br activity of e ector T cells by
activity of eﬀector T cells by producing inhibitory cytokines (Hinz et al., 2007). Therefore, evaluating the immune-cell population and Treg-re-lated factors such as CCR, CCL, and FOXP3 could be a simple metho-dology to test any therapeutic eﬀect of an agent on PC.
Tregs generally take part in the immune balance between immunity and tolerance to avoid any tissue damage by autoimmunity (Rothstein
& Camirand, 2015). Their suppressive mode of action includes highly expressed CD39 and CD73 ectoenzymes as well as upregulated CTLA-4 on the surface; CD39 and CD73 facilitate converting extracellular ATP to adenosine, which directly inhibits the proliferation of eﬀector T cells, whereas CTLA-4 can downregulate CD80 and CD86 on dendritic cells to impede their function (Plitas & Rudensky, 2016). Additionally, Tregs can directly produce anti-inflammatory cytokines such as IL-10 and TGF-β, which suppress eﬀector T cells thereby weakening the immune system as well as promoting tumor survival (Al Dulaijan, Mansouri, Karnyski, & Azzi, 2018; Ino et al., 2013).
Normal ssamchae (Normal Leaves: NL) is a line of Chinese cabbage and can be grown in the winter without packaging of the head. The appearance of NL can be described as an unbound cylindrical ensemble of leaves. Sweet and crunchy ssamchae is frequently consumed as an ingredient of a Korean-style salad. On the other hand, Amtak-ssamchae (anti-Tumor Leaves: TL) is a new cross of Chinese cabbage with turnip designed to increase the amounts of carotenoids, particularly β-car-otene. TL shares many characteristics with NL, e.g., its appearance and cultivation practices.
Dietary Kainic acid are known to benefit health by decreasing the risk of certain types of cancers and eye diseases (Johnson, 2002). Ac-cordingly, higher concentrations of carotenoids in TL could exert therapeutic eﬀects on cancers. Therefore, this study was aimed at in-vestigating the possible preventive eﬀects of TL against pancreatic cancer in an in vivo animal model by determining the tumor size and the immune-cell population in a tumor. In addition, the mechanisms un-derlying Treg regulation were studied.
2. Materials and methods
2.1. Extracts and Panc02 cell culture
NT and TL were obtained from Jeil Seed Bio Co., Ltd. (Jeungpyeong-gun, Chungbuk, Korea). They were ground into a fine powder after freeze-drying followed by extraction with 80% ethanol and removal of the solvent in a vacuum evaporator. Both samples were deposited in the Pharmacognosy Laboratory, College of Pharmacy at Dankook University.
Panc02 (which is murine pancreatic adenocarcinoma cell line) was employed in the experiments. The cells were cultured in the complete RPMI 1640 medium with 10% fetal bovine serum (FBS; Cellgro, Herndon, VA, USA), 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (Cellgro) at 37 °C and 5% CO2. Cytotoxicity of NL and TL extracts toward Panc02 cells was measured by a 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Panc02 cells were incubated with or without one of the extracts for 48 h, and viability was assessed.
Female C57BL/6 mice aged 6–8 weeks were purchased from KOATECH (Pyungtaek, South Korea). All experiments and procedures
were conducted in accordance with the National Institutes of Health Guide for the care and use of laboratory animals, and the protocol was approved by the Chung-Ang University’s Institutional Animal Care and Use Committee (IACUC) of the Laboratory Animal Research Center. All the mice were housed at 3–5 per cage at a pathogen-free animal facility at a controlled temperature of 21 ± 3 °C, relative humidity of 50% ± 10%, and on a 12/12 h light/dark cycle throughout the ex-periment, and a habituation period was added for at least 1 week before the start of every experiment. The mice were injected with 3 × 106 Panc02 cells (resuspended in 100 μL of saline) subcutaneously (s.c.) into the lower left back and treated or not treated with 80 mg/kg NF or TF intraperitoneally three times a week for 26 days; the size of tumors was measured once a week (Fig. 1). The mice were euthanized at the final time point using an overdose of CO2 and cervical dislocation ac-cording to the Chung-ang University IACUC guidelines, and tumor and spleen were collected and processed for ex vivo biochemical experi-ments.
2.3. Total RNA isolation and quantitative reverse-transcription PCR
Cancer cells were isolated from the biopsies of the tumor tissue, and total RNA was extracted using the TRIzol Reagent (Invitrogen, Waltham, MA, USA). Splenocytes (106) from mice were activated ex vivo by incubation with 1 μg/mL of anti-CD3 (BD Bioscience, Franklin Lakes, NJ, USA) for 48 h, and total RNA was isolated from each sample by means of TRIzol. RNA samples were transcribed into cDNA at 42 °C for 1 h in a 25-μL reaction mixture containing 5× reverse transcriptase buﬀer, 10 mM dNTPs, Maloney murine leukemia virus reverse tran-scriptase (MMLV-RT; 200 U, Sigma-Aldrich, St. Louis, MO, USA), and 100 pmoles of the oligo-dT primer. The concentration of cDNA was estimated by the quantitative reverse-transcription PCR method using 2× iQTM SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) to de-termine the mRNA levels of FOXP3, IL-10, TGFβ, CCR5, and CCL5. Amplification was performed on a CFX Connect™ Real-Time PCR Detection System (Bio-Rad) under the following conditions: 95 °C for 3 min, then 45 cycles at 95 °C for 10 s and 60 °C for 30 s. To confirm PCR specificity, the amplicons were subjected to a melting-curve analysis. The comparative threshold cycle method was used to calculate the re-lative amounts of mRNA in the experimental samples compared to the control samples. Gene expression was normalized to the expression levels of GAPDH.