Figure 1.
Structure of Dehydrocostuslactone (DCE), Costunolide (CS) and Dehydrocostunolide (HCS).
The α-β-unsaturated carbonyl group is marked with a dotted circle.
Figure 2.
Effect of DCE and CS on IL-6-elicited STAT3 activation in THP-1 cells.
(a) EMSA shows that DCE and CS dose-dependently decrease DNA-binding activity of STAT3 activated by 20 ng/ml IL6 in THP-1 cells. In the insert EMSA/supershift indicates that IL-6 induces prevalently the activation of STAT3. (b) Western Blot analysis shows that DCE and CS dose-dependently decrease tyrosine705 phosphorylation of STAT3 induced by 20 ng/ml IL-6 in THP-1 cells without changing the total amount of STAT3. Furthermore the sesquiterpens don't affect serine727 phosphorylation. The gels are representative of four independent experiments.
Figure 3.
Effect of DCE and CS on tyrosine phosphorylation of JAK tyrosine kinases.
Western Blot analysis shows that DCE and CS dose-dependentlydecreasetyrosine1022/1023 phosphorylation of JAK1 and tyrosine1007/1008 phosphorylation of JAK2 in THP-1 cells (a) and dose-dependently decreasetyrosine1054/1055 phosphorylation of Tyk2 in HeLa cells transiently transfected with Tyk2-pcDNA 3.0 (b). The compounds don't change the total amount of the corresponding non-phosphorylated proteins. The gelsarerepresentatives of four experiments performed separately.
Figure 4.
DCE and CS decrease intracellular GSH level in THP-1 cells.
DCE and CS time- and dose-dependently induce the drop in cellular GSH content without significantly affecting the amounts of GSSG. Data are presented as means ± SD of results from four independent experiments. Significant difference compared to control group (*p≤0.05).
Figure 5.
GEE reverts the inhibitory action of DCE and CS on the IL-6-elicited phosphorylation of STAT3.
(a) Western Blot analysis shows that inhibitory action of DCE and CS on IL-6 induced STAT3 Tyr705 phosphorylation is reverted by 1 mM glutathione monoethyl ester (GEE). The total amount of STAT3 is not affected during the experiments. The gels are representative of four experiments performed separately.
Figure 6.
DCE and CS induce ROS production.
Flow cytometric analysis of intracellular ROS generation. THP-1 cells loaded with CM-H2DCFDA and treated for 30 minutes (dark gray) and 1 hour (light gray) with 6, 12 and 25 µM (a) DCE or (b) CS shows and increment of fluorescence as respect to untreated cells (white). This increment is time-dependent but not dose-dependent. 500 µM diamide was used as positive control (inset). One representative experiment out of four is depicted.
Figure 7.
DCE and CS directly interact with GSH.
(a) HPLC analysis shows that GSH dose-dependently decrease the amounts of DCE and CS after 30 minutes of incubation at 37°C. (Standard deviation of date obtained from three independent experiments is shown). (b) Western Blot analysis shows that HCS, is not able to decrease IL-6 induced STAT3 Tyr705 phosphorylation as CS does in THP-1 cells. The total amounts of STAT3 are not affected during the experiments. Data are the representatives of four experiments performed separately. The gels are representative of four experiments performed separately.
Figure 8.
Effect of DCE and CS on glutathionylation of STAT3 in THP-1 cells.
Western Blot analysis of immunoprecipitated STAT3 (IP STAT3) shows that DCE and CS dose-dependently increase the amounts of glutathionylated STAT3. At 50 µM both lactones induce the same level of STAT3 glutathionylation as that induced by 500 µM diamide, a strong oxidant. The total amounts of STAT3 are not affecting during the experiments. The gels are representative of four experiments performed separately.