br Figure Suppression of NF
Figure 4. Suppression of NF-κB by NF-κB siRNA enhanced CX-induced apoptosis of A549RT-eto cells.
number of spheroids was counted at day 6. The data are representative of three experiments (***P < 0.001 compared with the control group). (D) The cell lysates from A549RT-eto treated with control or NF-κB siRNAs were prepared and separated on 12% SDS-PAGE gel. The ex-pressions of Oct4, Bmi1, Nanog, and Sox2 proteins were detected by immunoblotting with the corresponding antibodies.
In this study, we found that a purified compound, caged-xanthone (CX) from C. formosum ssp. pruniflorum, exhibited significant anti-pro-liferative effects against A549 cancer CT 99021 resistant to etoposide (A549RT-eto). Because our previous study has shown that A549RT-eto cells displayed drug resistance due to the overexpression of P-glyco-protein (P-gp) encoded by the MDR1 gene,16 it is meaningful that CX administration diminished the protein levels of P-gp, leading to reversal of MDR in A549RT-eto cells. The previous study showed that treatment with a NF-κB inhibitor (BAY) decreased the P-gp expression in A549RT-eto cells,16 which supports the hypothesis that the CX induced decrease of NF-κB levels might be ascribed to the decrease of P-gp expression, resulting in enhanced sensitivity to etoposide. Therefore, CX may be a highly effective candidate compound for an anti-MDR cancer drug. Although another study has shown that CX possessed in vitro cyto-toxicity against MCF-7, a breast cancer cell line,12 the molecular me-chanism by which this compound exerted cytotoxicity has rarely been revealed. We, for the first time, provide a line of evidence showing the cytotoxic mechanism of CX against A549RT-eto cancer cells. In A549RT-eto cells, CX-mediated induction of cell death is directly re-lated to NF-κB inhibition. Moreover, the CX treatment inhibited CSC-like phenotypes such as the decrease of sphere formation, expression of
stemness-related factors, and cell migration and invasion. We obtained the same inhibition of CSC-like phenotypes from NF-κB silencing with the CX treatment.
The levels of NF-κB proteins were enhanced in A549RT-eto cells compared with those in A549 parental cells. NF-κB activation has been associated with the initiation and progression of several human cancers, including breast, cervical, prostate, pancreatic, and lung cancer.20–22 It has also been involved in apoptosis resistance in cancer cells.23 In our study, the suppression of NF-κB induced apoptosis in A549RT-eto cells. This finding is in agreement with a previous report that NF-κB activated the overexpression of anti-apoptotic genes.24 Deactivation of NF-κB was reported to be associated with the down-regulation of BCL-2 through
caspase-3 activation.25 Moreover, NF-κB activation has been associated with the invasive and metastatic capabilities of cancers.26,27 The inva-
sion and metastasis process occurs when epithelial cancer cells lose their adherent properties and acquire the mesenchymal phenotype.26,27 This study demonstrated that the decrease of NF-κB levels by CX treatment diminished the expression of mesenchymal protein markers such as N-cadherin and vimentin, but enhanced the expression of an epithelial protein marker such as E-cadherin. The decrease of NF-κB levels by CX treatment also reduced the expression of stem cell tran-scription factors (Sox2, Nanog, Klf4, Bmi1, and Oct4) in A549RT-eto cells. The stemness-related transcription factors were found to be overexpressed in several cancers, including breast, prostate, and oral squamous cell carcinoma, and their expression levels are associated with tumor transformation, tumorigenicity, and tumor metastasis.28 On the basis of these studies, we propose that the decrease of NF-κB levels by CX treatment could be a potential resolution to treat other cancers besides A549 lung cancer with MDR.
Therefore, NF-κB molecules become one of the most suitable
Figure 5. Knockdown of NF-κB by NF-κB siRNA decreased cell invasion and spheroid formation in A549RT-eto cells.
candidates for therapeutic targets for cancer treatment.29 For instance, the inhibition of NF-κB using plant-derived agents such as triptolide reduced the colony- and sphere-forming capacity of pancreatic CSCs.30 Curcumin and epigallocatechin gallate as antitumor agents inhibit breast CSC phenotypes through the down-regulation of Stat3–NF-κB signaling. 31 In addition, resveratrol could suppress the adhesion, inva-sion, and migration of glioblastoma-initiating cells via the inhibition of PI3K/Akt/NF-κB signal transduction.32 However, we first provide a line of evidence showing that CX exerted cytotoxicity against A549RT-eto cancer cells through NF-κB down-regulation. Moreover, the CX treat-ment also reduced malignant cancer phenotypes, relevant to NF-κB activation in A549RT-eto cells. Therefore, these findings indicate that NF-κB can be a promising molecular target for MDR lung cancer. On the basis of these findings, down-regulation of NF-κB using CX may be a key to solve some major challenges in cancer therapy, namely cancer re-lapse and chemotherapy resistance.