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. 2012 Jul;97(7):1101-9.
doi: 10.3324/haematol.2011.049981. Epub 2012 Jan 22.

The human immunodeficiency virus-1 protease inhibitor nelfinavir impairs proteasome activity and inhibits the proliferation of multiple myeloma cells in vitro and in vivo

Camille Bono  1 , Lionel Karlin, Stephanie Harel, Enguerran Mouly, Sylvaine Labaume, Lionel Galicier, Sébastien Apcher, Hélène Sauvageon, Jean-Paul Fermand, Jean-Christophe Bories, Bertrand Arnulf
Affiliations

The human immunodeficiency virus-1 protease inhibitor nelfinavir impairs proteasome activity and inhibits the proliferation of multiple myeloma cells in vitro and in vivo

Camille Bono et al. Haematologica. 2012 Jul.

Abstract

Background: Multiple myeloma is characterized by the accumulation of tumor plasma cells in the bone marrow. Despite therapeutic improvements brought by proteasome inhibitors such as bortezomib, myeloma remains an incurable disease. In a variety of human cancers, human immunodeficiency virus protease inhibitors (e.g. nelfinavir) effectively inhibit tumor progression, but their impact on myeloma is unknown. We assessed the in vitro and in vivo effects of nelfinavir on multiple myeloma.

Design and methods: The effects of nelfinavir (1-10 μM) on proteasome activity, proliferation and viability of myeloma cell lines and plasma cells from patients were assessed by measuring PERK, AKT, STAT3 and ERK1/2 phosphorylation and CHOP expression with immunoblotting or flow cytometry. The in vivo effect was assessed in NOD/SCID mice injected with luciferase expressing human myeloma cell lines and treated with nelfinavir at a dose of 75 mg/kg/day. Tumor progression was evaluated using a bioluminescent system.

Results: Nelfinavir inhibited 26S chymotrypsin-like proteasome activity, impaired proliferation and triggered apoptosis of the myeloma cell lines and fresh plasma cells. It activated the pro-apoptotic unfolded protein response pathway by inducing PERK phosphorylation and CHOP expression. Cell death triggered by nelfinavir treatment correlated with decreased phosphorylation of AKT, STAT3 and ERK1/2. Nelfinavir enhanced the anti-proliferative activity of bortezomib, dexamethasone and histone deacetylase inhibitors and delayed tumor growth in a myeloma mouse model.

Conclusions: These results suggest that nelfinavir, used at a pharmacological dosage, alone or in combination, may be useful in the treatment of myeloma. Our data provide a preclinical basis for clinical trials using nelfinavir in patients with myeloma.

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Figures

Figure 1.
Figure 1.
Nelfinavir inhibits 26S proteasome activity. Histograms show the chymotrypsin-like (CT-like) and trypsin-like (T-like) activities of the 26S proteasome (A) Cellular lysates of LP1, U266 or MM1S cell lines were incubated with 5 μM nelfinavir (Nelf), 10 nM bortezomib (Bort) or 1 μM MG132 (MG) or cultured in medium alone (Med). (B) Cellular lysates of plasma cells from patients were incubated with nelfinavir (Nelf) 10 μM, bortezomib (Bort) 10 nM and MG132 (MG) 1 μM or cultured in medium alone (Med). (C) U266 cells were incubated with nelfinavir (5 μM) for the indicated times and the level of the ubiquitinylated proteins was determined by western blot. Error bars correspond to the standard deviation. The P value was calculated with the Student’s t-test, (*P≤0.05, **P≤0.01).
Figure 2.
Figure 2.
Nelfinavir inhibits proliferation of MM cell lines in vitro even in the presence of pro-survival cytokines. (A) (Right) Histograms show the proliferation of RPMI, LP1, U266, OPM2 and MM1S cell lines treated with nelfinavir (1 μM to 10 μM). A. (Left) Viability of plasma cells from three patients who received bortezomib (“P” and “M” were not responsive to bortezomib) the cells were cultured with the indicated concentrations of nelfinavir. (B) (Right) Histograms show the proliferation of U266 cells treated (filled) or not (open) with 5 μM nelfinavir in the presence of IGF-1 (100 ng/mL) or FGF (100 ng/mL) + heparin (100 μg/mL). (B) (Left) Histograms show the viability of the plasma cells from a patient treated (filled) or not (open) with 10 μM of nelfinavir in the presence of IGF-1 (100 ng/mL) or FGF (100 ng/mL) + heparin (100 μg/mL). Error bars correspond to the standard deviation. The P value was calculated with Student’s t-test, (*P≤0.05, **P≤0.01).
Figure 3.
Figure 3.
Nelfinavir cooperates with anti-myeloma agents to inhibit the proliferation of multiple myeloma cells. Bar graphs show the proliferation of U266 and LP1 cells cultured in the presence of nelfinavir (nelf) and (A) dexamethasone (Dex), (B) valproic acid (VA), and (C) bortezomib (BTZ). The concentrations at which each drug was used are indicated. Error bars correspond to the standard deviation. The P value was calculated with Student’s t-test, (*P≤0.05, **P≤0.01).
Figure 4.
Figure 4.
Nelfinavir induces the cleavage of pro-caspase 3 and apoptosis in MM cell lines. (A) Dot plots show apoptosis of LP1 and U266 cells cultured with nelfinavir at the indicated concentrations. The percentages of dead cells, measured by annexin V and propidium iodide staining, are indicated in each quadrant. (B). Western blot analysis of the kinetics of pro-caspase-3 cleavage in U266 cells cultured with nelfinavir (5 μM). Cleaved and native forms of the caspase-3 are indicated as is the actin control.
Figure 5.
Figure 5.
Nelfinavir decreases the phosphorylation of AKT, STAT3, ERK, and activates the pro-apoptotic pathway of the UPR system. (A) Right. U266 cells were starved for 4 h and incubated at the indicated times with 5 μM nelfinavir. The levels of AKT, phosphorylated AKT (P-AKT) and the control actin were detected by western blotting. Left. Histograms show the level of phosphorylated AKT in IGF1-stimulated U266 cells in the presence of nelfinavir (5 μM). (B) Histogram showing the intracellular FACS analysis of phosphorylated STAT3 (P-STAT3) and ERK1/2 (P-ERK1/2) in IL-6-stimulated U266 cells. Nelfinavir treated cells are indicated by the dotted lines (5 μM) or dashed line (20 μM), the bold line corresponds to non-treated cells and the isotype control is represented in filled gray. The mean intensity of fluorescence (MIF) of each treatment is indicated on the histograms. (C) Western blot analysis of the kinetics of P-PERK, ATF4 and CHOP expression in U266 cells treated with nelfinavir (5 μM). Actin is used as a loading control.
Figure 6.
Figure 6.
Nelfinavir decreases MM cell growth in NOD/SCID mice. U266-luc cells (8×106) were injected subcutaneously into the flank of NOD/SCID mice. Intraperitoneal administration of nelfinavir (75 mg/kg for 5 days/week) was initiated 24 h after cell inoculation and continued all through to the end of the experiment. The tumor burden was measured based on photon emission on the indicated day. (A) Representative bioluminescence images of mice on days 7, 14 and 21 are shown (days after the inoculation). (B) The mean photon emission readout indicated for each group (n=7).
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