Proteasome inhibitor PS-341 (bortezomib) induces calpain-dependent IkappaB(alpha) degradation

Abstract

The proteasome, a key component of the ubiquitin-proteasome pathway, has emerged as an important cancer therapeutic target. PS-341 (also called Bortezomib or Velcade) is the first proteasome inhibitor approved for newly diagnosed and relapsed multiple myeloma and is currently being tested in many clinical trials against other types of cancers. One proposed mechanism by which PS-341 exerts its anticancer effect is inactivation of nuclear factor-kappaB (NF-kappaB) through prevention of IkappaB(alpha) degradation. In this study, we show that PS-341 at concentrations that effectively inhibited the growth of human cancer cells, instead of increasing IkappaB(alpha) stability, paradoxically induced IkappaB(alpha) degradation. As a result, PS-341 facilitated p65 nuclear translocation and increased NF-kappaB activity. Moreover, IkappaB(alpha) degradation by PS-341 occurred early before induction of apoptosis and could not be inhibited by a pan-caspase inhibitor or caspase-8 silencing; however, it could be prevented with calpain inhibitors, calcium-chelating agents, calpain knockdown, or calpastatin overexpression. In agreement, PS-341 increased calpain activity. These data together indicate that PS-341 induces a calpain-mediated IkappaB(alpha) degradation independent of caspases. In the presence of a calpain inhibitor, the apoptosis-inducing activity of PS-341 was dramatically enhanced. Collectively, these unexpected findings suggest not only a novel paradigm regarding the relationship between proteasome inhibition and NF-kappaB activity but also a strategy to enhance the anticancer efficacy of PS-341.

FIGURE 1.

PS-341 induces IκBα degradation (A, C, and D) and inhibits proteasome activity (B) in human lung cancer cells. The given cells lines were treated with 50 nm PS-341 for the indicated time (A) or with the indicated concentrations of PS-341 for 8 h (C and D). After the aforementioned treatments, the cells were then subjected to preparation of whole-cell protein lysates and subsequent Western blot analysis. B, given cell lines were treated with 50 nm PS-341 for the indicated time and then subjected to proteasome activity assay. Columns, means of triplicate treatments; bars, ±S.D. LE, longer exposure.

FIGURE 2.

Induction of IκBα degradation by PS-341 in other types of cancer cells lines (A and B) and by other proteasome inhibitors in human lung cancer cells (C). A, given cell lines were treated with low (50 nm) or high (1000 nm) concentrations of PS-341 for 10 h. B, given cell lines were treated with 50 nm PS-341 for 8 h. C, given cell lines were treated with the indicated concentrations of MG132, epoxomycin (EPO), or ALLN for 8 h. After the aforementioned treatments, the cells were then subjected to preparation of whole-cell protein lysates and subsequent Western blot analysis. ΔIκBα, cleaved IκBα; LE, longer exposure; CF, cleaved fragment.

FIGURE 3.

PS-341 induces caspase-independent (A), calpain-mediated (B–D) IκBα proteolysis. A, top panel, the indicated cell lines were treated with 50 nm PS-341 alone or 50 nm PS-341 plus 50 μm Z-VAD-FMK, and 50 μm leupeptin, respectively, for 12 h. Bottom panel, H460 cells were transfected with control (Ctrl) or caspase-8 (Casp-8) siRNA. Forty eight hours later, the cells were treated with DMSO or 50 nm PS-341 for an additional 12 h. B, given cell lines were treated with 50 nm PS-341 alone or PS-341 combined with 200 μm EST, PD150606, and calpeptin, respectively, for 8 h. C, H460 cells were transfected with 20 nm control (Ctrl) or calpain regulatory (reg) siRNA. Thirty eight hours later, the cells were treated with DMSO or 50 nm PS-341 for an additional 10 h. D, given cell lines were treated with 50 nm PS-341 alone or PS-341 combined with 1.25 mm EGTA alone, 30 μm BAPTA alone, and EGTA plus BAPTA, respectively, for 8 h. After the aforementioned treatments, the cells were then subjected to preparation of whole-cell protein lysates and subsequent Western blot analysis. LE, longer exposure.

FIGURE 4.

PS-341 increases calpain activity (A and B), which can be blocked by a calpain inhibitor (C). A and B, H460 cells were exposed to the indicated concentrations of PS-341 for 4 h (A) or 25 nm PS-341 for 8 h (B) and then subjected to calpain activity assay with either a microplate spectrofluorometer (A) or fluorescent microscopy (B). Columns, means of triplicate determinations; bars, ± S.D. C, H460 cells were exposed to DMSO, 50 nm PS-341, 100 μm PD150606, and the combination of PD150606 and PS-341 for 8 h and then subjected to calpain activity assay with fluorescent microscopy.

FIGURE 5.

Calpastatin inhibition decrease IκBα levels (A) and mediates PS-341-induced IκBα degradation (B and C). A, indicated cell lines were transfected with control or calpastatin siRNA for ∼48 h. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. B, H460 cells were treated with 50 nm PS-341 for the indicated times. C, HEK293T cells were transiently transfected with empty vector or calpastatin plasmid for ∼36 h and then treated with the indicated concentrations of PS-341 for an additional 16 h. After the aforementioned treatments, the cells were subjected to preparation of whole-cell protein lysates for Western blot analysis.

FIGURE 6.

Proteasome inhibitors do not induce degradation of SRIκBα that carries serine to alanine mutations (A) and do induce p65 nuclear translocation (B) and NF-κB activity (C). The given cell lines were infected with 30 multiplicities of infection of Ad-SRIκBα for 48 h and then treated with 50 nm PS-341 or 10 μm MG132 for the indicated times. The cells were lysed for preparation of whole-cell protein lysates and subsequent Western blot analysis. The migration difference between the endogenous IκBα and SR-IκBα is likely due to mutation (i.e. amino acid change) and phosphorylation inability. B, given cell lines were treated with DMSO or 50 nm PS-341 for 4 h and then subjected to immunofluorescent staining of p65. C, indicated cell lines were transfected with pNF-κB-luc plasmid together with β-galactosidase plasmid as an internal control using FuGENE 6 transfection reagent. On the 2nd day, the cells were treated with the indicated concentrations of PS-341 for 8 h and then subjected to cell lysis and luciferase activity assay. Columns, means of triplicate determinations; Bars, ±S.D.

FIGURE 7.

Calpain inhibitor PD150606 augments effects of PS-341 on decreasing cell survival (A) and inducing apoptosis (B). A, H157 or H460 cells were treated with the given concentrations of PS-341 alone, 100 μm PD150606 alone, and the combination of PS-341 and PD150606. After 3 days, the cell numbers were estimated with the sulforhodamine B assay. Points, means of four replicate determinations; bars, ±S.D. B, given cell lines were treated with DMSO, 20 nm (H157) or 30 nm (H460) PS-341 alone, 100 μm PD150606 alone, or the combination of PS-341 and PD150606. After 48 h, the cells were harvested for estimating apoptotic cells with the annexin V assay. The percent positive cells in the upper right and lower right quadrants were added to yield the total of apoptotic cells.