Autophagy is a cellular process for the removal of abnormal intracellular organelles, pathogens and the recycling of cytoplasmic material to obtain energy (24
). This pathway also has been involved in immune responses, including antigen processing and presentation pathway (20
). Autophagy mediated intracellular lysosome-dependent degradation of abnormal cellular macromolecules and pathogens, and resulted in the accumulation of autophagolysosomes in the cytosol (24
). During the formation of autophagolysosome, LC3 and ATG12, two ubiquitin-like protein systems, play an important role for the autophagosomal membrane formation and expansion (26
). The conversion of soluble LC3-I to the membrane bound LC3 II is often used to monitor autophagy activation in the cell and GFP-LC3 constructs have been used successfully to track autophagy activation in vivo
and in vitro
). In the current study, we adopted GFP-LC3 mice to monitor whether oral administration of α-Mangostin would induce autophagy activation, and the increased GFP dots in intestinal epithelium suggesting the activation of autophagy.
The unfolded protein response (UPR) is an evolutionary conserved process to ER stress induced by accumulation of misfolded proteins in ER lumen (27
). UPR pathway is known to be important to the development and survival of cells, especially for highly secretory cells or cells that synthesize large quantities of proteins (27
). In mammals, the UPR is mediated by three transmembrane proteins that reside in the ER (27
): PKR-like ER-resident kinase (PERK), activated transcription factor 6 (ATF6) and inositol requiring enzyme 1 (IRE1) pathways. The accumulation of misfolded proteins within the ER attracts the chaperone protein, Grp78 and activates UPRs to facilitate protein folding, attenuate translation, and activate ER-associate degradation (ERAD) pathway. Briefly, ER stress-mediated dissociation of PERK from Grp78 leads to transphosphorylation of PERK and subsequently increases phosphorylation of eIF2α, which attenuate translation of CAP-dependent protein. At the same time, phosphorylated eIF2α induces transcription of ATF4 which activates a number of UPR genes including Grp78, CHOP and GADD34. ATF6 upon release from a Grp78 translocates to the Golgi and then induces transcription of several UPR genes including X-box protein 1 (XBP1), CHOP and GRP78. The most evolutionary conserved of the UPRs is IRE1. Activated IRE1 can splice the Xbp1 mRNA into the active form of Xbp1 (XBP1s) which regulates important genes involved in ER translocation, expansion and ERAD activation. Activated IRE1 also has kinase activity, and signals through TRAF2 that in turn activates JNK and its downstream targets that could promote inflammatory response and/or apoptosis. In the current study, we used XBP-1 reporter which express GFP fluorescence upon XBP-1 splicing by IRE-1 during ER stress. In addition, we detected phosphorylation of eIF2α to assess alteration of ER stress status in cells treated with thapsigargin and α-Mangostin. Consequently, we found that ER stress induced by thapsigargin can be reduced by cotreatment of α-Mangostin. In addition, although α-Mangostin was shown to inhibit SAECA like thapsigargin, it did not induce ER stress under concentration we used. Since thapsigargin specifically inhibits the fusion of autophagosome with lysosome, the last step in autophagic pathway (3
), it might induce the accumulation of ER stress by the defect in autophagic elimination of ER stress.
Interestingly, recent studies suggested that UPR mediated by ER stress was known to be linked with autophagy activation (6
). The activation of autophagy pathway by ER stress includes the following pathways: IRE1-mediated JNK activation and PERK/eIF2α phosphorylation (6
). Although both of UPR and autophagy are important for cellular homeostasis, unfolded protein responses triggered by ER stress have been shown in several tumors, particularly in area with hypoxia, and they help tumor cells survive in stressful tumor microenvironment (8
). Thus, the outcome of autophagy activation by small molecule drugs inducing autophagy activation on the survival of tumor was difficult to predict. Indeed, there was controversy about the pro-apoptotic or anti-apoptotic roles of autophagy (6
). Further, in several recent experiments done by others, it was suggested that ER stress can induce autophagy and the induction of autophagy by ER stress is beneficial to relieve ER stress and consequently ameliorate the disease progress (23
). However, the relationship between the two processes has not been fully elucidated yet. In the current study, activation of autophagy by α-Mangostin significantly inhibited tumor growth, suggesting it exerted proapoptotic function against tumor cells. Likewise, thapsigargin, a well-known ER stress inducer, which was recently proven to block autophagy flow, completely blocked the anti-tumor activity by α-Mangostin. Collectively, these results suggest that autophagy activation by α-Mangostin might be important for the successful antitumor activity.