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  • Our recent study reported that multidrug resistant MDR

    2020-08-28

    Our recent study reported that multidrug-resistant (MDR) human A549 lung cancer Acetylcysteine (A549RT-eto) with the elevated expression of NF-κB showed epithelial–mesenchymal transition (EMT), increasing spheroid formation and elevating the expression levels of stemness-related factors, including Oct4, Nanog, Sox2, Bmi1, and Klf4. Therefore, when new therapeutic agents targeting these malignant cancer cells were explored, we found that caged-xanthone (CX) isolated from the roots of Cratoxylum formosum ssp. pruniflorum diminished the expression of NF-κB, P-glycoprotein (P-gp) protein levels, cell migration and invasion, and sphere-forming ability of A549RT-eto cells. To address the role of NF-κB in these malignant cancer features, we treated A549RT-eto cells with NF-κB siRNAs in the present work. We found that the knockdown of NF-κB inhibited EMT and sphere formation. Furthermore, co-treatment with CX and NF-κB siRNA accelerated the death of apoptotic cells through the decrease of P-gp protein levels. These results suggest that NF-κB was involved in malignant cancer pheno-types and MDR in A549RT-eto cells. Taken together, our findings suggest that CX can be a potential therapeutic agent for the treatment of malignant tumor cells.
    1. Introduction
    Nowadays, a major hurdle in cancer therapy is multidrug resistance (MDR) due to overexpression of a drug efflux protein, P-glycoprotein (P-gp). As a membrane transporter protein, P-gp is an energy-dependent drug efflux pump that maintains intracellular drug concentrations below cytotoxic levels, thereby decreasing the cytotoxic effects caused by a variety of chemotherapeutic agents.1–4 In particular, recent results have shown that a transcription factor NF-κB is activated in a range of human cancers and thought to promote MDR cancers mainly because of its ability to protect transformed cells from apoptosis.5 Moreover, NF-κB plays a key role in cancer development and metastasis. These multistep processes involve local tumor growth and invasion, followed by dis-semination to and re-establishment at distant sites.6 Metastasis occurs through a series of steps: local invasion, intravasation, transport, ex-travasation, and colonization. A developmental program termed as epithelial–mesenchymal transition (EMT) has been demonstrated to play a critical role in promoting the progression of malignant tumors.7
    In addition, cancer stem cells (CSCs) represent a small subset of cancer cells that are characterized by their self-renewal capability and plur-ipotent differentiation potential similar to those of normal stem/pro-genitor cells. Therefore, CSCs are believed to be of great significance in carcinogenesis, tumor growth, metastasis, and cancer recurrence.8 Moreover, CSCs are capable of forming suspended spherical, clonal colonies, also called sarcospheres, and they preferentially express key marker genes of pluripotent embryonic stem cells such as Oct-3/4 and Nanog.9 Thus, the elucidation of the molecular pathways for the de-velopment of malignant cancer phenotypes provides a clue for treat-ments from natural products.
    Because many plants have been the rich source of compounds as therapeutic agents, currently, 75% of prescribed drugs worldwide are derived from plant sources.10 One of them is the Cratoxylum genus, whose biological activities have been evaluated for their secondary metabolite compounds, including xanthones, triterpenoids, and flavo-noids.11 Caged-xanthone (CX), cochinchinone C, can be isolated from the roots of C. formosum ssp. pruniflorum.12 CX has many biological
    Corresponding authors.
    E-mail addresses: [email protected] (C. Kaewpiboon), [email protected] (Y.-H. Chung).
    Available online 31 December 2018
    Figure 1. Up-regulation of NF-κB in A549RT-eto cells and the chemical structure of caged-xanthone (CX).
    activities such as antibacterial,13 anti-HIV-1,14 and cytotoxic activ-ities.12,15 However, the molecular mechanism on CX having anticancer activity has been rarely revealed.
    Therefore, in this study, we explored whether CX derived from the roots of C. formosum ssp. pruniflorum in Thailand can inhibit the pro-gression of malignant cancer cells through the down-regulation of NF-κB. The results suggest that CX could be a potent anticancer drug for the treatment of lung cancer overexpressing P-gp.
    2. Results
    2.1. CX induces cytotoxicity in A549RT-eto cells with higher levels and activity of NF-κB
    A549 lung cancer cells resistant to etoposide (A549RT-eto) exhibit enhanced P-gp protein levels, leading to MDR.16 Moreover, because A549RT-eto cells were found to exhibit resistance to etoposide, we wondered what feature A549RT-eto cells possess in comparison with A549 parental cells. When the expression level of NF-κB was compared, the levels of NF-κB proteins were enhanced in A549RT-eto cells com-pared with those in A549 parental cells (Fig. 1A). Furthermore, the localization of NF-κB was compared between A549RT-eto and their parental cells. The GFP-tagged NF-κB (p65; RelA) in A549 cells and A549RT-eto cells was detected in the cytoplasm and nucleus, respec-tively (Fig. 1B). These results suggest that A549RT-eto cells showed higher levels and activity of NF-κB compared with A549 parental cells.