ABSTRACT
Medulloblastomas arise in the cerebellum and are the most common pediatric primary malignant brain tumors. Currently, medulloblastoma patients are best treated with surgical removal of the tumor, adjuvant radiation therapy and chemotherapy. The chemotherapeutic agents that showed efficiency against medulloblastomas include lomustine and vincristine. However, the effects of these drugs on medulloblastomas as well as on other cell types is still not well defined. In the present report we present evidence that the cytotoxic effect of these drugs is not specific for medulloblastoma cells but includes also normal fibroblast and epithelial cells. We have also shown that vincristine and lomustine trigger apoptosis in all these cells through the mitochondrial pathway via decrease in the level of the anti-apoptosis proteins Bcl-2 and Bcl-xl, respectively. Intriguingly, the proportion of apoptotic cells induced in medulloblastoma and normal epithelial and fibroblastic cells was similar. In addition, vincristine induced low proportion of necrosis in medulloblastoma and normal fibroblast cells. Interestingly, while vincristine induced cell cycle delay in G2/M phase in normal as well as medulloblastoma cells, lomustine effect on the cell cycle was specific for medulloblastoma cells. Furthermore, we have shown that vincristine and lomustine up-regulated p21 protein level in a p53-independent manner. These results shed more light on the biological effects of vincristine and lomustine and show that lomustine is a more specific and potent anti-medulloblastoma agent.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cerebellar Neoplasms/drug therapy , Cyclin-Dependent Kinase Inhibitor p21/drug effects , Lomustine/pharmacology , Medulloblastoma/drug therapy , Vincristine/pharmacology , Cell Cycle/drug effects , Cell Line , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Epithelial Cells/drug effects , Fibroblasts/drug effects , Flow Cytometry , Humans , Immunoblotting , Proto-Oncogene Proteins c-bcl-2/drug effects , Up-Regulation , bcl-X Protein/drug effectsABSTRACT
PURPOSE: Phosphatidylinositol 3'-kinase (PI3'-kinase) can be activated by the K1 protein of Kaposi sarcoma-associated herpes virus (KSHV). However, the role of PI3'-kinase in KSHV-associated primary effusion lymphoma (PEL) is not known. To assess this, we studied survival and apoptosis in PEL cell lines following inhibition of PI3'-kinase. EXPERIMENTAL DESIGN: Constitutive activation of several targets of PI3-kinase and apoptotic proteins were determined by Western blot analysis using specific antibodies. We used LY294002 to block PI3'-kinase/AKT activation and assess apoptosis by flow cytometric analysis. RESULTS: Blocking PI3'-kinase induced apoptosis in PEL cells, including BC1, BC3, BCBL1, and HBL6, whereas BCP1 was refractory to LY294002-induced apoptosis. LY294002-induced apoptosis did not seem to involve Fas/Fas-L but had an additive effect to CH11-mediated apoptosis. We also show that AKT/PKB is constitutively activated in all PELs and treatment with LY294002 causes complete dephosphorylation in all cell lines except BCP1 where a residual AKT phosphorylation remained after 24 hours of treatment. FKHR and GSK3 were also constitutively phosphorylated in PELs and treatment with LY294002 caused their dephosphorylation. Although inhibition of PI3'-kinase induced cleavage of BID in all cell lines, cytochrome c was released from the mitochondria and caspase-9 and caspase-3 were activated in LY294002-induced apoptotic BC1 but not in resistant BCP1. Similarly, XIAP, a target of AKT, was down-regulated after LY294002 treatment only in sensitive PEL cells. CONCLUSIONS: Our data show that the PI3'-kinase pathway plays a major role in survival of PEL cells and suggest that this cascade may be a promising target for therapeutic intervention in primary effusion lymphomas.
