Inhibitory Effects of Verrucarin A on Tunicamycin-Induced ER Stress in FaO Rat Liver Cells

Endoplasmic reticulum (ER) stress is linked with development and maintenance of cancer, and serves as a therapeutic target for treatment of cancer. Verrucarin A, isolated from the broth of Fusarium sp. F060190, showed potential inhibitory activity on tunicamycin-induced ER stress in FaO rat liver cells. In addition, the compound decreased tunicamycin-induced GRP78 promoter activity in a dose dependent manner without inducing significant inhibition of luciferase activity and cell growth for 6 and 12 h. Moreover, the compound decreased the expression of GRP78, CHOP, XBP-1, and suppressed XBP-1, and reduced phosphorylation of IRE1α in FaO rat liver cells. This evidence suggests for the first time that verrucarin A inhibited tunicamycin-induced ER stress in FaO rat liver cells.


Introduction
The endoplasmic reticulum (ER) is an organelle present in all eukaryotic cells, and it plays a leading role in lipid and protein synthesis [1]. Membrane-spanning proteins or hydrosoluble proteins are folded by a chaperone in ER, in a manner parallel with protein synthesis at ER-bounded ribosomes. However, this folding process is disturbed by heat shock, inhibition of sugar chain modification, dysregulation of calcium dynamics, metabolic disorders, viral infections, etc. Under these conditions, proteins are misfolded or unfolded with subsequent aggregation of unusual proteins and apoptosis [2,3]. ER stress is implicated in various diseases such as inflammatory disorders, metabolic disease, and cancer [4,5]. ER stress is caused by various pathological or physiological condition such as ER calcium depletion, hypoxia, oxidative stress, and DNA damage, which can activate unfolded protein response (UPR), followed by restoration from stress conditions. The ER stress response is signaled in part through the dimerization of ER membrane-localized inositol requiring enzyme-1α (IRE1α). IRE1α is a serine-threonine protein kinase and endoribonuclease that, upon activation, initiates the unconventional splicing of the mRNA encoding X-box-binding protein-1 (XBP-1) [6][7][8]. This splicing reaction creates a translational frameshift to produce an active XBP-1 transcription factor. Activated XBP-1 has been reported to induce up-regulation of molecular chaperons such as GRP78 and ERdj4 to diminish the accumulation of unfolded proteins [9]. The 78-kDa glucose-regulated protein GRP78 is a central regulator of endoplasmic reticulum (ER) homeostasis, functioning in protein folding, ER calcium binding and modulation of transmembrane ER stress sensor activity [10]. Mice deficient in XBP-1, a transcription factor that modulates ER stress response, have been reported to develop insulin resistance [11]. Therefore, it has been suggested that candidates with reduced the ER chaperone expression levels, could not only be employed for the treatment of type 2 diabetes and obesity, but also for the treatment of diseases related with dysfunction of ER. Previously, there have been a number of reports on the design and development of synthetic ER stress inhibitors; however there exist only a few reports on ER stress inhibitors derived from fungi.
In the course of our screening program for identifying inhibitors of ER stress-induced XBP-1 activation and regulators of GRP78/Bip molecular chaperone expression using the microbial cultures libraries, we detected potent inhibitory effect in an extract of the soil fungus, Fusarium sp. F060190. Assay-guided fractionation of the EtOAc extract led to the isolation of the known compound, verrucarin A (1), from mass cultures of fungus strain Fusarium sp. F060190, as ER stress modulator. In the current study, we firstly reported that verrucarin A inhibited the ER stress-induced XBP-1 activation and GRP78/Bip molecular chaperone expression in FaO cells.

Isolation and Identification of Verrucarin A
In the course of the screening program for identifying ER stress inhibitors from microbial resources, a fungal isolate F060190 was selected based on its potent inhibitory effect against ER stress. To isolate the active compounds with an inhibitory effect on the GRP78 luciferase activity, the culture broth of Fusarium sp. F060190 was extracted with EtOAc. The EtOAc extract was separated on silica gel and ODS open-column chromatography, and then subjected to semi-preparative HPLC to yield compound 1. As shown in Figure 1, the structure of compound 1 was confirmed as verrucarin A (1) through NMR and MS spectra analyses, and comparison with published literature data [12,13].

Verrucarin A Inhibits Tunicamycin-Induced GRP78 Expression
GRP78 acts as a molecular chaperone in the endoplasmic reticulum (ER) to promote protein folding [14]. Thus, substances which either down-or up-regulate GRP78 transcription, are expected to be promising drugs for the treatment of type 2 diabetes and obesity [11]. In the course of our screening program for identifying regulators of GRP78 molecular chaperone expression, we isolated a known compound, designated as verrucarin A (Figure 1), which inhibited GRP78 expression in FaO cell reporter gene assay system.
To investigate the function of verrucarin A, we first examined the dose effect of verrucarin A on GRP78 promoter regulation by using a luciferase reporter gene under the control of the GRP78 promoter. Tunicamycin is a well-known as an inhibitor of protein glycosylation and a ER stress inducer. As shown in Figure 2, verrucarin A decreased the expression of the reporter gene at concentrations of 10, 20, and 30 nM when cells were co-stimulated with tunicamycin for 6 and 12 h.

Effect of Verrucarin A on Cell Viability
The effect of verrucarin A alone or in combination with tunicamycin on cell growth was evaluated in FaO rat liver cells. As shown in Figure 3, verrucarin A showed no significant variations in cell viability as compared with control at 1-10 nM and 5 μg·mL −1 of tunicamycin treatment. However, a significant increase in the cytotoxic effect of verrucarin A was noted at 30 nM as compared to cells treated with tunicamycin at 6 h and 12 h. The result of cell viability assay is presented in Figure 3.

Effect of Verrucarin A on ER Stress-Related Gene Expression
To investigate the effect of verrucarin A on ER stress-related gene expressions such as GRP78, XBP-1, and CHOP, this was studied by using RT-PCR analysis. As displayed in Figure 4, 5 nM of verrucarin A exhibited effects on GRP78, XBP-1, and CHOP mRNA expression. After 6 h treatment, 20 nM of verrucarin A dramatically reduced mRNA expression of GRP78, XBP-1, and CHOP genes. Accordingly, 30 nM of verrucarin A suppressed tunicamycin-induced XBP-1 splicing, GRP78, and CHOP gene expression after 6 h. These findings suggested that verrucarin A could inhibit ER stress in FaO cell ( Figure 4A). However, the expression of GRP78 and XBP-1 after treatment with 10 nM of verrucrin A exhibited a different tendency of downregulation at 3 h and subsequent increase at 12 h as compared with the control ( Figure 4B). These data suggest that 10 nM of verrucarin A has a short-term effect on suppression of tunicamycin-induced ER stress. Furthermore, inhibition of the XBP-1 splicing form presence of 10 nM of verrucarin A at 3, 6, and 12 h was noted. Interestingly, 10 nM of verrucarin A suppressed the 3 h treatment of the XBP-1 splicing form ( Figure 4B). In order to determine ER stress-related protein changes, following combination with verrucarin A exposure, western blotting was employed to analyze the expression of CHOP, XBP-1, and p-IRE1α. The protein expression of GRP78 and CHOP exhibited reduction in the presence of 10 nM of verrucarin A ( Figure 5A). These results suggest a mechanism by which early response of verrucarin A may inhibit the action of XBP-1 and CHOP expression after 6 h. The analysis also revealed that 30 nM of verrucarin A markedly decreased tunicamycin-induced IRE1α phosphorylation ( Figure 5B).

Figure 5. Verrucarin A inhibits ER stress in FaO cells. (A,B) Western blotting analysis.
Tm-induced XBP-1, CHOP, IRE1 expression were inhibited by VA. Cells pre-treated with 0, 10, and 30 nM of VA or not with each dose for 1 h was exposed to 5 μg·mL −1 Tm for 6 h in FaO cells. (+): treated with Tm, (−): untreated with.

Fermentation, Extraction and Isolation of Verrucarin A
Fusarium sp. F060190 was cultured in 1 L Erlenmeyer flask, each containing 200 mL of medium containing glucose 20 g, yeast extract 2 g, peptone 5 g, MgSO4·7H2O 0.5 g, and KH2PO4 1 g in 1 L of distilled water, pH 5.6-5.8. Flasks were individually inoculated with 2 mL seed cultures of Fusarium sp. F060190. Flask cultures were incubated at 28 °C and aerated by agitation on a rotary shaker at 140 rpm for a period of 7 days. Extraction of the filtered fermentation broth with EtOAc (5 L) provided an organic phase, which was then concentrated using a rotary evaporator to yield 1.2 g of crude extract. The EtOAc extract was subjected to C18 flash column chromatography (5 × 40 cm), eluting with a stepwise gradient of 20%, 40%, 60%, 80%, and 100% (v/v) MeOH in H2O (500 mL each). Among the seventeen pools (Fr.1-Fr.17) combined by their TLC profile, Fr. 8 eluted with 70% MeOH was found to have the highest activity. Thus, Fr. 8 was purified by semi-preparative reversed-phase HPLC eluting with a gradient from 50% to 70% CH3CN in H2O over 30 min to yield compound 1 (35 mg; tR = 26 min).

Chemicals
Dulbecco's modified Eagle's medium and fetal bovine serum were purchased from Gibco-BRL (Grand Island, NY, USA). Antibodies against CHOP were obtained from Cell Signaling Technology (Beverly, CA, USA) and antibody against p-IRE1α was from Abcam (Cambridge, UK). In addition, antibodies against IRE1 α, XBP-1, GAPDH, and PCNA were purchased from Santa Cruz Biotechnology (Dallas, CA, USA). Enhanced Cell Viability Assay Kit (EZ-CyTox) was obtained from Daeil Lab Service (Seoul, Korea), and luciferase assay kit was from Promega (Madison, WI, USA). Chemiluminescence reagent was obtained from Thermo Scientific (Burlington, IL, USA).

Cell Culture and Transfection
FaO rat liver cells were maintained in Dulbecco's modified Eagle's medium supplemented with 2 mM L-glutamine, and 10% heat-inactivated fetal bovine serum, and were cultured in a humidified CO2 incubator at 37 °C. For luciferase reporter assays, cells (80%-90% confluence) were incubated in 12-well plates for 24 h. Using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), cells were subsequently transfected with 0.8 µg of pGRP78-luc. After 24 h, cells were treated with verrucarin A and tunicamycin (Sigma-Aldrich, St. Louis, MO, USA) for various times and luciferase activity was measured with a detection kit.

Cell Viability Assay
For the cell viability assay, semi-confluent cells in a 96-well plate were treated with tunicamycin and/or verrucarin A. After adding 10 µL of the EZ-CyTox solution to each well of the culture plate for 2 h. The absorbance was measured using a micro-plate reader at 450 nm (reference wavelength at 650 nm).

Plasmid
Based on the published sequence of the human GRP78 gene, a 378-bp fragment of the GRP78 promoter region (-371 to region; relative to the transcriptional initiation site) was amplified by PCR from genomic DNA with the following sets of primers and cloned into the KpnI and HindIII sites of the pGL3-basic vector (Promega), which contains the luciferase reporter gene: sense strand (5′-CGGGGTACCGTCACTCCTGCTGGA-3′) and antisense strand (5′-CATCCCAAGCTTTCGACCTC ACCGTCGCCTACTC-3′).

Western Blot Analysis
After different treatments, cells were washed three times with ice-cold phosphate-buffered saline (PBS) and collected by scraping in lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Nonidet P-40 (NP-40), 0.5% sodium deoxycholate, 0.1% SDS, 1X protease inhibitor cocktail set III (Calbiochem, Darmstadt, Germany), 0.5 µM Cantharidin (Calbiochem), 1 mM Na3VO4 and 1 mM DTT. The mixture was disrupted by three freeze-thaw cycles and subsequently centrifuged at 15,000 rpm at 4 °C for 30 min. The supernatant was collected and preserved at −70 °C until use. Total protein was transferred to a polyvinylidene difluoride (PVDF) membrane and was subjected to western blot analysis with specific antibodies. Immune complexes were detected with enhanced chemiluminescence reagents.

Conclusions
Verrucarin A (1) was isolated from the culture broth of Fusarium sp. F060190. The compound showed potential inhibitory activity on tunicamycin-induced GRP78 promoter activity assay using FaO rat liver cells. Also, the compound decreased the gene expression of GRP78, CHOP, and XBP-1, and it reduced phosphorylation of IRE1α in FaO rat liver cells by tunicamycin. Therefore, these results provide evidence that verrucarin A is a potential ER stress regulator, and it could be useful candidate for therapeutic use in cases involving diabetes, obesity, and disorders related with dysfunction of ER-stress.