Glioma is the most aggressive form of primary brain tumor, with dismal patient outcome and no effective therapeutic approaches available. Targeting the ubiquitin-proteasome pathway has recently emerged as a potent rational anticancer strategy. Bortezomib, a specific proteasome inhibitor, has been approved for the treatment of relapsed or refractory multiple myeloma and other hematological malignancies as a single agent or as part of a combination therapy. However, bortezomib alone or in combination showed only minimal effects in the treatment of solid tumors. Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic protein which protects tumor cells against spontaneous and chemotherapy-induced apoptosis. In multiple myeloma, specific downregulation of Mcl-1 induces apoptosis. Furthermore, previous studies demonstrated that proteasome inhibitors induce Mcl-1 accumulation that, in turn, slows down their pro-apoptotic effects, and the cell survival in multiple myeloma is highly dependent on Mcl-1. In the present study, we investigated the role of Mcl-1 downregulation in bortezomib-induced apoptosis in gliomas. We observed that bortezomib triggers caspase-3 and PARP activation, upregulates cytochrome
The proteasome is critical for the maintenance of homeostasis of the majority of intracellular proteins and is considered as a promising target for anticancer therapy. Bortezomib is the first proteasome inhibitor used for the treatment of relapsed or refractory multiple myeloma (
Mcl-1 is a short-lived protein which opposes several apoptotic stimuli. These features distinguish it from other anti-apoptotic Bcl-2 members (
Gliomas are the most common and highly invasive primary brain tumors in humans. Despite some recent advances in surgical, radiation and chemotherapy approaches, the prognosis remains poor (
Bortezomib is successfully used for treating hematological malignancies. Its reported benefits in the treatment of solid tumors, however, are less than encouraging (
In the present study, we examined the effect of Mcl-1 and/or the proteasome inhibitors on glioma cell growth and survival. We aimed to assess the specific role of Mcl-1 in the response to bortezomib.
The glioblastoma cell lines U251 and U87 were purchased from the Chinese Academy of Medical Sciences (CAMS) and were grown as a monolayer in Dulbecco’s modified Eagle’s medium (DMEM) with 10% heat-inactivated fetal calf serum, 100 U/ml penicillin and 100 mg/ml streptomycin and were maintained in a humidified incubator at 37°C and 5% CO2. Bortezomib was obtained from Sigma (St. Louis, MO, USA). Antibodies against caspase-3 (human mAb), PARP (F7 human mAb), cytochrome
U87 and U251 cells were plated on 96-well plates (2,000 cells/well) and treated with bortezomib (100 ng/ml; Sigma) for 24 h. Following the treatment, thiazolyl blue tetrazolium bromide (MTT, 5 mg/ml; Sigma) was added to each well, and the cells were incubated in the dark at 37°C. MTT produces a yellowish solution that is converted to dark blue, water-insoluble MTT formazan by mitochondrial dehydrogenases of the living cells. After 4 h, the medium was aspirated and the dark blue crystals were dissolved in dimethyl sulfoxide (DMSO) (150
Western blot analysis was performed on lysates prepared from U251 and U87 cells treated as indicated. The cells were homogenized in the lysis buffer containing 0.5 mM Tris-Cl (pH 6.8), 2% SDS (w/v), 10% glycerin (w/v) and protease inhibitor cocktails (Sigma-Aldrich, Dublin, Ireland). After determining the protein concentration of the samples using the BCA protein assay (Pierce, Rockford, IL, USA), 20
U251 and U87 cells were plated in 24-well plates (15,000 cells/well) and were treated with bortezomib for 48 h. Following the treatment, the monolayer cells were harvested with trypsin-EDTA and washed with PBS. Cells were then incubated at room temperature in binding buffer (10 mM HEPES, 135 mM NaCl, 5 mM CaCl2) which contained Annexin V-FITC conjugate (1
The sequence of the small interfering RNA (siRNA) used to silence
All data are given as the mean ± SD. N is the number of the samples. Western blot data are expressed relative to the control, assigning a value of 1 to the control group baseline mean. Data were analyzed using Student’s t-test or 2-way ANOVA by SPSS 17.0 software as appropriate. P-value <0.05 was considered as a significant difference between the data sets.
Previous research has shown that proteasome inhibitor bortezomib can cause apoptosis
Since the MTT results showed that bortezomib can reduce the U251 and U87 cell viability, we investigated the effect of bortezomib on apoptosis in glioma cells. We recorded the influence of different bortezomib concentration on the apoptosis by Annexin V and PI double dye staining using flow cytometry. Different concentrations of bortezomib can cause apoptosis of U251 and U87 cells (
Several members of the Bcl-2 family are known to be targets for bortezomib. In order to further determine the effect of bortezomib on the apoptosis in the two glioma cell lines, we checked the expression levels of apoptosis-related proteins by using western blot analysis. Under normal circumstances, caspase-3 can exist in an inactive form. PARP, as a DNA repair enzyme, is also the substrate of caspase. After being cleaved, the activated form of caspase-3 mediates cell apoptosis. Cytochrome
Studies have shown that anti-apoptotic protein Mcl-1 is degraded by the ubiquitin-proteasome pathway. As a proteasome inhibitor, bortezomib can induce apoptosis in glioma cells. Using western blot analysis, we explored the effect of Mcl-1 on the apoptosis induced by bortezomib in two types of glioma cells, U251 and U87. Bortezomib increases the protein expression level of Mcl-1 in both cell lines (
To definitely establish the role of Mcl-1 in the bortezomib-induced apoptosis in glioma cells, we made use of RNA interference (RNAi) technology. We transfected U87 and U251 cells with Mcl-1 siRNA and control siRNA to evaluate the potential effects on the cell viability due to Mcl-1 down-regulation. The MTT assay demonstrated that the combined treatment with bortezomib and Mcl-1 siRNA can significantly inhibit U251 and U87 cell viability. The inhibition of Mcl-1 expression can increase the sensitivity of both cell types to bortezomib (
Based on previous experimental results which demonstrated that Mcl-1 siRNA can increase the inhibition of cell growth, we used siRNA to knock down Mcl-1 expression in order to determine whether Mcl-1 played an essential role in bortezomib-induced apoptosis. Using flow cytometry, we observed that Mcl-1 siRNA can significantly enhance bortezomib-induced apoptosis compared with the control siRNA in U251 and U87 glioma cells (
The ubiquitin-proteasome pathway represents the major pathway for intracellular protein degradation (
Bortezomib has been observed to target many molecules that are associated with tumor progression and treatment resistance, such as nuclear factor-κB (NF-κB), pro-apoptotic and anti-apoptotic Bcl-2 family proteins and p53 (
The pivotal role Mcl-1 plays in protecting cancer cells from apoptosis is well documented (
Collectively, the present study demonstrates the efficient induction of apoptosis by proteasome inhibitor bortezomib in the glioma cell lines and provides novel evidence that a combinational therapeutic treatment regime modulating the Mcl-1 expression levels may be an efficacious approach to sensitize glioma to the apoptosis-inducing effects of bortezomib. Bortezomib can inhibit the expression of Mcl-1 and increase the bortezomib sensitivity in the glioma U251 and U87 cells. The combined use of bortezomib and Mcl-1 inhibitor can effectively inhibit the proliferation of glioma cells. Elucidation of the pathways of cell death induced by Mcl-1 downregulation in combination with bortezomib treatment may help to identify key molecular targets that govern the viability of glioma cells. These targets could be further manipulated to provide antitumor effects and warrant further investigation.
Bortezomib reduces cell viability in a dose-dependent manner. U87 and U251 cells were cultured in DMEM supplemented with 10% FBS in the presence of various doses of bortezomib. Cell viability was measured by an MTT assay at 24 h relative to the control. Values are the mean ± SD; n=6. **P<0.05 compared with the control group.
Bortezomib induces apoptosis in glioma cell lines. (A) The effect of bortezomib on U251 cell apoptosis, as assessed by flow cytometry. (B) The effect of bortezomib on U87 cell apoptosis, as assessed by flow cytometry. The percentage (%) of cells in each fraction was determined by positivity for Annexin V and PI staining at the different doses of bortezomib.
Bortezomib triggers a dose-dependent activation of caspase-3 and PARP and upregulates the cytochrome
Bortezomib upregulates Mcl-1 protein, and Mcl-1 siRNA downregulates Mcl-1 expression. U251 and U87 cells were transfected with Mcl-1 siRNA or control siRNA. (A) The effect of bortezomib on the expressions of Mcl-1 in U251 and U87 cells. (B) The effect of Mcl-1 siRNA on the expressions of Mcl-1 in U251 and U87 cells. Values are the mean ± SD; n=3. **P<0.01 compared with the control group.
Knocking down Mcl-1 expression increases glioma cell sensitivity to the inhibition of cell growth in U251 and U87 cells. U251 and U87 cells were transfected with Mcl-1 siRNA or control siRNA. Two hours after transfection, U251 and U87 cells were treated with bortezomib (10 nM) for 72 h. After treatment, the MTT assays were carried out to assess cell viability. Values are the mean ± SD; n=3. *P<0.05 compared with the control group.
Knockdown of Mcl-1 augments apoptosis induced by bortezomib in U251 and U87 glioma cells. U251 and U87 cells were transfected with Mcl-1 siRNA or control siRNA. Two hours after transfection, U251 and U87 cells were treated with bortezomib (10 nM) for 24 h. The induction of apoptosis was determined by Annexin V and PI staining using flow cytometry. The percentage (%) of cells in each fraction was determined by positivity for Annexin V and PI staining at the different combination of bortezomib and siRNA treatment.
Altered expression of apoptosis-associated proteins in U251 and U87 cells following the treatment with bortezomib and Mcl-1 siRNA. U251 and U87 cells were transfected with Mcl-1 siRNA or control siRNA. Two hours after transfection, U251 and U87 cells were treated with bortezomib (10 nM) for 24 h. Western blot analysis of expression of the apoptosis-associated proteins was carried out on U251 and U87 cells. Tubulin was used as a loading control. (A) The effect of Mcl-1 siRNA on the expression of cleaved caspase-3 in U251 and U87 induced by bortezomib. (B) The effect of Mcl-1 siRNA on the expression of PARP in U251 and U87 induced by bortezomib. (C) The effect of Mcl-1 siRNA on the expression of cytochrome