Biseugenol (Eug) is known to antiproliferative of cancer cells; however, to date, the antiperitoneal dissemination effects have not been studied in any mouse cancer model. In this study, Aryl hydrocarbon receptor (AhR) expression was associated with lymph node and distant metastasis in patients with gastric cancer and was correlated with clinicolpathological pattern. We evaluated the antiperitoneal dissemination potential of knockdown AhR and Biseugenol in cancer mouse model and assessed mesenchymal characteristics. Our results demonstrate that tumor growth, peritoneal dissemination and peritoneum or organ metastasis implanted MKN45 cells were significantly decreased in shAhR and Biseugenol-treated mice and that endoplasmic reticulum (ER) stress was caused. Biseugenol-exposure tumors showed acquired epithelial features such as phosphorylation of E-cadherin, cytokeratin-18 and loss mesenchymal signature Snail, but not vimentin regulation. Snail expression, through AhR activation, is an epithelial-to-mesenchymal transition (EMT) determinant. Moreover, Biseugenol enhanced Calpain-10 (Calp-10) and AhR interaction resulted in Snail downregulation. The effect of shCalpain-10 in cancer cells was associated with inactivation of AhR/Snail promoter binding activity. Inhibition of Calpain-10 in gastric cancer cells by short hairpin RNA or pharmacological inhibitor was found to effectively reduced growth ability and vessel density in vivo. Importantly, knockdown of AhR completed abrogated peritoneal dissemination. Herein, Biseugenol targeting ER stress provokes Calpain-10 activity, sequentially induces reversal of EMT and apoptosis via AhR may involve the paralleling processes. Taken together, these data suggest that Calpain-10 activation and AhR inhibition by Biseugenol impedes both gastric tumor growth and peritoneal dissemination by inducing ER stress and inhibiting EMT.

BACKGROUND We have shown previously that honokiol (HNK), a bioactive component of the medicinal plant Magnolia officinalis, inhibits growth of human prostate cancer cells in vitro and in vivo. However, the effect of HNK on androgen receptor (AR) signaling is not known. METHODS LNCaP, C4-2, and TRAMP-C1 cells were used for various assays. Trypan blue dye exclusion assay or clonogenic assay was performed for determination of cell viability. The effects of HNK and/or its analogs on protein levels of AR and its target gene product prostate specific antigen (PSA) were determined by western blotting. RNA interference of p53 was achieved by transient transfection. Reverse transcription-polymerase chain reaction was performed for mRNA expression of AR. Nuclear translocation of AR was visualized by microscopy. Apoptosis was quantified by DNA fragmentation assay or flow cytometry after Annexin V-propidium iodide staining. RESULTS HNK and its dichloroacetate analog (HDCA) were relatively more effective in suppressing cell viability and AR protein level than honokiol epoxide or biseugenol. Nuclear translocation of AR stimulated by a synthetic androgen (R1881) was markedly suppressed in the presence of HNK. Downregulation of AR protein resulting from HNK exposure was attributable to transcriptional repression as well as proteasomal degradation. HNK-mediated suppression of AR protein was maintained in LNCaP cells after knockdown of p53 protein. HNK-induced apoptosis was not affected by R1881 treatment. CONCLUSIONS The present study demonstrates, for the first time, that HNK inhibits activity of AR in prostate cancer cells regardless of the p53 status.

Angiogenesis is regulated by hyperglycemic conditions, which can induce cellular stress responses, reactive oxygen species (ROS), and anti-oxidant defenses that modulate intracellular signaling to prevent oxidative damage. The RUNX2 DNA-binding transcription factor is activated by a glucose-mediated intracellular pathway, plays an important role in endothelial cell (EC) function and angiogenesis, and is a target of oxidative stress. RUNX2 DNA-binding and EC differentiation in response to glucose were conserved in ECs from different tissues and inhibited by hyperglycemia, which stimulated ROS production through the aldose reductase glucose-utilization pathway. Furthermore, the redox status of cysteine and methionine residues regulated RUNX2 DNA-binding and reversal of oxidative inhibition was consistent with an endogenous Methionine sulfoxide reductase-A (MsrA) activity. Low molecular weight MsrA substrates and sulfoxide scavengers were potent inhibitors of RUNX2 DNA binding in the absence of oxidative stress, but acted as antioxidants to increase DNA binding in the presence of oxidants. MsrA was associated with RUNX2:DNA complexes, as measured by a sensitive, quantitative DNA-binding ELISA. The related RUNX2 protein family member, RUNX1, which contains an identical DNA-binding domain, was a catalytic substrate of recombinant MsrA. These findings define novel redox pathways involving aldose reductase and MsrA that regulate RUNX2 transcription factor activity and biological function in ECs. Targeting of these pathways could result in more effective strategies to alleviate the vascular dysfunction associated with diabetes or cancer.