The Journal of Toxicological Sciences
Online ISSN : 1880-3989
Print ISSN : 0388-1350
ISSN-L : 0388-1350
Letter
Dihydropyrazine induces endoplasmic reticulum stress and inhibits autophagy in HepG2 human hepatoma cells
Shinji TakechiMadoka SawaiYuu Miyauchi
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2024 Volume 49 Issue 7 Pages 313-319

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Abstract

Dihydropyrazines (DHPs) are formed by non-enzymatic glycation reactions in vivo and in food. We recently reported that 3-hydro-2,2,5,6-tetramethylpyrazine (DHP-3), which is a methyl-substituted DHP, caused severe oxidative stress and cytotoxicity. However, the molecular mechanisms underlying the cytotoxic pathways of the DHP response remain elusive. Because oxidative stress induces endoplasmic reticulum (ER) stress and autophagy, we investigated the ability of DHP-3 to modulate the ER stress and autophagy pathways. DHP-3 activated the ER stress pathway by increasing inositol-requiring enzyme 1 (IRE1) and PKR-like ER kinase (PERK) phosphorylation and transcription factor 6 (ATF6) expression. Moreover, DHP-3 increased the expression of activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP), which are downstream targets of PERK. In addition, DHP-3 inhibited the autophagy pathway by increasing the accumulation of microtubule-associated protein 1 light chain 3 alpha-phosphatidylethanolamine conjugate (LC3-II) and p62/sequestosome 1 (p62), while decreasing autophagic flux. Taken together, these results indicate that DHP-3 activates the ER stress pathway and inhibits the autophagy pathway, suggesting that the resulting removal of damaged organelles is inadequate.

INTRODUCTION

The relationship between the accumulation of glycation products and lifestyle-related diseases has recently received substantial attention. Glycation products are formed non-enzymatically in vivo and in food via condensation between the carbonyl groups of reducing sugars and free amine groups, and they comprise a chemically diverse group of compounds. Glycation products are generally slow to develop, and they accumulate in the body with age. Additionally, their diverse and complex structures make their toxicity difficult to assess and predict.

Dihydropyrazines (DHPs) are formed by the dimerization of either D-glucosamine (Kashige et al., 1995) or 5-aminolevulinic acid (Teixeira et al., 2001), and do not belong to the category of advanced glycation end products (AGEs). DHPs are ubiquitous in the body, as many compounds with pyrazine skeletons, which are potential metabolites of DHPs, have been detected in foods (Joo and Ho, 1997; Maga, 1982) and human urine (Zlatkis et al., 1973). We previously reported that DHPs cause the production of various radicals (Yamaguchi et al., 2012), DNA strand breaks (Kashige et al., 2000; Yamaguchi et al., 1996), growth inhibition and mutagenesis in Escherichia coli (Takechi et al., 2004), and SH-compounds (cysteine, dithiothreitol (DTT), 2-mercaptoethanol, 2-mercaptoethylamine, and N-acetyl-cysteine) were found to suppress the inhibition of GAPDH by DHP-3 in vitro (Takechi et al., 2010). Experiments in mammalian cells revealed that 3-hydro-2,2,5,6-tetramethylpyrazine (DHP-3, Fig. 1) is highly cytotoxic, especially among methyl-substituted DHPs, and it induces cytotoxicity via oxidative stress (Ishida et al., 2012, 2014; Takechi et al., 2011, 2015) and exerts anti-inflammatory effects by suppressing Toll-like receptor 4 signaling (Esaki et al., 2020; Sawai et al., 2022). In a recent study, we compared the cytotoxicity of DHP-3 to other glycation products, including Nε-(carboxymethyl)-L-lysine (CML), an AGE, and acrylamide, a highly toxic glycation product noted for carcinogenicity but not AGE. The results demonstrated that the cytotoxicity of DHP-3 was higher than that of the other tested glycation products (Miyauchi et al., 2023). Furthermore, we observed that DHP-3 induces cytotoxicity independently of the receptor for AGE (RAGE) (Miyauchi et al., 2021). These properties are often associated with oxidative stress, but the molecular mechanisms underlying the cytotoxic pathways of the DHP response via oxidative stress have remained elusive.

Fig. 1

Chemical structure of DHP-3.

Oxidative stress can induce the unfolded protein response (UPR) to cope with ER stress (Rashid et al., 2015). The UPR is mediated by the ER chaperone protein, glucose-regulated protein 78/binding immunoglobulin protein (Grp78/Bip) and the ER-resident transmembrane sensor proteins inositol-requiring enzyme 1 (IRE1), PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF6) (Ron and Walter, 2007; Schröder, 2008). Each sensor triggers unique downstream responses when activated. IRE1 possesses both endoribonuclease and kinase domains that regulate X-box binding protein 1 (XBP1) processing and mitogen-activated protein kinase 8 (MAPK8/JNK1) activation, respectively (Urano et al., 2000). XBP1 functions as a potent transcription factor by binding to UPR elements and activating a broad spectrum of UPR-related genes (Lee et al., 2003). Activated MAPK8 controls cell survival and death by regulating autophagy and apoptosis (Urano et al., 2000). PERK phosphorylates eukaryotic translation initiation factor 2 subunit alpha to activate the transcription of specific gene subsets, including ATF4 and C/EBP homologous protein (CHOP) (Harding et al., 2000), which transcriptionally regulate autophagy-related genes (ATGs) (B’chir et al., 2013). ATF6 translocates to the Golgi, where it is proteolytically processed to release its cytosolic transcriptional activator domain, and then it translocates to the nucleus to activate ER stress target genes (Shen et al., 2002).

Autophagy is the major lysosomal degradation pathway that delivers proteins, cytoplasmic components through autophagosomes, and organelles to lysosomes for degradation and recycling. More than 30 ATGs controls autophagy, including ATG12 and ATG5, which complex with ATG16L to initiate autophagosome formation (Mizushima et al., 2003; Hanada et al., 2007; Matsushita et al., 2007). The ATG5-dependent conversion of microtubule-associated protein 1 light chain 3 (LC3/ATG8) from LC3-I (free form) to LC3-II (LC3-phosphatidylethanolamine conjugate: membrane-bound form) is a key step in the induction of autophagy (Mizushima et al., 2003; Hanada et al., 2007; Matsushita et al., 2007). ATF4 and CHOP have been shown to transcriptionally regulate more than a dozen ATG genes in addition to the p62/sequestosome 1 gene, including driving the expression of the autophagy proteins ATG12 and ATG5, respectively (B’chir et al., 2013). p62 can interact with multiple signaling molecules through several protein-protein interaction motifs, including the ubiquitin-associated domain (UBA) and the LC3-interacting region (LIR). p62 binds directly to ubiquitinated proteins through its UBA domain and acts as a receptor for the ubiquitinated proteins or organelles, in addition to binding to LC3 through the LIR. p62, a selective substrate for autophagy, serves as a link between LC3 and ubiquitinated substrates, and then p62, LC3, and ubiquitinated substrates are incorporated into the completed autophagosome and degraded in autolysosomes (Pankiv et al., 2007).

In this study, we hypothesized that the endoplasmic reticulum (ER) stress pathway and the autophagy pathway mediate DHP-3-induced cytotoxicity. The results reported in this study show that DHP-3 activates the ER stress pathway by increasing IRE1 and PERK phosphorylation and ATF6 expression, and by increasing the expression of ATF4 and CHOP, which are downstream targets of PERK. In addition, we found that DHP-3 inhibited the autophagy pathway by increasing LC3-II and p62 accumulation and reducing autophagic flux. These results indicate that DHP-3 activates the ER stress pathway and inhibits the autophagy pathway. It has been suggested that this leads to an inadequate removal of the damaged organelles or proteins and thus exerts a cytotoxic effect.

MATERIALS AND METHODS

Materials

DHP-3 was synthesized as previously described (Yamaguchi et al., 1996). Antibodies against IRE1 (sc-390960), PERK (sc-377400), phospho-PERK (sc-32577), LC3 (sc-271625), p62 (sc-28359), ATF6 (sc-166659), ATF4 (sc-390063), CHOP (sc-7351), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, sc-32233) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). An antibody against phospho-IRE (AP0878) was obtained from ABclonal (Wuhan, China). Horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin G (IgG) antibody (A4416) and HRP-conjugated goat anti-rabbit IgG peroxidase antibody (A6154) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Bafilomycin A1 was obtained from LKT Laboratories (Saint Paul, MN, USA). All other reagents and chemicals used were of the highest grade commercially available.

Cell culture

HepG2 cells (JCRB1054) were obtained from the Human Science Research Resources Bank (Osaka, Japan). Dulbecco’s modified Eagle medium (DMEM) was purchased from Wako (Osaka, Japan). Cells were cultured in DMEM supplemented with 10% (v/v) fetal bovine serum in a humidified atmosphere with 5% CO2 at 37°C. The cells were grown to 80%–90% confluence before being treated with various concentrations (100–400 µM) of DHP-3 in 0.1% (v/v) dimethyl sulfoxide (DMSO).

Immunoblotting assay

After treatment, the cells were lysed with RIPA buffer. Cell lysates were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The membrane was blocked with PVDF Blocking Reagent (Toyobo, Tokyo, Japan) and then incubated with primary antibodies. After washing with phosphate-buffered saline with 0.1% Tween 20, the membranes were treated with HRP-conjugated secondary antibodies. SuperSignal™ West Dura Extended Duration Substrate (Thermo Fisher Scientific, Waltham, MA, USA) was used as the HRP substrate. The signals were detected using the VersaDoc™ Imaging System (Bio-Rad Laboratories, Hercules, CA, USA) and iBright system (Thermo Fisher Scientific).

Autophagic flux assay

Autophagic flux was measured by inhibiting LC3-II turnover by immunoblotting (Klionsky et al., 2008; Mizushima et al., 2010). To inhibit autophagic flux, cells were treated with 100 nM bafilomycin A1 in 0.1% (v/v) DMSO and then subjected to immunoblotting. The autophagic flux index was calculated as follows: autophagic flux index = (LC3-II expression in the presence of bafilomycin A1)/(LC3-II expression in the absence of bafilomycin A1). LC3-II expression was normalized by GAPDH expression. In each experiment, the autophagic flux index of the control group was normalized to 1.

Statistical analysis

Data analysis was performed using JASP (Version 0.18.3) software. Statistical significance was determined by ANOVA, followed by Dunnett’s test. Statistical significance was set at p < 0.05.

RESULTS AND DISCUSSION

Previously, the viability of HepG2 cells exposed to DHP-3 was assessed. The results demonstrated that DHP-3 had a significant impact on cell cytotoxicity at concentrations of 500 µM or higher for 24 hr (Ishida et al., 2012). Therefore, a 100-400 µM exposure for 24 hr was chosen for the following experiments. To investigate the activation of the ER stress pathway by DHP-3, we analyzed the activation of ER-resident transmembrane sensor proteins in DHP-3-treated HepG2 cells by immunoblotting to assess the phosphorylation of IRE1 and PERK and expression of the cytoplasmic transcriptional activation domain of ATF6. The phosphorylation of IRE1 and PERK and the expression of the cytosolic transcriptional activator domain of ATF6 were increased by DHP-3 in a dose-dependent manner (Fig. 2A and B). Moreover, PERK–ATF4–CHOP pathway activation was examined by immunoblotting for ATF4 and CHOP. ATF4 and CHOP expression was increased by DHP-3 in a dose-dependent manner (Fig. 2A and B). These results suggest that the ER stress pathway and the PERK–ATF4–CHOP pathway, which is responsible for the induction of autophagy, are activated by DHP-3. Furthermore, we conducted an immunoblotting analysis to assess the effects of DHP-3 on autophagy in HepG2 cells. This analysis involved examining the expression of LC3-II, which is the LC3-phosphatidylethanolamine conjugate and an indicator of autophagosomes. In the typical process of autophagy induction, the amount of LC3-II increases transiently (Mizushima and Yoshimori, 2007). However, the results indicated that DHP-3 induced an increase in LC3-II expression in a dose-dependent and time-dependent manner (Fig. 3A, B, C and D). Furthermore, p62, a selective substrate for autophagy, also accumulated in a dose-dependent manner in response to DHP-3 treatment (Fig. 3A and B). These results suggest that DHP-3 treatment may instead suppress net autophagy. To confirm this hypothesis, we compared LC3-II expression in the presence and absence of bafilomycin A1, which inhibits both lysosomal enzyme activity and autophagosome-lysosome fusion, and measured autophagic flux (Klionsky et al., 2008; Mizushima et al., 2010). Bafilomycin A1 treatment was observed to significantly increase LC3-II expression in control and DHP-3-treated cells (Fig. 4A and B). However, no significant difference in accumulation was observed between bafilomycin A1-treated and DHP-3/bafilomycin A1-treated cells (p = 0.16). Furthermore, the autophagic flux index was significantly reduced in DHP-3-treated cells (Fig. 4C). These data suggest that DHP-3 treatment has the potential to inhibit autophagy flux. This is likely to be due to the prevention of a downstream step in autophagy and/or decreased autophagic degradation rather than an upstream of autophagosome formation (Mizushima et al., 2010). Thus, our results indicate that DHP-3 activates the ER stress pathway and inhibits the autophagy pathway, which could lead to the inadequate removal of damaged organelles and thereby cause cytotoxicity.

Fig. 2

DHP-3 activates the ER-resident transmembrane sensor proteins and the PERK–ATF4–CHOP pathway in HepG2 cells. The cells were treated with various concentrations (100–400 µM) of DHP-3 for 24 hr. Control cells were treated without DHP-3. (A) Immunoblot analysis of phospho-IRE1 (pIRE1), IRE1, phospho-PERK (pPERK), PERK, the cytosolic transcriptional activator domain of ATF6 (ATF6), ATF4 and CHOP. GAPDH served as an internal control. (B) The relative ratios of pIRE1/IRE1, pPERK/PERK, ATF6/GAPDH, ATF4/GAPDH and CHOP/GAPDH. The value in the control group was normalized to 1. The values represent the mean ± S.E. of three independent experiments (*p < 0.05 and **p < 0.01 indicate significant differences from the control).

Fig. 3

DHP-3 induces the accumulation of LC3-II and p62 in HepG2 cells. Immunoblot analysis of LC3-II and p62. GAPDH served as an internal control. (A and B) The cells were treated with various concentrations (100–400 µM) of DHP-3 for 24 hr. Control cells were treated without DHP-3. (C and D) The cells were treated with 400 µM of DHP-3 for 1 hr, 3 hr, 6 hr, 12 hr and 24 hr. Control cells were treated without DHP-3. (B and D) The relative ratios of LC3-II/GAPDH and p62/GAPDH. The value in the control group was normalized to 1. The values represent the mean ± S.E. of three independent experiments (**p < 0.01 and ***p < 0.001 indicate significant differences from the control).

Fig. 4

DHP-3 inhibits autophagic flux in HepG2 cells. The cells were treated with or without 400 µM DHP-3 or 100 nM bafilomycin A1 (BafA1) for 24 hr. (A) Immunoblot analysis of LC3-II. GAPDH served as an internal control. (B) The relative ratios of LC3-II/GAPDH. The value in the control group was normalized to 1. The values represent the mean ± S.E. of three independent experiments (*p < 0.05 and ***p < 0.001 indicate significant differences from the control). (C) Comparison of the autophagic flux index in the presence and absence of DHP-3. The value in the control group was normalized to 1. The values represent the mean ± S.E. of three independent experiments (**p < 0.01 indicates significant differences from the control).

In research on the relationship between glycation products and disease, AGEs have been the most extensively analyzed and are present in various pathologies (Sruthi and Raghu, 2021). AGEs and their receptor, RAGE, induce cellular stresses such as endoplasmic reticulum stress, hypoxia, and oxidative stress. These stresses stimulate autophagy, which leads to the removal of damaged organelles or proteins (Sruthi and Raghu, 2021). In contrast, DHP-3, a non-AGE, induced cytotoxicity in a RAGE-independent manner (Miyauchi et al., 2021); as noted above, DHP-3 activated the ER stress pathway but inhibited the autophagy pathway. Therefore, it is suggested that autophagy contributes little to the reduction of DHP-3-induced cytotoxicity and that the stress response pathway induced by DHP-3 is different from that induced by AGEs. Further studies are needed to identify the underlying molecular mechanisms of DHP-3-mediated cytotoxicity.

ACKNOWLEDGMENT

We thank Joe Barber Jr., PhD, from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.

Conflict of interest

The authors declare that there is no conflict of interest.

REFERENCES
 
© 2024 The Japanese Society of Toxicology
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