The acyclic diterpenoid acid geranylgeranoic acid (GGA) has been reported to induce autophagic cell death in several human hepatoma-derived cell lines; however, the molecular mechanism for this remains unknown. In the present study, several diterpenoids were examined for ability to induce XBP1 splicing and/or lipotoxicity for human hepatoma cell lines. Here we show that three groups of diterpenoids emerged: 1) GGA, 2,3-dihydro GGA and 9-cis retinoic acid induce cell death and XBP1 splicing; 2) all-trans retinoic acid induces XBP1 splicing but little cell death; and 3) phytanic acid, phytenic acid and geranylgeraniol induce neither cell death nor XBP1 splicing. GGA-induced ER stress/ unfolded protein response (UPR) and its lipotoxicity were both blocked by co-treatment with oleic acid. The blocking activity of oleic acid for GGA-induced XBP1 splicing was not attenuated by methylation of oleic acid. These findings strongly suggest that GGA at micromolar concentrations induces the so-called lipid-induced ER stress response/UPR, which is oleate-suppressive, and shows its lipotoxicity in human hepatoma cells.
Mutant p53 proteins in human hepatoma cell lines such as HuH-7 (Y220C) and PLC/PRF/5 (R249S) accumulate in the cytoplasm, and lose their transcriptional function. Geranylgeranoic acid (GGA) is a naturally occurring acyclic diterpenoid that induces cell death in both cell lines, but not in HepG2 cells harboring wild-type p53. Here, we demonstrate that micromolar concentrations of GGA induce a rapid nuclear translocation of cytoplasmic p53 in both p53-mutant cell lines and p53 knockdown attenuates GGA-induced cell death in HuH-7 cells. Cell-free experiments demonstrate that GGA is able to release 670-kD p53-containing complexes from putative huge macromolecular aggregates in post-mitochondrial fractions as revealed on blue-native gradient PAGE. Among several p53-target genes tested, GGA upregulates PUMA gene expression, and ivermectin, an inhibitor for importin α/β, blocks GGA-induced nuclear translocation of cytoplasmic p53 and suppresses GGA-induced upregulation of PUMA mRNA levels in HuH-7 cells. Taken together, these data suggest that GGA treatment stimulates a nuclear translocation of mutant p53 through its dissociation from cytoplasmic aggregates, which may be essential for GGA-induced cell death.
Metabolic alternation in cancer cells is one of the most common characteristics that distinguish malignant cells from normal cells. Many studies have explained the Warburg hypothesis that cancer cells obtain more energy from aerobic glycolysis than mitochondrial respiration. Here, we show that a branched-chain C-20 polyunsaturated fatty acid, geranylgeranoic acid (GGA), induces upregulation of the cellular protein levels of TP53-induced glycolysis and apoptosis regulator (TIGAR) and synthesis of cytochrome c oxidase 2 (SCO2) in human hepatoma-derived HuH-7 cells harboring the mutant TP53 gene, suggesting that GGA may shift an energetic state of the tumor cells from aerobic glycolysis to mitochondrial respiration. In addition, UPLC/TOF/MS-based metabolomics analysis supported the GGA-induced energetic shift, as it revealed that GGA induced a time-dependent increase in the cellular contents of fructose 6-phosphate and decrease of fructose 1,6-diphosphate. Furthermore, metabolomics analysis revealed that GGA rapidly induced spermine accumulation with slight decrease of spermidine. Taken together, the present study strongly suggests that GGA may shift HuH-7 cells from aerobic glycolysis to mitochondrial respiration through the immediate upregulation of TIGAR and SCO2 protein levels.
Background and Aim: Geranylgeranoic acid (GGA) is a natural polyprenoic acid that is expected to prevent a second primary hepatoma. In human hepatoma-derived HuH-7 cells, GGA induces cell death as well as the initial phase of autophagy, but blocks the maturation process of autolysosomes, which may be linked to cell death. However we have not yet revealed the molecular mechanism of GGA-induced initial autophagy. In this meeting, we present a possible mechanism of GGA-induced initial autophagy, which may involve mTOR signaling pathway and unfolded protein response (UPR). Methods: To analyze the cellular process of autophagy, we observed multicolor fluorescent puncta by live-cell imaging in HuH-7/mRFP-GFP-LC3 cells after GGA (10 μM) treatment. We also examined cellular protein levels of phospho-mTOR (Ser2448 or Ser2481), mTOR, Raptor, Rictor, phospho-AKT, Beclin1, LC3β and FoxO1 by western blotting. The cellular levels of XBP1 mRNA splicing, CHOP and PDIA4 mRNAs were measured by RT-qPCR to estimate UPR. Results: In HuH-7 cells, phospho-mTOR (Ser2448) protein expression level was high under normal culture condition. GGA treatment further upregulated phospho-mTOR (Ser2448) and induced a transient surge of phospho-mTOR (Ser2481) at 30 min. However from 1 h to 8 h after GGA treatment, phosphorylation of mTOR was suppressed down to sub-basal levels. The similar up-and-down movements were observed also in the cellular levels of mTOR, Raptor and Rictor, and at 4 h these proteins became undetectable. However phospho-AKT levels did not show significant change after GGA treatment. Rapid and dramatic induction of XBP1 splicing was observed after GGA treatment. In other words, after 15 min of GGA treatment, the upregulation of XBP1s mRNA (spliced form) was observed and the upregulation continued until 4 h. In contrast, XBP1u mRNA (unspliced form) was downregulated in a time dependent manner until 4 h. However at 24 h, these changes were reversed and XBP1u became dominant. CHOP mRNA was upregulated after 1 h GGA treatment and the upregulation continued to 4 h. The cellular level of PDIA4 mRNA was not changed by GGA. The cellular protein levels of Beclin1 and FoxO1 were upregulated by GGA treatment. The accumulations of LC3β-II and autophagosome were also induced with GGA. GGA-induced accumulation of LC3β-II was partially attenuated by co-treatment with 4μ8C, a specific inhibitor of IRE1 ribonuclease activity. Discussion: In the literature, it has been repeatedly reported that signaling pathway of mTOR is activated at a basal level in HuH-7 cells. Therefore, we were interested what would happen on mTOR signaling when GGA induced initial autophagy in HuH-7 cells. As a result, signaling pathway of mTOR, either mTORC1 or mTORC2, was unexpectedly further upregulated by GGA treatment, but this upregulation at 30 min was transient. After 1-h treatment, GGA induced rapid unfolded protein response (UPR), which may trigger suppression of mTOR signaling. Hence, we speculate that UPR-mediated suppression of mTOR signaling may provide permissive effects on GGA-induced initial autophagy. Citation Format: Chieko Iwao, Yoshihiro Shidoji. Geranylgeranoic acid (GGA) induced autophagic cell death through induction of unfolded protein response (UPR) and suppression of mTOR signaling pathway. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A18.
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