The peptide derived from the Ig-like domain of human herpesvirus 8 K1 protein induces death in hematological cancer cells

Abstract

Background: Although significant progress has been made in the treatment of lymphomas, many lymphomas exhibit resistance to cell death, suggesting a defective Fas signaling, which remains poorly understood. We previously reported that cells expressing the K1 protein of human herpesvirus 8 (HHV-8) resist death through the complex formation of the Ig-like domain of K1 with Fas. Recently, we investigated whether peptides derived from the Ig-like domain of the K1 protein may affect cell death.

Methods: K1 positive and negative cell lines were incubated with the K1-derived peptides, and cell death (apoptotic and necrotic) was assessed by flow cytometry and LDH assay. Activation of caspases was assessed by fluorometric assay and flow cytometry. Fas receptor-independent, peptide-mediated cell killing was tested in the Fas-resistant Daudi cell line and Jurkat cell clones deficient in caspase-8 and FADD functionality. Activation of TNF receptors I and II was blocked by pre-incubation with corresponding blocking antibodies. The effect of the K1 peptide in vivo was tested in a mouse xenograft model.

Results: We observed that the peptide S20-3 enhanced cell death in K1-positive BJAB cells and HHV-8 positive primary effusion lymphoma (PEL) cell lines. Similar effects of this peptide were observed in B-cell lymphoma and T-lymphoblastic leukemia cells without K1 expression but not in normal human peripheral blood mononuclear cells. A single intratumoral injection of the S20-3 peptide decreased the growth of Jurkat xenografts in SCID mice. The mechanism of tumor cell death induced by the S20-3 peptide was associated with activation of caspases, but this activity was only partially inhibited by the pan-caspase inhibitor z-VAD. Furthermore, the K1 peptide also killed Fas-resistant Daudi cells, and this killing effect was inhibited by pre-incubation of cells with antibodies blocking TNFRI.

Conclusion: Taken together, these findings indicate that the S20-3 peptide can selectively induce the death of malignant hematological cell lines by Fas- and/or TNFRI-dependent mechanisms, suggesting the K1-derived peptide or peptidomimetic may have promising therapeutic potential for the treatment of hematological cancers.

Figure 1

A human herpesvirus 8 K1 peptide induces dose-dependent cell death and activates caspase cascade in BJABK1 cells. BJABK1 cells were pulse-treated for 1 hour with 100 μM concentration of indicated Ig-like domain-derived peptides and 200 ng/mL of FasL (A), 100 μM concentration of the indicated peptide alone (B), and increasing concentrations of S20-3 or S8-2 peptides (C). Cells were, subsequently, incubated in a complete medium for 24 hours, stained with AnnexinV/PI, and examined by flow cytometry. (D) BJABK1 cells were treated with 100 μM peptide or DMSO for 1 hour. The cells were then washed and incubated in complete medium for 4 hours. Fluorometric caspase activity was analyzed by flow cytometry. The results are presented as means ± SD of triplicate wells. Asterisks indicate statistically significant differences compared with control treatment; *P < 0.05.

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 2 experiments with samples analyzed in triplicates.

Figure 3

The S20-3 peptide–induced cell death is only partially dependent on caspases and involves necroptosis. (A) Jurkat (wild-type), Jurkat I9.2 (caspase-8–deficient), and Jurkat I2.1 (FADD-dominant-negative mutant) cell lines were incubated with 100 μM peptide S20-3. (B) BJAB cells were incubated with 100 μM peptide S20-3 in the presence or absence of 20 μM pan-caspase inhibitor z-VAD-FMK. (C) Daudi cells were incubated with 100 μM peptide S20-3 or buffer in the presence or absence of 20 μM pan-caspase inhibitor z-VAD-FMK. After 1 hour of incubation, cells were washed and incubated in complete medium for 24 hours before flow cytometry analysis. Data in (A) and (B) are shown as means ± SD of triplicate wells; *P < 0.01.

Figure 4

The S20-3 peptide–induced cell death involves TNFRI. (A) Immunoblot analysis of total cellular levels of TNF receptors I and II in BJAB, Jurkat, and Daudi cells. Numbers represent expression levels relative to GAPDH (loading control). (B) Daudi cells were pre-incubated for 1 hour with 5 μg/mL of TNFRI- or TNFRII-blocking antibodies, followed by 1 hour of treatment with 5 ng/mL of TNF-α or 100 μM peptide S20-3, and immediately analyzed for necrosis by LDH release assay. (C) BJABK1 cells (left panel) and BJAB cells (right panel) were pre-incubated for 1 hour with 5 μg/mL of TNFRI-blocking antibody, subsequently treated with 100 μM peptide S20-3 or 5 ng/mL of TNF-α for 1 hour, and analyzed as in (B). The relative cytotoxicity values in (B) and (C) were calculated as LDH release in [(treated-control)/(high control-control)]x100 and are shown as means ± SD of triplicate wells; *P < 0.005, **P < 0.02.

Figure 5

The S20-3 peptide, but not the structurally similar TCR-derived peptide, significantly suppresses growth of Jurkat cell xenografts. (A) Sequence alignment of the relevant regions of the Ig-V domains based on the known structures (http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?hslf=1&uid=cd00099&#seqhrch) and the sequence comparison of S20-3 with the corresponding human TCR-α-derived peptide. (B) Predicted structures of S20-3, S10-2, and S8-2 peptides extracted from the structure of TCR-α (Protein Database ID 1FYT) using Cn3D 4.3 software (www.ncbi.nlm.nih.gov/Structure/CN3D/cn3d.shtml). (C) Jurkat cells were treated with 100 μM peptides (S20-3, TCR) or buffer for 1 hour and, subsequently, incubated in complete medium for 24 hours. Cell killing was analyzed by flow cytometry, and background death (buffer) was subtracted. Values are presented as the means of the percentage of activity relative to the activity of S20-3 ± SE from 3 independent experiments. (D) Flanks of SCID mice were injected with 5 × 10⁶ Jurkat cells. Two weeks later, tumors were injected with a single dose of S20-3, TCR peptide, or vehicle (DMSO) in 50 μL of saline (4 mice each). Eight days after treatment, mice were killed and the tumors were harvested and measured. Tumor measurements are reported as means ± SD; *P < 0.05.