Background: Grifolic acid is a derivative of grifolin, an antitumor natural compound, and it was reported as an agonist of free fatty acid receptor GPR120. Little is known about its antitumor effects and the involvement of GPR120. Methods: GH3 cells, the rat anterior pituitary adenoma cells, were cultured and the cell death was measured by MTT assay and Annexin V/PI staining. The mitochondrial membrane potential (MMP) of GH3 cells was measured by JC-1 staining. Cellular ATP levels and the intracellular NAD/NADH ratio were measured. GPR120 expression in GH3 cells was observed by RT-PCR and Western Blot, and siRNA was used to inhibit GPR120 expression in GH3 cells. Results: Grifolic acid dose- and time-dependently induced the necrosis of GH3 cells. Grifolic acid significantly reduced the mitochondrial membrane potential (MMP) and decreased cellular ATP levels in GH3 cells. In contrast, the MMP of isolated mitochondria was not decreased by grifolic acid. The intracellular NAD/NADH ratio was significantly increased by grifolic acid. GPR120 is expressed in GH3 cells, but GPR120 agonists such as EPA, GW9508 and TUG891 did not affect the viability of GH3 cells. Moreover, GPR120 siRNA knockdown showed no significant influence on grifolic acid-induced GH3 cell death. Conclusion: Grifolic acid induces GH3 cell death by decreasing MMP and inhibiting ATP production, which may be due to the inhibition of NADH production through a GPR120-independent mechanism. Keywords: Grifolic acid, GH3 cells, Mitochondria, Cell death Background established. Some reports showed that grifolic acid was Some natural compounds, which are isolated from bot- an agonist of free fatty acid receptor GPR120 , and any, fungi and marine organism, are putative candidates grifolic acid was able to activate extracellular regulated for antitumor drugs . It is well known that grifolin, protein kinases (ERK1/2), causing increased secretion of one of the natural compounds isolated from the fresh glucose-dependent insulinotropic polypeptide (GIP) fruiting bodies of the mushroom Albatrellus confluens from GPR120-expressing enteroendocrine cells . It was , exhibits antitumor effects on nasopharyngeal carcin- also showed that GPR120 activation might produce pro- oma, osteosarcoma, and gastric tumor cells [3–5]. Grifo- tective effects on murine enteroendocrine cell line STC-1 lic acid (the structure in Additional file 1) is a derivative cells . The effects of grifolic acid on tumor cells and the of grifolin, but its effects on tumor cells are not well involvement of GPR120 warrants further study. Anterior pituitary adenomas, one of the common intracranial tumors, is increasingly diagnosed due to the * Correspondence: firstname.lastname@example.org; email@example.com † advances in neuroimaging technology . Besides trans- Yufeng Zhao and Lei Zhang contributed equally to this work. The institute of Basic Medical Sciences, Xi’an Medical University, Xi’an sphenoidal surgery, medical therapies are important 710021, China treatments for anterior pituitary adenomas . New Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhao et al. BMC Pharmacology and Toxicology (2018) 19:26 Page 2 of 9 effective antitumor drugs may significantly improve the hours later, the media were discarded and 100 μl isopro- therapy of anterior pituitary adenomas. In this study, we panol with 0.01 mol/L HCl was added to each well. After observed the effects of grifolic acid on the viability of the formazan crystals were fully solubilized, the absorb- GH3 cells, the rat anterior pituitary adenoma cells that ance values at 560 nm were measured by ELISA reader secret growth hormone and prolactin . (Thermo Fisher, USA). The background absorbance The death of tumor cells is often related to the dys- values at 630 nm were also measured and subtracted function of mitochondria. Mitochondria are essential to from that of 560 nm. Then the absorbance values were produce ATP and play a dominant role in cellular viabil- used for statistical analysis. The experiments were per- ity, apoptosis and death . Intracellular ATP at the formed in triplicate. normal level is required for cell survival, and the reduc- tion of ATP level results in the apoptosis or necrosis of Flow cytometry analysis of cell death living cells [13, 14]. Mitochondrial membrane potential After being treated by grifolic acid in serum-free (MMP), which is generated during the procedure of medium, GH3 cells were detached from the dishes by 0. redox energy transfer from NADH to oxygen via the 05% trypsin/EDTA and stained using AnnexinV-FITC/PI electron transport chain in mitochondria, represents the staining kits . Briefly, the cells were re-suspended function of mitochondria and is critical for ATP produc- into the binding buffer at 1×10 cells/ml, and AnnexinV- tion. The actions of grifolic acid on mitochondria func- FITC/PI was added to cell suspension in a dilution of 1:20. tion such as MMP and ATP production were also The cells were gently mixed and incubated for 15 min at investigated in this study. In addition, we found GPR120 room temperature in the dark. Finally, the cells were expression in GH3 cells, and the role of GPR120 in the diluted into binding buffer and went through the flow cy- effects of grifolic acid on GH3 cells was studied. tometry to measure AnnexinV- and PI-staining positive cells (BD Biosciences, USA). The experiments were per- Methods formed in triplicate. Chemicals Grifolic acid and TUG891 were obtained from R&D Inc. Cellular ATP measurement (Minnneapolis, USA). GW9508, EPA, GPR120 poly- Cellular ATP levels in GH3 cells were measured using clonal antibody, MTT and Cellular ATP assay kits were ATP detection assay kits . Briefly, GH3 cells after bought from Sigma-Aldrich (St. Louis, USA). Annexin being treated by grifolic acid in serum-free medium V-FITC/PI staining kits were the products of BD Phar- were lyzed by detergent under shaking at 700 rpm for mingen (San Jose, USA). Rat GPR120 siRNA, lipofecta- 5 min. The constituted substrate solutions were added mine RANiMAX, DMEM, FBS, JC-1 and Mitochondria for incubation for 5 min in a dark place. Then the lumi- Isolation Kit for Culture Cells were obtained from nescence of each sample was recorded using the lumi- Thermo Fisher Scientific (Waltham, USA). NAD/NADH nescence plate reader (Thermo Fisher, USA). The Assay Kits were the products of Abcam (Cambridge, standard curves were constructed and the ATP level of UK). Protein extraction kits were bought from Bio-Rad each sample was calculated. The total protein levels (Hercules, USA). RNA isolation kits, reverse transcrip- were quantified by BCA assay and used to correct the tion kits and PCR kits were the products of Takara Bio- cellular ATP levels for data analysis. The experiments technology (Dalian, China). were performed in triplicate. Cell culture NAD/NADH measurement GH3 cells were obtained from American Type Culture GH3 cells (4×10 cells per sample) were washed in cold Collection (ATCC Number: CCL-82.1™) and cultured in PBS and treated with NAD/NADH Extraction Buffer. The DMEM containing 10% FBS, 100 U/ml penicillin and buffer was centrifuged at 12,000 g for 5 min at 4 °C and 100 μg/ml streptomycin. The media were changed every the supernatants were collected for NAD/NADH assay 2 days, and GH3 cells were sub-cultured after 80% con- . As indicated by the instruction, each sample was fluence and seeded to plates or dishes for the following divided into two parts for NADt (total NAD measurements. including NAD and NADH) assay and NADH assay respectively. The samples were mixed with Reaction Cell viability assay Mix firstly and incubated for 5 min at room temperature. GH3 cells grew up to 90% confluence in 96-well plates Then NADH Developer was added to incubate for 4 h. and then were changed to serum-free medium with re- The OD value of each sample was read at 450 nm. gent treatment including grifolic acid, EPA, GW9508 NAD/NADH ratio was calculated as: NAD/NADH and TUG891. At the end of treatment, MTT was added ratio = (NADt-NADH) / NADH. The experiments were into media at a final concentration of 0.5 mg/ml. Four performed in triplicate. Zhao et al. BMC Pharmacology and Toxicology (2018) 19:26 Page 3 of 9 MMP measurement ChemiDoc MP gel imaging analysis system. The experi- GH3 cells were stained with JC-1 (a final concentration ments were performed in triplicate. of 5 μg/ml) in the serum-free medium for 15 min at 37 °C in a humidified 5% CO incubator. Then the cells were RT-PCR washed with the serum-free medium and put onto the The total RNA from GH3 cells were extracted and re- stage of confocal microscope (Leica TCS SP8). The fluor- verse transcription was performed using the kits from escence intensity was recorded every 5 min before and Takara. cDNA was used to run PCR with rat GPR120 after the treatment with grifolic acid. The red fluorescence primers. The forward primer is 5′- GCA TAG GAG (excitation wavelength at 560 nm and emission wave- AAA TCT CAT GG-3′ and the reverse primer is 5′- length at 600 nm) and green fluorescence (excitation GAG TTG GCA AAC GTG AAG GC-3′. The PCR wavelength at 488 nm and emission wavelength at product size was 340 bp. The reaction mixture was de- 535 nm) were measured synchronously, and the ratio of natured for 5 min at 94 °C for 30 s, and then it went to fluorescent intensity (red/green) for each cell was analyzed 30 cycles of denaturing at 94 °C for 30 s, annealing at using the LAS LITE software . The experiments were 58 °C for 15 s and extending at 72 °C for 15 s. PCR performed in triplicate. products were analyzed by electrophoresis on 1.4% agar- ose gel with ethidium bromide. The experiments were Mitochondria isolation performed in triplicate. The mitochondria were extracted from GH3 cells using the Mitochondria Isolation Kit for the Culture Cells Data analysis under the instruction . GH3 cells (1×10 cells per The data were expressed as Mean ± S.EM. D’Agostino- sample) were loaded with JC-1 firstly and then were har- Pearson omnibus test was used to test the normality of vested to isolate mitochondria. Mitochondria isolation data distribution. For the normal distribution data, com- reagent A was added to incubate on ice for 2 min, parisons of means of multiple groups with each other or followed by the adding of mitochondria isolation reagent against one control group were analyzed with one way B and C. Then the mixtures were centrifuged at 700 g ANOVA followed by the appropriate post-hoc tests. For for 10 min at 4 °C. The supernatants were collected and the abnormal distribution data, Kruskal-Wallis H test centrifuged at 12,000 g for 15 min at 4 °C, and the pel- was used to analyze the significance of groups. P < 0.05 lets were collected and incubated in mitochondria isola- was considered to be significantly different. tion reagent C for MMP measurement. Results GPR120 siRNA transfection Effects of grifolic acid on GH3 cell viability GH3 cells grew to 70% confluent at the time of transfec- Grifolic acid (20 μmol/L) inhibited the viability of GH3 tion. The transfection complexes for rat GPR120 Silen- cells after 1 h incubation and resulted in total cell death cer Select siRNA (siRNA ID: S148233, Invitrogen) and 6 h later. It showed a dose- and time-dependent inhib- Lipofectamine RNAiMAX were prepared by mixture of ition of GH3 cell viability from 2.5 μmol/L to 20 μmol/L siRNA (100 pmol in 50 μl of Opti-MEM medium) and in 24 h incubation (Fig. 1). The IC50 of grifolic acid at Lipofectamine RNAiMAX (1 μlin50 μl of Opti-MEM 24 h treatment was 4.25 μmol/L. AnnexinV/PI staining medium). The complexes were added to the medium at and the flow cytometry analysis was also used to observe a dilution of 1:4. The cells were cultured for 48 h and the death of GH3 cells. The percentage of Annexin V then GPR120 knockdown was measured by Western and PI-positive GH3 cells in control was 2.45% ± 0.83%, blot. The cells with transfection were used to measure and it increased to 42.51% ± 7.86% after treatment with cell death, cellular ATP levels and MMP. grifolic acid (10 μmol/L) for 6 h. The represent analysis results were shown in Fig. 2a and b. GH3 cells in control Western blot showed normal shape (Fig. 2c), but they were signifi- In brief, GH3 cells were homogenized and total protein cantly swollen and mostly broken after grifolic acid was extracted using ReadyPrep protein extraction kits treatment (Fig. 2d). and quantified by BCA assay. The protein (40 μg) was analyzed by SDS-PAGE on a 10% Peptide Criterion Gel Effects of grifolic acid on MMP and ATP production in and transferred to nitrocellulose membranes using a GH3 cells Trans-Blot SD semi-dry electrophoresis transfer cell MMP was indicated by JC-1 fluorescence . JC-1 exhib- (Bio-Rad). The membranes were then probed with ited membrane potential-dependent accumulation in rabbit-anti GPR120 antibody (1:1000) and the blot was mitochondria, showing the shift of emission wavelength developed with the chemiluminescent reagents. The lu- from green to red. Mitochondrial depolarization was indi- minescence of the membranes was imaged by the cated by a decrease in the red/green fluorescence intensity Zhao et al. BMC Pharmacology and Toxicology (2018) 19:26 Page 4 of 9 attenuated MMP in a longer 60 min. Grifolic acid at the concentration of 1.25 μmol/L did not attenuate MMP in 60 min compared to control (Fig. 3). The cellular ATP levels were then measured. The cel- lular ATP level was 38.18 ± 4.23 nmol/mg protein in control GH3 cells, and it began to significantly drop after the treatment with 20 μmol/L grifolic acid for 0.5 h and dropped to 17.76 ± 3.23 nmol/mg protein in 2 h (Fig. 4a). Grifolic acid at concentration of 10μmol/L also significantly resulted in the decrease in cellular ATP levels from 37.67 ± 4.89 nmol/mg protein in control to 22.56 ± 2.49 nmol/mg protein after 2 h of the treatment and to 8.15 ± 2.03 nmol/mg protein after 6 h (Fig. 4b). Fig. 1 Grifolic acid reduces GH3 cells viability. GH3 cells in serum-free Role of GPR120 in grifolic acid-induced GH3 cell death medium were treated by grifolic acid for different time, and then MTT As shown in Fig. 5a, RT-PCR showed the expression of assay was used to measure cell viability. The absorbance values of MTT assay at 560 nm were analyzed. * means P <0.05 vs control. ** means GPR120 in GH3 cells. The negative control using RNA P < 0.01 vs control, n =16 without reverse transcription did not show the amplifi- cation of GPR120 gene, confirming the specificity of RT- ratio . We found that grifolic acid resulted in a signifi- PCR. In accordant to it, western blot showed that the cant decrease in the red/green fluorescence intensity ratio proteins from GH3 cells were stained positively by in a dose-dependent and time-dependent manner. Grifolic GPR120 antibody and the molecular size of the stained acid (20 μmol/L) significantly attenuated MMP after band was 41KDa, the exact molecular size of rat 5 min incubation and caused the maximal effect in GPR120 (Fig. 5b). GPR120 agonists including EPA (20 20 min. Grifolic acid (10 μmol/L) also took effects in μmol/L), GW9508 (20 μmol/L) and TUG891 (20 μmol/ 10 min and achieved maximal effect in 40 min. Grifolic L) did not show significant effects on the viability of acid at the concentration of 5 μmol/L and 2.5 μmol/L also GH3 cells after the incubation for 24 h. In contrast, Fig. 2 Grifolic acid induces cell death of GH3 cells. a Flow cytometry measurement showed that GH3 cells in control had low level of cell death as indicated by low staining of Annexin V and PI; b GH3 cell after grifolic acid treatment (10 μmol/L for 6 h) showed a significant increase in cell necrosis as indicated by high percentage of Annexin V and PI-positive staining cells; c The normal shape of GH3 cells in control; d GH3 cells showed swelling and broken after grifolic acid treatment. The photos were representative results of 3 independent experiments Zhao et al. BMC Pharmacology and Toxicology (2018) 19:26 Page 5 of 9 Fig. 3 Grifolic acid diminishes MMP of GH3 cells. The fluorescent intensity of MMP indicator JC-1 in control (a) and after 20 μmol/L grifolic acid treatment for 5 min (b), 10 min (c) and 20 min (d). The statistical analysis of fluorescent intensity ratio of JC-1 in each cell was shown in (e)to reflect MMP levels. ** P <0.01 vs control, n =80 grifolic acid (20 μmol/L) had a significant inhibitory ef- isolated mitochondria in the experiments maintained nor- fect on the viability of GH3 cells (Fig. 5c). mal MMP during the measurement. Although grifolic acid A significant reduction of GPR120 protein level was inhibited MMP in the whole cells, it did not induce any achieved in GH3 cells with GPR120 siRNA transfection change in MMP in the isolated cell-free mitochondria compared to the control (Fig. 6a). GPR120 knockdown during the incubation for 20 min (Fig. 7a). It is suggested did not significantly influence grifolic acid-induced in- that grifolic acid-induced decrease in MMP in GH3 cells hibition of the GH3 cell viability (Fig. 6b). In addition, is initiated by the changes prior to electron transport the decrease in ATP levels and MMP by grifolic acid chain inside mitochondria. treatment was not significantly influenced by GPR120 In the next, NAD/NADH ratio in GH3 cells was mea- knockdown too (Fig. 6c and d). sured. The NAD/NADH ratio was significantly increased after 5 min of the grifolic acid treatment and reached a Effects of grifolic acid on electron transport chain of higher level after 20 min, indicating that the reduction mitochondria and NADH production of NADH may be the reason for the decreased MMP in The mitochondria from GH3 cells were stained with GH3 cells after grifolic acid treatment (Fig. 7b). JC-1. JC-1 intensity of mitochondrial sample that was extracted from GH3 cells remained stable during the Discussion measurement. The uncoupler carbonyl cyanide 3- Grifolic acid is a farnesyl phenolic compound first iso- chlorophenylhydrazone (CCCP) significantly reduced lated from the mushroom Albatrellus confluens and can JC-1 intensity of the isolated mitochondria to 25.67 ± 4. be totally synthesized now . It has been shown that 81% of the control. This result indicated that the grifolin, an analog of grifolic acid, has inhibitory effects Zhao et al. BMC Pharmacology and Toxicology (2018) 19:26 Page 6 of 9 Fig. 5 GPR120 is expressed in GH3 cells and did not influence GH3 cell viability. a: GPR120 transcription in GH3 cells was shown by RT-PCR. Fig. 4 Grifolic acid reduces cellular ATP levels in GH3 cells. a The Lanes 1-3 were cDNA marker, GPR120 amplification products from GH3 cellular ATP levels of GH3 cells being treated by 20 μmol/L grifolic cells and negative control, respectively. The size of PCR product is acid; b The cellular ATP levels of GH3 cells being treated by 10 μmol/L 340 bp. b: The protein expression of GPR120 in GH3 cells was shown grifolic acid. ** means P < 0.01 vs control, n =12 by western blot. Lane 1 was the immunostaining of GPR120 and lane 2 was the protein marker. c: MTT assay of GH3 cell viability in response to GPR120 agonists and grifolic acid. ** means P < 0.01 vs control, n =12 on variety of tumor cells [4, 22–24]. In this study, we demonstrated that grifolic acid induced the death of GH3 pituitary adenoma cells in a dose- and time- [11, 25–28]. We also found that PI3K inhibitor Wort- dependent manner. Grifolic acid in concentration of 20 mannin, ERK1/2 inhibitor U-0126, and p53 inactivator μmol/L induced total death of GH3 cells after 6 h treat- cyclic pifithrin-α, p-nitro, respectively did not induce ment. In contrast, grifolin in concentration of 50 μmol/L cell death in GH3 cells (Additional file 2). Therefore, it induced only 15% of cell death after the incubation for is suggested that grifolic acid-induced GH3 cell death 6 h on U2OS and MG63 osteosarcoma cells . The dis- may not be mediated by PI3K, ERK1/2 and p53. crepancy may result from the difference in the cell types The cellular ATP level is a critical factor maintaining and the culture conditions. In this study, GH3 cells were cellular viability. The reduction rate of ATP production treated with grifolic acid in the serum-free medium to determines the cell death types [13, 29, 30]. Rapid falling exclude the influence of serum ingredients. Serum-free of intracellular ATP leads to cell death through necrosis medium may increase the sensitivity of GH3 cells to gri- pathways such as oncosis . Grifolic acid at 10 μmol/ folic acid treatment. L induced a significant increase in the number of It was indicated that PI3K/Akt, ERK1/2 and p53 were Annexin V and PI -positive cells, indicating that GH3 the possible intracellular signaling molecules that medi- cells died mainly through necrosis. Grifolic acid also re- ated the antitumor effects of grifolin in different types sulted in a significant and fast decrease in the cellular of tumor cells [3, 22, 24]. Although PI3K, ERK1/2 and ATP levels in GH3 cells. The rapid deprivation of ATP p53 were involved in the regulation of cell viability and may be responsible to the necrosis of GH3 cells. growth in many tumor cell types, the inhibition of Mitochondria are vital to cellular viability, with its PI3K, ERK1/2 and p53 did not acutely induce cell death dominant role in the production of ATP as cell energy Zhao et al. BMC Pharmacology and Toxicology (2018) 19:26 Page 7 of 9 Fig. 6 GPR120 does not mediate the effects of grifolic acid on GH3 cell viability. a The inhibition of GPR120 expression was achieved by siRNA transfection for 48 h; b Grifolic acid-induced cell death was not affected by GPR120 knockdown; c Grifolic acid-induced decrease in ATP production was not affected by GPR120 knockdown; d Grifolic acid-induced attenuation of MMP was not affected by GPR120 knockdown source [32, 33]. The fuel energy is mainly transported to The MMP reduction observed in the cells may be due to NADH during oxidation, and the redox energy from the deficiency of NADH and consequent rundown of NADH is transferred to oxygen via the electron trans- proton pump. As expected, it was found that the cellular port chain in mitochondria. This procedure generates NADH levels decreased and NAD/NADH ratio in- MMP, which drives the protons into mitochondrial creased acutely after the grifolic acid treatment. There- matrix through ATP synthase to produce ATP or fore, it is concluded that grifolic acid blocks fuel through uncoupling proteins to produce energy. Mito- metabolism and NADH production, which in turn de- chondria dysfunction may be the reason for grifolic acid- creases MMP and ATP production and leads to GH3 induced decrease in cellular ATP levels. We then investi- cell death. Because fuel metabolism is executed by a gated the effects of grifolic acid on mitochondrial func- series of complex enzyme reactions, the accurate targets tion. MMP is usually measured using JC-1 because it of grifolic acid need to be further clarified in detail. accumulates in mitochondria driven by MMP to emit It was reported that grifolic acid is an agonist of red fluorescence in the mitochondria and emits green GPR120 [7, 19, 38]. GPR120 is considered to be a prom- fluorescence when it releases into the cytoplasm under ising pharmaceutical target for the treatment of meta- the condition of MMP reduction [34, 35]. Grifolic acid bolic diseases . Recently, several non-FFA agonists of induced a fast decrease in the red/green intensity ratio GPR120 including GW9508, TUG891, and grifolic acid of JC-1 in GH3 cells, indicating MMP reduction or less have been discovered [38, 40, 41]. In this study, the JC-1 kept within mitochondria. Accordingly, the ATP mRNA and protein expression of GPR120 in GH3 cells levels in GH3 cells significantly decreased after the grifo- was confirmed by RT-PCR and western blot. To our lic acid treatment. The direct action of grifolic acid on knowledge, this is the first report of GPR120 expression mitochondria was then studied. In the isolated mito- in GH3 cells. It was reported that GPR120 activation chondria, the uncoupler CCCP, which leads to proton protects mouse enteroendocrine cell line STC-1 cells leak and MMP reduction [36, 37], significantly dimin- against serum deprivation-induced apoptosis , show- ished MMP as expected, indicating that the isolated ing protective effects of GPR120 on cellular viability. mitochondria may function well. Grifolic acid, however, However, we found that grifolic acid induced cell death did not reduce MMP in the isolated mitochondria of of GH3 cells in serum-free culture condition. To clarify GH3 cells. It indicated that grifolic acid did not act dir- the role of GPR120 in the action of grifolic acid on GH3 ectly on the electron transport chain of mitochondria. cells, we tested the effects of the other putative GPR120 Zhao et al. BMC Pharmacology and Toxicology (2018) 19:26 Page 8 of 9 Additional files Additional file 1: The structure of grifolic acid. (JPG 153 kb) Additional file 2: The effects of PI3K inhibitor, ERK1/2 inhibitor and p53 inactivator on GH3 cells. PI3K inhibitor Wortmannin (0.1μmol/L), ERK1/2 inhibitor U-0126 (1 μmol/L), and p53 inactivator cyclic pifithrin-α, p-nitro (1 μmol/L) did not induce cell death in GH3 cells respectively, as measured by MTT assay. (P =0.58, n = 12) (JPG 2759 kb) Abbreviations Cyclic pifithrin-α-p-nitro: 2-(4-Nitrophenyl) imidazo[2,1-b]-5,6,7,8- tetrahydrobenzothiazole)ATCCAmerican Type Culture Collection; ATP: Adenosine triphosphate; CCCP: Carbonyl cyanide 3- chlorophenylhydrazone; ELISA: Enzyme linked immunosorbent assay; EPA: Eicosapentaenoic acid; ERK1/2: Extracellular regulated protein kinases; FBS: Fetal bovine serum; GIP: Glucose-dependent insulinotropic polypeptide; GPR120: G protein-coupled receptor 120; JC-1: 5,5′,6,6′-tetrachloro-1,1′,3,3′- tetraethylbenzimi- dazolylcarbocyanine iodide; MMP: Mitochondrial membrane potential; MTT: 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H- tetrazolium bromide; NADH: Nicotinamide adenine dinucleotide; PBS: Phosphate buffered solution; TUG891: 3-(4-((4-fluoro-49-methyl-[1,19- biphenyl]-2-yl)methoxy)phenyl) propanoic acid Funding This work was supported in by the grant from Shaanxi Province (2015KTCQ03-03) and Xi’an Medical University (2015RCYJ02). Availability of data and materials The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request. Authors’ contributions YZ and LZ designed the study, analyzed the data and drafted the manuscript. Fig. 7 Grifolic acid does not reduce MMP of isolated mitochondria AY, DC, RX and YL contributed to acquisition of data, analysis of data and but reduces cellular NAD/NADH ratio in GH3 cells. a MMP of isolated critical revision of the manuscript. XL, YZ, LW and JY designed the experiment mitochondria from GH3 cells was represented by JC-1 intensity. Grifolic for mitochondrial membrane potential assay and flow cytometry and did the acid (20 μmol/L) did not decrease MMP of isolated mitochondria in work of data analysis and manuscript writing. XX and XS were accountable for 20 min incubation. The uncoupler CCCP significantly inhibited MMP; all aspects of the work and contributed to the formation of experimental concept and the final approval of the manuscript for publication. All authors b Grifolic acid (20 μmol/L) acutely induced a significant increase in gave the final approval of the version to be published. NAD/NADH ratio in GH3 cells. ** P <0.01, n =6 Ethics approval and consent to participate Not applicable. agonists on the viability of GH3 cells. Applications of EPA, GW9508 and TUG891 did not show any cytotoxic Competing interests effects on GH3 cells under same conditions. In The authors declare that they have no competing interests. addition, GPR120 knockdown in GH3 cells did not affect the cytotoxic effects of grifolic acid. Taken to- Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in gether, GPR120 is not involved in the action of grifolic published maps and institutional affiliations. acid on GH3 cells. Grifolic acid was used within a range of 10-100 μmol/L in other studies to activate GPR120 Author details The institute of Basic Medical Sciences, Xi’an Medical University, Xi’an [7, 19, 42, 43]. Considering that the concentration of 710021, China. Department of Gerontological Surgery, The First Affiliated grifolic acid used in this study is within the same range, Hospital, Xi’an Medical University, Xi’an 710061, China. Medical Research it is more likely that grifolic acid is not a pure GPR120 Center, The Second Affiliated Hospital, Xi’an Medical University, Xi’an 710038, China. agonist and have other acting targets. Received: 4 September 2017 Accepted: 4 May 2018 Conclusions Grifolic acid induces GH3 cell death by inhibiting ATP References production. Inhibition of mitochondrial fuel metabolism 1. Gerhauser C. Cancer chemoprevention and nutriepigenetics: state of the art and NADH production may be the reason for the inhib- and future challenges. 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Published: May 30, 2018