Bortezomib-mediated downregulation of S-phase kinase protein-2 (SKP2) causes apoptotic cell death in chronic myelogenous leukemia cells

Bortezomib (Velcade^®) and antibodies against Bax, tubulin, cytochrome c, GAPDH, caspase-3, caspase-9, and PARP were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibodies against BID and cleaved caspase-3 were purchased from Cell Signaling Technologies (Beverly, MA, USA). XIAP, cIAP1, and caspase-8 antibodies were purchased from R and D (USA). BD Cytofix/Cytoperm plus fixation and permeabilization solution kit with BD GolgiPlug, propidium iodide (PI) staining solution, annexin V binding buffer, mitochondrial membrane potential detection (JC-1) kit, stain buffer (FBS), annexin V-FITC antibody, H2AX (pS139)-Alexa Fluor 647 antibody, rabbit anti-active caspase-3- Bv605 antibody and PARP cleaved form-AF700 antibody were obtained from BD Biosciences (NJ, USA). Apoptotic DNA-ladder kit was obtained from Roche (Penzberg, Germany).

K562, AR230, and LAMA-84 CML cells were grown in RPMI 1640 medium supplemented with 10 % (v/v) fetal bovine serum, 100 U/ml penicillin and 100 U/ml streptomycin at 37 °C in humidified atmosphere containing 5 % CO₂.

1 × 10⁴ cells were treated with the indicated doses of bortezomib in a final volume of 0.1 ml for 24 h. The ability of bortezomib to suppress cell growth was determined by MTT cell proliferation assays, as previously described [29]. Replicates of three wells for each dosage including vehicle control were analyzed for each experiment.

To determine whether bortezomib can induce cell cycle arrest in CML cells, K562, and AR230 cells were exposed to various doses of bortezomib for 24 h. Cells were washed, fixed with 70 % ethanol, and incubated for 30 min at 37 °C with 0.1 % RNase A in phosphate-buffer saline (PBS). Cells were washed again, resuspended, and stained with 25 μg/ml propidium iodide (PI) for 30 in PBS min at room temperature. Cell distribution across the cell cycle was analyzed by flow cytometry using BD LSRFortessa analyzer (BD Biosciences) as described previously [30].

K562 and AR230 cells were incubated with the indicated concentrations of bortezomib. The cells were collected by centrifugation, washed with PBS and stained with fluorescein-conjugated annexin V and propidium iodide (BD Biosciences) and the percentage of cells undergoing apoptosis was measured by flow cytometry as previously described [31].

SKP2 siRNA and scrambled control siRNA were obtained from Qiagen. K562 and AR230 cells were transfected using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. Six hours post-transfection, the lipid and siRNA complex were removed, and fresh RPMI medium with serum was added. After 48 h of incubation, cells were lysed and immunoblotted with antibodies against SKP2, p27Kip1, and GAPDH as a loading control.

CML cells were treated with bortezomib for various time periods as described in the figure legends and lysed as described previously [32]. Proteins (25–50 µg) were resolved by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane (Immobilon, Millipore, Billerica, MA). Immunoblotting was performed using various antibodies, the blots were developed and further visualized under a ChemiDoc system (Amersham, Bio-Rad, USA).

K562 cells were treated with various doses of bortezomib for 24 h. After the incubation period, the cells were fixed and permeabilized using BD Cytofix/Cytoperm plus fixation and permeabilization solution kit, as per protocol from the manufacturer. Approximately 0.3 × 10⁶ cells in FBS were stained with 5.0 µl of H2AX (pS139)-Alexa Fluor 647 antibody for 30 min at room temperature. The cells were washed once with FBS (300 g for 5 min) and then resuspended in FBS. The DNA breaks were measured by flow cytometry using BD LSRFortessa analyzer.

10⁵ cells of K562 CML cell line were incubated in the presence of different concentrations of bortezomib (10, 25 and 50 nm) in a final volume of 1 ml per condition for 24 h. The cells were then counted using hemacytometer in duplicates and 6 × 10⁴ cells were washed with 1 × PBS, centrifuged at 1200 rpm for 5 min. The pellet then was resuspended in 150ul of 1× PBS and using the Cytofunnels set we did cytospining of the samples on the microscopic slides at 800 rpm for 5 min. Furthermore, the cells were fixed with 100 % methanol for 30 min at −20 °C followed by permeabilization for 5 min at 4 °C in 0.2 % Triton X-100 solution in PBS. Cells were then washed and nuclei were counterstained with 1 µg/ml DAPI in PBST for 5 min and rinsed 3 times in PBST and once in H₂O to remove salts. Slides were then mounted using Vectashield antifade mounting medium (Vector Laboratories, CA, USA) and covered with cover glasses. Excess of fluid was removed with paper towel and slides were sealed with transparent histofluid and kept overnight at 4 °C. Imaging was performed the next day on Zeiss axio imager microscope using 63×/1.25 oil objectives.

K562 cells were treated with bortezomib for 24 h, briefly stained with JC-1 stain for 15 min in dark at 37 °C as per instructions from the kit manufacturer. The cells were washed with 1× assay buffer, re-suspended using 500 µl of the same buffer. After that mitochondrial membrane potential was measured by flow cytometry using a BD LSRFortessa analyzer (BD Biosciences, USA) [33].

K562 cells were treated with various doses of bortezomib for 24 h, cells were fixed and permeabilized using BD Cytofix/Cytoperm plus fixation and permeabilization solution kit, as per protocol from the manufacturer. Approximately 0.3 × 10⁶ cells in FBS were stained with 5.0 µl each of anti- Active Caspase-3-BV605 and PARP Cleaved Form-AF700 antibodies for 30 min at room temperature. Cells were then washed with FBS and then resuspended using the same buffer. Quantification of the active caspase-3 and cleaved PARP in the cells were analyzed by flow cytometry using BD LSRFortessa analyzer.

To measure the release of cytochrome c from mitochondria, we performed the assay as reported earlier [34]. K562 cells were treated with 10, 25 and 50 nm bortezomib for 24 h, cells were harvested and resuspended in hypotonic buffer (1 mM Tris–HCl, pH 7.4, 0.13 M NaCl, 5 mM KCl, 7.5 mM MgCl₂). Cells were homogenized and centrifuged to obtain the cytosolic as well as mitochondrial fractions. Twenty to twenty-five microgram of protein from cytosolic and mitochondrial fractions of each sample were analyzed by immunoblotting using an anti-cytochrome c and tubulin antibody.

K562, AR230 and LAMA84 (1 × 10⁴) cells were treated with and without bortezomib as described in the figure legends and mixed with 1.0 mL of MethoCult H4034 Optimum (Stem Cell Technologies). Colonies were counted based on morphology after 10 days.

Comparisons between groups were made using the paired Student’s t test. The software GraphPad Prism (version 5.0 for Windows, GraphPad Software Inc., San Diego, CA, http://​www.​graphpad.​com). Values of * p < 0.05 were considered statistically significant.

To assess the effect of bortezomib on cell viability, a panel of human CML cell lines (AR230, LAMA-84, and K562) were treated with increasing concentrations (10, 25 and 50 nm) of bortezomib for 24 h. A dose-dependent decrease in cell proliferation was observed in all the treated cell lines (Fig. 1a). Bortezomib-mediated inhibition of cell viability was also observed in a time-dependent manner (data not shown).

To investigate whether the inhibition of cell viability induced by bortezomib is due to cell cycle arrest or apoptosis K562 and AR230 cells were treated with different doses of bortezomib for 24 h as indicated. An increase in subG0 population was observed in a dose-dependent manner with the cell lines, K562, and AR230 (Fig. 1b). The sub-G0 population of cells was found to increase from 6.48 % in control cells to 19.5, 33.8 and 49.8 % at 10, 25 and 50 nm bortezomib-treated K562 cells respectively. Similar results were obtained in AR230 cells with an increase of sub-G0 population from 6.56 % in control cells to 16.2, 27.6 and 38.4 % in cells treated with 10, 25 and 50 nm of bortezomib respectively. The increase in sub-G0 population was accompanied by decreased G0/G1 and G2/M phases in bortezomib-treated CML cells.

To investigate whether the increased sub-G0 population in response to bortezomib treatment in CML cells was a resultant of induction of apoptosis, K562, and AR230 cells were treated with 10, 25 and 50 nm bortezomib for 24 h and apoptosis was measured by annexin-V-FITC/PI dual staining. As shown in Fig. 1c, Additional file 1: Figure S1a, b, treatment of CML cells with bortezomib resulted in a dose-dependent increase in apoptosis. Furthermore bortezomib treatment resulted in an increase in early (annexin +ive and PI −ive cells) and late (annexin +ive and PI +ive cells) stage apoptotic cell fractions in K562 and AR230 respectively. There was significant bortezomib-mediated apoptosis in K562 cell (18 % at 10 nm, p < 0.05; 40.1 % at 25 nm, p < 0.001, 81.1 % at 50 nm, p < 0.001) and in AR230 cells (13.5 % at 10 nm, p < 0.001; 33.2 % at 25 nm, p < 0.001, 38.4 % at 50 nm, p < 0.001) with respect to the untreated control cells. Bortezomib treatment was found to be more effective towards K562 cells line when compared to AR230 cells with all tested doses (Additional file 1: Figure S1c, d). To further confirm the bortezomib-mediated apoptosis in K562 and AR230 cells, we performed the DNA fragmentation analysis as described in the “Methods” section. An increase in DNA fragmentation was observed upon bortezomib treatment of both cell lines in a dose-dependent manner (Fig. 1d). These results correlated with the quantification of phospho-γ-H2AX (S139) by flow cytometry as shown in Additional file 2: Figure S2a. Bortezomib treatment causes a significant increase in DNA double strand breaks as evident by increase in phosphorylation of H2AX. DNA damage leads to double-stranded breaks that are always followed by the phosphorylation of the histone H2A variant, H2AX. Since phosphorylation of H2AX at Serine 139 is abundant, fast, and directly correlates well with each double-stranded break, it is used as a sensitive marker for DNA damage [35]. Furthermore, we performed DAPI immunofluorescence staining to visualize apoptotic nuclear bodies—a common hallmark of apoptosis. As shown in Additional file 2: Figure 2b, bortezomib treatment of K562 cells causes a dose-dependent malformation of nuclei architecture followed by subsequent formation of apoptotic bodies. The results suggest that the antiproliferative activity of bortezomib in CML cells is via induction of apoptosis.

During growth and division, the cell requires a periodic activation of a family of protein kinases known as several cyclin-dependent kinases (CDK), whose expression levels are highly regulated by ubiquitination and proteasomal degradation [36‐39]. Several studies have reported that the expression of p27Kip1, a CDK inhibitor, is dysregulated by proteasome pathway [40‐42]. The F-box protein, SKP2 is an ubiquitin ligase that plays a vital role in the degradation of p27Kip1 [15, 19, 20]. In accordance with these facts, we intend to check whether bortezomib-mediated apoptosis is due to proteasome inhibition and degradation of SKP2. The proteasome inhibition in K562 and AR230 cells mediated by bortezomib treatment after 24 h was evidenced by the accumulation of polyubiquitinated proteins (Fig. 2a).

Moreover, it was observed that bortezomib treatment resulted in downregulation of SKP2 in both cell lines, with a concomitant up-regulation of p27Kip1 (Fig. 2b). Similar results were obtained using another CML cell line, LAMA-84 (Additional file 3: Figure S3). This antagonistic effect observed for SKP2 and p27Kip1 suggests that bortezomib induced- downregulation of SKP2 forms an important mechanism to induce apoptosis in CML cell lines via up-regulation of p27Kip1. Since the p27Kip1 abundance increased as a result of bortezomib treatment of CML cells, we investigated the effect of bortezomib on the stability of p27Kip1 using cycloheximide chase assay. As indicated in Fig. 2c, bortezomib treatment increased the stability of p27Kip1 in K562 cells. These findings suggest that bortezomib-mediated p27Kip1 up-regulation is a result of p27Kip1 stabilization.

To further confirm the antagonistic effect observed for SKP2 and p27Kip1, we sought to silence the SKP2 expression in K562 cells by specific siRNA against SKP2. We observed that the knockdown of SKP2 resulted in the increased expression of p27Kip1 in CML cells (Fig. 2d). Altogether, the results suggest that the bortezomib-induced apoptosis is mediated by the downregulation of SKP2 and concomitant accumulation of p27Kip1 in CML cells.

Activation of caspase-8 followed by truncation of Bid leads its translocate to the mitochondrial membrane which results in activation of the intrinsic apoptotic pathway. In accordance to that, we sought to determine, whether bortezomib-induced apoptosis involves the mitochondrial-mediated activation of caspases. Our data suggest that K562 cells treated with bortezomib for 24 h showed activation of caspase-8 (Fig. 3a), followed by the truncation of Bid, which led to the conformational change of Bax protein as shown by the increase in activated form of Bax (6A7) in Fig. 3b, c.

The altered conformation of Bax protein has been shown to reduce the mitochondrial membrane potential (MMP), leading to matrix remodeling resulting in cytochrome c release. We, therefore, investigated whether the bortezomib has an effect on the MMP in K562 cells. Cells were treated with increasing concentration of bortezomib and labeled using JC1 dye and MMP was measured by flow cytometry. JC-1 is a lipophilic, cationic dye that can specially enter into mitochondria. In control cells with elevated mitochondrial membrane potential, the dye forms J-aggregate complex with intense red fluorescence, while in apoptotic cells with reduced MMP, it gives a green fluorescence. Figure 3d illustrates a dose-dependent loss of MMPs (p < 0.001 in at all doses) in CML cells as indicated by the increase in JC1 green fluorescence—a marker of apoptotic cells. Similar results were obtained by using Rhodamine 6G, another dye utilized for the detection of mitochondrial membrane potential (data not shown). We then investigated the release of cytochrome c from the mitochondria in bortezomib-treated cells. As is evident in Fig. 3e cytochrome c was found to be released into the cytosol after bortezomib treatment. There was a concomitant decrease in cytochrome c level in the mitochondrial fraction of K562 cells. The results indicate that bortezomib-mediated inhibition of proteasome pathways led to the depolarization of MMP, which in turn led to the increased flux of cytochrome c in the cytosol. We further investigated whether the bortezomib-mediated release of cytochrome c can activate caspase-9, subsequently caspase-3 and eventually PARP cleavage. Figure 4a shows that bortezomib treatment of K562 and AR320 cells resulted in the activation of caspase-9, caspase-3, and cleavage of PARP. Similar results were obtained with LAMA-84 cells (Additional file 4: Figure S4). Furthermore, activation of caspase-3 and PARP cleavage was determined by flow cytometry. The data was in accordance with Western blot analysis showing significant caspase-3 activation and PARP cleavage in bortezomib-treated CML cells (Fig. 4b). In addition, pre-treatment of K562 cells with z-VAD-FMK, a generalized inhibitor of caspases, prevented bortezomib-mediated activation of caspase-3 and apoptosis (Fig. 4c), suggesting the involvement of caspases in bortezomib-mediated apoptosis.

Inhibitors of apoptosis proteins (IAP) have been shown to have direct effects on caspases [43]. Therefore, we investigated whether bortezomib-induced cell death involved modulation of IAP members which determines the cell’s response to the apoptotic signal. Therefore, we sought to determine whether bortezomib has any effect on the expression of IAP members such as XIAP and CIAP-1. As shown in Fig. 4d, bortezomib treatment resulted in down-regulation of XIAP cIAP-1 and survivin. These findings suggest that bortezomib-mediated apoptosis involved these IAP proteins in CML cells.

To assess the anti-leukemic effects induced by bortezomib treatment in a more patho-physiologically relevant approach, we evaluated the effects on leukemic progenitors (CFU-L) using clonogenic assays in methylcellulose. Treatment of K562 and AR230 cells with bortezomib resulted in greater inhibition of CFU-L colony growth of leukemic precursors in both cell lines. The effect was dose-dependent, and a significant decrease was observed at 10 nm treatment with of bortezomib (Fig. 5).