# Galile Acid Induces S and G2 Phase Arrest and Apoptosis in Human Ovarian Cancer Cells In Vitro
## 4 Discussion  
GA is a natural polyphenol with antibacterial, antiviral, and antitumor properties. Its tumor suppressive properties has, thus far, been demonstrated in prostate cancer [11], breast cancer [20], bladder cancer [21], cervical cancer and lung cancer [9]. In prior experiments, we demonstrated that GA produced the largest suppression of human OC cells, among eight natural phenols used in TCM [13; 14]. In this paper, the IC50 values of GA exposure in human OC cell lines OVCAR 3 and A2780/CP70 with 24, 48, 72 h exposure were further evaluated. As in our previous experiments, the suppressive effects of GA on OVCAR-3 cells were stronger, relative to A2780/CP70 cells. The specific mechanism is still unclear. In addition, a decrease of IC50 values of GA in both cell lines was observed when the treatment time increased from 24 h to 72 h. Our findings are in accordance to previously published reports [22; 23]. Many polyphenols inhibit the viability of cancer cells due to their induction of apoptosis [24]. Recent research showed that polyphenols induce apoptosis by suppress or activate mitochondrial death system and affect mitochondrial function and structure [25]. In this study, GA showed significant activity of apoptosis induction at 10 uM and 20 uM in OVCAR 3 and A2780/CP70 cells, respectively. In all, these data suggest that GA suppresses OC cell growth by promoting apoptosis. This form of apoptotic stimulation has been demonstrated in other cancerous cells. GA showed significant apoptosis induction at 10 uM in lymphoblastic leukemia cell line C121 [26] and at 50 uM in human breast cancer cell MDA-MB-231 [27]. Stimulating apoptosis in cells is a key mechanism by which anticancer therapy works. In addition, GA was also shown to have enhanced bioavailability in humans, relative to other polyphenols [28]. In fact, oral intake of dietary herbal supplement with 800 mg GA resulted in 8-10 uM GA in the serum of healthy volunteers [29]. According to results from these studies, GA demonstrated a strong potential as an anti-tumor agent. The mitochondrial intrinsic pathway is the main pathway for polyphenols to induce cell apoptosis [30; 31]. In this research, we demonstrated marked elevation in pro-apoptotic proteins; namely, cleaved caspase-3 (4.53-fold), Bad (1.33-fold) and Bax (1.53-fold). This result indicated that GA activated caspase-3 mediated intrinsic apoptotic pathways in OVCAR-3 cells via the upregulation of proapoptotic proteins (Bax and Bad). Tumor suppressor p53 protein modulates cell apoptosis [32]. Our previous research found by kaempfer, a natural flavonoid, induced intrinsic apoptosis via activation of p53 in OVCAR-3 cells [33]. To examine the effect of p53 on GA-mediated apoptosis, p53 protein levels was assessed via Western blotting. We demonstrated that the basal expression of p53 protein was markedly elevated (\(p<0.05\)) post GA exposure in OVCAR-3 cells. This result suggests that the GA-mediated apoptosis occurs via the regulation of p53 protein. The p53 point mutation R248Q, found in OVCAR3 cells, affects DNA binding and has been shown to reduce the ability to induce apoptosis [34]. Therefore, the role of p53 gene in the GA-mediated apoptosis of OVCAR-3 cells needs to be further confirmed, such as the use of chip technology to determine the binding sites of p53 protein on the promoter of apoptosis genes. This is the first paper to document the mechanism of promoting apoptosis with GA in OC cells. Cell cycle arrest is an important factor affecting cell growth. Our previous research found that many natural compounds such as saponins [17], proanthocyanidins [35] and theaflavin-3,3-digallate [36], inhibit the viability of cancer cells via promoting cell cycle arrest. In this research, cell cycle analysis revealed that GA stimulated an increase in the population of S and G2 phase cells in two examined OC cell lines. It is reported that the Cdc2/cyclin B pathway is related to G2 cell cycle arrest [37]. In this research, Cdc2, p-Cdc2 and cyclin B were decreased.