Peptides containing LANA caspase cleavage sites inhibit caspase activity, decrease PARP cleavage and increase cell viability.

Abstract

<p>Caspase activity assays were performed as described in Materials and Methods. Peptides containing the N-terminal caspase cleavage site of LANA (LP-Nterm) or the C-terminal caspase cleavage site of LANA (LP-Cterm) were dissolved in PBS and tested as potential caspase inhibitors at a concentration equal to the commercial caspase-1 substrate (Ac-YEVD-pNa, 200 μM, Sigma) or caspase-3 substrate (Ac-DEVD-pNa, 200 μM, Sigma). PBS was used as the negative control. (A) Effect of LP-Nterm and LP-Cterm on caspase-1 activity. (B) Effect of LP-Nterm and LP-Cterm on caspase-3 activity. In both A and B, the linear caspase activity rate (rfu/min) is plotted with the assay run in triplicate in two separate experiments with one representative experiment shown. Note: The increase in activity of caspase-1 by the LP-Nterm may be due to the presence of a cysteine in this peptide which could protect loss of activity of the enzyme due to oxidation over time. (C) HEP3B cells were pre-treated with DMSO, ZVAD, Tat-LP-Nterm (T-LPN) or Tat-LP-Cterm (T-LPC) peptide for two hrs and then exposed to DMSO alone (control) or etoposide for 16 hrs at 50 μm to induce apoptosis and PARP cleavage. Cell lysates were analyzed by western blot for cleaved PARP and β-actin (loading control). The percent inhibition of PARP cleavage as determined using the LiCor system is indicated below the blot. The immunoblot shown is a representative of 3 different experiments with Tat-LP-Nterm and Tat-LP-Cterm at 50 μM (Tat-LP-Cterm at 100 μM done once). (D) Cell viability following pretreatment with DMSO, ZVAD or T-LPC followed by DMSO or etoposide treatment. Cells were pretreated with DMSO (control), Tat-LP-Cterm or ZVAD (50 μM) for two hrs followed by etoposide treatment at 50 μM. To assess cell viability total cellular protein obtained from the adherent cells following 3 PBS washes was determined by BCA assay. The results shown are the average of 4 independent experiments. ** P< 0.01, * P< 0.05, for two tailed Student’s t-test.</p