Guo-Fong Dai

Hepatocellular carcinoma (HCC) is the most common liver or hepatic cancer, accounting for 80% of all cases. The majority of this cancer mortality is due to metastases, rather than orthotopic tumors. Therefore, the inhibition of tumor metastasis is widely recognized as the key strategy for successful intervention. A cembrane-type diterpene, flaccidoxide-13-acetate, isolated from marine soft coral Sinularia gibberosa, has been reported to have inhibitory effects against RT4 and T24 human bladder cancer invasion and cell migration. In this study, we investigated its suppression effects on tumor growth and metastasis of human HCC, conducting Boyden chamber and Transwell assays using HA22T and HepG2 human HCC cell lines to evaluate invasion and cell migration. We utilized gelatin zymography to determine the enzyme activities of matrix metalloproteinases MMP-2 and MMP-9. We also analyzed the expression levels of MMP-2 and MMP-9. Additionally, assays of tissue inhibitors of metalloproteinase-1/2 (TIMP-1/2), the focal adhesion kinase (FAK)/phosphatidylinositide-3 kinases (PI3K)/Akt/mammalian target of the rapamycin (mTOR) signaling pathway, and the epithelial-mesenchymal transition (EMT) process were performed. We observed that flaccidoxide-13-acetate could potentially inhibit HCC cell migration and invasion. We postulated that, by inhibiting the FAK/PI3K/Akt/mTOR signaling pathway, MMP-2 and MMP-9 expressions were suppressed, resulting in HCC cell metastasis. Flaccidoxide-13-acetate was found to inhibit EMT in HA22T and HepG2 HCC cells. Our study results suggested the potential of flaccidoxide-13-acetate as a chemotherapeutic candidate; however, its clinical application for the management of HCC in humans requires further research.

(+)-Bornyl p-coumarate is an active substance that is abundant in the Piper betle stem and has been shown to possess bioactivity against bacteria and a strong antioxidative effect. In the current study, we examined the actions of (+)-bornyl p-coumarate against A2058 and A375 melanoma cells. The inhibition effects of (+)-bornyl p-coumarate on these cell lines were assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay and the underlying mechanisms were identified by immunostaining, flow cytometry and western blotting of proteins associated with apoptosis and autophagy. Our results demonstrated that (+)-bornyl p-coumarate inhibited melanoma cell proliferation and caused loss of mitochondrial membrane potential, demonstrating treatment induced apoptosis. In addition, western blotting revealed that the process is mediated by caspase-dependent pathways, release of cytochrome C, activation of pro-apoptotic proteins (Bax, Bad and caspase-3/-9) and suppression of anti-apoptotic proteins (Bcl-2, Bcl-xl and Mcl-1). Also, the upregulated expressions of p-PERK, p-eIF2α, ATF4 and CCAAT/enhancer-binding protein (C/EBP)-homologous protein (CHOP) after treatment indicated that (+)-bornyl p-coumarate caused apoptosis via endoplasmic reticulum (ER) stress. Moreover, increased expressions of beclin-1, Atg3, Atg5, p62, LC3-I and LC3-II proteins and suppression by autophagic inhibitor 3-methyladenine (3-MA), indicated that (+)-bornyl p-coumarate triggered autophagy in the melanoma cells. In conclusion, our findings demonstrated that (+)-bornyl p-coumarate suppressed human melanoma cell growth and should be further investigated with regards to its potential use as a chemotherapy drug for the treatment of human melanoma.

Flaccidoxide-13-acetate, an active compound isolated from cultured-type soft coral Sinularia gibberosa, has been shown to have inhibitory effects against invasion and cell migration of RT4 and T24 human bladder cancer cells. In our study, we used an 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), colony formation assay, and flow cytometry to determine the mechanisms of the anti-tumor effect of flaccidoxide-13-acetate. The MTT and colony formation assays showed that the cytotoxic effect of flaccidoxide-13-acetate on T24 and RT4 cells was dose-dependent, and the number of colonies formed in the culture was reduced with increasing flaccidoxide-13-acetate concentration. Flow cytometry analysis revealed that flaccidoxide-13-acetate induced late apoptotic events in both cell lines. Additionally, we found that flaccidoxide-13-acetate treatment upregulated the expressions of cleaved caspase 3, cleaved caspase 9, Bax, and Bad, and down-regulated the expressions of Bcl-2, p-Bad, Bcl-x1, and Mcl-1. The results indicated that apoptotic events were mediated by mitochondrial dysfunction via the caspase-dependent pathway. Flaccidoxide-13-acetate also provoked endoplasmic reticulum (ER) stress and led to activation of the PERK-eIF2α-ATF6-CHOP pathway. Moreover, we examined the PI3K/AKT signal pathway, and found that the expressions of phosphorylated PI3K (p-PI3K) and AKT (p-AKT) were decreased with flaccidoxide-13-acetate concentrations. On the other hand, our results showed that the phosphorylated JNK and p38 were obviously activated. The results support the idea that flaccidoxide-13-acetate-induced apoptosis is mediated by mitochondrial dysfunction, ER stress, and activation of both the p38 and JNK pathways, and also relies on inhibition of PI3K/AKT signaling. These findings imply that flaccidoxide-13-acetate has potential in the development of chemotherapeutic agents for human bladder cancer.

Metastasis of cancer is the cause of the majority of cancer deaths. Active compound flaccidoxide-13-acetate, isolated from the soft coral Cladiella kashmani, has been found to exhibit anti-tumor activity. In this study, Boyden chamber analysis, Western blotting and gelatin zymography assays indicated that flaccidoxide-13-acetate exerted inhibitory effects on the migration and invasion of RT4 and T24 human bladder cancer cells. The results demonstrated that flaccidoxide-13-acetate, in a concentration-dependent manner, reduced the levels of matrix metalloproteinase-2 (MMP-2), MMP-9, urokinase-type plasminogen activator receptor (uPAR), focal adhesion kinase (FAK), phosphatidylinositide-3 kinases (PI3K), p-PI3K, AKT, p-AKT, mammalian target of rapamycin (mTOR), p-mTOR, Ras homolog gene family, member A (Rho A), Ras, mitogen-activated protein kinase kinase 7 (MKK7) and mitogen-activated protein kinase kinase kinase 3 (MEKK3), and increased the expressions of tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 in RT4 and T24 cells. This study revealed that flaccidoxide-13-acetate suppressed cell migration and invasion by reducing the expressions of MMP-2 and MMP-9, regulated by the FAK/PI3K/AKT/mTOR pathway. In conclusion, our study was the first to demonstrate that flaccidoxide-13-acetate could be a potent medical agent for use in controlling the migration and invasion of bladder cancer.