DPBA did not induce EGFR dimerization, phosphorylation, and ubiquitination, but it significantly promoted EGFR degradation and repressed downstream survival pathways. and EGFR. DPBA did not induce EGFR dimerization, phosphorylation, and ubiquitination, but it significantly promoted EGFR degradation and repressed downstream survival pathways. Further analyses showed that DPBA induced clathrin-independent EGFR Tandospirone endocytosis mediated by flotillin-dependent lipid rafts and unaffected by EGFR TKIs. Activation of the early and late endosome markers rab5 and rab7 but not the recycling endosome marker rab11 was involved in DPBA-induced EGFR lysosomal degradation. The present study offers a new EGFR ligand for EGFR pharmacological degradation and proposes it as a potential treatment for EGFR-positive NSCLC, particularly NSCLC with innate or acquired EGFR TKI resistance. DPBA can also serve as a chemical probe in the studies on EGFR trafficking and degradation. by our group, has been found to exhibit anti-tumour activity in vitro and in vivo. In order to improve its biological activity, we carried out structural modifications and obtained a series of derivatives with improved activities.24 The anti-tumour mechanisms of 23-HBA or its derivatives mainly involve mitochondrial ROS burst, mitochondrial membrane potential depolarization, non-classical mitochondrial autophagy, telomerase activity inhibition, and multidrug resistance reversal.55C58 So far, there have been no reports on 23-HBA or its derivatives, whose modes of action resembles that of DPBA. Our study sheds a new insight into the molecular mechanism of 23-HBA and its derivatives. The present study identifies one 23-HBA derivative, DPBA, a promising anticancer drug candidate that binds to EGFR ECD and promotes EGFR endocytosis and lysosomal degradation in the treatment of EGFR-positive NSCLC (Fig. ?(Fig.5h).5h). Here, we unveiled a new class of Tandospirone small-molecule EGFR degraders directly targeting EGFR ECD. In this way, we provide a strategy to inhibit EGFR kinase-independent functions and suppress innate or acquried EGFR TKI-mediated NSCLC resistance. Materials and methods Cell culture A431, A549, NCI-H1299, NCI-H1650, NCI-H1975, NCI-H522, MDA-MB-231, MDA-MB-435, MDA-MB-453, MDA-MB-468, MCF-7, HepG2, HT-29, HCT116, SW620, HEK-293T, and BEAS-2B were purchased from the American Type Culture Collection (Manassas, VA, USA). HaCaT was obtained from Beina Chuanglian Biotechnology Research Institute (Beijing, China). HepG2/ADM cells were generously provided by Prof. Kwok-Pui Fung (Chinese University of Hong Tandospirone Kong, Hong Kong, China). H1299, HNPCC1 H1650, and H1975 were cultured in RPMI-1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% (v/v) foetal bovine serum (FBS, Thermo Fisher Scientific) and 1% (v/v) penicillinCstreptomycin (PS, Thermo Fisher Scientific). All other cell lines were cultured in Dulbeccos modified Eagles medium supplemented with 10% (v/v) FBS and 1% (v/v) PS. Cells were maintained at 37?C in a humidified atmosphere incubator with 5% CO2. Cell line authentication and detection of mycoplasma contamination were performed before usage. Reagents and antibodies DPBA (98% purity) was synthesized as described previously.24 DAPI (4,6-diamidino-2-phenylindole), CHX, leupeptin, E-64, Ca074Me, pepstatin A, pitstop2, MCD, FITC-dextran, Tris (2-carboxyethyl) phosphine hydrochloride (TCEP), Tris [(1-benzyl-1for 10?min, while the precipitated pellet was lysed again in the same way in 0.5?ml lysis buffer and the post-nuclear supernatant was combined with the first. One millilitre of lysis buffer with 50% (v/v) OptiPrep was Tandospirone added to the supernatants and transferred to an ultra-centrifuge tube. Three millilitres of 0C20% gradient OptiPrep in lysis buffer was placed on the top of the mixture. After centrifugation at 25,000?r.p.m. for 90?min in a Tandospirone Beckman ultra-centrifuge, 10 fractions were collected from top to bottom of the gradients. EGFR, TfR, flotillin-1, and caveolin-1 in different fractions were analysed by Western blot..
