br Epithelial to mesenchymal transition EMT is a
Epithelial-to- mesenchymal transition (EMT) is a primary process characterized by losing polarity, reduced cell-to-cell adhesion and advanced migratory capacity . Notably, Twist-related protein 1 (Twist1) is a basic helix- loop-helix (bHLH) transcription factor and a known regulator for EMT, which inhibits the Puromycin of an epithelial marker (E-cadherin) while promotes mesenchymal marker (e.g. N-cadherin and vimentin) expression to facilitate EMT or metastasis . Overexpression of Twist1 is also associated with cisplatin resistance in lung cancer and several other types of cancers . Accordingly, EMT induced by Twist1 can also lead to cell transformation which possesses similar properties with cancer stem cells
. Twist1 also negative affects apoptotic cell death via serine (Ser 68) phosphorylation by p38, c-Jun N-terminal kinases (JNK) and Erk1/2 to promote EMT and metastasis . Consistently, increased expression of Twist1 by c-ros oncogene 1 (ROS1) G2032R mutation can result in elevated malignant phenotypes such as enhanced migration, invasion and resistance to therapeutic agents . Therefore, Twist1 is an important mediator during cancer progression and targeting Twist1 might be an effective strategy to eliminate cancer cells.
Large-scale screening for lipidomics have dramatically advanced our understanding about the small molecule-protein interaction . By introducing
chemical proteomic methods in this field, a novel LiPIP (Lipid-Protein Interaction Profiling) approach has been identified and can be successfully coupled with mass spectrometry (MS) to assess drug binding . The flavonoid atalantraflavone (AFL), is a new natural product isolated from leaves of Atalantia monophylla (L.) DC . However, the function of AFL in lung cancer remains totally unknown.
In current work, we have unraveled a novel function for AFL. We found that AFL displayed anti-tumor activity towards lung cancer cells. AFL dose-dependently decreased the viability, migration and colony formation of NSCLC cells probably by inducing apoptosis. Furthermore, we have identified that Twist1 might be the target for AFL by LiPIP. AFL markedly decreased Twist1 stability by promoting ubiquitin mediated proteasomal degradation of Twist1. Given the critical role on Twist1 stability, we found that AFL could reverse cisplatin resistance in some NSCLC cell lines. Our findings have identified a tumor suppressive activity for AFL in NSCLC and provided a promising clue to anti-tumor therapy.
2. Materials and Methods
2.1 Cell culture and reagents
H1299, H1155, H838, 95C, 95D and A549 cells were obtained from Shanghai Institute of Cell Biology, Chinese Academy of Sciences. Tumor cells were maintained in high glucose DMEM supplemented with 8% FBS, 100 µg/mL penicillin and 100 U/mL streptomycin. Cell lines were verified by PCR and were not contaminated by mycoplasma. The Atalantraflavone (AFL) was generously supplied by Dr. J. Suthiwong from Department of Chemistry and Center of Excellence for Innovation in Chemistry, Khon Kaen University. For antibody details, see Table S1.
2.2 Viability assay
The Cell Counting Kit-8 (CCK-8; Dojindo) was used to measure the viability. Following treatment with AFL for 24 h, cells were re-suspended and seeded into a 12-well plate (~105 cells/well) for 5 days. 20 µl/well CCK-8 solutions were added and
incubated for 1.5 h. Optical density at 450 nm was evaluated using the MRX II microplate reader (Dynex) according to the manufacturer’s instructions.
2.3 Quantitative PCR (qPCR)
The total RNAs were extracted with Trizol reagent according to the manufacturer’s instructions. Primers were designed, synthesized and purchased from Life Technologies (Shanghai). For details, see Table S1. The qPCR assays were performed with an miScript SYBR Green PCR Kit (Qiagen). GAPDH was the control.