ABSTRACT
Arsenic trioxide (ATO) induces apoptosis in a range of solid tumors and leukemia cells, and has been clinically applied for the treatment of acute promyelocytic leukemia with confirmed efficacy. Acute megakaryocytic leukemia (AMKL) is an aggressive malignancy with poor prognosis, if bone marrow transplantation is not possible. In this study, we applied flow cytometry, Western blot analysis and microarray techniques to investigate the effects of ATO on apoptosis and the cell division cycle of AMKL cell lines CHRF-288-11 and MEG-01. Our data demonstrated that ATO is a potent agent against AMKL as indicated by apoptotic markers, Annexin V and caspase-3. ATO activated the intrinsic (mitochondrial) pathway of apoptosis, which involved disrupting mitochondrial membrane potential, increased Bax/Bcl-2 ratio and caspase-9 activation, as well as the extrinsic (death receptor) pathway mediated by Fas and caspase-8 activation. We provided the first evidence that ATO stimulated expressions of CD137 mRNA and protein, which might be relevant to the extrinsic mechanism. ATO induced delays of cell cycle progression at S phase and arrest at G2/M phase of AMKL cells, but caspase-3 expression appeared not to be phase-specific. The multiple-signaling mechanism of ATO warrants it a potential agent to incorporate in the treatment regimen of AMKL.
Subject(s)
Apoptosis/drug effects , Arsenicals/pharmacology , Cell Cycle/drug effects , Oxides/pharmacology , Antigens, CD/genetics , Antigens, CD/metabolism , Apoptosis/genetics , Arsenic Trioxide , Caspase 8 , Caspase 9 , Caspases/genetics , Caspases/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Flow Cytometry , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/genetics , Humans , Intracellular Membranes/drug effects , Intracellular Membranes/physiology , Leukemia, Megakaryoblastic, Acute/genetics , Leukemia, Megakaryoblastic, Acute/metabolism , Leukemia, Megakaryoblastic, Acute/pathology , Membrane Potentials/drug effects , Mitochondria/drug effects , Mitochondria/physiology , Oligonucleotide Array Sequence Analysis/methods , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Receptors, Nerve Growth Factor/genetics , Receptors, Nerve Growth Factor/metabolism , Receptors, Tumor Necrosis Factor/genetics , Receptors, Tumor Necrosis Factor/metabolism , Signal Transduction/drug effects , Time Factors , Tumor Necrosis Factor Receptor Superfamily, Member 9 , fas Receptor/genetics , fas Receptor/metabolismABSTRACT
Thrombospondin-1 (TSP-1) is a naturally occurring anti-angiogenic compound that induces apoptosis of endothelial and cancer cells via its receptor CD36. The objectives of our study were to investigate the in vitro effects of TSP-1 on the apoptosis of primary human leukemia cells as well as leukemia cell lines and the possible mechanism involving CD36. Our results demonstrated that TSP-1 induced apoptosis in CD36 positive cell lines CHRF-288-11, Meg-01 and HL-60, but not CD36 negative K562, at a dose-dependent manner as demonstrated by DNA ladder formation, Annexin V and propidium iodide (PI) stainings. The addition of anti-CD36 antibody FA6-152 or thrombopoietin (TPO) significantly nullified the effects of TSP-1. TSP-1-mediated apoptosis was consistently associated with the up-regulation of active Caspase-3. Responses of 2 CD36 positive primary AML samples to TSP-1 and FA6-152 were similar with those of leukemia cell lines. TSP-1 significantly induced apoptosis in B-ALL but the counter-effects of FA6-152 were less apparent. CD36 negative AML cells appeared less susceptible to TSP-1 and FA6-152. Our data provided strong evidence that TSP-1 exerted direct apoptotic effects on leukemia cells and could be developed as an adjunct to conventional therapy, particularly for leukemia cells that express CD36 or other TSP-1 receptors.
