phenanthrenes and Myelodysplastic-Syndromes

Although certain combination therapies comprising arsenic trioxide (As2O3) with other agents exist for the treatment of several types of human cancer, few As2O3 combination therapies are clinically effective for myelodysplastic syndromes (MDS). Triptolide (TL) may be an effective therapeutic agent for the treatment of MDS. However, to date, there is no combination therapy for MDS with As2O3 and TL. Therefore, the aim of the present study was to investigate this combination therapy on the apoptosis of MDS SKM‑1 cells. The MDS SKM‑1 cells were treated with As2O3, TL or the two in combination at various concentrations, or were mock‑treated. Cell viability, cell apoptosis, levels of reactive oxygen species (ROS) and the expression of the cell apoptosis‑associated genes, B cell lymphoma‑2 (Bcl‑2), Bcl‑2‑associated X protein (Bax) and caspase‑3, were determined using an MTT assay, flow cytometric analysis of annexin V‑fluorescein isothiocyanate/propidium iodide double‑stained cells, flow cytometic analysis of intracellular 2',7'‑dichlorodihydrofluorescein diacetate fluorescence and reverse transcription‑quantitative polymerase chain reaction analysis, respectively. Combination index (CI) analysis was performed to determine whether effects were synergistic (CI<1). The combination treatment was found to synergistically inhibit MDS SKM‑1 cell growth, induce cell apoptosis, increase ROS levels, upregulate the expression levels of Bax and caspase‑3, and downregulate the mRNA expression of Bcl‑2. In conclusion, the combination treatment of As2O3 and TL synergistically induced apoptosis in the MDS SKM‑1 cells.

Topics: Apoptosis; Arsenic Trioxide; Arsenicals; bcl-2-Associated X Protein; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cell Survival; Diterpenes; Drug Synergism; Epoxy Compounds; Gene Expression Regulation, Neoplastic; Humans; Myelodysplastic Syndromes; Oxides; Phenanthrenes; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species

Background Aberrant or impaired repair of double-strand DNA breaks is a common feature of de novo acute myeloid leukemia and myelodysplastic syndromes. Since poly (ADP-ribose) polymerase (PARP) inhibitors have been recently shown to selectively target cells with defects in double-strand DNA repair, the aim of this study was to explore the possibility of exploiting defects in DNA repair in leukemic cells using PARP inhibitors.. Leukemic cell lines were exposed to various PARP inhibitors alone and in combination with non-cytotoxic concentrations of DNA methyltransferase inhibitor, 5' aza-2'-deoxycytidine and/or the histone deacetylase inhibitor, MS275, to test for potentiation of apoptosis with these agents.. PARP inhibitors, KU-0058948 and PJ34, induced cell cycle arrest and apoptosis of primary myeloid leukemic cells and myeloid leukemic cell lines in vitro. Immunofluorescence analysis also revealed that PARP inhibitor sensitivity in these leukemic cells was due to a defect in homologous recombination DNA repair. Addition of 5' aza-2'-deoxycytidine failed to increase the cytotoxicity of PARP inhibitors. In contrast, MS275 potentiated the cytotoxic effect of KU-0058948 and PJ34 in all PARP inhibitor-sensitive leukemic cells. Immunofluorescence analysis supported the idea that histone deacetylase inhibitors potentiate cytotoxicity by inhibiting DNA repair processes. Conclusions On the basis of the data presented here, we suggest that PARP inhibitors can potentially exploit defects in double-strand DNA break repair in leukemic cells, paving the way for testing the therapeutic potential of these agents in myelodysplastic syndromes and acute myeloid leukemia.

Topics: Apoptosis; Azacitidine; Benzamides; Butyrates; Cell Cycle; Cell Line; Cell Line, Tumor; Cell Survival; Decitabine; DNA Modification Methylases; DNA Repair; Drug Synergism; Flow Cytometry; Fluorescent Antibody Technique; Fluorobenzenes; Histone Acetyltransferases; HL-60 Cells; Humans; Hydroxamic Acids; Immunohistochemistry; K562 Cells; Leukemia, Myeloid; Myelodysplastic Syndromes; Phenanthrenes; Phthalazines; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Pyridines; U937 Cells

To investigate the apoptotic effect of triptolide on MDS cell line MUTZ-1 cells and its mechanism, MUTZ-1 cells were incubated with indicated concentrations of triptolide. The growth of MUTZ-1 cells was observed by MTT assay and apoptosis was detected by DNA fragmentation analysis and flow cytometry using Annexin V-FITC/PI staining. The gene and protein expressions were determined by reverse transcription polymerase chain reaction (RT-PCR) and Western blot, respectively. The results showed that MUTZ-1 cell viability in presence of triptolide decreased markedly in a dose- and time-dependent manner. The growth-inhibitory IC50 value for triptolide treatment was 55.06 ng/ml. A DNA ladder pattern of internucleosomal fragmentation was observed. The translocation of phosphatidylserine at the outer surface of the cell plasma membrane could be induced by triptolide and its level increased following the augmentation of the drug concentration. Treatment of MUTZ-1 cells with triptolide for 12 hours resulted in the activation of caspase-3, cleavage of PARP and decrease of c-IAP2 mRNA. The expressions of pro-caspase 3 and c-IAP2 were inversely correlated with the incidence of apoptosis. (r = -0.907, P = 0.000; r = -0.919, P = 0.000 respectively). In conclusion, Triptolide inhibits MUTZ-1 cell growth by inducing apoptosis. The apoptotic effect of triptolide in MUTZ-1 cells is mediated by the caspase-3 activation and PARP cleavage. Moreover, the activation of caspase-3 may be associated with the down-regulation of c-IAP2.

Topics: Antineoplastic Agents, Alkylating; Apoptosis; Baculoviral IAP Repeat-Containing 3 Protein; Blotting, Western; Caspase 3; Cell Line; Diterpenes; Epoxy Compounds; Flow Cytometry; Gene Expression; Humans; Inhibitor of Apoptosis Proteins; Myelodysplastic Syndromes; Phenanthrenes; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Ubiquitin-Protein Ligases