Figure 5
Figure 5. Statins diminish UCN-01–induced ERK1/2 activation and enhance Akt inactivation while reciprocally promoting JNK activation. (A) U937 cells (upper panels) were exposed for 18 hours to 100 nM UCN-01 (UCN) with or without 20 μM lovastatin (LV), 40 μM fluvastatin (FV), or 30 μM simvastatin (SV), respectively; alternatively, Jurkat cells (lower panels) were exposed to 150 nM UCN-01 with or without 20 μM lovastatin, after which Western blot analysis was performed to monitor the phosphorylation status of ERK1/2. (B) RPMI8226 cells (8226) were treated with 100 nM UCN-01 with or without 10 μM lovastatin for 24 hours, and U266 cells were exposed to 150 nM UCN-01 with or without 10 μM lovastatin for 48 hours, after which ERK1/2 phosphorylation was assessed by Western blot analysis. (C) U937 cells were incubated with lower concentrations of UCN-01 (50 to 75 nM) with or without 2.5 to 5μM lovastatin for 48 hours or 72 hours, after which cells were lysed and subjected to Western blot analysis to monitor ERK1/2 phosphorylation. (D) U937 (upper panels) and Jurkat cells (lower panels) were treated as described in panel A, after which phosphorylation/activation of JNK and Akt was monitored by Western blot analysis. (E) RPMI8226 (upper panels) and U266 cells (lower panels) were treated as described in panel B, after which phosphorylation of JNK and Akt was assessed by Western blot analysis. For panels A-E, 30 μg protein was loaded in each lane. The results are representative of 3 separate experiments. (F) U937 cells were exposed (18 hours) to 100 nM UCN-01 + 20 μM lovastatin in the presence or absence of 10 μM SP600125, after which the percentage of annexin V+ cells was determined by flow cytometry. The results represent the means ± SD for 3 separate experiments performed in triplicate. *Significantly lower than values for treatment without SP600125 (P < .05). Veh indicates vehicle. (G) U266 cells ectopically expressing JNK1-APF (inset, Western blot), a dominant negative form of JNK1, or its corresponding empty vector were exposed to 150 nM UCN-01 + 10 μM lovastatin for 48 hours, after which the percentage of apoptotic cells was assessed by annexin V staining and flow cytometry. The results represent the means ± SD for 3 separate experiments performed in triplicate. **Significantly lower than values for empty vector controls (U266/neo) (P < .02). The blots shown (inset) were obtained from same films, and vertical lines indicate where they were cut.

Statins diminish UCN-01–induced ERK1/2 activation and enhance Akt inactivation while reciprocally promoting JNK activation. (A) U937 cells (upper panels) were exposed for 18 hours to 100 nM UCN-01 (UCN) with or without 20 μM lovastatin (LV), 40 μM fluvastatin (FV), or 30 μM simvastatin (SV), respectively; alternatively, Jurkat cells (lower panels) were exposed to 150 nM UCN-01 with or without 20 μM lovastatin, after which Western blot analysis was performed to monitor the phosphorylation status of ERK1/2. (B) RPMI8226 cells (8226) were treated with 100 nM UCN-01 with or without 10 μM lovastatin for 24 hours, and U266 cells were exposed to 150 nM UCN-01 with or without 10 μM lovastatin for 48 hours, after which ERK1/2 phosphorylation was assessed by Western blot analysis. (C) U937 cells were incubated with lower concentrations of UCN-01 (50 to 75 nM) with or without 2.5 to 5μM lovastatin for 48 hours or 72 hours, after which cells were lysed and subjected to Western blot analysis to monitor ERK1/2 phosphorylation. (D) U937 (upper panels) and Jurkat cells (lower panels) were treated as described in panel A, after which phosphorylation/activation of JNK and Akt was monitored by Western blot analysis. (E) RPMI8226 (upper panels) and U266 cells (lower panels) were treated as described in panel B, after which phosphorylation of JNK and Akt was assessed by Western blot analysis. For panels A-E, 30 μg protein was loaded in each lane. The results are representative of 3 separate experiments. (F) U937 cells were exposed (18 hours) to 100 nM UCN-01 + 20 μM lovastatin in the presence or absence of 10 μM SP600125, after which the percentage of annexin V+ cells was determined by flow cytometry. The results represent the means ± SD for 3 separate experiments performed in triplicate. *Significantly lower than values for treatment without SP600125 (P < .05). Veh indicates vehicle. (G) U266 cells ectopically expressing JNK1-APF (inset, Western blot), a dominant negative form of JNK1, or its corresponding empty vector were exposed to 150 nM UCN-01 + 10 μM lovastatin for 48 hours, after which the percentage of apoptotic cells was assessed by annexin V staining and flow cytometry. The results represent the means ± SD for 3 separate experiments performed in triplicate. **Significantly lower than values for empty vector controls (U266/neo) (P < .02). The blots shown (inset) were obtained from same films, and vertical lines indicate where they were cut.

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