Apoptotic effect of 3,4-dihydroxybenzoic acid on human gastric carcinoma cells involving JNK/p38 MAPK signaling activation.

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.