Materials
A. arguta (sarunashi) fruits were purchased from local stores in Shin-jo village in Okayama prefecture (Japan). The average weight of a sarunashi fruit was 7.27 ± 1.72 g (mean ± standard deviation). The fruits were processed using a juicer, centrifuged at 2600×g for 20 min at 20 °C, and the supernatant was collected. Sarunashi fruits (200 g) yielded 65.5 g (63 mL) of supernatant (hereafter referred to as sarunashi juice or sar-j). Sar-j was stored at − 20 °C until use. IsoQ (CAS 21637–25-2, MW 464.38) was purchased from Kanto Chemical Co. Inc. (Tokyo, Japan). NNK was purchased from Toronto Research Chemicals (North York, ON, Canada) and MNNG was purchased from Nacalai Tesque (Kyoto, Japan). Vitamin C was purchased from Wako Pure Chemical Co. Ltd. (Osaka, Japan). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was purchased from Dojindo Laboratories (Kumamoto, Japan). For metabolic activation, the supernatant fraction of rat liver homogenate (S9) prepared using phenobarbital and 5,6-benzoflavone was obtained from FUJIFILM Wako Pure Chemical. Akt (pan) (C67E7) rabbit mAb (#4691), phospho-Akt (Ser473) (D9E) rabbit mAb (#4060), and LY294002 (#9901) were purchased from CST, Japan (Tokyo, Japan). Salmonella enterica subspecies I, serovar Typhimurium (Salmonella typhimurium) strain TA1535 [hisG46 ΔuvrB gal bio chl1005 rfa1001], was a gift from Dr. B. N. Ames of the University of California, Berkeley [19]. S. typhimurium YG7108 [hisG46 ΔuvrB gal bio chl1005 rfa1001 Δadast::Kmr Δogtst::Cmr], a strain lacking O6-methylguanine DNA methyltransferases (ogtST and adaST), was a kind gift from Dr. M. Yamada of the National Institute of Health [20]. The human lung epithelial-like cell line A549 (ATCC CCL185), derived from lung carcinoma, was provided by RIKEN BRC through the National BioResource Project of the MEXT/AMED, Japan (Tsukuba, Japan).
The vitamin C content in sar-j was quantitatively determined by iodine titration [21] and average vitamin C content in sar-j samples was found to be 1.48 ± 0.030 g/L. The total amount of polyphenolics in the juice was measured using the method of Singleton and Rossi [22] using the Folin-Ciocalteu method in terms of the gallic acid equivalent (mg/mL), and the average total phenolics amount in sar-j was found to be 4.40 ± 0.041 mg/mL.
Animals
Mice (A/J jms Slc female, 3-weeks old, average weight 8–13 g) were purchased from Japan SLC Inc. (Hamamatsu, Japan). Five mice were housed per cage in the animal room and randomly separated into treatment groups at least 1 week prior to the commencement of experiments. The mice had free access to murine chow pellets (MF powder, Oriental Yeast Co. Ltd., Tokyo, Japan) and water, and were maintained on a 12-h light/12-h dark cycle with optimum air exchange and a constant room temperature of 20 °C. All experiments were performed in accordance with the Guidelines for Animal Experiments of the Okayama University Advanced Science Research Center (permission no. OKU-2018028, 2,018,030, 2,019,671, 2,021,460, 2,021,461, 2,022,299, and 2,022,336) based on the Act on Welfare and Management of Animals (Act of Japan, No. 105 of October 1, 1973, and Amendment of Act No. 68 of 2005), Standards Relating to the Care, Management, and Alleviation of Pain and Distress of Laboratory Animals (Notice of the Ministry of the Environment No. 88 of 2006).
Anti-tumorigenesis study in mice with Sar-j and isoQ
NNK-induced tumorigenesis experiments were performed as described in our previous report [5]. In experiment 1, mice (A/J, 4-weeks old) were divided into three groups of 15–16 animals each (groups I–III). Prior to the experiments, sar-j was defrosted, centrifuged at 9000×g for 20 min at 20 °C, and the supernatant was collected. Potassium disulfite (0.1 g) was added to 1 L of the supernatant to prevent fermentation. Mice in group I received water containing potassium disulfite (0.1 g/L). Mice in groups II and III received sar-j in lieu of water from 4 weeks of age to the time of sacrifice. Tumors were induced in groups I and II with a single intraperitoneal (i.p.) injection of NNK (100 mM in 0.1 ml saline) at 8 weeks of age. The mice in group III were injected with 0.1 mL of saline as a substitute for NNK (control). The mice were sacrificed at 30 weeks of age.
In experiment 2, two isoQ doses of 25 mM (11.6 mg/L) and 10 mM (4.64 mg/L) were tested. Because the average total phenolics content in sar-j was 4.40 ± 0.041 g/L, we selected approximately 0.5 and 0.1% of the total phenolics content in sar-j. Mice (A/J, 4-weeks old) were divided into five groups of 5–15 animals each (groups IV–VIII). At 8 weeks of age, mice in groups IV, V and VI were injected with NNK (100 mM in 0.1 ml saline), while the mice in groups VII and VIII were administered a single i.p. injection of 0.1 mL saline. The mice received 10 mM isoQ (groups V) and 25 mM of isoQ (group VI and VII) in lieu of water from 4 weeks of age until sacrifice. The mice were sacrificed at 30 weeks of age. In both experiments, the number of surface lung nodules of the left lung lobe was counted using a loupe and digital calipers (AS ONE Corporation, Osaka, Japan). Tumor nodules that were > 1 mm in diameter were counted.
Anti-mutagenicity test
The inhibitory effects of sar-j and isoQ on NNK- and MNNG-induced mutagenicity were investigated using the Ames test [19]. Sar-j was defrosted, centrifuged at 9000×g for 20 min at 20 °C, sterilized by filtration of the supernatant through a 0.45 mM filter, then used for the Ames assay. NNK was assayed with S. typhimurium TA1535 or YG7108 in the presence of rat liver homogenate (hereafter refer to as +S9). MNNG was assayed with S. typhimurium TA1535 or YG7108 in the absence of rat liver homogenate (hereafter refer to as -S9). The effects of sar-j and isoQ on mutagenicity were examined as previously described [23]. Briefly, the preincubation mixture was prepared by mixing the components in the following order: 100 μL of a sample (sar-j with water, or isoQ solution), 450 μL of S9 mix or Na-phosphate buffer (0.1 M, pH 7.40), 100 μL of an overnight culture of bacteria, and finally 50 μL of a solution of mutagen (NNK or MNNG). Following incubation for 20 min at 37 °C, molten agar was added and the mixture was poured onto a minimal agar plate. The plates were incubated for 48 h, and the resulting revertant colonies were counted manually. When the number of colonies per plate exceeded 3000, the colonies in a specific square area were counted, and the total number of colonies in the plate was estimated from the average count in five such areas. The sample amounts used for the assay are referred to as mL eq. of the original juice. The results of the pilot Ames tests revealed that ≥20 mL of sar-j was cytotoxic on the Ames bacteria (S. typhimurium). Therefore, the anti-mutagenicity tests were performed with ≤10 mL of sar-j. Because the total phenolics content in 10 mL of sar-j was 44 mg, up to 116 mg (250 nmol) of isoQ was used for the antimutagenicity test. The experiments were performed in triplicate.
The mutagenic activity (%) was derived using the following equation:
$$100\times \left[\left(\textrm{revertants}\ \textrm{in}\ \textrm{the}\ \textrm{presence}\ \textrm{of}\ \textrm{juice}\ \textrm{or}\ \textrm{isoQ}\right)-\left(\textrm{spontaneous}\ \textrm{revertants}\right)\right]/\left[\left(\textrm{revertants}\ \textrm{in}\ \textrm{the}\ \textrm{absence}\ \textrm{of}\ \textrm{juice}\ \textrm{or}\ \textrm{isoQ}\right)-\left(\textrm{spontaneous}\ \textrm{revertants}\right)\right]$$
Effects on phosphorylation of Akt in A549 cells
To determine the concentration of sar-j and isoQ for phosphorylation studies, cell viability was measured using the MTT assay as previously described [5]. To evaluate the cytotoxic effects of sar-j and isoQ, A549 cells (1 × 104 cells in 90 μL) were seeded in 96-well plates. Sar-j was diluted to obtain various concentrations ranging from 1 to 10%, of the original juice, hereafter referred to as 0.01 to 0.1 equivalent (eq.), respectively. After 24 h of culture, various concentrations of sar-j or isoQ (10 μL/ well) were added and the cells were incubated for another 24 h. MTT solution (0.05 g in 10 μL) was added to each well and incubated for 2 h. MTT containing medium was then removed and dimethyl sulfoxide (DMSO) was added to the wells to dissolve the formazan complexes. The optical density of the cells were measured and that of the control (untreated) cells was set at 100% viability.
Akt phosphorylation in A549 cells was determined as follows [5]. Briefly, A549 cells were cultured in 35 mm dishes for 24 h and then 0.3 mL of isoQ (final conc., 0.1 mM) or sar-j (final conc., 0.05 equivalent of original juice) dissolved in DMEM were added, and the cells were incubated for 24 h. For the negative control (NC), A549 cells were cultured and 0.3 mL DMEM (without sar-j or isoQ) was added to the cells. For the positive control (PC), A549 cells were cultured and positive kinase-inhibitor control (without sar-j or isoQ) was added to the cells. LY294002 (final 50 mM) served as positive kinase-inhibitor control was added to the PC dish 1 h prior to termination of incubation. For the epidermal growth factor (EGF)-stimulation experiments, EGF (final concentration, 100 ng/mL) was added 10 min before the termination of incubation. Following incubation, the cells were harvested, lysed, and protein concentrations were quantified using a DC Protein Assay kit (Bio-Rad, Hercules, CA, USA). For western blotting, equal amounts of protein samples were electrophoretically separated on a sodium-dodecyl sulfate-polyacrylamide (12%) gel and transferred onto a polyvinylidene difluoride membrane. The membranes were blocked with 5% skim milk in tris buffered saline containing 0.1% Tween 20 (TBST) for 1 h, and then probed with specific antibodies at 4 °C for 16 h. After washing with TBST, the membranes were incubated with appropriate HRP-conjugated secondary antibodies (1:10000) for 1 h at room temperature. Protein bands were detected using ImmunoStar® LD (Fujifilm Wako Pure Chemical, Osaka, Japan) chemiluminescent reagent and imaged with the biomolecular imager ImageQuant LAS 4000 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). β-Actin was used as a loading control.
Statistical analyses
Data are expressed as mean ± standard deviation (SD) for each data point as indicated in each fig. SD is indicated with a bar. Statistical significance was set at P < 0.05. Statistical analyses were performed using KaleidaGraph (Synergy Software, Reading, PA, USA) and Excel add-in software, Excel statistics (SSRI Co. Ltd., Tokyo, Japan).