Herbal medicines are currently attracting attention as potential cancer therapeutics and preventive agents. Phyllanthus niruri L. is a well-known medicinal plant that has been used as a hepatoprotective, antiviral, antibacterial, analgesic, antispasmodic and antidiabetic medicine; however, there are few reports describing its anti-tumor activity. Our group isolated components of Phyllanthus niruri L. by chromatographic fractionation and mass spectrometry. Of the two major isolated components (ethyl brevifolin carboxylate and Corilagin), Corilagin demonstrated better anti-tumor potential and lower toxicity in normal cells [unpublished data, Ming et al.]. Corilagin is a gallotannin that has been identified in several plants, including Phyllanthus niruri L. . Corilagin has been shown to exhibit versatile medicinal activity including anti-inflammatory effects as well as hepato-protective activity [2, 10, 11]. Recently, an anti-tumor effect on hepatocellular carcinoma was reported ; however, the anti-tumor mechanism is still unclear.
In this study, we confirmed the antitumor effect of Corilagin on ovarian cancer cells and further investigated the mechanism of this effect. Corilagin induced cell cycle arrest at the G2/M stage and enhanced apoptosis in ovarian cancer cells. Cyclin B1, Myt1, Phospho-cdc2 and Phospho-Weel were down-regulated after Corilagin treatment. Importantly, we found that Corilagin inhibited TGF-β secretion into the culture supernatant of all tested ovarian cancer cell lines and blocked the stabilization of Snail induced by TGF-β. The reduction of TGF-β secretion was specific to Corilagin treatment; Corilagin also targeted TGF-β-related signaling molecules, such as pAKT, pERK and pSmads. Other natural products, such as genistein and curcumin, can also alter the TGF-β pathway. Both of these agents can abrogate the enhancement of u-PA levels induced by TGF-β1 and also inhibit the TGF-β1-induced synthesis of fibronectin , inferring that some natural products have the potential to be effective in the treatment of cancer.
G2/M checkpoint-based anti-cancer strategies have focused on targeting and inactivating the G2/M checkpoint, thus forcing the cancer cells into mitosis with increased DNA damage and finally into mitotic catastrophe and cell death. The Cyclin B/cdc2 complex performs an important function in controlling the G2/M phase by rapidly phosphorylating the target protein to induce progression into the M phase [13, 14]. The phosphorylation and dephosphorylation of specific amino acids in cdc2 are responsible for the control of G2/M cell cycle progression by the Cyclin B1/cdc2 complex [13, 14]. More specifically, in the G2 phase, cdc2 is phosphorylated at Thr14 and Tyr15 by the protein kinases Myt1 and Wee1, thereby converting it into an inactive precursor. Consistent with these reports, in the present study, we observed that Corilagin decreases the protein level of Cyclin B1, p-cdc2 (Tyr15) in both Hey and SKOv3ip cells, which might be the molecular mechanism responsible for Corilagin’s efficacy in inducing G2/M arrest. We also observed down-regulation of p-Wee1 (Ser642) and Myt1 in Hey and SKOv3ip cells, indicating that the efficacy of Corilagin in inducing G2/M arrest in ovarian cancer cells is possibly due to the down-regulation of cdc2 and Cyclin B1 through Wee1 and Myt1 regulation.
Akt is suggested to function as a G2/M initiator. The activity of PI3K/Akt is required at multiple points during the cell cycle. Downstream functions of the PI3K/Akt pathway during G2/M transitions may include inhibition of the Chk1 G2 checkpoint protein or activation of cdc25C, which promotes cdc2 activation and entry into mitosis in primary oocytes from the starfish Asterina pectinifera. Akt was reported to inhibit Myt1 through Akt-dependent phosphorylation and down-regulation at the G2/M transition . In the present study, we observed that Corilagin inhibited both pAKT (Ser473) and Myt1 expression in Hey and SKOv3ip cells after stimulation with EGF, suggesting that the inhibition of Akt/Myt1 also contributes to the G2/M arrest resulting from Corilagin treatment. Further studies will be required to support these assumptions and to determine the role of upstream events, such as Chk1 and Chk2, in ovarian cancer cell responses to Corilagin.
Corilagin has been reported as a TNF-α-releasing inhibitor in inflammatory scenarios [5, 6]. In this study, we observed that the secretion of TGF-β was inhibited by Corilagin in a dose-dependent manner in all ovarian cancer cells evaluated, indicating that Corilagin also disturbed the expression and efficacy of TGF-β. Our results further demonstrated that Corilagin not only targets the classical Smad pathway via pSmad2 but also down-regulates MAPK signaling. The thing that most intrigued us is that Corilagin treatment induced a dramatic decline in the expression of the Snail protein, especially at higher doses, which indicates that Corilagin not only exerts its effects on cell cycle control but also contributes to epithelial-mesenchymal transition (EMT) in ovarian cancer.
As with all cancer cells, ovarian cancer cells undergo an EMT to disseminate within the intraperitoneal cavity or metastasize to distant sites . TGF-β signaling plays a critical role in ovarian cancer EMT and metastasis. Ovarian cancer is thought to arise from normal ovarian surface epithelium (OSE). TGF-β has been shown to inhibit human OSE proliferation and induce apoptosis, which may prevent the over-proliferation of cells during a normal ovulatory cycle . Although TGF-β can act as a tumor suppressor by inhibiting cell proliferation in the early stages of tumor development, it can also promote metastasis in various cancer models [19, 20]. It appears that at later stages, cancer cells protect themselves and tend to acquire increasing resistance to TGF-β growth inhibitory signals, which is an important reason for the shift of TGF-β from tumor suppressor to tumor promoter . Much remains to be elucidated about how TGF-β contributes to ovarian cancer progression, particularly in the regulation of EMT. A high concentration of TGF-β has been detected in ascites, blood and other bodily fluids of ovarian cancer patients . When ovarian cancer cells were cultured, various TGF-βs, including TGF-β1, TGF-β2 and TGF-β3, induced pro-matrix metalloproteinase (MMP) secretion, the loss of cell-cell junctions, down-regulation of E-cadherin, up-regulation of N-cadherin, and the acquisition of a fibroblastoid phenotype, all of which are consistent with EMT [23–25]. In addition, our recent studies identified that TGF-β is the most important inflammatory factor in ovarian cancer. TGF-β stabilizes the protein level of Snail, an inducer of EMT, and further enhances Snail expression when combined with other inflammatory factors (Jin et al. unpublished data). However, how Corilagin has this effect on TGF-β and thus undermines the stability of Snail still needs to be elucidated.
TGF-β binds to type I (ThRI) and type II (ThRII) receptors. Upon ligand binding to ThRII, ThRI is activated and phosphorylates the receptor-regulated Smads. The phosphorylated receptor-regulated Smads then bind to the co-Smad, Smad4, and translocate to the nucleus to modulate gene expression. TGF-β also initiates Smad-independent pathways, including those mediated by the mitogen-activated protein kinase family members (TAK1, extracellular signal-regulated kinase, p38, and c-Jun-NH2-kinase) and phosphatidylinositol 3-kinase . In this study, we found that Corilagin not only inhibits the secretion of TGF-β but also blocks the TGF-β-related signaling proteins pSmads, pAKT, and pERK (Figure 8). Our research provides evidence that TGF-β/Smad/AKT/ERK signaling is the target of Corilagin and that this herbal medicine could be an effective ovarian cancer therapeutic agent.