BACKGROUND AND PURPOSE Protocatechuic 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 APPROACH We 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 RESULTS PCA 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‐κB. All these effects of PCA could be mediated via the RhoB/ protein kinase Cε (PKCε) and Ras/Akt cascade pathways, as demonstrated by inhibition of PKCε and transfection of PKCε siRNA and ras overexpression vector. Finally, PCA inhibited metastasis of B16/F10 melanoma cells to the liver in mice.CONCLUSION AND IMPLICATIONS Our data imply that PCA down‐regulated the Ras/Akt/NF‐κB 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.

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 PCA‐mediated 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.

Obesity is associated with a great diversity of diseases including non-alcoholic fatty liver disease. Our previous report suggested that Hibiscus sabdariffa extracts (HSE) had a metabolic-regulating and liver-protecting potential. In this study, we performed a clinical trial to further confirm the effect of HSE. Subjects with a BMI ≧ 27 and aged 18–65, were randomly divided into control (n = 17) and HSE-treated (n = 19) groups, respectively, for 12 weeks. Our data showed that consumption of HSE reduced body weight, BMI, body fat and the waist-to-hip ratio. Serum free fatty acid (FFA) was lowered by HSE. Anatomic changes revealed that HSE improved the illness of liver steatosis. Ingestion of HSE was well tolerated and there was no adverse effect during the trial. No alteration was found for serum α-amylase and lipase. The clinical effect should mainly be attributed to the polyphenols of HSE, since composition analysis showed that branched chain-amino acids, which is associated with obesity, is not obviously high. In conclusion, consumption of HSE reduced obesity, abdominal fat, serum FFA and improved liver steatosis. HSE could act as an adjuvant for preventing obesity and non-alcoholic fatty liver.

Our previous study has revealed that dioscin, a compound with anti-inflammatory, lipid-lowering, anticancer and hepatoprotective effects, may induce autophagy in hepatoma cells. Autophagy is a lysosomal degradation pathway that is essential for cell survival and tissue homeostasis. In this study, the role of autophagy and related signaling pathways during dioscin-induced apoptosis in human lung cancer cells was investigated. Results from 4′-6-diamidino-2-phenylindole and annexin-V/PI double-staining assay showed that caspase-3- and caspase-8-dependent, and dose-dependent apoptoses were detected after a 24-h dioscin treatment. Meanwhile, autophagy was detected as early as 12 h after an exposure to low-dose dioscin, as indicated by an up-regulated expression of LC3-II and beclin-1 proteins. Blockade of autophagy with bafilomycin A1 or 3-methyladenine sensitized the A549 and H1299 cells to apoptosis. Treatment of A549 and H1299 cells with dioscin caused a dose-dependent increase in ERK1/2 and JNK1/2 activity, accompanied with a decreased PI3K expression and decreased phosphorylation of Akt and mTOR. Taken together, this study demonstrated for the first time that autophagy occurred earlier than apoptosis during dioscin-induced human lung cancer cell line apoptosis. Dioscin-induced autophagy via ERK1/2 and JNK1/2 pathways may provide a protective mechanism for cell survival against dioscin-induced apoptosis to act as a cytoprotective reaction.