Construction and characterization of a new TRAIL soluble form, active at picomolar concentrations

Abstract

Apoptosis induction has emerged as a treatment option for anticancer therapy. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a type II transmembrane protein, is a potent and specific pro-apoptotic protein ligand, which activates the extrinsic apoptosis pathway of the cell death receptors. Here we describe the construction and characterization of a new soluble TRAIL, sfTRAIL, stabilized with the trimerization Foldon domain from the Fibritin protein of the bacteriophage T4. Supernatants of 0.22 μM-filtered supernatants were produced in Vero-transduced cells with HSV1-derived viral amplicon vectors. Experiments were undertaken in two known TRAIL-sensitive (U373 and MDA.MB.231) and two TRAIL-resistant (MCF7 and A549) cell lines, to determine (i) whether the sfTRAIL protein is synthetized and, (ii) whether sfTRAIL could induce receptor-mediated apoptosis. Our results showed that sfTRAIL was able to induce apoptosis at concentrations as low as 1899.29 pg/mL (27.71 pM), independently of caspase-9 activation, and reduction in cell viability at 998.73 fM.

Keywords: TRAIL; amplicon vectors; apoptosis; cancer treatment.

Figure 1

(A) Schematic representation of the sfTRAIL protein; (B) Western blot analysis of sfTRAIL protein expression. Vero cells were transduced with pA.EUA1 or pA.sfTRAIL amplicon vectors, at MOI1. Anti-TRAIL antibody and anti-β-actin were used.

Figure 2. Cell viability (MTS) of cells incubated with sfTRAIL supernatants

Values shown are the mean value (± SDs) of triplicates. (SN) means supernatants. sfTRAIL concentration in supernatants was 1899.29 pg/mL. Data represents three experiments performed in triplicate. p-value were assessed by Student’s t-test.

Figure 3. Western blot analysis of sfTRAIL-mediated apoptosis induction. sfTRAIL-mediated apoptosis induction of A549, MCF7, U373 and MDA-MB-231 cells, cultured in conditioned media, at 2h, 4h and 8h post-induction (PI)

UTC, untreated control.

Figure 4. Cell viability (MTS) of cells incubated with sfTRAIL supernatants. Values shown are the mean value (± SDs) of three experiments performed in triplicate (Stock A, B and C)

(SN) means supernatants; NTC (SN) represents supernatants of non-transduced cells; ^* indicates p-value (Student’s t-test), where ^** means p-value=0.0032 and ^*** p-value<0.0001.