Apoptosis in medulloblastoma: in vitro analysis
The poor prognosis associated with medulloblastoma (MB) has stimulated the evaluation of improved treatment strategies. The potential of lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, as a possible anticancer drug against MB was evaluated in vitro. Analysis of cell morphologic changes, cell viability, DNA fragmentation and flow cytometry demonstrated growth inhibition and induction of apoptosis in MB cells and lovastatin 'resistant' MB cells after lovastatin treatment. Cell cycle arrest in G1 was concomitant with apoptosis. Mevalonate prevented lovastatin-induced changes in MB, thus confirming that blockage of the mevalonate pathway is a critical step in the mechanism of lovastatin-induced MB apoptosis, and that HMG-CoA reductase activity is necessary for MB survival. Within the mevalonate pathway, blocking protein farnesylation with manumycin A and blocking G-protein function by the depletion of GTP using mycophenolic acid (MPA) were followed by MB cell proliferationinhibition and apoptosis. Cell death induced by manumycin A was uniformly more rapid and efficient than that induced by lovastatin. The cell cycle was also arrested in G1 by MPA. At low concentration lovastatin, manumycin A and MPA did not induce apoptosis individually, but when combined, they synergistically induced NM apoptosis. These results indicate that blocking farnesylation of proteins, especially G-proteins, at least partially accounts for MB apoptosis induced by blocking the mevalonate pathway. The molecular mechanisms involved in MB apoptosis were studied. The followings were demonstrated: (1) regulation of HMG-CoA reductase gene expression was perturbed; (2) the caspase-3 pathway was activated; (3) apoptosis was p53-independent; (4) Bcl-2 and Bax did not regulate apoptosis; (5) up-regulation of the CKIs p21WAF1 and P27KIP1 accompanied cell cycle arrest and growth inhibition; (6) c-myc overexpression did not influence cell sensitivity to undergo apoptosis; and (7) the regulation of ras gene expression did not account for MB apoptosis induced by these compounds. The efficient induction of apoptosis by lovastatin favors this drug as a potential new therapeutic intervention for MB.