To evaluate whether DHW-208 is able to induce apoptotic cell death, cells were treated with DHW-208, and Annexin V-positive cells were detected by FACS analysis. an antitumor effect by inhibiting the PI3K/AKT/mTOR-signaling pathway with a high degree of safety in breast cancer. Above all, we report for the first time that DHW-208 suppressed the growth of human breast cancer cells by inhibiting the PI3K/AKT/mTOR-signaling pathway both in vivo and in vitro. Our study may provide evidence for the use of DHW-208 as an effective, novel therapeutic candidate for the treatment of human breast cancers in clinical trials. strong class=”kwd-title” Subject terms: Breast cancer, Breast cancer, Targeted therapies, Targeted therapies, Apoptosis Introduction Improvements in the detection and treatment of breast cancer have led to better prognosis and survival, with a 5-year survival rate of nearly 90%1,2. However, breast cancer is still one of the most frequent malignant diseases in women worldwide and the second leading cause of mortality in females1,3,4. Moreover, there is still no effective treatment strategy for advanced breast cancer that has metastasized3,5. To date, many therapeutic targets have been verified for treating breast cancers, including CDK4/6 inhibitors, HDAC inhibitor, Estrogen pathway antagonists, VEGF inhibitors, PI3K inhibitors, mTOR inhibitors, etc.6C8. Among these, the phosphate idylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway was found to play a central role in the cell physiology of breast cancer9C11. Mutations in the PI3K/AKT/mTOR pathway are frequently detected in breast cancer. Approximately 60% of breast cancer tumors have genetic alterations that activate the PI3K/AKT/mTOR pathway. Because of the important role that the PI3K/AKT/mTOR pathway plays in tumors, many inhibitors that target this pathway have been developed12C14. The first PI3K inhibitor for breast tumors, Alpelisib tablets, was approved by the FDA in May 2019 for the treatment of advanced or metastatic breast cancer15. Despite advances in the development of drugs targeting the PI3K/AKT/mTOR pathway, much safer and more effective targeted drugs are still needed in the Amylmetacresol clinic. The PI3K/AKT/mTOR pathway controls numerous cellular functions such as growth, proliferation, survival, motility, and metabolism16C19. Activated by upstream signaling molecules, phosphatidylinositol 4,5-diphosphate (PIP2) in the plasma membrane is converted by the catalytic subunit of P13K to phosphatidylinositol 3,4,5-triphosphate (PIP3). PIP3 brings phosphoinositide-dependent protein kinase 1 (PDK1) to the cell membrane where the latter phosphorylates Thr308 in the catalytic domain of AKT. Subsequently, Ser473, in the regulatory domain of AKT is phosphorylated by mammalian rapamycin complex Amylmetacresol 2 (mTORC2), and AKT kinase activity becomes fully activated13,16,20. Activated mTORC1 can also activate ribosomal protein S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein (4EBP), promoting protein synthesis and cell proliferation21C23. The roles AKT played in the cell are numerous and various, but all result in anti-apoptosis, or pro-cell proliferation effects24. The physiological functions of AKT included involvement in metabolism, protein synthesis, apoptotic pathways, cell cycle, and transcription factor regulation25C27. These processes are identified as key factors in establishing and maintaining oncogenic phenotypes28,29. Recently, a number of 4-amino-quinazoline derivatives have been developed as selective inhibitors of tyrosine kinase, such as Gefitinib, Erlotinib, and Lapatinib. Moreover, some 4-aminoquinazoline derivatives have ever been reported as PI3K inhibitors30. Previously, we had designed and synthesized a series of 4-aminoquinazoline derivatives targeting the PI3K/AKT/mTOR-signaling pathway31. Among them, 2,4-difluoro-N-(5-(4-((1-(2-hydroxyethyl)-1H-pyrazol-4-yl)amino)quinazolin-6-yl)-2-methoxypyridin-3-yl)benzenesulfonamide (DHW-208) (Fig. ?(Fig.1a)1a) showed optimal anti-breast cancer activity and significant inhibitory activity against Amylmetacresol four main subunits of PI3K (p110/p85, p110/p85, p120, p110/p85). In the present study, we determined the effects of DHW-208 on the growth, proliferation, migration, and invasion of breast cancer cells in vitro and those related molecular mechanism. We then studied the effect of DHW-208 on tumor Rabbit Polyclonal to Adrenergic Receptor alpha-2A growth in nude mice xenografted with human breast cancer cells. Open in a separate window Fig. 1 Effects of DHW-208 on T47D and MDA-MB-231 cell viability and proliferation.a Chemical structure of DHW-208. b SRB assay for cell viability of T47D, MDA-MB-231, BT474, and MCF-7 cells treated with different concentrations of DHW-208 for 24, 48, and 72?h. c SRB assay to determine cytotoxicity toward normal breast cell MCF-10A at 72?h. d Growth inhibition effects of DHW-208 on T47D and MDA-MB-231 cells were measured by colony formation assay. Bar graphs showed the quantitative results of the colony formation assay (down). Each value is the mean (SD) from triplicate samples. * em p /em ? ?0.05, ** em p /em ? ?0.01 vs. control. Materials and methods Reagents and antibodies DHW-208 was synthesized by Pharmaceutical chemistry laboratory, Shenyang Pharmaceutical University, Shenyang, China. BEZ235 was obtained from MedChem Express (NJ,.