Effects on metabolic activity (WST-1 assay) After treatment with 5-aza-dC, we observed an enhanced reduction of metabolic activity in all cell lines treated for six days versus three
days (Figure 1). As 5-aza-dC incorporation depends on cell cycle progression and proliferation frequency , the longer incubation period allows more 5-aza-dC to be incorporated into DNA. Surprisingly, 5-aza-dC exhibited the strongest inhibitory effect in slowly proliferating D283-Med cells, whereas DAOY cells, showing the shortest replication time, were much more resistant. Although 5-aza-dC-induced inhibition was stronger after 6 versus 3 days of treatment, leading to a total loss of metabolic activity in D283-Med and MEB-Med8a, about 20% of metabolic activity remained in DAOY cells. The relative 5-aza-dC resistance of DAOY cells versus MEB-Med8a and D283-Med PF-4708671 supplier cells in mortality and cell growth arrest has already been shown by our workgroup . This indicates that, beside the incubation period-dependent incorporation
rate, other mechanisms, like repair efficiency or DNMT activity, are involved in 5-aza-dC-induced cytotoxicity. Figure 1 Time- and dose-dependent inhibition of metabolic activity by 5-aza-dC. Metabolic activity of three medulloblastoma cell lines was measured by WST-1 assay after 5-aza-dC treatment for three or six days. Raw values were normalized to untreated control. Data from one experiment are shown as means ± SEM of triplicate samples. VPA led to a strong dose-dependent decrease of metabolic activity in all three MB cell lines (Figure 2a). Z-VAD-FMK price The individual VPA concentrations leading to 30% inhibition (IC 30) were between 0.27 mM (MEB-Med8a) and 0.9 mM (D283-Med) after VPA treatment for three days. After combinatorial treatment with 5-aza-dC, additive effects on the reduction of metabolic activity in two cell lines (DAOY, D283-Med) with a significant synergistic response in DAOY
cells were observed. This is in accordance with data obtained from Yang et al. showing synergistic Verteporfin cost effects on inhibition of cell growth and induction of apoptosis in human leukemic cell lines . In contrast, combined 5-aza-dC/VPA treatment of MEB-Med8a cells revealed a significant increase of 25% in metabolic activity compared to 5-aza-dC monotherapy (Figure 3a). Conceivably in MEB-Med8a cells, VPA mainly induces G1 arrest by induction of p21 selleck chemicals llc expression  and, therefore, prevents cytotoxic 5-aza-dC incorporation into the DNA molecule. Figure 2 Dose-dependent inhibition of metabolic activity by valproic acid, SAHA, abacavir, retinoic acid, and resveratrol. Metabolic activity of three medulloblastoma cell lines was measured by WST-1 assay after treatment with the indicated modulators for three days. Raw values were normalized to untreated control. Data are presented as mean ± SEM from at least three independent experiments done in triplicates.