Heme oxygenase-1 nuclear translocation regulates bortezomibinduced cytotoxicity and mediates genomic instability in myeloma cells

Abstract

Multiple myeloma (MM) is a clonal B-cell malignancy characterized by an accumulation of clonal plasma cells in the bone marrow leading to bone destruction and bone marrow failure. Several molecular mechanisms underlie chemoresistance among which heme oxygenase-1 (HO-1) could play a major role. The aim of the present research was to evaluate the impact of HO-1 in MM following bortezomib (BTZ) treatment and how HO-1 is implicated in the mechanisms of chemoresistance. MM cells were treated for 24h with BTZ (15 nM), a boronic acid dipeptide inhibitor of the 26S proteasome used in the treatment of patients with MM as first-line therapy. We evaluated cell viability, reactive oxygen species (ROS) formation, endoplasmic reticulum (ER) stress, HO-1 expression and compartmentalization and cellular genetic instability. Results showed that BTZ significantly reduced cell viability in different MM cell lines and induced ER-stress and ROS formation. Concomitantly, we observed a significant overexpression of both HO-1 gene and protein levels. This effect was abolished by concomitant treatment with 4-phenybutirric acid, a molecular chaperone, which is known to reduce ER-stress. Surprisingly, inhibition of HO activity with SnMP (10μM) failed to increase BTZ sensitivity in MM cells whereas inhibition of HO-1 nuclear translocation by E64d, a cysteine protease inhibitor, increased sensitivity to BTZ and decreased genetic instability as measured by cytokinesis-block micronucleus assay. In conclusion, our data suggest that BTZ sensitivity depends on HO-1 nuclear compartmentalization and not on its enzymatic activity and this finding may represent an important tool to overcome BTZ chemoresistance in MM patients.

Keywords: endoplasmic reticulum stress; genomic instability; heme oxygenase; multiple myeloma; oxidative stress.

Figure 1

A. Cell viability following BTZ (15 nM for 24h) treatment in different multiple myeloma cell lines (**p<0.001 vs untreated control). Results are presented as percentage of the control; B. HMOX-1 gene expression following BTZ (15 nM) treatment at different time points and in different multiple myeloma cell lines (**p<0.001 and ***p<0.0001 vs untreated control). Results are expressed as relative expression level (2^−ΔΔCt); C. Reactive Oxygen Species formation following BTZ (15 nM) treatment at different time points and in U266 cell line (**p<0.001 and ***p<0.0001 vs untreated control). Results are expressed as median fluorescence intensity (**p<0.001 and ***p<0.0001 vs untreated control). All values are mean ± SE of four experiments in duplicate.

Figure 2. PERK, IRE1α, BiP and HO-1 protein levels in U266 cell cultures treated with BTZ (15 nM for 24h)

A. or thapsigargin B. treatments were visualized by immunoblotting with specific antibodies. Densitometric analysis was performed after normalization with actin. Blots shown are representative of Western blot analysis from four separate experiments. (***p<0.0001 vs untreated control; τ p<0.05 vs BTZ alone; ττ p<0.01 vs BTZ alone). All values are mean ± SE of four experiments in duplicate.

Figure 3. Cell viability following treatment with BTZ (15 nM for 24h) alone or in combination with SnMP (10 μM)

Results are presented as percentage of the control. (***p<0.0001 vs untreated control). All values are mean ± SE of four experiments in duplicate.

Figure 4. CLSM analysis of HO-1 localization in untreated U266 cell cultures (A, E, I, M) and following BTZ (15 nM for 24h) treatment alone (B, F, J, N) and/or in combination with E64d (20 μM) (C, G, K, O; and D, H, L, P)

HO-1 detection was performed by incubation with anti-rabbit monoclonal antibody followed by secondary antibody conjugated to TRITC (red). Counterstaining of cells was performed by using the nuclear dye, DAPI (blue). The photographs result from sequential analysis of the same microscopic field, followed by merging of different images with specific staining. (Scale bars 10μm).

Figure 5

A. HO-1 nuclear protein levels in U266 cell cultures treated with BTZ (15 nM for 24h) was visualized by immunoblotting with specific antibodies; B. HO-1 nuclear protein levels in U266 sensible (U266/BTZ-S) and resistant (U266/BTZ-R) to BTZ. Laminin b1 shows equal amount of nuclear protein loading in all lanes.

Figure 6

A. Cell viability following BTZ (15 nM for 24h) treatment alone or in combination with E64d (20 μM). Results are presented as percentage of the control. (*p<0.05 vs BTZ alone; ***p<0.0001 vs untreated control). All values are mean ± SE of four experiments in duplicate. B. HO-1 nuclear protein levels in U266 cell cultures treated with BTZ (15 nM for 24h) treatment alone or in combination with E64d (20 μM). Laminin b1 shows equal amount of nuclear protein loading in all lanes.

Figure 7

A. Representative images showing micronuclei in binucleated cells following Giemsa staining. B. Quantification (CBMN assay) of binucleated cells containing micronuclei (MN), nuclear bridge (NB), tetra (TETRA) and trinucleation (TRI). Results were obtained in untreated U266 cell cultures and following E64d (20 μM); C. Quantification of binucleated/mononucleated cells (CBPI). Results were obtained in untreated U266 cell cultures and after E64d (20 μM) following exposure to UV light. All values are mean ± SE of four experiments in duplicate. Percentage of cells were calculated over a 1000 counted cells.