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  • CT-99021 br We then purified CX derived from C formosum ssp

    2020-08-28


    We then purified CX derived from C. formosum ssp. pruniflorum as described in the Materials and Methods section and treated A549RT-eto CT-99021 with CX. The molecular structure of CX is shown in Fig. 1C.
    (A) Cell lysates from A549 and A549RT-eto cells were prepared and separated on 8% SDS-PAGE gel. The expression of NF-κB was detected by immunoblotting, and the protein level of NF-κB was compared by immunoblotting with the corresponding antibody. (B) A549 and A549RT-eto cells were transfected with a pEGFP-RelA (NF-κB tran-scription factor subunit) vector. Green fluorescence was then analyzed using a fluorescence microscope at 24 h post-transfection. (C) The chemical structure of caged-xanthone (CX), which was isolated as 
    2.2. CX reduces NF-κB and P-gp levels, leading to apoptosis in A549RT-eto cells
    A549RT-eto cells were found to exhibit up-regulation of NF-κB, leading to elevated levels of P-glycoprotein (P-gp) encoded by MDR 1.17 Therefore, we searched and isolated compounds derived from medic-inal plants in Thailand to reverse MDR. As shown in Fig. 2A, CX was found to efficiently induce the death of A549RT-eto cells in a dose-dependent manner after 72 h of exposure of A549RT-eto cells with CX at two-fold serial dilutions from 25 to 1.56 μg/mL. After various con-centrations of CX were treated in A549 parental and A549RT-eto cells for 72 h, IC50 values were examined. The results showed that the IC50 values of A549 parental cells and A549RT-eto cells were 2.51 ± 0.20 and 4.96 ± 0.01 μg/mL, respectively (Fig. 2B). Therefore, A549 par-ental cells are more sensitive to growth inhibition with CX treatment. Because it has been reported that NF-κB is involved in the regulation of MDR through P-gp protein levels,18 the protein levels of NF-κB and P-gp in A549RT-eto cells treated with CX (1 and 5 μg/mL) were then ex-amined. As shown in Fig. 2C, CX treatment reduced the expression le-vels of NF-κB and P-gp. In addition, after treatment with CX (1 and 5 μg/mL) at 24 h, we found reduced levels of pre-caspase 9 and pre-caspase 8 as well as increased levels of cleaved PARP, as a substrate of
    Figure 2. Optimization of CX concentration for cytotoxicity and apoptosis induced by CX treatment in A549RT-eto cells.
    active caspase 3 and 7 in A549RT-eto cells (Fig. 2C), indicating that CX induced not only extrinsic but also intrinsic apoptosis.
    (A) A549RT-eto cells were treated with CX (20–0.16 μg/mL) for 72 h, and then, the percentage of cell viability was measured by MTT. IC50 values were calculated by the MTT assay. Results were averaged from triplicate wells, and the error bars indicate S.D. (**p < 0.001; A549 vs A549RT-eto). (B) A549RT-eto cells were treated with CX (0, 0.1, 2, and 5 μg/mL) for 24 h. After treatment, the morphological changes of cells and cell viability were observed under an optical mi-croscope (100× and 400× ). (C) The cell lysates from A549RT-eto treated with CX were prepared and separated on 12% SDS-PAGE gel. The expressions of pre-caspase 8, 9, cleaved PARP, NF-κB, and P-gp proteins were detected by immunoblotting with the corresponding antibodies.
    2.3. CX decreases migration, invasion and spheroid formation of A549RT-eto cells
    To evaluate the effects of CX on the migration of A549RT-eto cells, we performed wound-healing assay. For this experiment, the cells were co-incubated with non-cytotoxicity doses (0 and 1.5 μg/mL) of CX for 24 h. The treatment with CX (1.5 μg/mL) for 24 h significantly de-creased the cell migration rates in A549RT-eto cells (Fig. 3A). We also examined cell invasion capacity using a three-dimensional matrigel-coated filter after A549RT-eto cells were treated with CX. As shown in Fig. 3B, the treatment of A549RT-eto cells with CX significantly de-creased the cell invasion rates (approximately two-fold) compared with mock treatment.
    Because CX inhibited cell migration and invasion, we next examined the inside of cells during the inhibition of EMT by measuring the levels of relevant proteins. Because it is well known that EMT is characterized by the down-regulation of E-cadherin and up-regulation of N-cadherin, CX treatment increased the expression of E-cadherin as well as 
    inhibited the expression of N-cadherin and vimentin. Therefore, CX can potentially inhibit EMT by modulating the expression of E-cadherin, N-cadherin, and vimentin (Fig. 3C).
    CSCs play a major role in cancer initiation, progression, metastasis, and drug resistance.19 Therefore, it is a challenge to search for antic-ancer drugs to overcome CSCs. We thus examined whether CX treat-ment can interrupt A549RT-eto sphere formation because the sphere formation assay is considered a useful method to evaluate self-renewal of CSCs in vitro. As shown in Fig. 3D, CX treatment inhibited the size and number of spheroid formation compared with mock treatment. The expression of transcription factors or co-activators responsible for stemness such as Oct4, Bmi1, Nanog, and Sox2 was measured. As shown in Fig. 3E, CX treatment reduced the protein levels of Oct4, Bmi1, Nanog, and Sox2 in a dose-dependent manner. These results suggest that CX possessed an inhibitory capability on CSC phenotypes of A549RT-eto cells.