A decreased TMRE Ruxolitinib order signal corresponding

to decreased membrane potential was observed in a significant number of S20-3 peptide-treated (20%) and CH-11–treated (22%) cells as early as 4 hours after treatment, relative to treatment with buffer or the control S8-2 peptide (Additional file 1: Figure S1). The S20-3 peptide is effective against various hematological cancer cell lines We further investigated whether the S20-3 peptide would be effective in inducing cell death in HHV-8–positive cancer cell lines (KS-1, BC-3, BCBL-1), which have been shown to express K1 [10]. All HHV-8–infected cell lines tested were sensitive to the S20-3 peptide, which induced death in about 20–35% of cells, whereas no significant effect on cell death was detected with the S8-2 control peptide (Figure 2A). Figure 2 The HHV-8 K1-derived peptide S20-3 induces cell death

in K1-positive and K1-negative hematological cancer cells but not in PBMCs from healthy donors. Indicated cell lines (1 × 106 cells/mL) were incubated with 100 μM peptide S20-3 or buffer for 1 hour. Cells were washed and incubated in complete medium for 24 hours before flow cytometry analysis. (A) HHV-8– and K1-positive cell lines KS-1, BC-3, BCBL-1; (B) HHV-8 and K1-negative cell lines BJAB, Jurkat, Daudi; (C) Jurkat cells and PBMCs from healthy donors. Data in (A) and (B) are shown as the means ± SD of triplicate wells. Double asterisks indicate significant differences compared with control treatments; **P < 0.01. Panel (C) shows representative results of selleck chemical 2 experiments

with samples Ketotifen analyzed in triplicates. To evaluate whether the peptides were able to modulate the interaction between Fas and K1, 293T cells were transiently transfected with the vector expressing Flag-tagged K1 protein, lysed, and subjected to co-immunoprecipitation analysis used previously to show a direct physical interaction of Fas with K1 [8]. We observed that K1-Fas interaction was not disrupted by incubation of cells with the S20-3 or other K1-derived peptides with the exception of the shorter peptide S10-1 (Additional file 1: Figure S2). The lack of S20-3 peptide’s effect on the K1-Fas interaction suggested a possible cell-killing mechanism independent of K1. To confirm this hypothesis, we tested the peptide’s ability to kill K1-negative cell lines. The S20-3 peptide was able to induce significant levels of cell death in K1-negative BJAB cells (30%) and in the T-cell leukemia Jurkat cell line (25%) (Figure 2B). Quite surprisingly, the S20-3 peptide was equally effective in killing Daudi cells (35%), which express low levels of Fas on the cell surface and are considered Fas-resistant [17]. In contrast, human PBMCs from healthy donors, treated with S20-3 peptide, showed no significant amount of cell death (Figure 2C). Overall, S20-3 peptide treatment induced a 4.6 ± 1.