Luteolin overcomes acquired resistance to osimertinib in non-small cell lung cancer cells by targeting the HGF-MET-Akt pathway.

Osimertinib, a third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), has overcome the acquired resistance of first- and second-generation EGFR-TKIs due to the EGFR T790M mutation in non-small cell lung cancer (NSCLC). However, acquired resistance to osimertinib remains a significant clinical challenge. Luteolin, a natural flavonoid from traditional Chinese medicine, has exerted antitumor effects in various tumors. In this study, we investigated whether the natural flavonoid luteolin can enhance the antitumor effects of osimertinib in NSCLC cells. We established an acquired osimertinib-resistant cell line, H1975/OR, and evaluated the effects of luteolin and osimertinib alone and in combination on proliferation, migration, invasion, and apoptosis of H1975/OR cells. The potential mechanisms by which the combination of luteolin and osimertinib exert their effects were investigated by PCR, western blot, gene silencing, molecular docking, SPR and kinase activity analysis. The combination of luteolin and osimertinib inhibited the proliferation, migration, and invasion of H1975/OR cells and promoted apoptosis. We identified mesenchymal-epithelial transition factor (MET) amplification and overactivation as important resistance mechanisms of H1975/OR cells. The combination downregulated the gene and protein expression of MET and inhibited its protein phosphorylation, thereby blocking the activation of the downstream Akt pathway. Additionally, the mediated effects of MET on the synergistic effect of luteolin and osimertinib were confirmed by silencing of MET. Luteolin strongly bound with nonphosphorylated MET by occupying the active pocket of MET and inhibiting its activation. Notably, the combination also downregulated the expression of autocrine hepatocyte growth factor (HGF), the sole ligand of MET. In conclusion, luteolin can synergize with osimertinib to overcome MET amplification and overactivation-induced acquired resistance to osimertinib by suppressing the HGF-MET-Akt pathway, suggesting the clinical potential of combining luteolin with osimertinib in NSCLC patients with acquired resistance.

Aphthous ulcer drug inhibits prostate tumor metastasis by targeting IKKɛ/TBK1/NF-κB signaling.

Tumor metastasis is the major cause of death for prostate cancer (PCa) patients. However, the treatment options for metastatic PCa are very limited. Epithelial-mesenchymal transition (EMT) has been reported to be an indispensable step for tumor metastasis and is suggested to associate with acquisition of cancer stem cell (CSC) attributes. We propose that small-molecule compounds that can reverse EMT or induce mesenchymal-epithelial transition (MET) of PCa cells may serve as drug candidates for anti-metastasis therapy. Methods: The promoters of CDH1 and VIM genes were sub-cloned to drive the expression of firefly and renilla luciferase reporter in a lentiviral vector. Mesenchymal-like PCa cells were infected with the luciferase reporter lentivirus and subjected to drug screening from a 1274 approved small-molecule drug library for the identification of agents to reverse EMT. The dosage-dependent effect of candidate compounds was confirmed by luciferase reporter assay and immunoblotting. Wound-healing assay, sphere formation, transwell migration assay, and in vivo intracardiac and orthotopic tumor xenograft experiments were used to evaluate the mobility, metastasis and tumor initiating capacity of PCa cells upon treatment. Possible downstream signaling pathways affected by the candidate compound treatment were analyzed by RNA sequencing and immunoblotting. Results: Drug screening identified Amlexanox, a drug used for recurrent aphthous ulcers, as a strong agent to reverse EMT. Amlexanox induced significant suppression of cell mobility, invasion, serial sphere formation and in vivo metastasis and tumor initiating capacity of PCa cells. Amlexanox treatment led to downregulation of the IKK-ɛ/ TBK1/ NF-κB signaling pathway. The effect of Amlexanox on EMT reversion and cell mobility inhibition can be mimicked by other IKK-ɛ/TBK1 inhibitors and rescued by reconstitution of dominant active NF-κB. Conclusions: Amlexanox can sufficiently suppress PCa metastasis by reversing EMT through downregulating the IKK-ɛ/TBK1/NF-κB signaling axis.

A cell culture condition that induces the mesenchymal-epithelial transition of dedifferentiated porcine retinal pigment epithelial cells.

The pathological change of retinal pigment epithelial (RPE) cells is one of the main reasons for the development of age-related macular degeneration (AMD). Thus, cultured RPE cells are a proper cell model for studying the etiology of AMD in vitro. However, such cultured RPE cells easily undergo epithelial-mesenchymal transition (EMT) that results in changes of cellular morphology and functions of the cells. To restore and maintain the mesenchymal-epithelial transition (MET) of the cultured RPE cells, we cultivated dedifferentiated porcine RPE (pRPE) cells and compared their behaviors in four conditions: 1) in cell culture dishes with DMEM/F12 containing FBS (CC dish-FBS), 2) in petri dishes with DMEM/F12 containing FBS (Petri dish-FBS), 3) in cell culture dishes with DMEM/F12 containing N2 and B27 supplements (CC dish-N2B27), and 4) in petri dishes with DMEM/F12 containing N2 and B27 (Petri dish-N2B27). In addition to observing the cell morphology and behavior, RPE specific markers, as well as EMT-related genes and proteins, were examined by immunostaining, quantitative real-time PCR and Western blotting. The results showed that dedifferentiated pRPE cells maintained EMT in CC dish-FBS, Petri dish-FBS and CC dish-N2B27 groups, whereas MET was induced when the dedifferentiated pRPE cells were cultured in Petri dish-N2B27. Such induced pRPE cells showed polygonal morphology with increased expression of RPE-specific markers and decreased EMT-associated markers. Similar results were observed in induced pluripotent stem cell-derived RPE cells. Furthermore, during the re-differentiation of those dedifferentiated pRPE cells, Petri dish-N2B27 reduced the activity of RhoA and induced F-actin rearrangement, which promoted the nuclear exclusion of transcriptional co-activator with PDZ-binding motif (TAZ) and TAZ target molecule zinc finger E-box binding protein (ZEB1), both of which are EMT inducing factors. This study provides a simple and reliable method to reverse dedifferentiated phenotype of pRPE cells into epithelialized phenotype, which is more appropriate for studying AMD in vitro, and suggests that MET of other cell types might be induced by a similar approach.

Optimization of reprogramming culture conditions for the generation of induced pluripotent stem cells from Col1a1 4F2A-Oct4-GFP mice with high efficiency.

A reprogrammable transgenic mouse strain, called Col1a1 4F2A-Oct4-GFP, was bred for the present study. Because the somatic cells of this mouse strain contain only two copies of each Yamanaka factor, these animals are inefficient at producing induced pluripotent stem cells (iPSCs; approx. 0.005%) under traditional culture conditions. With an optimized culture condition, the iPSC production rate of mouse embryonic fibroblasts (MEFs) of Col1a1 4F2A-Oct4-GFP mice (MEFCol1a14F2A-Oct4-GFP ) was increased to approximately 8%. Further, promotion of cell proliferation by serum supplementation did not enhance iPSC production. Inhibition of transforming growth factor ß (TGF-ß) in the serum by SB431542 neither affected the growth rate of MEFCol1a14F2A-Oct4-GFP nor promoted iPSC production. However, the use of the gamma-irradiated STO-NEO-LIF (γSNL) cells to serve as feeders for iPSC production resulted in a 5-fold higher rate of iPSC production than the use of γMEFICR feeders. Interestingly, the use of SB431542 with the γMEFICR -adopted system could eliminate this difference. RT-PCR-based comparative analysis further demonstrated that TGF-ß expression was 10-fold higher in γMEFICR than in γSNL cells. Consistent with previous reports, mesenchymal to epithelial transition was found to participate in the initial steps of reprogramming in the specific context of MEFCol1a14F2A-Oct4-GFP . Moreover, we found that the initial seeding density is one of the pivotal factors for determining a high efficiency of iPSC generation. The iPSCs efficiently generated from our MEFCol1a14F2A-Oct4-GFP resembled mouse embryonic stem cells (mESCs) in aspects of teratoma formation and germline transmission. Depending on the culture conditions, our Col1a1 4F2A-Oct4-GFP mouse system can generate bona fide iPSCs with variable efficiencies, which can serve as a tool for interrogating the route taken by cells during somatic reprogramming.

Lipid Supplement in the Cultural Condition Facilitates the Porcine iPSC Derivation through cAMP/PKA/CREB Signal Pathway.

Large numbers of lipids exist in the porcine oocytes and early embryos and have the positive effects on their development, suggesting that the lipids may play an important role in pluripotency establishment and maintenance in pigs. However, the effects of lipids and their metabolites, such as fatty acids on reprogramming and the pluripotency gene expression of porcine-induced pluripotent stem cells (iPSCs), are unclear. Here, we generated the porcine iPSCs that resemble the mouse embryonic stem cells (ESCs) under lipid and fatty-acid-enriched cultural conditions (supplement of AlbuMAX). These porcine iPSCs show positive for the ESCs pluripotency markers and have the differentiation abilities to all three germ layers, and importantly, have the capability of aggregation into the inner cell mass (ICM) of porcine blastocysts. We further confirmed that lipid and fatty acid enriched condition can promote the cell proliferation and improve reprogramming efficiency by elevating cAMP levels. Interestingly, this lipids supplement promotes mesenchymal-epithelial transition (MET) through the cAMP/PKA/CREB signal pathway and upregulates the E-cadherin expression during porcine somatic cell reprogramming. The lipids supplement also makes a contribution to lipid droplets accumulation in the porcine iPSCs that resemble porcine preimplantation embryos. These findings may facilitate understanding of the lipid metabolism in porcine iPSCs and lay the foundation of bona fide porcine embryonic stem cell derivation.