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3,4-Dihydroxybenzoic acid (protocatechuic acid, PCA) is discussed to represent antioxidative food components in a human diet rich in fruits and vegetables, and has been shown to prevent carcinogenesis or antitumor growth in vivo. However, the molecular mechanisms involved in chemopreventive activity of PCA are poorly understood. In this study, investigations were conducted to examine the detailed signaling pathway involved in PCA-induced apoptosis in human gastric adenocarcinoma (AGS) cells. The data from cell viability assay showed that PCA exhibited the antiproliferation effect on AGS cells in a time-and dose-dependent manner. The occurrence of apoptosis induced by PCA was confirmed by morphological and biochemical features, including apoptotic bodies formation and an increase in the distribution of hypodiploid phase. Molecular data showed the effect of PCA in AGS cells might be mediated via sustained phosphorylation and activation of JNK and p38 mitogen-activating protein kinases (MAPK), but not ERK. Treatment with pharmacological inhibitors or transfection with the mutant p38 or/and JNK expression vector reduced PCAmediated apoptosis and the JNK/p38 MAPK-related proteins phosphorylation and expression, including ATF-2, c-Jun, FasL, Fas, p53 and Bax. Preincubation with Nok-1 monoclonal antibody, which is inhibitory to Fas signaling, interfered with PCA-induced cleavage of procaspase and sensitization to anti-APO-induced apoptosis. These results suggest the possible involvement of multiple signaling pathways from the MAPK to the subsequent mitochondria-and/or Fas-mediated caspase activation are potential requirements for PCA-induced AGS apoptosis. Further, PCA effectively induced JNK/p38 activation in PCA-response cell lines. Taken together, our data present the first evidence of PCA as an apoptosis inducer in AGS cells, even in tumor cells of digestive organs, and provide a new mechanism for its anticancer activity. ' 2007 Wiley-Liss, Inc.

BACKGROUND AND PURPOSEProtocatechuic acid (PCA) is plentiful in edible fruits and vegetables and is thus one anti-oxidative component of normal human diets. However, the molecular mechanisms involved in the chemopreventive activity of PCA are poorly understood. Here, we investigated the mechanism(s) underlying the anti-metastatic potential of PCA. EXPERIMENTAL APPROACHWe used AGS cells in a wound healing model and Boyden chamber assays in vitro and injection of B16/F10 melanoma cells in mice (metastasis model in vivo) to analyse the effect of PCA on cancer cell invasion and metastasis. The activities and expression of molecular proteins were measured by zymographic assay, real-time RT-PCR and Western blotting. KEY RESULTSPCA inhibited cell migration and invasion at non-cytotoxic concentrations. Decreased expression of matrix metalloproteinase (MMP)-2 and a coincident increase in tissue inhibitor of MMP followed treatment with PCA. The PCA-inhibited MMP-2 activity and expression was accompanied by inactivation of NF-kB. All these effects of PCA could be mediated via the RhoB/ protein kinase Ce (PKCe) and Ras/Akt cascade pathways, as demonstrated by inhibition of PKCe and transfection of PKCe siRNA and ras overexpression vector. Finally, PCA inhibited metastasis of B16/F10 melanoma cells to the liver in mice. CONCLUSION AND IMPLICATIONSOur data imply that PCA down-regulated the Ras/Akt/NF-kB pathway by targeting RhoB activation, which in turn led to a reduction of MMP-mediated cellular events in cancer cells and provides a new mechanism for the anti-cancer activity of PCA. Abbreviations

In view of the continuing need for effective anticancer agents, and the association of diet with reduced cancer risk, edible plants are increasingly being considered as sources of anticancer drugs. Hibiscus sabdariffa Linne (Malvaceae), an attractive plant believed to be native to Africa, is cultivated in the Sudan and Eastern Taiwan. Polyphenols had been demonstrated previously to possess antioxidative and antitumor promoting effects. In this study, investigations were conducted to examine the mechanism of the anticancer activity of H. sabdariffa L., Hibiscus polyphenol-rich extracts (HPE). Using HPLC assay, HPE was demonstrated to contain various polyphenols. HPE induced cell death of eight kinds of cell lines in a concentration-dependent manner. Among them human gastric carcinoma (AGS) cells were the most susceptible to HPE (0.95 mg/mL HPE inhibited its growth by 50%). Our results revealed that AGS cells underwent DNA fragmentation, and had an increase in the distribution of hypodiploid phase (apoptotic peak, 52.36%) after a 24-h treatment with HPE (2.0 mg/mL). This effect of HPE in AGS cells might be mediated via p53 signaling and p38 MAPK/FasL cascade pathway, as demonstrated by an increase in the phosphorylation of p53 and the usage of a specific p38 inhibitor, SB203580. Thus, our data present the first evidence of HPE as an apoptosis inducer in AGS cells and these findings may open interesting perspectives to the strategy in human gastric cancer treatment.

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Hibiscus sabdariffa Linne is a traditional Chinese rose tea and has been effectively used in folk medicines for treatment of hypertension, inflammatory conditions. H. sabdariffa aqueous extracts (HSE) were prepared from the dried flowers of H. sabdariffa L., which are rich in phenolic acids, flavonoids and anthocyanins. In this review, we discuss the chemopreventive properties and possible mechanisms of various H. sabdariffa extracts. It has been demonstrated that HSE, H. sabdariffa polyphenol-rich extracts (HPE), H. sabdariffa anthocyanins (HAs), and H. sabdariffa protocatechuic acid (PCA) exert many biologic effects. PCA and HAs protected against oxidative damage induced by tert-butyl droperoxide (t-BHP) in rat primary hepatocytes. In rabbits fed cholesterol and human experimental studies, these studies imply HSE could be pursued as atherosclerosis chemopreventive agents as they inhibit LDL oxidation, foam cell formation, as well as smooth muscle cell migration and proliferation. The extracts also offer hepatoprotection by influencing the levels of lipid peroxidation products and liver marker enzymes in experimental hyperammonemia. PCA has also been shown to inhibit the carcinogenic action of various chemicals in different tissues of the rat. HAs and HPE were demonstrated to cause cancer cell apoptosis, especially in leukemia and gastric cancer. More recent studies investigated the protective effect of HSE and HPE in streptozotocin induced diabetic nephropathy. From all these studies, it is clear that various H. sabdariffa extracts exhibit activities against atherosclerosis, liver disease, cancer, diabetes and other metabolic syndromes. These results indicate that naturally occurring agents such as the bioactive compounds in H. sabdariffa could be developed as potent chemopreventive agents and natural healthy foods.

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