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  • T-5224 br In recent years Chinese medicine has emerged as


    In recent years, Chinese medicine has emerged as an attractive source for a new generation of autophagy inhibitors (Fu et al., 2018; Song et al., 2017). Ginsenoside Rg3, an active ingredient of Panax ginseng, exerts anticancer effects on some cancers (Lee et al., 2015; Wu et al., 2015). Ginsenoside Rg3 can divide into two isomers: 20(S) and
    Fig. 1. Icotinib treatment inhibits growth and increases autophagic flux in PC-9 and HCC827 cells. A and B, Inhibitory effect of icotinib on PC-9 and HCC827 cells. The percentage of vi-able T-5224 was measured by MTS assay. Data are presented as the mean ± S.D. (n = 3). C, Cells were treated with 1 μM icotinib for 24 h before being labeled with fluorescence and imaged by fluorescence microscopy. Green, FITC-labeled LC3. Red, LysoTracker-labeled lysosome. Blue, DAPI-labeled nuclei. D, The percentage of puncta-positive cells was quan-tified by automated image acquisition and analysis using a threshold of > 5 dots/cell.
    ***P < 0.001. The data are representative of three independent experiments. E, Immunoblotting for LC3 and p62 using lysates from cells treated with 1 μM icotinib in the presence or absence of CQ for 24 h. (For in-terpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
    20(R). The 20(R) isomer is poorly soluble, while the 20(S) isomer has a much higher solubility and is more suitable for pharmaceutical devel-opment (Fan et al., 2017). Kim et al. reported that 20(S)-Rg3 (Rg3) can inhibit doxorubicin-induced autophagy by suppressing autolysosome formation and degradation to sensitize hepatocellular carcinoma cells to doxorubicin (Kim et al., 2014). So far, few studies of the relationship of Rg3 and autophagy in NSCLC have been conducted. In the present study, we investigated the effects of Rg3 on the regulation of autophagy in NSCLC cell lines. We further confirmed that Rg3 enhanced the sen-sitivity of NSCLC cells to icotinib via autophagy inhibition in vitro and in vivo.
    2. Materials and methods
    2.1. Chemicals and antibodies
    2.2. Cell lines and animals
    HCC827 and PC-9 cells were purchased from the Cell Bank of the Chinese Academy of Science (Shanghai, China). Cells were cultured in RPMI-1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% FBS (Gibco) at 37 °C under a 5% CO2 and 90% humidified atmo-sphere. The experiments with these cells were carried out within 6 generations after resuscitation. Female athymic BALB/c nude mice (Shanghai Institute of Material Medicine, Chinese Academy of Science, China) were maintained in a specific pathogen-free facility and were treated with humane care after approval from the Animal Care and Use
    Fig. 2. PC-9 and HCC827 cells resistant to icotinib showed increased basal autophagy. A, Inhibitory effects of icotinib on PC-9R and HCC827R cells. The percentage of viable cells was measured by the MTS assay. Data are presented as the mean ± S.D. (n = 3). B LC3 II levels were evaluated by immunoblotting using lysates from cells treated with 1 µM icotinib (parental cells) or 20 μM icotinib (resistant cells) for 24 h. C Immunoblotting for LC3 and p62 using lysates from cells treated with 20 μM icotinib in the presence or absence of CQ for 24 h.
    Committee of Hangzhou Cancer Hospital.
    2.3. Proliferation assay
    Cell proliferation was determined by MTS assay. Cells were seeded into 96-well plates and treated with icotinib, CQ, Rg3 or a combination. After treatment, 10 μl MTS (Promega, G1111) was added to each well and incubated for 2 h. Absorbance was measured using a model ELX800 Microplate Reader (Bio-Tek Instruments, Inc, Winooski, USA) at 490 nm to calculate cell proliferation. Three independent experiments were performed to determine the half-maximal inhibitory concentra-tion (IC50) values.