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  • br To evaluate the basal toxicity of naringenin and hesperetin

    2022-09-15


    To evaluate the basal toxicity of naringenin and hesperetin com-bined treatment in normal cells, we used HUVECs and human normal fibroblast Detroit551 cells. Treatment with the most 6-Hydroxydopamine effective εCUP mimic condition (narigenin: 7.5 µM, hesperetin: 2.5 µM) significantly inhibited cell viability of Panc-1 cell. However, it had no anti-viability effect on HUVEC and Detroit551 (Fig. 4A). Treatment with εCUP mimic condition also significantly inhibited Panc-1 cell migration, but it had no inhibitory effect on HUVEC and Detroit551 (Fig. 4B). We further assessed the toxicity of gemcitabine, a conventional chemotherapeutic reagent in HUVECs and Detroit551 6-Hydroxydopamine to compare with εCUP mimic condition. Gemcitabine significantly induced an anti-viability effect on Panc-1 cells even at the lower concentration than εCUP mimic condi-tion. However, gemcitabine also significantly reduced HUVECs and Detroit551 viability at the same dosages (Fig. 4C). These results suggest that combined treatment of naringenin and hesperetin effectively in-duces anti-cancer effects in human pancreatic cancer cells without af-fecting normal cells.
    Intracellular signaling modulated by naringenin and hesperetin combined treatment in pancreatic cancer
    In silico target proteins of naringenin or hesperetin were demon-strated from The Human Protein Reference Database (Lee et al., 2016d). We selected various pancreatic cancer cell targets of naringenin or hesperetin using the Human Protein Reference Database; they in-cluded functional receptors, kinases, and unclassified-proteins (Fig. 5A and S1).
    Previously, we showed that exogenous εCUP treatment decreased the phosphorylation of FAK and p38 in human pancreatic cancer cells (Lee et al., 2017a; Lee et al., 2018). Based on the signal transduction by εCUP, phosphorylation of FAK and p38 were examined using combined or single-agent treatment of naringenin and hesperetin in a time de-pendent manner. Exogenous treatment with εCUP mimic condition decreased the phosphorylation of FAK and p38, whereas there was no particular decrease in phosphorylation in single-agent treatment with naringenin or hesperetin (Fig. 5B). These results suggest stomata combined treatment of naringenin and hesperetin effectively inhibits the in-tracellular signaling in human pancreatic cancer cells.
    In vivo effect of naringenin and hesperetin combined treatment
    In vivo, anti-growth effects of naringenin, hesperetin, εCUP mimic condition, and ƒCUP mimic condition were examined in Panc-1
    A mAU
    b
    a
    c
    d
    B
    mAU
    C
    ƐCUP
    fCUP
    Yield (ppm) LOD (LOQ) Yield (ppm) LOD (LOQ)
    Fig. 2. Contents of constituent elements of enzymatic hydrolysis of Citrus unshiu peel (εCUP) and fermented extraction of Citrus unshiu peel (ƒCUP). (A) After enzymatic hydrolysis, Citrus unshiu peel (εCUP) was dissolved in methanol and injected into a HPLC system at a column flow rate of 1 ml/min; a, b, c, and d indicate narirutin, hesperidin, naringenin, and hesperetin, respectively. (B) After fermented extraction, Citrus unshiu peel (ƒCUP) was dissolved in methanol and injected into a HPLC system at a column flow rate of 1 ml/min; a’, b’, c’, and d’ indicate narirutin, hesperidin, naringenin, and hesperetin, respectively. (C) Extracted yields and LOD and LOQ values of narirutin, hesperidin, naringenin, and hesperetin in εCUP and ƒCUP were calculated using HPLC chromatograms.
    xenograft model. PBS-treated Panc-1 xenograft tumors grew to an average size of 97.13 ± 24.45 mm3 by 25 days after transplantation. In contrast, ƒCUP mimic condition-treated Panc-1 xenograft tumors grew to an average size of 65.28 ± 23.41 mm3 and εCUP mimic condition-treated Panc-1 xenograft tumors grew to an average size of 44.49 ± 22.03 mm3 by 25 days after transplantation. Naringenin or 
    hesperetin treated Panc-1 xenograft tumors grew to an average size of 117.99 ± 35.88 or 138.33 ± 41.98 mm3, respectively, under the same condition (Fig. 6A). There was no significant weight loss in all xenograft models (Fig. 6B). To ascertain the anti-growth effect of ƒCUP mimic or εCUP mimic treatment at the molecular level, we investigated caspase-3-mediated apoptosis in ƒCUP mimic or εCUP mimic-treated