However, programmed cell death has been updated and expanded to 12 types in the last 20?years, and the barriers between them are not strict. long-term TAM use can induce serious drug resistance, leading to breast cancer recurrence and death in patients. Further, it is almost useless among patients with estrogen receptor negative (ER??) breast cancer. Shikonin (SK) is a natural product broadly explored in cancer therapy. Some studies have demonstrated the combined treatment of SK and clinical anticancer drugs including TAM on various tumors. However, the combined effect of SK and 4-hydroxytamoxifen (4-OHT) on ER- breast cancer is not known. The current study aimed to assess the combination effects of SK and 4-OHT on human breast cancer cells, MCF-7 (ER?+) and MDA-MB-435S (ER??), in vitro and in vivo and to investigate the underlying mechanisms. Methods CCK-8 assays and flow cytometry were conducted to determine the cell viability and apoptotic profiles of human breast cancer cell lines (MCF-7 and MDA-MB-435S) treated with SK, 4-OHT, and the combination. ROS and JC-1 assays were used to determine ROS level and mitochondrial membrane potential. Western blot analysis was performed to investigate proteins that are associated with apoptosis. Haematoxylin & Eosin (HE) staining was used to detect the tumor and kidney morphology of mice. TUNEL and immunohistochemical staining were performed to detect Ki67 expression level and cell apoptotic profile in tumor tissues. Results SK and 4-OHT synergistically inhibited MCF-7 and MDA-MB-435S cell proliferation and promoted apoptosis by reducing mitochondrial membrane potential and increasing the intracellular ROS level. The combination of SK and 4-OHT activated the mitochondrial-dependent apoptosis and the death receptor pathways, significantly regulating the PI3K/AKT/Caspase 9 signaling pathway. Compared with SK and 4-OHT alone, the combination of SK and 4-OHT could better inhibit tumor growth in mice. Conclusion The combination of SK and 4-OHT shows highly efficient anticancer effects on breast cancer therapy. SK may be a promising candidate as an adjuvant to 4-OHT for breast cancer treatments, especially for ER- breast cancer. at 4?C for 15?min using Eppendorf 5810R centrifuge. Protein concentrations were measured using the BCA Protein Assay kit (#23227 Thermo Fisher Scientific, USA). Approximately 80?g of total protein were separated by 10% SEMSCpolyacrylamide gels and transferred to PVDF membranes. After blocked with 5% defatted milk solution, membranes were incubated with primary antibodies, such as Smac/Diablo (#10434-1-AP, rabbit polyclonal IgG, 1: 1000 dilution); PI3K (#13329-1-AP, rabbit polyclonal IgG, 1: 1000 dilution), AKT (#10176-2-AP, rabbit polyclonal IgG, 1: 1000 dilution), Caspase 9 (#10380-1-AP, rabbit polyclonal IgG, 1: 1000 BIRT-377 dilution), PARP-1 (#66520-1-Ig, mouse monoclonal IgG, BIRT-377 1: 600 dilution), Bad (#10435-1-AP, rabbit polyclonal IgG, 1: 1000 dilution), Bcl-2 (#12789-1-AP, rabbit polyclonal IgG, 1:1000 dilution), Bax (#50599-2-Ig, rabbit polyclonal IgG, 1:1000 dilution), Caspase 8 (#13423-1-AP, rabbit polyclonal IgG, 1: 1000 dilution), Fas (#13098-1-AP, rabbit polyclonal BIRT-377 IgG, 1:1000 dilution), Bid (#10988-1-AP, rabbit polyclonal IgG, 1: 1000 dilution), Caspase-3 (#19677-1-AP, rabbit polyclonal IgG, 1:1000 dilution) and GAPDH (#10494-1-AP, rabbit polyclonal IgG, 1: 1000 dilution) at 4?C overnight. After thrice washing in TBST for each 5?min, membranes were incubated with HRP-conjugated secondary antibodies (#SA00001-1, #SA00001-2, 1: 5000 dilution) at room temperature for 2?h. Detection was performed by an enhanced chemiluminescent reagent (Thermo Fisher Scientific, USA) according to the manufacturers instructions. Bands were then recorded by a digital camera (Tanon 5200, Shanghai, China). Finally, the results were analyzed with Image J Software (National Institutes of Health, BetheSEMa, Maryland, USA), and all the targeted proteins were normalized to GAPDH. Animals and treatment Female nude mice at 6C8?weeks old were purchased from Model Animal Research Center of Nanjing University (Nanjing, China). They were kept at 22?CC24?C with a 12?h light/12?h dark cycle in a pathogen-free isolation facility. They were BIRT-377 allowed to adapt for 1?week prior to experimentation. Cultured MCF-7 cells were washed and re-suspended in ice-cold PBS. Portions of the suspension (6??106 cells in 0.1?mL) were subcutaneously injected into the right flanks of each mouse. After 2?weeks, the mice bearing tumors (100?mm3 on average) were randomly Mouse monoclonal to CD32.4AI3 reacts with an low affinity receptor for aggregated IgG (FcgRII), 40 kD. CD32 molecule is expressed on B cells, monocytes, granulocytes and platelets. This clone also cross-reacts with monocytes, granulocytes and subset of peripheral blood lymphocytes of non-human primates.The reactivity on leukocyte populations is similar to that Obs grouped into four (n?=?8 mice per group) in accordance with tumor volumes. SK (1.5?mg/kg) and 4-OHT (3?mg/kg) were dissolved in DMSO and administered once every 2?days for six times via intraperitoneal injection. The vehicle (DMSO)-treated group was included as a control. The body weight and tumor volumes were measured and recorded every 2?days. The long (is the tumor wet weight of drug group, and is the tumor wet weight of the control.