Cytotoxicity |
➢ 0.5-10 µM dGGA induced cell death (trypan blue) in HuH-7 cells in 16 h (Nakamura 1995) [ 40].
➢ 10 µM GGA induced cell death (trypan blue) in HuH-7 cells at 16 h, but not in normal hepatocytes (Shidoji 1997) [ 18].
➢ Cell death in HuH-7 cells induced by 10 µM GGA was prevented by co-treatment with 100 µM α-tocopherol (Shidoji 1997) [ 18].
➢ Cell death in HuH-7 cells induced by 50 µM GGA was prevented by co-treatment with 25 µM oleic acid (OA) (Iwao 2015) [ 15].
➢ Cell death in HuH-7 cells induced by 20 µM GGA was prevented by pretreatment with TLR4 siRNA or VIPER, a TLR4 inhibitor (Yabuta 2020) [ 16].
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➢ 500-3000 µM PA induced cell death (MTT) in HepG2 cells in 24 h (Saha 2022) [ 45].
➢ 500-1000 µM PA induced cell death (SYTOX Green) in HepG2 cells in 24 h, which was suppressed by co-treatment with the mitochondrial ROS quencher, MitoTEMPO, or the mPT inhibitor, olesoxime (Oh 2023) [ 46].
➢ 200 & 400 µM PA induced cell death in HepG2 cells in 24 h (Li 2021) [ 43], which was suppressed by co-treatment with OA (Zeng 2020) [ 44].
➢ 1000 and 2000 µM PA induced hypoxia-dependent cell death in HepG2 cells in 24 h (Hwang 2015) [ 11].
➢ 200 µM PA induced hypoxia-dependent cell death in HepG2 and Hep3B cells in 48 h, which was enhanced by co-treatment with L-carnitine (Matsufuji 2023) [ 12].
✧1000 µM PA induced TLR4-dependent inflammation in primary macrophages in 4 h (Lancaster 2018) [51]. |
➢ 10-20 µM ATRA or 9CRA induced no cell death in HuH-7 cells in 24 h (Nakamura 1995) [ 40].
➢ 1-10 µM ATRA or 9CRA decreased cell viability (trypan blue) in Hep3B cells (Hsu 1998) [ 38].
➢ 10 µM ATRA inhibited the proliferation of Hepa1-6 and HepG2 cells in 5 days (Fang 2020) [ 39].
➢ 5-20 µM ATRA protected HepG2 or HuH-7 cells and 1-5 µM ATRA protected Hep3B cells from serum starvation-induced cell death in 5-10 days (Wang 2013) [ 42].
➢ 0.1-10 µM ATRA did not inhibit the proliferation of HepG2 or Hep3B cells in 3 days (Lin 2005) [ 41].
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Mitochondrial morphology |
- a perinuclear clustering of mitochondria (Okamoto 2011) [14]. - a fragmentation of mitochondria (unpublished, Shidoji). |
➢ 200 µM PA induced mitochondrial fragmentation in HepG2 cells in 2 h (Eynaudi 2021) [ 54].
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- reduced the number of mitochondria (Sun 2022) [49]. - reduced mitochondrial volume (Sun 2022) [49]. - reduced or eliminated mitochondrial cristae (Sun 2022) [49]. ✧1 µM ATRA did not alter the mitochondrial mass for 24-72 h in the acute-promyelocytic-leukemia (APL)-derived NB4 cell line (Gianni 2022) [55]. |
Mitochondrial membrane potential (ΔΨm) |
- a dissipation of Rhodamine 123 fluorescence 1 h, which was prevented by cotreatment with 100 µM α-tocopherol (Shidoji 1997) [18]. - a dissipation of MitoTracker Red fluorescence in 2 h (Okamoto 2011) [14]. |
➢ 500 µM PA induced dissipation of TMRE red fluorescence (mitochondrial permeability transition or mPT) in HepG2 cells in 24 h, which was abrogated by cotreatment with BAPTA-AM chelating intracellular Ca 2+ (Oh 2023) [ 46].
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✧1 µM ATRA reduced Mito-Tracker Red/Green fluorescence in NB4 cells for 24-96 h (Gianni 2022) [55]. |
Mitochondrial ROS production |
- MitoSox Red fluorescence of hepatoma cells in 15 min, which was prevented by cotreatment with wortmannin (Okamoto 2011) [14]. - MitoSox Red fluorescence of hepatoma cells in 2 h, which was prevented with either 50 µM oleic acid (OA), 100 µM α-tocopherol or VIPER (Yabuta 2020) [16]. |
➢ 500-1000 µM PA upregulated MitoSox fluorescence in HepG2 cells in 24 h, which was suppressed by cotreatment with MitoTEMPO, a mitochondrial ROS quencher (Oh 2023) [ 46].
➢ 500 µM PA increased ROS production at 3 h in both mouse primary hepatocytes and mouse hepatocyte-derived AML12 cells (Huang 2023) [ 56].
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- H2-DCF-DA fluorescence (Sun 2022) [49]. - lipid peroxides (C11-BODIPY fluorescence) (Sun 2022) [49]. |
ER stress response (UPRER) |
- translational downregulation of cyclin D1 (CCND1) in 30 min (Shimonishi 2012) [17]. - splicing of XBP1 mRNA in 15 min (Iwao 2015) [15], which was prevented by cotreatment with either 50 µM OA or VIPER, but not with 100 µM α-tocopherol (Yabuta 2020) [16]. - accumulation of XBP1 protein in the nucleus in 8 h (Iwao 2015) [15]. - upregulation of DDIT3 (CHOP) mRNA in 8 h (Iwao 2015), which was prevented by cotreatment with 50 µM OA or VIPER, but not with 100 µM α-tocopherol (Yabuta 2020) [16]. |
- splicing of XBP1 mRNA in 16 h (Qi 2015) [58]. - upregulation of DDIT3 (CHOP) protein in 16 h (Qi 2015) [58]. - phosphorylation of IRE1α in 8 h (Qi 2015) [58].
➢ 400 µM PA enhanced expressions of DDIT3 ( CHOP) mRNA after 24 h in HepG2 cells, which was prevented by co-treatment with 200 µM OA (Zeng 2020) [ 44].
➢ 500 µM PA enhanced cellular expression of p- PERK, ATF4, p- eIF2α, DDIT3 ( CHOP), and TXNIP in both mouse primary hepatocytes and mouse hepatocyte-derived AML12 cells (Huang 2023) [ 56].
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➢ 10 - 20 µM ATRA induced splicing of XBP1 mRNA in 1 h in HuH-7 cells (Iwao 2015) [ 15].
➢ 10 - 20 µM ATRA induced accumulation of XBP1 protein in the nucleus of HuH-7 cells in 8 h (Iwao 2015) [ 15].
➢ ✧In P19 embryonic carcinoma cells, ATRA induced the upregulation of several UPR-related genes ( Atf6, Xbp1, Chop) (Saito 2023) [ 59].
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Pyroptosis |
- nuclear translocation of cytoplasmic NF-κB in 3 h, which was blocked by cotreatment with 50 µM OA, 100 µM α-tocopherol, BI605906 or VIPER (Yabuta 2020) [16]. - upregulation of TLR2 expression in 3 h, which was prevented with 50 µM OA or VIPER (Yabuta 2020) [16]. - upregulation of NLRP3 expression in 3 h, which was prevented with 50 µM OA, 100 µM α-tocopherol or VIPER (Yabuta 2020) [16]. - appearance of GSDMD-N terminal fragment in 1 h (Yabuta 2020) [16]. - localization of GSDMD to the plasma membrane in 3 h, which was prevented by co-treatment with VIPER (Yabuta 2020) [16].
➢ Cell death induced by 10 µM GGA was completely prevented by pre-treatment with acYVAD-cmk, a specific inhibitor against CASP1 (Shidoji 1997) [ 18].
➢ 20 µM GGA induced activation of CASP1 activity in 8 h, which was prevented by Z-LEVD-fmk, a CASP4-specific inhibitor (Yabuta 2020) [ 16].
➢ 20 µM GGA induced rapid and transient activation of CASP4 from 1 h to 5 h (Yabuta 2020) [ 16].
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➢ 300 µM PA induced nuclear NF-κB activity in HepG2 cells in 15 min (Joshi-Barve 2007) [ 63].
➢ 400 µM PA induced nuclear translocation of NF-κB in HepaRG cells in 40 min (Sharifnia 2015) [ 64].
➢ 400 µM PA increased mRNA and protein expressions of inflammasome marker NLRP3, CASP1, and IL-1β, as well as GSDMD after 24 h in HepG2 cells (Zeng 2020) [ 44].
➢ 1000 µM PA in normal human liver LO2 cells for 24 h induced activation of CASP1, release of IL-1β, and GSDMD-NT (Yao 2022) [ 78].
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➢ 10 µM ATRA increased the content of Fe 2+ in both HepG2 and Hep3B cells (Sun 2022) [ 49].
➢ 10 µM ATRA downregulated the expression of GPX4 and FTH1 proteins in both HepG2 and Hep3B cells (Sun 2022) [ 49].
➢ 5 µM ATRA upregulated one of the ATRA-responsive genes, OTUD7B, which inhibits NF-κB activity in HepG2 and HuH-7 cells in 6 h (Kanki 2013) [ 47].
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Cytoplasmic Ca2+
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➢ 10 µM GGA induced a transient increase of cytoplasmic-free Ca 2+ in HuH-7 cells at 20 min and 6 h (Yabuta 2020) [ 16].
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➢ 500-1000 µM PA induced lysosomal Ca 2+ release to increase cytosolic Ca 2+ in HepG2 cells in 6 h, which was suppressed by cotreatment with MitoTEMPO, a mitochondrial ROS quencher (Oh 2023) [ 46].
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➢ 1-10 µM ATRA increased [Ca 2+]i from 24 to 48 h in HepG2 cells (Wei 2014) [ 65].
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LysoPLs |
- upregulation of a group of lysophospholipids including lysophosphatidylcholine (lysoPC) with C16:0, C20:4, or C20:3 fatty acids in 24 h (Shidoji 2021) [19]. - the most rapid (2 h) upregulation of lysoPC (C20:4) and lysophosphatidylethanolamine (C20:4) (Shidoji 2021) [19]. |
➢ 40 µM lysoPC induced cell death in human primary hepatocytes in 24 h to the same extent as 400-800 µM PA-induced cell death (Han 2008) [ 69].
➢ 42.5-85 µM lysoPC induced cell death both in HuH-7 and human primary hepatocytes in 16 h to the same extent as 800 µM PA-induced cell death (Kakisaka 2012) [ 79].
➢ 40-80 µM lysoPC induced cell death in HepG2 and HuH-7 cells in 18 h (Chen 2022) [ 80].
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✧Liver lysoPC (16:1 or 18:1) was decreased by oral administration (10 mg/kg, 1 wk) of a synthetic ligand (AM580) for RAR to mice and increased by oral administration (30 mg/kg, 1 wk) of a synthetic ligand for RXR (LG268) to female C57BL6 mice (Weiss 2011) [70]. |
Autophagy |
- massive accumulation of GFP-LC3-labeled autophagosomes (Okamoto 2011) [14]. - time-dependent accumulation of LC3β-II and p62/SQSTM (Okamoto 2011) [14], which was not prevented by co-treatment with 4μ8C (IRE1 Inhibitor III) that inhibits XBP1 mRNA splicing (Iwao 2015) [15]. - upregulation of ATG4B and BECN1 in 30 min (Okamoto 2011) [14]. - nuclear translocation of cytoplasmic p53 in 3 h (Iwao 2014) [62]. |
➢ 500 µM PA impaired autophagic flux indicated by accumulation of LC3β-II and p62/SQSTM through downregulation of DDX58 in HepG2 cells (Frietze 2022) [ 71].
➢ 500-1000 µM PA induced dysfunction of lysosomes in HepG2 cells in 6 h (Oh 2023) [ 46].
✧100 µM PA reduced autophagic flow in embryonic mouse hypothalamus cell line N43/5 (Hernandez 2019) [75]. |
- up-regulation of LC3-II and decrement of p62/SQSTM (Wang 2021) [48].- up-regulation of ATG7 protein and ATG7 mRNA (Wang 2021) [48].
➢ The downregulation of ATG7 gene expression by siRNA enhanced 40 µM ATRA-induced cell death in hepatoma cells (Wang 2021) [ 48].
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CASP3 |
➢ Cell death induced by 10 µM GGA was delayed by pre-treatment with acDEVD-CHO, a specific inhibitor against CASP3 (Shidoji 1997) [ 18].
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➢ 500 µM PA increased activation of CASP3 in mouse hepatocyte-derived AML12 cells and mouse primary hepatocytes in 24 h (Huang 2023) [ 56].
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➢ 40 µM ATRA induced activation of CASP3 activity in PLC/PRF/5 and HLE cells in 24 h (Wang 2021) [ 48].
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