Each cell line is the mean of three independent Camptothecin experiments performed in triplicate. Error bars represent s. e. m. IC50 values after HL cell lines were incubated with increasing doses cell lines that expressed at least one active phosphorylated member of the JAK f that HL cell lines are addicted to the JAK/STAT pathway, and selective inhibition of STAT proteins by RNA interference has been shown to result in antiproliferative effects in HL cell lines. 2 Because AZD1480 inhibited p STAT3, p STAT5 and p STAT6, we investigated the antiproliferative effect of AZD1480 in HL cells by using the MTS assay. AZD1480 induced antiproliferative effects in a time and dose dependent manner. After 72 h of incubation, the half maximal inhibitory concentration values for AZD1480 ranged from 1 to 8 mM, L 540 cells were the most sensitive to AZD1480.
Thus, although submicromolar concentrations of AZD1480 inhibited STAT phosphorylation, these low concentrations were not sufficient to induce a significant antiproliferative activity, especially in the HD LM2 and L 428 cells. In contrast, AZ 960 at a higher concentration, AZD1480 had a stronger effect in all cell lines, including KM H2, which lacked active JAK/STAT proteins. These data suggest that at this higher concentration, AZD1480 induced antiproliferative effects in HL cell lines in a JAK/ STAT independent manner. To further investigate the mechanisms of the antiproliferative activity of AZD1480, we determined the inhibitor,s effect on apoptosis. Consistent with the MTS data in Figure 1c, we found that AZD1480 induced apoptotic cell death in a dosedependent manner.
At the lower concentration of AZD1480, L 540 cells were the most sensitive and L 428 cells were modestly sensitive. At the higher concentration, AZD1480 induced apoptosis in all four cell lines, but L 540 cells remained the most sensitive. Consistent with induction of apoptosis, AZD1480 activated caspases 9 and 3, and induced poly polymerase Figure 2 AZD1480 induces apoptosis in HL cell lines. Representative experiment demonstrating the effect of two different doses of AZD1480 on apoptosis as determined by annexin V binding assay. The percentage of dead cells is shown in the upper right quadrant. Summary of the results of dual annexin V and propidium iodide staining. Each value is the mean of three independent experiments performed in triplicate. Po0. 05, Po0.
005, NS, not significant. Error bars represent s. e. m. Immunoblotting showing activation of the intrinsic apoptotic pathway in HL cell lines incubated with AZD1480 for 72 h. Consistent with the data in and, cleavage of poly polymerase and activation of caspases 9 and 3 were observed in all the cell lines exposed to 5 mm AZD1480. In L 540 and L 428, caspase cleavage was observed also with 1 mm AZD1480. cleavage. This was observed as soon as after 24 h of incubation with AZD1480 in all cell lines, when high doses were used. AZD1480 induces paradoxical hyperphosphorylation of JAK2 and TYK2 at the activation loop in HL cells Although AZD1480 inhibited phosphorylation of STATs, its effect on the phosphorylation of the JAK family members in HL cells is unknown. Only the Tyr phosphorylated forms of JAKs are known to exhibit kinase activity, and phosphorylation at two tandem Tyr residues in the activat
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