cancer cells has been implicated as a key factor for cancer recurrence. The slow progression of prostate cancer allows a window of opportunity for intervention through diet. Therefore, the aim of this thesis was to evaluate the potential use of citrus peel extract in halting the cell cycle re-entry of quiescent prostate cancer cells. Firstly, water extract and hexane extract of citrus peel were prepared using a maceration technique. The anticancer activity of the extracts was evaluated on prostate cancer PC-3 and LNCaP cells using in-vitro assays. The cell viability analysis showed that hexane extract was more effective in reducing cell viability compared to water extract in both cell lines. For cell cycle analysis, quiescent prostate cancer PC-3 and LNCAP cells were released to re-enter cell cycle in the presence of extract. Water extract completely inhibited the entry of quiescent cells from G0/G1 phase to S phase upon release from quiescence. In contrast, the hexane extract completely allowed the cells progress to S and G2/M phase. However, both extracts significantly decreased the DNA synthesis rate in PC-3 cells compared to the control when analysed using EdU incorporation assay. Hesperidin and naringin, the predominant flavonoid compounds in water extract, exhibited negligible cell cycle re-entry inhibitory effect on quiescent PC-3 cells suggesting the contribution of other bioactive compounds in the extract. More importantly, the water extract showed no toxicity when tested on the normal human fibroblast cells. Subsequently, solvent extraction and chromatography techniques were used to identify the compounds in water extract that are responsible for the cell cycle re-entry inhibitory effect. The results showed that the water fraction exhibited the highest inhibitory effect among the fractions tested. Not only that, the inhibitory activity of fractions was lower than the water extract suggesting the compounds exhibited a greater effect in combination and the presence of a synergic effect. Using various analytical methods, citric acid was identified as one of the cell cycle re-entry inhibitor compounds present in water extract. The cell viability assay showed the IC50 of citric acid for prostate cancer PC-3 cells and normal epithelial prostate RWPE-1 cells were almost similar. However, citric acid significantly exhibited higher cell toxicity effect on PC-3 cells than RWPE-1 cells at a concentration above 1 mg/mL indicating the specific toxicity of citric acid on cancer cells. Not only that, the citric acid showed no significant toxicity effect on human normal fibroblast GM3348 cells when tested on similar concentration. As the mechanisms of cell cycle re-entry of quiescent cells remain largely unclear, a study was attempted to elucidate the protein changes by the citric acid at the cellular level. The proteomic experiment results revealed several proteins were regulated differently in the citric acid treated cells that were possible to act with the inhibition of cell cycle re-entry. The Ingenuity Pathway Analysis software predicted that citric acid possibly inhibited PC-3 cells from re-entering cell cycle mainly by suppressing PI3K/AKT and ERK/MAPK signalling pathways and activating PTEN pathway. Modulation of these signalling pathways by diet or drug has the potential to reduce the cell cycle re-entry of quiescent prostate cancer cells and preventing cancer recurrence. The outcomes of this study have widened the applications of citrus peel extract as a chemopreventive agent for post-therapy cancer patients. The recovery and utilization of bioactive compounds from citrus peel not only reduce the waste but also will open an avenue for the development of affordable fortifying food products with potential in reducing the risk of cancer recurrence.