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Objective: Choriocarcinoma is the most common epithelial cancer among gestational trophoblastic diseases (GTDs); the mechanism of trophoblastic carcinogenesis is unknown. This study aimed to examine the expression of NF-κB family proteins in GTDs and placental tissues as well as the roles of c-Rel in choriocarcinoma. Methods: We examined the expression of NF-κB family proteins in normal placenta and hydatidiform mole tissues as well as extravillous trophoblast (EVT) and choriocarcinoma cell lines by Western blot and immunohistochemistry. Immunoprecipitation was performed to determine which proteins can bind with c-Rel in choriocarcinoma cells. To investigate the roles of c-Rel in choriocarcinoma, we examined the effects of c-Rel knockdown and overexpression on cell proliferation, migration, and invasion using small interfering RNAs and gene activation plasmid. Results: The expression of c-Rel was strong in choriocarcinoma and EVTs, but very weak in villi of normal placenta and hydatidiform mole. Immunoprecipitation suggested that c-Rel heterodimerizes with p65 in choriocarcinoma. c-Rel knockdown reduced invasion, migration, and AKT phosphorylation in choriocarcinoma cells. c-Rel overexpression in choriocarcinoma increased migratory and invasive abilities, and the effect on invasion was inhibited by a PI3K inhibitor. Conclusion: These findings suggest that c-Rel might play a role in promoting the invasion of choriocarcinoma cells through PI3K/AKT signaling.

-Naturally occurring low-molecular weight compounds with a chemical structure like that of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, such as 1-benzyl-1,2,3,4-tetrahydroisoquinoline (1BnTIQ), are candidates for the endogenous neurotoxins that cause Parkinson's disease (PD). 1BnTIQ is an endogenous amine in human CSF and increases in the CSF of patients with PD. It inhibits complex I and elicits PD-like behavioral abnormalities in monkey and mouse. In this study, we searched metabolites of 1BnTIQ by rat liver S9 using liquid chromatography-tandem mass spectrometry, and identified a dehydrated metabolite, 1-benzyl-3,4-dihydroisoquinoline (1BnDIQ). 1BnDIQ was identified by corresponding mass spectra and precursor ion scans in authentic and complete enzyme samples. Multiple reaction monitoring analysis showed microsome-dependent 1BnDIQ production. We previously reported that 1BnDIQ is more toxic than 1BnTIQ in cytotoxicity study in SH-SY5Y neuroblastoma cells. In addition, 1BnTIQ is reported to pass through the blood-brain barrier of the rat brain, and 1BnDIQ is supposed to be more lipophilic than 1BnTIQ. 1BnDIQ may easily reach the brain, and it might contribute to PD-related neurotoxicity.

Abstract. Gestational trophoblastic neoplasia (GTN)results from the malignant transformation of placental trophoblasts which secrete human chorionic gonadotropin (hCG) as do normal placenta or hydatidiform mole. N-acetylglucosaminyltransferase IV (GnT-IV) is a glycosyltransferase which catalyses the formation of β1,4GlcNAc branches on the mannose core of N-glycans. Previous studies reported that β1,4GlcNAc branches on hCG were detected in GTN but not in normal pregnancy or hydatidiform mole. The aim of the present study was to understand the role of GnT-IVa in choriocarcinoma and find the target proteins for GnT-IVa glycosylation which contribute to the malignancy of choriocarcinoma. Immunohistochemistry showed that Griffonia simplicifolia lectin-II staining and GnT-IVa staining were intense in trophoblastic cells of invasive mole and choriocarcinoma. We established a choriocarcinoma cell line with GnT-IVa overexpression (Jar-GnT4a), and examined its malignant potential and target proteins for GnT-IVa glycosylation. GnT-IVa overexpression increased the cell migration and invasion (2.5-and 1.4-fold) as well as the ability to adhere to the extracellular matrix (ECM) components, including fibronectin and collagen type I and IV. The tumour formation potential of Jar-GnT4a in mice was significantly higher than that of control (P= 0.0407), and the cumulative survival rate of mice with Jar-GnT4a was relatively lower than those with control. Immunoprecipitation studies showed that β1,4GlcNAc branches of N-glycans on integrin β1 in choriocarcinoma cells were increased by GnT-IVa overexpression. Nano-LC/MS/MS analysis suggested that lysosome-associated membrane glycoprotein 2 (LAMP-2) was a target protein for glycosylation by GnT-IVa. The increase in β1,4GlcNAc branches on LAMP-2 by GnT-IVa overexpression was confirmed by lectin blot analysis using whole cell lysate and conditioned medium. Our results suggest that highly branched N-glycans generated by the action of GnT-IVa are present in trophoblastic cells of GTN in proportion to GnT-IVa expression level, and that GnT-IVa may contribute to the malignancy of choriocarcinoma by promoting cell adhesion, migration and invasion through glycosylation of integrin β1 and LAMP-2.