title: Effects of the combination of RAD001 and docetaxel on breast cancer stem cells.
abstract: Recent evidence has suggested that breast cancer contains a rare population of cells called cancer stem cells (CSCs), which have an extensive self-renewal ability and contribute to metastasis and therapeutic resistance. This study evaluated the in vitro and in vivo effects of RAD001 (Everolimus) alone or in combination with docetaxel on stem cells from primary breast cancer cells and two breast cancer cell lines (MCF-7 and MDA-MB-231). In In vitro studies, we sorted ESA(+)CD44(+)CD24(-/low) cells as stem cells using flow cytometry from primary breast cancer cells, MCF-7 and MDA-MB-231 cell lines. MTT assays were used to quantify the inhibitory effect of the drugs on total cells and stem cells. Apoptosis and the cell cycle distributions of stem cells were examined by flow cytometry. The tumourigenicity of stem cells after treatment was investigated by soft agar colony formation assays. In In vivo studies, the BALB/c mice were injected with MDA-MB-231 stem cells and the different treatments were administered. After necropsy, the expression of Ki67, CD31, AKT1, and phospho-AKT (Thr308) was analysed by immunohistochemistry. In In vitro studies, all three populations of stem cells were resistant to the standard treatment doses of docetaxel compared with total cells treated with the same drug. Treatment with RAD001 resulted in growth inhibition of all stem cells in a dose-dependent manner. An additive growth inhibitory effect of the combination treatment on the three stem cells was observed in in vitro compared with treatment with RAD001 alone (P<0.001). In addition, an increase in G2/M cell cycle arrest and an increased population of cells in early apoptosis were seen in the combination treatment group compared with either single-agent group (P<0.01). In vivo, the volumes of the xenograft tumours significantly decreased in RAD001 alone group compared to control group (P=0.008), and RAD001 plus docetaxel therapy was much more effective at reducing tumour volume in mice compared with either single-agent alone (P<0.05). Compared with RAD001 alone, the combination of RAD001 and docetaxel reduced the expression of Ki67, CD31, AKT1 and phospho-AKT (Thr308) (P<0.05). We conclude that the combination treatment of RAD001 and docetaxel can inhibit the growth of stem cells in vitro and in vivo by inhibiting cell proliferation, inducing apoptosis, cell cycle arrest and reducing the expression of Ki67, CD31, AKT1 and phospho-AKT (Thr308). These data indicate that combination treatment with RAD001 and docetaxel may represent an effective therapy for breast cancer. However, further studies are required to evaluate the drug interaction between RAD001 and docetaxel in the clinical setting.
authors.affiliation: 3rd Department of Breast Cancer, China Tianjin Breast Cancer Prevention, Treatment and Research center, Tianjin Medical University Cancer Institute and Hospital, Tianjin, PR China.
authors.forename: Xiaobei
authors.initials: X
authors.lastname: Zhang
authors.forename: Sheng
authors.initials: S
authors.lastname: Zhang
authors.forename: Yan
authors.initials: Y
authors.lastname: Liu
authors.forename: Jingjing
authors.initials: J
authors.lastname: Liu
authors.forename: Yi
authors.initials: Y
authors.lastname: Ma
authors.forename: Yuanxi
authors.initials: Y
authors.lastname: Zhu
authors.forename: Jin
authors.initials: J
authors.lastname: Zhang
mesh_terms: D000818:Animals; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use*; D001943:Breast Neoplasms / Q000188:drug therapy*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D000077143:Docetaxel; D004305:Dose-Response Relationship, Drug; D000068338:Everolimus; D005260:Female; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D007166:Immunosuppressive Agents / Q000627:therapeutic use; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D020123:Sirolimus / Q000008:administration & dosage / Q000031:analogs & derivatives*; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects; D043823:Taxoids / Q000008:administration & dosage*; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology
chemical_list: D007166:Immunosuppressive Agents; D043823:Taxoids; D013778:Tetrazolium Salts; D013844:Thiazoles; D000077143:Docetaxel; D000068338:Everolimus; C022616:thiazolyl blue; D020123:Sirolimus