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  • br Conclusions br Caged xanthone

    2020-08-28


    4. Conclusions
    Caged-xanthone (CX), cochinchinone C, from the roots of C. for-mosum ssp. pruniflorum in non-cytotoxicity concentration (1.5 µg/ml) diminished cell migration, invasion, and sphere-forming ability of A549RT-eto cells through down-regulation of NF-κB. Moreover, NF-κB was involved in the CHIR-99021 resistance to etoposide in A549RT-eto cells. Therefore, these observations suggest that CX may be a key to solve some major challenges in obstacle of cancer therapy. 
    5. Experimental scetion
    5.1. Plant materials
    Roots of C. formosum ssp. pruniflorum were collected in May 2004 from Nong Khai Province in the northeastern part of Thailand. Identification was made by Prof. Puangpen Sirirugsa, Department of Biology, Faculty of Science, Prince of Songkla University. A specimen (No. 0012677) was deposited at Prince of Songkla University Herbarium.
    5.2. Preparation of caged-xanthone cochinchinone C (CX)
    Optical rotations were measured on a JASCO P-1020 polarimeter. A SPECORD S 100 (Analytikjena) spectrophotometer was used to measure Ultraviolet (UV) CHIR-99021 spectra. Infrared spectra (IR) were recorded on a Perkin-Elmer 783 FTS FT-IR spectrometer, and 1H and 13C NMR spectra were recorded on a 300 MHz Bruker FTNMR Ultra Shield in CDCl3, with TMS as the internal standard. Chemical shifts are reported in δ (ppm), and coupling constants (J) are expressed in hertz. Quick Column chromatography (QCC) and column chromatography (CC) were carried out on silica gel (Merck) type 100 (0.063–0.20 mm) and silica gel 60 F254 (Merck) with a gradient system of acetone-n-hexane or as stated otherwise, or silica gel 60 RP-18 (Merck) with pure MeOH.
    Air-dried roots of C. formosum ssp. pruniflorum (5.30 kg) were ex-tracted using CH2Cl2 (2 × 20 L, for 5 days) at room temperature. The crude CH2Cl2 extracts were allowed to evaporate under reduced pres-sure to afford brownish crude (60.0 g) extracts. Such extracts were subjected to QCC on silica gel using n-hexane as the first eluent and
    C. Kaewpiboon et al.
    using acetone with higher polarity to give six fractions (F1–F6). Fraction F2 was then separated by QCC eluting with a gradient of CH2Cl2-n-hexane to afford 11 subfractions (F2A–F2K). Subfraction F2B was further purified by QCC with a gradient of EtOAc-n-hexane to give five subfractions (F2B1–F2B5). Next, subfraction F2B4 was purified by
    A549RT-eto cells were developed and kindly provided by the Laboratory of Biochemistry, Chulabhorn Research Institute, Thailand, as described elsewhere.33 The A549RT-eto cells and their parental A549 cells were cultured in RPMI-1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% FBS, 1% penicillin, and streptomycin (Gibco) at 37 °C in a humidified atmosphere of 5% CO2 in air.
    5.4. Cell viability assay
    The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed for the measurement of cell survival as previously described.34 Both A549RT-eto cells and their parental A549 cells were seeded onto a 96-well plate separately at a density of 5 × 104 cells/ml. After 24 h of cell culture, 2.0 μL of each CX solution in DMSO at various concentrations was added to both of the cell lines to obtain 200 μL of a cell solution with CX. Therefore, the final concentrations of CX were 20–0.16 µg/mL. Samples free of the CX/DMSO solution served as control samples and the μg/mL samples only contained 2.0 μL of DMSO. After this, the cells were cultured for 48 h. In order to eliminate the interference of DMSO, a smaller amount (1%) was selected through experimentation (ie, observing no change in cell viability through using DMSO alone; data not shown) for the cytotoxicity assays.
    Thereafter, the media in the wells were removed and replaced with 100 μL fresh medium containing 5 mg/mL MTT and incubated at 37 °C for 2 h to allow the formation of the insoluble formazan crystal by the mitochondrial active (living) cells. The media were then removed, 100 μL DMSO was added to lyse the cell membranes and solubilize the formazan crystals and the absorbance of the formazan produced by living cells was measured at 570 nm. The relative percentage of cell survival was calculated by the mean absorbance of the treated cells (ODT) and the mean absorbance of control cells (ODC) using the fol-lowing formula: % Cell survival = (ODT/ODC).  Bioorganic & Medicinal Chemistry 27 (2019) 2368–2375
    5.5. Antibodies and reagents
    20) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-Bmi1 (EPR3752) and Oct4 (EPR2054) were pur-chased from Abcam (Abcam, England).
    5.6. Immunoblotting
    Cells were harvested and lysed with a lysis buffer (150 mM NaCl,