title: [Everolimus combined with all-trans retinoid acid reverses drug resistance in acute promyelocytic leukemia NB4-R1 cells].
abstract: To investigate the effect of everolimus(RAD001)combined with all-trans retinoid acid(ATRA) on drug resistance of ATRA-resistance acute promyelocytic leukemia(APL) cell line NB4-R1 and its molecular mechanism.
  
  
  
  APL NB4-R1 cells were treated with different concentrations of RAD001(1 nmol/L, 10 nmol/L and 100 nmol/L) with ATRA(1μmol/L) for 24, 48 and 72 h, respectively. The differentiation of NB4-R1 cells was analyzed by flow cytometry with CD11b staining and nitro blue tetrozolium(NBT) reduction test. Cell cycle was detected by cell cycle staining kit and apoptosis was detected by flow cytometry with Annexin V/PI staining. Protein expressions of LC-3II, PML-RARα, P-P70S6K and P-4E-BP1 were determined by Western blotting.
  
  
  
  RAD001 combined with ATRA significantly induced NB4-R1 cells differentiation, but RAD001 or ATRA alone did not enhance NB4-R1 differentiation. The co-treatment induced accumulation of cells in G1 phase and decreased the proportion of cells in S phase. The combined treatment had no effect on cell apoptosis. The differentiation rate of NB4-R1 cells in 100 nmol/L RAD001, 1μmol/L ATRA, RAD001 combined with ATRA and control groups was(2.29±0.57)%,(17.06±2.65)%,(54.47±4.91)% and(2.54±0.53)%, respectively; the proportion of cells in G1 phase was(35.20±11.97)%,(33.54±6.25)%,(53.70±8.73)% and(27.40±6.01)%, respectively; cells apoptosis rate was(2.30±0.14)%,(2.25±0.21)%,(2.40±0.28)% and(1.95±0.07)%, respectively. The combination of RAD001 with ATRA significantly inhibited mTOR signaling downstream proteins P-P70S6K, P-4E-BP1 and enhanced autophagy-related protein LC3-II and Beclin 1. The co-treatment also induced degradation of fusion protein PML-RARα.
  
  
  
  RAD001 combined with ATRA can induce cell differentiation, inhibit cell cycle, resulting the reverse of drug resistance in NB4-R1 cells, which is associated with increase of autophagy level and degradation of PML-RARα.
authors.affiliation: Bone Marrow Transplantation Center, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China.
authors.forename: Wei-Chao
authors.initials: WC
authors.lastname: Liao
authors.affiliation: Bone Marrow Transplantation Center, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: He
authors.affiliation: Bone Marrow Transplantation Center, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China.
authors.forename: Bin-Sheng
authors.initials: BS
authors.lastname: Wang
authors.affiliation: Bone Marrow Transplantation Center, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China.
authors.forename: He
authors.initials: H
authors.lastname: Huang
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D002454:Cell Differentiation; D045744:Cell Line, Tumor / Q000187:drug effects; D019008:Drug Resistance, Neoplasm; D000068338:Everolimus / Q000494:pharmacology*; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000473:pathology*; D015514:Oncogene Proteins, Fusion / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism; D015398:Signal Transduction; D014212:Tretinoin / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000970:Antineoplastic Agents; D018797:Cell Cycle Proteins; C087000:EIF4EBP1 protein, human; D015514:Oncogene Proteins, Fusion; D010750:Phosphoproteins; C093476:promyelocytic leukemia-retinoic acid receptor alpha fusion oncoprotein; D014212:Tretinoin; D000068338:Everolimus; D038762:Ribosomal Protein S6 Kinases, 70-kDa; C448959:ribosomal protein S6 kinase, 70kD, polypeptide 2