Pyrrolidine dithiocarbamate sensitizes U251 brain glioma cells to temozolomide via downregulation of MGMT and BCL‑XL
- Authors:
- Published online on: August 28, 2017 https://doi.org/10.3892/ol.2017.6849
- Pages: 5135-5144
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Copyright: © Tang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Brain glioma is the most common type of primary tumor in the central nervous system and is one of the leading causes of mortality in patients with cancer (1,2). Currently an integrated therapeutic method of surgery, radiotherapy and chemotherapy has been adopted in clinical high-grade glioma treatment (3). Patients receiving radiotherapy with concomitant and adjuvant temozolomide (TMZ), demonstrated certain advances in progression-free survival (PFS) and 5-year survival; however, the rate of total survival has not improved (4). TMZ is an oral administered alkylating chemotherapeutic drug, capable of crossing the blood-brain barrier and has been widely used to treat refractory anaplastic astrocytoma and newly diagnosed glioblastoma multiforme (5,6). The therapeutic benefits of TMZ treatment primarily depend on its ability to methylate the O-6 positions of guanine residues (7). This methylation induces irreversible damage to the DNA and triggers abnormal activation of the repair system, leading to cycle arrest and cell death (7). However, certain tumor cells are able to repair this type of DNA damage by expression of the protein, O6-alkylguanine DNA alkyltransferase (AGT), encoded in humans by the O-6-methylguanine-DNA methyltransferase (MGMT) gene (8). A previous study revealed that the presence of MGMT protein diminishes the therapeutic efficacy of TMZ in brain tumors; the study also indicated that high MGMT expression predicts poor response to TMZ and little benefit from chemotherapy with TMZ (9). Conversely, in specific cases, suppressed synthesis of MGMT due to methylation of the MGMT gene promoter is considered a good prognostic factor in TMZ treated patients with glioma (10). At the molecular level, several mechanisms, including nuclear factor-κB (NF-κB) (11), tumor protein 53 (12), specificity protein 1 (12), clone of myelocytomatosis viral oncogene in cancer (Myc) (13) and c-Jun N-terminal kinase (JNK) (14) mediated signaling pathways, have been suggested to be involved in the transcription regulation of MGMT. As a consequence, modifying MGMT expression via its transcription factors has been proposed as a means to sensitize tumor cells to TMZ (15).
NF-κB represents a family of ubiquitous transcription factors that modulate the expression levels of genes by binding to specific κB sites (16). The activity of NF-κB is regulated by the NF-κB inhibitory protein (IκB) (17). In the inactive state, IκB binds to and sequesters NF-κB family members in the cytoplasm (17). Following NF-κB signaling pathway activation by various stimuli, including hypoxia, cytokines and chemotherapeutic drugs, IκB is phosphorylated by IκB kinase (IKK) (17); phosphorylated IκB is subjected to ubiquitination and kinase proteasome-mediated degradation, which results in the activation and translocation of NF-κB to the nucleus (17). Excluding its roles in innate immunity and inflammation, the NF-κB signaling pathway was revealed to regulate a number of cellular processes, including cell proliferation, differentiation and apoptosis (16–18). Furthermore, it has been reported that activation of the NF-κB signaling pathway may also contribute to tumor initiation, progression and resistance to radiotherapy or chemotherapy (19,20). High constitutive NF-κB activity has been observed in numerous lymphoid and myeloid tumors (21), in addition to various solid tumors, including pancreatic cancer (22), glioblastoma (23) and breast cancer (24). Certain recent studies demonstrated that hypoxia may activate the NF-κB signaling pathway, induce the epithelial-mesenchymal transition of breast cancer cells (24), cause invasion of pancreatic cancer cells and contribute to gemcitabine mediated resistance (25).
The present study focused on the association between NF-κB activity and glioma cell progression and chemotherapy. The critical roles which NF-κB serves in the progression and chemoresistance of gliomas have been demonstrated by accumulating experimental evidence (26). Similar to MGMT, NF-κB also contributes to the development of glioma resistance to alkylating agents (27). Furthermore, certain studies indicated that NF-κB associated chemoresistance was partially mediated by the NF-κB/MGMT signaling pathway, which may be activated by alkylating drugs (28,29). In order to determine the effect of inhibiting NF-κB activity on glioma cell viability and sensitivity to alkylating drugs, in addition to clarifying the underlying molecular mechanisms, the present study compared the proliferation inhibiting, cell apoptosis inducing and cell cycle arresting effects of TMZ, pyrrolidine dithiocarbamate (PDTC) and TMZ + PDTC combined. This was followed by determining the expression level alterations of MGMT and other associated genes, including B-cell lymphoma extra large (BCL-XL) and survivin. The present study aimed to further current understanding of the effects and the underlying molecular mechanisms of inhibiting NF-κB activity in glioma therapy, and aid the development of a clinical strategy with combined TMZ and NF-κB inhibitors.
Materials and methods
Chemicals and reagents
Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and TRIzol® were obtained from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA); temozolomide and dimethyl sulfoxide (DMSO) were purchased from Sigma Aldrich (Merck Millipore, Darmstadt, Germany); the MTT, cell cycle and apoptosis analysis kit, Annexin V-fluorescein isothiocyanate (FITC) kit, Bradford protein assay kit, radioimmunoprecipitation assay, lysis buffer, phenyl methanesulfonyl fluoride and BeyoECL Plus were all purchased from Beyotime Institute of Biotechnology (Haimen, China). A Prime Script™ RT Master Mix was obtained from Takara Bio, Inc. (Otsu, Japan); a KAPA SYBR Fast qPCR kit was purchased from Kapa Biosystems, Inc. (Wilmington, MA, USA). The primary antibodies used for western blot analysis were mouse anti MGMT, BCL-XL, survivin and β-actin, and the secondary antibodies used were horseradish peroxidase-conjugated goat anti-mouse IgG (H + L), all supplied by Abcam (Cambridge, UK).
Cell lines and cell culture
The U251 human glioblastoma cell line was purchased from the National Institute of Biological Sciences (Beijing, China) and cultured in DMEM (DMEM basic 1X 1199500) supplemented with 10% FBS (10099141) (both from Gibco; Thermo Fisher Scientific, Inc.). Cells were incubated for about 6 months at 37°C in a humidified chamber with 5% CO2.
MTT assays for cell proliferation
Exponentially growing U251 cells were digested and re-plated into 96-well plates (4×103 cells; 100 µl/well; six repeat wells in each column). These plates were randomly divided into four groups: TMZ group, PDTC group, TMZ + PDTC group and control group. Following an incubation of 24 h, the groups were cultured as follows: Medium of the TMZ group was replaced with fresh medium (DMEM basic 1X with 10% FBS) containing 200 µmol/l TMZ; PDTC group was divided into three subgroups and medium of the three subgroups were replaced with fresh medium (DMEM basic 1X with 10% FBS) containing 20, 50 and 80 µmol/l PDTC; TMZ + PDTC group was divided into three groups and medium of the three subgroups were replaced with fresh medium (DMEM basic 1X with 10% FBS) containing 200 µmol/l TMZ and 20 µmol/l PDTC, 200 µmol/l TMZ and 50 µmol/l PDTC or 200 µmol/l TMZ and 80 µmol/l PDTC. DMSO was added into the control group. A column from each group was evaluated every 24 h and 10 µl/well MTT (5 mg/ml) was added to six wells in one column. Following incubation for 4 h, 100 µl/well formazan solution was added and incubated for an additional 4 h. Absorbance was determined at 570 nm, using an Ultra multi-functional microplate reader (Tecan Group, Ltd., Durham, NC, USA). The evaluation was performed for five consecutive days and the cell inhibition rates were calculated as follows: Rate of proliferation inhibition = [mean optical density (OD) value of control cells - mean OD value of treated cells]/mean OD value of control cells. The results were confirmed by ≥3 repetitions.
Flow cytometry detecting cell apoptosis and cell cycle distribution
U251 cells (8×105) were re-plated in 6-well plates (9.6 cm2). Fresh medium (DMEM basic 1X with 10% FBS) was added with the following supplements: 200 µmol/l TMZ into the TMZ group; 20 µmol/l PDTC, 50 µmol/l PDTC and 80 µmol/l PDTC into the three subgroups of the PDTC group, respectively; 200 µmol/l TMZ and 20 µmol/l PDTC, 200 µmol/l TMZ and 50 µmol/l PDTC or 200 µmol/l TMZ and 80 µmol/l PDTC, into the three subgroups of the TMZ + PDTC group, respectively; 200 µmol/l TMZ and 100 µmol/l PDTC into the fourth subgroup of the TMZ + PDTC group (only added in this part of the experiment); and the solvent DMSO was added into control group. Following incubation for 24 h, cells were digested by trypsinization and detached cells in the medium were collected. Cells were harvested and washed with cold PBS, 5×105 cells were collected in 1.5 ml microtubes (MCT-150-C; Axygen, Union City, CA, USA). Subsequently, cells were gently resuspended in 100 µl 1X binding buffer with 5 µl Annexin V-FITC and 5 µl propidium iodide (PI) staining solution. Following incubation for 15 min in the dark at 25°C, cells were mixed gently with 400 µl 1X binding buffer and filtered with a 0.45 µm filter unit Millex-HV (Merck Millipore), prior to flow cytometry analysis (FACSCalibur™; BD Biosciences, San Jose, CA, USA). Subsequent analyses of flow cytometry data were performed using CellQuest Pro software (version 5.1; BD Biosciences). For cell cycle distribution evaluation, cells (groups with 100 µmol/l PDTC were removed) were collected and resuspended with cold ethanol (70%) and fixed in ethanol for 12 h at 4°C, prior to being harvested. Following washing with cold PBS twice, cells were mixed gently with 500 µl PI and incubated for 30 min in the dark at 35°C. Cell cycle distribution was detected using flow cytometry.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis
Cells (5×106 cells initially seeded) were cultured in 6-well plates and the following treatment was administered for 48 h at 37°C, in a humidified chamber containing 5% CO2: 80 µmol/l PDTC to the PDTC group, 200 µmol/l TMZ to the TMZ group, 200 µmol/l TMZ and 50 µmol/l PDTC to the TMZ + PDTC group, and DMSO to the control groups. Total RNA was extracted using TRIzol® reagent, according to the manufacturer's instructions. First-strand cDNA was reverse transcribed from 1 mg total RNA using the Prime Script™ RT Master Mix, and target gene mRNA was amplified by the KAPA SYBR Fast qPCR kit, using a Bio-Rad CFX96™ real-time system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The reaction system (15 µl), PCR conditions and gene specific primers are presented in Tables I and II.
Western blot analysis
Cells were cultured in 6-cm dishes, following grouping and treatment as in RT-qPCR analysis; the cell lysates were harvested and protein level was evaluated using Bradford Protein Assay kit (P0006C; Beyotime Institute of Biotechnology). Equal amounts of total protein (80–200 µg) were separated using 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. Subsequent to blocking with 5% fat-free milk and 0.1% Tween-20 in PBS-T for 1 h at room temperature, the membranes were incubated with a dilution of anti-MGMT (mouse monoclonal MT3.1 to MGMT; ab39253; 1:500), anti-BCL-XL (mouse monoclonal MT3.1 to MGMT; ab39253; 1:500), anti-survivin (mouse monoclonal 60.11 to survivin; ab93274; 1:800) and anti-β-actin (anti-β-actin antibody mAbcam 8226, ab8226; 1:600) (all from Abcam) primary antibodies. Horseradish peroxidase-conjugated anti-mouse secondary antibodies (goat anti-mouse IgG H&L horseradish peroxidase pre-adsorbed; ab97040; 1:1200; Abcam) were used, and bound antibodies were detected using the BeyoECL system (P0018; Beyotime Institute of Biotechnology).
Statistical analysis
Data are presented as the mean ± standard deviation. All statistical analyses were performed using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA). The Student's t-test (one sample or independent-samples t-test) was used to analyze the difference between the means of the treatment group and the control group. One-way analysis of variance (ANOVA) was used to analyze the significance among ≥3 groups and Fisher's least significant difference method for multiple comparisons was used when the probability for ANOVA was statistically significant. Methods of nonparametric statistical analysis including the Mann-Whitney U Test and Kruskal-Wallis ANOVA, were used when the variances did not pass the Levene test for normality or homogeneity. P<0.05 was considered to indicate a statistically significant difference.
Results
Combining TMZ and PDTC induces the highest proliferation inhibition rate
TMZ, PDTC and TMZ + PDTC treatments all significantly suppressed the proliferation of U251 cells (P<0.05). Cell OD values decreased with increasing PDTC concentrations (P<0.05; Fig. 1A). The TMZ and PDTC treatment combination exhibited a greater significant inhibiting effect and lower OD values compared with the corresponding PDTC group (P<0.05; TMZ + PDTC vs. PDTC). However, TMZ + 80 µmol/l PDTC did not induce lower OD values compared with TMZ + 50 µmol/l PDTC (P<0.05; Fig. 1A). The proliferation inhibition rates in the TMZ + PDTC 50 µmol/l PDTC group (61.56% at 72 h) were higher, compared with in the TMZ group (29.88% at 72 h) and 80 µmol/l PDTC group (44.02% at 72 h; P<0.05; Fig. 1B).
Combination of TMZ and PDTC induces the most significant cell apoptosis
Following an incubation of 24 h, TMZ, PDTC and TMZ + PDTC markedly induced cell apoptosis compared with the control (Fig. 2). TMZ + PDTC induced higher apoptosis rates as compared with TMZ and the corresponding concentrations of PDTC (Table III). The rate of cell apoptosis increased with increasing PDTC concentrations (P<0.05; Table III). However, when treated with 100 µmol/l PDTC or TMZ + ≥50 µmol/l PDTC, cell apoptosis rates reached >93%, and no significant difference had been identified among these groups (P<0.05; Table III).
PDTC enhances the cell cycle arresting effect of TMZ treatment
The combination of TMZ and PDTC led to a significant G0/G1 cell cycle arresting effect in U251 cells (Fig. 3). The percentage of cells in the G0/G1 stage in TMZ + PDTC groups was markedly higher than in the TMZ group (42.4±1.39%) and the corresponding PDTC groups (P<0.05), whereas the percentage of cells in the G2 stage in TMZ + PDTC groups were markedly lower compared with that in the TMZ group (25.1±1.14%) and the corresponding PDTC groups (P<0.05; Table IV). A higher concentration of PDTC induced a higher proportion of cells in the G0/G1 stage; however, TMZ + 80 µmol/l PDTC induced no more significant G0/G1 arrest than TMZ + 50 µmol/l PDTC (P<0.05; Table IV).
PDTC suppresses the expression of MGMT, BCL-XL and survivin
Following incubation with PDTC for 48 h, the mRNA levels of MGMT, BCL-XL and survivin were significantly reduced (P<0.05; Fig. 4A-C). The protein levels of MGMT, BCL-XL and survivin were also reduced following PDTC treatment (Fig. 4D).
PDTC counteracts MGMT and BCL-XL upregulation induced by TMZ
MGMT, BCL-XL and survivin expression levels increased significantly, subsequent to TMZ treatment for 24 h (P<0.05; Fig. 5). However, this upregulation was abrogated by simultaneous treatment with PDTC. MGMT and BCL-XL expression levels in the PDTC + TMZ group were markedly lower compared with that in the TMZ group, whereas survivin expression was not altered significantly (P<0.05; Fig. 5).
Discussion
NF-κB serves a number of critical roles in the development, invasion, recurrence and chemoresistance of malignant brain glioma (30). Abnormal constitutive activation of the NF-κB signaling pathway may be identified in a large number of clinical glioma specimens (31). Wang et al (32) revealed that >90% of the 259 human diffuse gliomas included in the study exhibited high activation of NF-κB, and the extent of activation represented by the protein expression level of the p65 subunit was positively associated with glioma grade and malignancy. Highly activated NF-κB may lead to increased expression levels of matrix metalloproteinases (MMPs) (33) and vascular endothelial growth factor (VEGF), which facilitates microvascular invasion and distal metastases of glioblastoma stem cells (GSCs) (34). In conjunction with the high level of constitutive activity, it has previously been demonstrated that numerous alkylating agents may activate NF-κB in glioma cells (35). Contributing to the anti-apoptotic effect of the NF-κB signaling pathway and blocking constitutive or stimulated NF-κB activation may be a potential approach to suppressing glioma cell viability and enhance the efficacy of alkylating drugs (36).
Furthermore, the inhibition of NF-κB by genetic or chemical inhibitors induces apoptosis of various glioma cells and restores the apoptotic response following treatment with ionizing radiation or chemotherapeutic agents, thus reversing NF-κB linked radioresistance or chemoresistance in a number of models (37). For example, when NF-κB function was profoundly suppressed in U87 and U251 glioma cells via the overexpression of non-degradable IκB, chemical agents including carmustine (BCNU), carboplatin, SN38 glucuronide and tumor necrosis factor-α, induced significant proliferation inhibition and apoptosis in these refractory cell lines (29). SN50, a specific NF-κB inhibitor, may induce differentiation and reduce malignant characters (including neuro-sphere formation, motility, invasion and tumor initiation in vivo) of GSCs. The GSCs sensitivity to TMZ and radiotherapy was markedly enhanced by a low dose of SN50 (38). In the present study, another specific NF-κB inhibitor, PDTC, was used: PDTC is membrane permeable and an antioxidant (39). PDTC may reduce the phosphorylation and degradation of IκB and subsequently block NF-κB activation and nuclear translocation (40). In conjunction with previous studies (28,41,42), the results of the MTT assay and flow cytometry conducted in the present study demonstrated that a low concentration of PDTC may markedly inhibit proliferation and induce apoptosis in U251 cells. Furthermore, the combined treatment of TMZ and PDTC led to a significant increase in proliferation-inhibition rate, apoptosis rate and a more significant cell cycle arrest, as compared with TMZ or PDTC treatment alone (Figs. 1–3). These results indicated a synergistic effect between TMZ and NF-κB inhibitors; a comprehensive understanding of the molecular mechanisms underlying this synergistic action may facilitate the exploration of more efficacious NF-κB inhibitors and a practical combination strategy of NF-κB inhibitors and alkylating agents.
As previously described, MGMT is a key factor of chemoresistance in gliomas (10) and inhibiting the NF-κB signaling pathway may markedly enhance TMZ chemotherapeutic efficacy in U251 cells (37). The results of current study suggest that NF-κB inhibitors mediate their killing effects by inhibiting a potential NF-κB/MGMT signaling pathway. In order to further support this suggestion, evidence is required which indicates that an NF-κB/MGMT signaling pathway exists in glioma cells and that NF-κB inhibitors, including PDTC, significantly reduce MGMT expression in glioma cell lines following treatment with alkylating agents. A number of studies have previously demonstrated the existence of an NF-κB/MGMT signaling pathway in glioma cells (15,43,44). Two putative NF-κB binding sites within the MGMT promoter region have been discovered by Lavon et al (28), demonstrated a specific and direct interaction of NF-κB at each of these sites. They further revealed that forced expression of the NF-κB subunit p65 in HEK293 cells and glioma cells induced an increase in MGMT expression levels, whereas the addition of the NF-κB inhibitor IκB completely abrogated this induction (28). MGMT expression was revealed to be attenuated by fluoxetine via disrupting the NF-κB signaling pathway and consequently sensitized 98 G, SF767 and U251 glioma cells to TMZ treatment (44). Similarly, the present study identified markedly lower MGMT expression levels in PDTC-treated U251 cells (Fig. 4). As aforementioned, alkylating agents may activate the NF-κB signaling pathway, and NF-κB serves an important role in MGMT regulation. Also, it has previously been revealed that alkylating drugs stimulate MGMT expression in glioma cells (45). Therefore, an NF-κB/MGMT signaling pathway may be activated by alkylating drugs, in addition to stimulating MGMT expression. Other anti-apoptotic mechanisms may also serve major roles in NF-κB-mediated chemoresistance to alkylating agents (31). In order to confirm these suggestions, the present study treated U251 cells with 100 µmol/l TMZ. As a notable result, significant increases in MGMT expression levels and in two additional NF-κB downstream genes, BCL-XL and survivin, were demonstrated.
Additional evidence derives from a previous study by Lavon et al (28), which revealed that the suppression of MGMT activity with O6-benzylguanine eradicates the chemoresistance acquired by glioma cell lines, with ectopic p65 or high constitutive NF-κB activity. Despite the understanding that high MGMT expression in resistant glioma cells is stimulated by constitutive or drug-activated high NF-κB activity (29,32), evidence from previous studies supports the present study's hypothesis of the existence and the contribution of an NF-κB/MGMT signaling pathway towards chemoresistance (28,43,44). However, the questions remain about whether this signaling pathway may be exploited to combat fatal gliomas, and if the efficacy of combined TMZ and PDTC treatment is a result of the downregulation of MGMT. In order to investigate these considerations, the present study compared the expression levels of MGMT in cells treated with TMZ + PDTC and in cells treated with TMZ alone, it was revealed that the expression level of MGMT was significantly reduced by the addition of PDTC (Fig. 5). These results are supported by a number of similar previous studies (28,44). For instance, in glioblastoma initial cells, resveratrol was identified to block MGMT upregulation induced by TMZ treatment via inhibiting NF-κB activation (15). Therefore, downregulation of the NF-κB/MGMT signaling pathway is a feasible strategy in order to overcome TMZ-resistance. For example, BAY 11–7082, a specific IκB kinase (IKK) inhibitor leading to the de-activation of the NF-κB signaling pathway, sensitized selected U251 TMZ-resistant cells (TR/U251) to TMZ treatment, resulting in substantial cell death (37). However, Bay 11–7082 may induce cell death independent from inhibiting the activation of the NF-κB signaling pathway (46), and this is the reason for the selection of PDTC, instead of Bay 11–7082 in the present study.
In addition to the regulation of MGMT expression, the impact of NF-κB activity on its canonical target genes, including BCL-XL, survivin, BCL-2, inhibitor of apoptosis 1/2 (IAP1/IAP2), TNF receptor associated factor 1/2, VEGF, matrix metallopeptidase 9 (MMP9) and cyclin D1, are also worth further study for glioma therapy. BCL-XL, BCL-2, survivin and IAP1/IAP2 are key anti-apoptotic factors contributing to cell survival, and are closely associated with the progression and chemotherapy or radiotherapy resistance of various types of cancer (47–49). BCL-XL upregulation by signal transducer and activator of transcription 3, contributes to mutant Kirsten rat sarcoma-mediated apoptosis resistance in colorectal cancer (49). In a recent study with 117 ovarian epithelial neoplasms, survivin overexpression was identified in the majority of malignant cases and was associated with patient prognosis, indicating a crucial role in the development of epithelial ovarian neoplasms (50). Furthermore, it has previously been revealed that survivin expression may be upregulated by treatment in certain lymphoid or myeloid tumors (48). As a consequence, a large number of novel antagonist and inhibitors of these anti-apoptotic factors have been developed, and a number of them demonstrated good clinical results (47,51). The overexpression of these anti-apoptotic factors may be induced by highly activated upstream NF-κB; therefore, inhibiting NF-κB activity may be a potential method by which to suppress tumors with overexpressed BCL-XL, BCL-2 or survivin (52). In conjunction, the present study identified that the expression of BCL-XL and survivin are significantly inhibited by PDTC treatment in U251 cells (Fig. 4). Furthermore, NF-κB activity and, consequently, cyclin D1, COX-2, BCL-XL and BCL-2 expression levels may be suppressed in vivo in prostate tumors by Apigenin, which has been administrated to transgenic adenocarcinoma mouse prostates (53). Similarly, MGMT, BCL-XL, BCL-2 and survivin serve important roles in NF-κB mediated therapeutic resistance (54). Conversely, combining traditional agents with novel inhibitors of these survival and anti-apoptotic factors may be a promising cancer therapy strategy (55). Downregulated anti-apoptotic factors may be an additional mechanism underlying PDTC-enhanced TMZ efficacy in the present study, particularly considering that NF-κB and its downstream anti-apoptotic factors are often stimulated by radiotherapy and chemotherapy (54). The present study also demonstrated TMZ induced the upregulation of BCL-XL and survivin in U251 cells. Additionally, a lower level of BCL-XL, not survivin, was demonstrated in PDTC + TMZ treated cells, as compared with TMZ only treated cells (Fig. 5). Therefore, a reduction BCL-XL expression may serve a role in PDTC induced U251 cell sensitivity to TMZ, whereas the expression level of survivin may not be altered significantly in TMZ treated cells by PDTC, at a low concentration of ~50 µmol/l.
The results of the present study in regards to survivin were in accordance with the findings of Elhag et al (56), which revealed that the ability of silibinin, a natural plant component, to potentiate the cytotoxic efficacy of TMZ in human LN229, U87 and A172 glioblastoma cells was unrelated to survivin protein levels, whereas silibilin may attenuate metastatic processes by suppressing NF-κB and the downstream gene, MMP9. Additionally, it was revealed that expression levels of cyclin D1 were not significantly altered by PDTC treatment, although the addition of PDTC led to an enhanced G0/G1 phase arresting effect (56).
The present study demonstrated that PDTC, a widely tested inhibitor of NF-κB activation as well as an antioxidant and metal chelator, may inhibit cell proliferation, induce apoptosis and cell cycle arrest and enhance sensitivity to TMZ in U251 glioma cells. It was also revealed that PDTC sensitized U251 cells to TMZ, mainly via blocking the NF-κB and NF-κB/BCL-XL signaling pathways that were activated by TMZ treatment. Currently, a large number of anti-NF-κB drugs have been developed for cancer therapy (57); the results of the present study may provide specific aid in furthering the understanding of the chemotherapeutic effects, and the underlying mechanisms involved in inhibiting NF-κB in glioma cells, thus providing an important theoretical basis for developing NF-κB inhibitors with greater efficacy and improved methods of cancer treatment.
Acknowledgements
The present study was supported by The National Natural Science Foundation of China (grant no. NSFC: 81272783) and The National Key Technology Research and Development Program of the Ministry of Science and Technology of China (grant no. 2014BAI04B02). The authors thank Mrs Xiaohong Yang (The National Institute of Biological Sciences, Beijing, China) for her contributions and Dr Weiliang Jiang (Shanghai Jiaotong University School of Medicine, Shanghai, China) for his comments on the manuscript.
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