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  • br of tumor cell apoptosis MSCs have

    2022-05-09


    of tumor cell apoptosis [18–20]. MSCs have also been reported to prevent tumor growth through apoptosis induction, 5-BrdU and cel-lular signaling regulation, and immune cell infiltration induction [21].
    Interferons (IFNs) are pleiotropic cytokines that regulate the innate and acquired immunity, activate host defenses against viral and bac-terial infections, and function in tumor surveillance. IFNs comprise two main families: type I (IFN-α, -β, -ε, -κ, and -ω) and type II (IFN-γ) [22]. IFN-α and IFN-β are secreted by many cell types, including lymphocytes (natural killer [NK] cells, B-cells, and T-cells), macrophages, fibroblasts, endothelial cells, and osteoblasts [23]. They stimulate both
    Abbreviations: 40K-ASC-CM, CM recovered from 40K-ASCs; 7-AAD, 7-aminoactinomycin D; APC, allophycocyanin; ASCs, adipose tissue-derived MSCs; Cas, caspase; CFSE, carboxyfluorescein diacetate succinimidyl ester; CM, condition medium; DMEM, Dulbecco's modified Eagle's medium; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; H460*, CFSE-labeled H460; HBSS, Hank's Balanced Salt solution; IFNs, interferons; mbTRAIL, membrane-bound TRAIL; MSCs, mesenchymal stem cells; MTT, methylthiazolyldiphenyl-tetrazolium bromide; PBS, phosphate-buffered saline; RT-PCR, reverse transcription-polymerase chain re-action; SD, standard deviation; sTRAIL, secreted TRAIL; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand
    ∗ Corresponding author. ∗∗ Corresponding author. E-mail addresses: [email protected] (K.S. Bae), [email protected] (Y.W. Eom).
    macrophages and NK cells to elicit potent anti-tumor, anti-viral, and immunomodulatory functions [24,25]. Moreover, MSCs that are primed or genetically modified with type I and II IFNs exert antitumor effects via nuclear factor-κB (NF-κB), signal transducer and activator of tran-scription (Stat), or tumor necrosis factor-related apoptosis-inducing li-gand (TRAIL) pathways [26–30].
    Previously, we reported that adipose tissue-derived MSCs (ASCs) cultured at a high density expressed type I IFNs and suppressed tumor growth in MCF-7 breast cancer cells [31]. Herein, we investigated TRAIL expression in ASCs cultured at a high density, analyzed the mechanisms controlling IFN-β and TRAIL expression, and evaluated the tumor suppression potential of ASCs against H460 lung cancer cells in vitro and in vivo.
    2. Materials and methods
    In accordance with the procedures approved by the Institutional Review Board of the Yonsei University Wonju College of Medicine, human adipose tissues from three healthy donors (24–38 years of age) were obtained through elective liposuction procedures under anesthesia at the Wonju Severance Christian Hospital (Wonju, Korea). Informed consent was obtained from all donors. Mononuclear cells were isolated using a modified protocol as described by Zuk et al. [32]. Briefly, li-poaspirates were thoroughly washed with phosphate-buffered saline (PBS) to remove contaminated blood cells and local anesthetics. Mononuclear cells were then obtained by digestion with 0.075% type IA collagenase (Sigma-Aldrich, St. Louis, MO, USA) in PBS, cen-trifugation at 1200×g for 5 min, and resuspension in low-glucose Dulbecco's modified Eagle's medium (DMEM; Gibco, Rockville, MD, USA) containing 10% fetal bovine serum (FBS) and penicillin/strepto-mycin. Debris was removed by filtration through a 100-μm mesh filter (Cell Strainer; Becton Dickinson, Franklin Lakes, NJ, USA). Mono-nuclear cells (5 × 106) were seeded in a 100-mm culture dish with low-glucose DMEM containing 10% FBS and penicillin/streptomycin. After 2 days, the medium was replaced to remove non-adherent cells. Thereafter, the cell culture medium was changed twice weekly, and the cells were passaged with 0.25% trypsin/0.1% EDTA (Gibco) until they reached 90% confluence. For experiments, ASCs were seeded at 5,000, 10,000, 20,000, or 40,000 cells/cm2 (5K-ASCs, 10K-ASCs, 20K-ASCs, and 40K-ASCs, respectively) and cultured for the indicated times (1–5 days).