ABSTRACT
Antibody-mediated rejection (AMR) is one of the major causes of graft loss after transplantation. Recently, the regulation of B cell differentiation and the prevention of donor-specific antibody (DSA) production have gained increased attention in transplant research. Herein, we established a secondary allogeneic in vivo skin transplant model to study the effects of romidepsin (FK228) on DSA. The survival of grafted skins was monitored daily. The serum levels of DSA and the number of relevant immunocytes in the recipient spleens were evaluated by flow cytometry. Then, we isolated and purified B cells from B6 mouse spleens in vitro by magnetic bead sorting. The B cells were cultured with interleukin-4 (IL-4) and anti-clusters of differentiation 40 (CD40) antibody with or without FK228 treatment. The immunoglobulin G1 (IgG1) and IgM levels in the supernatant were evaluated by enzyme-linked immunosorbent assay (ELISA). Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and western blotting were conducted to determine the corresponding levels of messenger RNA (mRNA) and protein expression in cultured cells and the recipient spleens. The results showed that FK228 significantly improved the survival of allogeneic skin grafts. Moreover, FK228 inhibited DSA production in the serum along with the suppression of histone deacetylase 1 (HADC1) and HDAC2 and the upregulation of the acetylation of histones H2A and H3. It also inhibited the differentiation of B cells to plasma cells, decreased the transcription of positive regulatory domain-containing 1 (Prdm1) and X-box-binding protein 1 (Xbp1), and decreased the expression of phosphorylated inositol-requiring enzyme 1 α (p-IRE1α), XBP1, and B lymphocyte-induced maturation protein-1 (Blimp-1). In conclusion, FK228 could decrease the production of antibodies by B cells via inhibition of the IRE1α-XBP1 signaling pathway. Thus, FK228 is considered as a promising therapeutic agent for the clinical treatment of AMR.
Subject(s)
Animals , Mice , Depsipeptides , Endoribonucleases , Hematopoietic Stem Cell Transplantation , Histone Deacetylase Inhibitors/pharmacology , Protein Serine-Threonine Kinases , Skin TransplantationABSTRACT
[ ABSTRACT] AIM:To investigate the depressant effect of FK228 combined with rapamycin on the human breast cancer cell line MCF-7 and MDA-MB-435.METHODS:FK228, a new histone deacetylase inhibitor, and rapamycin, the specific inhibitor of the mammalian target of rapamycin ( mTOR) protein, were used in the study.MCF-7 cells and MDA-MB-435 cells were exposed to different concentrations of FK228 and rapamycin.The inhibitory rate of cell growth was de-termined by SRB assay.Combination index ( CI) was used to evaluate the interaction between FK228 and rapamycin.The expression of the apoptotic proteins, cycle proteins and nucleic acid proteins were detected by Western blotting.The cell cycle was analyzed by flow cytometry.RESULTS: Both FK228 and rapamycin showed growth inhibitory effects on the breast cancer cell lines in a time-and dose-dependent manner.CI of the 2 drugs was less than 1 when the inhibitory rate of the cell growth was 50%effective dose (ED50)~ED70, indicating a synergistic effect.The combination therapy of FK228 with rapamycin increased the apoptotic proteins, and induced the down-regulation of phosphorylated Akt and over-expres-sion of caspase-3 compared with a single use of the drugs.The combination therapy of FK228 with rapamycin reduced the cycle proteins, and the cell cycle was arrested in G2/M.The levels of phosphorylated H2AX and acetylated H3 were ob-viously increased after combination therapy.CONCLUSION:The combination therapy of FK228 with rapamycin inhibits the cell proliferation and increases apoptosis with a synergistic effect, which may become a new trend for treating endometri-al cancer.
ABSTRACT
Objective:To investigate the underlying mechanism of histone deacetylase (HDAC) inhibitor FK228-in- duced apoptosis of the prostate cancer cell line DU145.Methods:The inhibitory effect of FK228 on DU145 cell growth and its cytotoxicity were determined by MTT assay;cell cycle arrest was detected by flow cytometry assay;morphological change was observed by Giemsa staining;and defined kinase protein levels were determined by Western blot analysis.Re- suits:FK228 obviously inhibited DU145 cells growth,arrested cell cycle at G_0/G_1 phase,induced cells morphological changes and degraded several kinase proteins,including EGFR,Her2,Raf-1,Src,Cdk4 and IAP member Survivin.The degradation of these kinases blocked Raf-Mek-Erk and PI3K/Akt survival signal pathways,inducing apoptosis.Condu- sion:FK228 may induce DU145 cell apoptosis through depletion of multiple kinase proteins and blockade of survival sig- nal pathways of DU145 cells.