All-Trans Retinoic Acid Induces Apoptosis In N-Acetylglucosaminyltransferase V Repressed Human Hepatocarcinoma Cells Via Endoplasmic Reticulum Stress

N-Acetylglucosaminyltransferase V (GnT-V), which localizes in Golgi appatatus, is a key enzyme in the processing of asparagine-linked glycans (N-glycans) during the synthesis of glycoproteins. It is well known as a molecule that is associated with tumorigenesis of normal cells and metastasis of tumor cells. We previously demonstrated that GnT-V repression sensitized the human hepatocarcinoma SMMC7721 cells to apoptosis induced by all-trans retinoic acid (ATRA). Furthermore, endoplasmic reticulum (ER) stress was triggered in GnT-V repressed SMMC7721 cells. In the present study, we further investigate the molecular mechanism by which ATRA induces apoptosis in GnT-V repressed SMMC7721 cells.We previously found that chronic ER stress was induced in GnT-V-AS/7721 cells (SMMC7721 cells transfected with antisense cDNA of GnT-V). In the first part of this work, we report that ATRA induces apoptosis in GnT-V-AS/7721 cells via ER stress. We show here that ER stress is intensified in GnT-V-AS/7721 cells with 80μM ATRA treatment for 24 h, which is evidenced by the increase of GRP78/Bip (glucose regulated protein 78/immunoglobulin chain binding protein), CHOP (C/EBP homologus protein) and spliced XBP1 (X box binding protein 1). Additionally, activation of caspase-12, caspase-9, and-3 was detected, and apoptosis morphology was observed in GnT-V-AS/7721 cells with ATRA treatment. These results suggest that ATRA intensifies the ER stress triggered in GnT-V-AS/7721 cells, which represents a novel mechanism of ATRA to induce apoptosis. We further observed that GnT-V was significantly repressed in GnT-V-AS/7721 cells with 80μM ATRA treatment for 24 h, suggesting that repression of GnT-V by ATRA causes the intensified ER stress and ER stress-mediated apoptosis in GnT-V-AS/7721 cells.In the second part of this work, we confirm the findings that ATRA induces apoptosis in GnT-V-AS/7721 cells via ER stress. Additionally, we found that ATRA repressed the expression of betaine-homocysteine methyltransferase (BHMT) and cystathionine-β-synthase (CBS), which are key enzymes that are involved in homocysteine metabolism, increased the level of intracellular homocysteine, and decreased the glutathione (GSH) level in GnT-V-AS/7721 cells. To investigate the effect of ATRA on homocysteine metabolism, cells were challenged with exogenous homocysteine. In GnT-V-AS/7721 cells with ATRA treatment, a significant elevation of intracellular homocysteine levels suggests that ATRA perturbs homocysteine metabolism in GnT-V-AS/7721 cells and, therefore, sensitizes the cells to homocysteine-induced ER stress. An obvious increase in the levels of GRP78/Bip protein and spliced XBP1 mRNA were observed. Furthermore, we observed that ATRA blunted the homocysteine-induced increase of GSH only in GnT-V-AS/7721 cells. These results demonstrate that ATRA intensifies ER stress and induces apoptosis in GnT-V-AS/7721 cells by disturbing homocysteine metabolism through the down-regulation of CBS and BHMT, depleting the cellular GSH and, in turn, altering the cellular redox status. In addition, we showed that ATRA did not trigger ER stress, induce apoptosis, or affect homocysteine metabolism in L02 cells, which is a cell type that is derived from normal liver tissue. These results provide support for the hypothesis that ATRA is an anticancer agent.In summary, ATRA perturbs the homocysteine metabolism in GnT-V-AS/7721 cells and in turns, increases intracellular homocysteine levels and depletes the cells of GSH. Thus, ATRA intensifies ER stress and induces apoptosis in GnT-V-AS/7721 cells.