Abstract
BackgroundGambogic acid (GA) was extracted from the dried yellow resin of gamboge (Garcinia hanburyi) which is traditionally used as a coloring material for painting and cloth dying. Gamboge has been also used as a folk medicine for an internal purgative and externally infected wound. We focused on the mechanisms of apoptosis induction by GA through the unfold protein response (ER stress) in HeLa cells.MethodsThe cytotoxic effect of GA against HeLa cells was determined by trypan blue exclusion assay. Markers of ER stress such as XBP-1, GRP78, CHOP, GADD34 and ERdj4 were analyzed by RT-PCR and Real-time RT-PCR. Cell morphological changes and apoptotic proteins were performed by Hoechst33342 staining and Western blotting technique.ResultsOur results indicated a time- and dose-dependent decrease of cell viability by GA. The ER stress induction is determined by the up-regulation of spliced XBP1 mRNA and activated GRP78, CHOP, GADD34 and ERdj4 expression. GA also induced cell morphological changes such as nuclear condensation, membrane blebbing and apoptotic body in Hela cells. Apoptosis cell death detected by increased DR5, caspase-8, −9, and −3 expression as well as increased cleaved-PARP, while decreased Bcl-2 upon GA treatment. In addition, phosphorylated JNK was up-regulated but phosphorylated ERK was down-regulated after exposure to GA.ConclusionsThese results suggest that GA induce apoptosis associated with the ER stress response through up-regulation of p-JNK and down-regulation of p-ERK in HeLa cells.
Highlights
Gambogic acid (GA) was extracted from the dried yellow resin of gamboge (Garcinia hanburyi) which is traditionally used as a coloring material for painting and cloth dying
We have shown for the first time that GA can induce apoptosis in cervical cancer HeLa cells associated with the endoplasmic reticulum (ER) stress response by up-regulation of C/ EBP homologous protein (CHOP), p-Jun N-terminal kinase (JNK) and down-regulation of p-extracellular regulated protein kinase (ERK)
The result showed GA induced X-box protein 1 (XBP1) splicing at 4 h and the XBP1 mRNA was elevated by approximately 6 fold compared to the level observed in control cells (0 h) (Figure 2)
Summary
Gambogic acid (GA) was extracted from the dried yellow resin of gamboge (Garcinia hanburyi) which is traditionally used as a coloring material for painting and cloth dying. The ER stress response is regulated by three ER transmembrane receptors; pancreatic ER kinase (PKR)-like ER kinase (PERK), inositol requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6) [13]. These ER transmembrane proteins are kept in an inactive state through their association with the ER chaperone BiP/ GRP78 (glucose-related protein, 78kD). Activated PERK blocks general protein synthesis by phosphorylating eukaryotic initiation factor 2 (eIF2α) This phosphorylation enables translation of ATF4 which translocates to the nucleus and induces the transcription of genes required to restore ER homeostasis. It has been showed that CHOP is a critical ER stress-induced apoptosis molecule through regulating the expression of Bcl, Bim and DR5 [19,20]
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