Data are expressed as means SE Statistical significance was

Data are expressed as means±SE. Statistical significance was determined by the Student's -test or ANOVA with post hoc Bonferroni test. A value of <0.05 was considered statistically significant. To characterize the role of FPPS in the paclitaxel-induced apoptotic cell death of human glioblastoma U87MG cells, we established stable transfectants of U87MG pde-5 inhibitors expressing human FPPS. Western blot analysis showed a clear band, which indicated the overexpression of FPPS, in U87MG cells (A, insert). Exposure of U87MG control cells to paclitaxel resulted in a dramatic increase in the number of apoptotic cells, as assessed by the number of cells with sub-G DNA content and by the number of annexin-V-positive cells in a dose- and time-dependent manner (A and B). However, paclitaxel-induced apoptotic cell death was significantly attenuated in U87MG cells overexpressing FPPS. These effects of FPPS was not observed in the -(4-hydroxyphenyl)retinamide (4-HPR)-treated cells, suggesting that action of FPPS is the specific to paclitaxel-induced apoptotic cell death in U87MG (). This was also confirmed by the evaluation of IC values of paclitaxel and 4-HPR (). To clarify further the molecular mechanisms underlying the suppression of paclitaxel-induced apoptotic cell death by FPPS, we examined the activation of key proteins associated with apoptosis in paclitaxel-treated cells. As shown in C and D, Bax expression and cytochrome c release were increased at 24h after treatment with paclitaxel in cells transfected with the Flag vector alone, whereas they were markedly attenuated in cells overexpressing FPPS. Overexpression of FPPS also enhanced the expression of Bcl-X in paclitaxel-treated cells compared to cells transfected with the Flag vector alone. The tumor suppressor p53 is a key regulator of apoptosis, and acts as a transcription factor under stressed conditions, followed by expression of pro-apoptotic target genes such as Bax . We therefore examined the level of p53 protein expressed in U87MG cells treated with paclitaxel. Consistent with inhibition of apoptotic proteins, the overexpression of FPPS blunted the elevated expression of p53 induced by paclitaxel (E). These results suggest that FPPS blocks paclitaxel-induced apoptotic cell death through the regulation of key apoptotic proteins. In order to verify the significance of FPPS in paclitaxel-induced cellular damage, we examined the effect of inhibiting FPPS activity. The addition of pamidronate, a specific inhibitor of FPPS, abolished the FPPS-mediated improvement in cell viability and reduction in apoptotic cell death (A–C). The inhibition of FPPS enzyme activity thus rendered the cells more susceptible to paclitaxel-induced cellular damage. These findings suggest that the augmented expression of FPPS inhibits apoptotic cell death in U87MG cells treated with paclitaxel. To clarify further the molecular mechanisms underlying the suppression of paclitaxel-induced apoptotic cell death by FPPS, we examined the involvement of FPP, a catalytic product of FPPS, in paclitaxel-induced apoptotic cell death. As shown in D and E, the addition of FPP partially suppressed paclitaxel-induced apoptosis in both Flag only and Flag-FPPS overexpressed cells as assessed by the accumulation of sub-G DNA content. This result suggests that the FPPS-catalyzed metabolite is involved in the regulation of paclitaxel-induced apoptosis. To identify the signaling cascades activated by paclitaxel, we investigated the effect of paclitaxel treatment on the PI3K/Akt pathway and the three mitogen-activated protein kinase (MAPK) cascades, p38, JNK, and ERK. U87MG cells were treated with paclitaxel for the indicated time periods, and immunoblotting was performed with specific antibodies. As shown in A, paclitaxel activated the three MAPK cascades, p38, JNK, and ERK, but not the PI3K/Akt pathway. Overexpression of FPPS almost completely abolished the paclitaxel-mediated activation of p38 and JNK pathways. On the other hand, the ERK signaling cascade was less affected in cells overexpressing FPPS. To elucidate further the signaling pathways involved in paclitaxel-induced apoptosis, we examined the effects of specific inhibitors of the three MAPK cascades. Among the inhibitors used, only SP600125 markedly suppressed the paclitaxel-induced accumulation in sub-G DNA, indicating that the JNK-mediated signaling pathway is involved in the FPPS-mediated suppression of apoptotic cell death induced by paclitaxel (B). The viability of cells treated with SB203580 (20μM), SP600125 (20μM), or PD98059 (20μM) was >90%, as determined by the MTT assay (data not shown).