Bortezomib sensitizes human renal cell carcinomas to TRAIL apoptosis through increased activation of caspase-8 in the death-inducing signaling complex

Abstract

Bortezomib (VELCADE) could sensitize certain human renal cell carcinoma (RCC) lines to the apoptotic effects of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Analysis of seven human RCC showed a clear increase in the sensitivity of four of the RCC to TRAIL cytotoxicity following bortezomib (5-20 nmol/L) treatment, whereas the remaining three remained resistant. Tumor cell death following sensitization had all the features of apoptosis. The enhanced antitumor activity of the bortezomib and TRAIL combination was confirmed in long-term (6 days) cancer cell outgrowth assays. The extent of proteasome inhibition by bortezomib in the various RCC was equivalent. Following bortezomib treatment, neither changes in the intracellular protein levels of various Bcl-2 and IAP family members, nor minor changes in expression of TRAIL receptors (DR4, DR5), correlated well with the sensitization or resistance of RCC to TRAIL-mediated apoptosis. However, enhanced procaspase-8 activation following bortezomib pretreatment and subsequent TRAIL exposure was only observed in the sensitized RCC in both cell extracts and death-inducing signaling complex immunoprecipitates. These data suggest that the molecular basis for bortezomib sensitization of RCC to TRAIL primarily involves early amplification of caspase-8 activity. In the absence of this increased caspase-8 activation, other bortezomib-induced changes are not sufficient to sensitize RCC to TRAIL-mediated apoptosis.

Figure 1

Bortezomib sensitizes some human renal cancer cell lines to caspase dependent apoptosis by rhTRAIL. (a) MTS growth inhibition assay (b) Annexin V and propidium iodide (PI) staining; Cells were treated overnight with 20 nM Bortezomib, and then 8 hours with 100 ng/ml rhTRAIL (c) Cells were treated for 2 hours with 20 uM zVAD or zIETD-FMK, and then for 2 hours with 20 nM Bortezomib followed by an overnight treatment with 50 ng/ml rhTRAIL.

Figure 2

Long-term cell survival assays also demonstrate bortezomib sensitization of RCC to TRAIL apoptosis. RCC were treated overnight with bortezomib and rhTRAIL alone or in combination. The following morning drugs were washed out and replaced with fresh media. After 6 days cells were fixed with methanol and stained with crystal violet.

Figure 3

The proteasome was inhibited equally in all RCC tested. (a) Cells were treated overnight with 1, 10 or 20 nM Bortezomib and then assayed with the Proteo-glo assay (Promega). (b) Cells were treated overnight with 20 nM Bortezomib; cell lysates were used for western blotting for ubiquitinated proteins.

Figure 4

Bortezomib causes an increase in the surface expression of some death receptors and cFLIPs in some RCC lines. (a) Death receptor expression on tumor cell lines was analyzed after overnight treatment with 20nM bortezomib or media alone by flow cytometry using anti-TRAIL Receptor-1 to 4 Flow Cytometry Set (Axxora). (b) DISC protein expression was measured by western blotting of cell lysates. (c) MTS assay; Cells were treated with TRAIL for 90 minutes, and then washed three times with media to remove unbound TRAIL. Media was replaced in wells and bortezomib was added for an overnight treatment. For the control plate cells were washed with media then TRAIL and/or bortezomib were added for the overnight incubation.

Figure 5

Bortezomib caused changes in expression of proteins related to apoptosis in some RCC cell lines. (a) Western blot analysis was performed on both pro and anti-apoptotic proteins. (b) Western blot analysis was performed on Akt and caspase-9.

Figure 6

Bortezomib causes increased caspase 8 activation at the DISC. (a) Caspase 8 activation was measured using the Caspase-Glo^® 8 Assay System (Promega). (b) Cells were treated overnight with 20 nM Bortezomib followed by 60 minute treatment with biotinylated rhTRAIL. Lysates were immunoprecipitated with strepavidin coated magnetic beads and western blot analysis was performed on eluates for DISC components. (c) Cells were treated overnight with 20 nM bortezomib followed by 60 minutes with rhTRAIL. Lysates were prepared and analyzed by western blot. (d) MCF-7 cells were treated overnight with 20 nM bortezomib followed by 2 hours with rh TRAIL and lysates were prepared and analyzed by western blotting.