Abstract
Purpose:
Retinoblastoma (Rb) is an aggressive childhood cancer of the developing retina that is associated with epigenetic deregulation of several cancer pathways. In addition to the significantly upregulation of the proto-oncogene spleen tyrosine kinase (SYK), the angiogenic potential of Rb correlates with invasive growth and metastasis. Lymphocyte-derived microparticles (LMPs) possess strong antiangiogenic effect against pathological ocular angiogenesis and potent inhibitory effect on cell viability. This study is designed to elucidate the mechanisms underlying the anti-Rb effect of LMPs.
Methods:
LMPs were produced from human T cell lymphoblast-like cell line cells (CEM T cells) after 0.5 μg/mL actinomycin D stimulation and isolated LMPs were characterized with annexin V staining and gated using 1.0 µM beads. Rb cell line (Y-79) and primary cultured Rb cells (isolated from primary site intraocular retinoblastoma of Rb patients) were subjected to WST-1, cellular senescent, apoptotic assay and FACS cell cycle analysis after treated with LMPs. Quantitative RT-PCR, immunohistochemistry and Western blot were performed to detect interest gene expression and protein levels in LMPs and in Rb cells.
Results:
LMPs significantly reduced Rb cell viability in a dose-dependent manner. LMPs in a concentration of 10µg/ml significantly induced Rb cell-cycle arrest at G0/G1 phase with associated increases of the senescence-associated β-galactosidase activity. LMPs in higher concentrations (≥20µg/ml) induced cell death (46% apoptotic cells in LMPs-treated primary cultured Rb cells compared to 14% in control cells). LMPs treatment has a significant suppression effect on the expression of SYK in primary Rb cells. Moreover, LMPs contain a considerable amount of p21(cip1), a cell cycle inhibitor. Inhibition of p21(cip1) significant induced pro-apoptotic effect of LMPs.
Conclusions:
LMPs dose-dependently induced Rb cell senescence or apoptosis; the mechanism favouring this effect is directly mediated by p21(cip1) pathway. These data may open unexpected avenues for the development of novel therapeutic strategies that are particularly useful and relevant for the treatment of Rb cancer.
Keywords: 503 drug toxicity/drug effects •
555 immunomodulation/immunoregulation •
703 retinoblastoma