# A Comparison Study on the Metabolites in PC-3, RWPE-1, and Chrysin-Treated PC-3 Cells
## 4 Discussion  
This study investigated the metabolome as a valuable source of prostate cancer biomarkers by distinguishing between normal prostate and prostate cancer cells. Additionally, metabolomic applications for monitoring cancer treatment were explored by comparing the metabolome of PC-3 cells with that of chrysin-treated cells by GC-MS analysis. GC-MS-based metabolomic analysis in cancer research is an important platform for evaluating cancer development through metabolic profiling and earlier identification of more sensitive diagnostic makers [12; 13; 14; 15]. GC-MS-based metabolomic analysis has been developed for metabolite extraction from cell cultures, tissues, and serum/plasma because GC-MS can analyze water-soluble cellular materials, water-insoluble fatty acids, and phospholipids [16]. This study found significant differences in the metabolite profiles between normal prostate cells and prostate cancer cells. As shown in Figure 2, the average levels of 18 metabolites differed significantly between the normal prostate and prostate cancer cell groups. In addition, PC-3 Chr cells showed a difference in metabolic profiles from PC-3 cells (Figures 3 and 4), whereas the PC-3 Chr cell group was similar to the RWPE-1 cell group in the metabolome. Chrysin has been shown to exert anticancer activities through emerging cellular and molecular mechanisms, including the inhibition of invasion and metastasis [17], oxidative stress [18], inflammation, angiogenesis [19], proliferation, and induction of apoptosis [9] in prostate cancers [20]. Recently, we showed that chrysin has anticancer effects, including the inhibition of epithelial-mesenchymal transition (EMT), vasculogenic mimicry, angiogenesis, and the induction of apoptosis by inhibiting the SPHK/HIF-lalpha signaling pathway in hypoxia-induced PC-3 cells [10]. Chrysin, which has anticancer effects, was used to evaluate the application of GC-MS-based metabolic analysis for monitoring cancer treatment. This study demonstrated the metabolic changes induced by chrysin in cancer cells. Figure 3: Bar graphs of the 15 most significant metabolites in the analysis of variance results comparing the two groups (PC-3 and chrysin-treated PC-3 cells (PC-3 Chr)). Data represent the mean \(\pm\) SD. * \(p<0.05\), ** \(p<0.01\), and *** \(p<0.001\) versus the PC-3 cell group. This study showed that the average levels of amino acids, alcohols, carboxylic acids, organic acids, sugars, and urea were significantly different in cancer and normal cells. Furthermore, multivariate analysis and comparison of metabolite concentration levels showed that the normal and chrysin-treated cell groups were highly similar and differed significantly from the PC-3 group. Predominantly, proline, isoleucine, and glycine amino acids and urea were not detected in RWPE-1 and PC-3 Chr cells, while these amino acids were highly detected in the PC-3 cell group. Similar to our study, a pilot clinical study showed that the amino acid profiles of serum and urine might provide valuable and clinically useful markers for prostate cancer [21]. Glycine is essential for the synthesis of proteins, nucleic acids, and lipids that are important for cell growth and tumor homeostasis [22]. Furthermore, glycine metabolism correlates with cell proliferation and poor prognosis in several tumors [23]. Proline metabolism triggers with conversion into \(\Delta\)1- pyrroline-5-carboxylate (P5C) by proline dehydrogenase enzyme (PRODH) in tumor growth and metastatic pro  
Figure 4: Bar graphs of the 23 most significant metabolites in the analysis of variance results comparing the three groups (RWPE-1, PC-3, and chrysin-treated PC-3 cells (PC-3 Chr)). Data represent the mean \(\pm\) SD. a\(\sim\)c means in a row by different superscripts are significantly different by LSD (least significant difference) at \(p<0.05\). gression.