14-3-3 proteins have important roles in several cellular processes such as cell cycle progression, the DNA-damage checkpoint and apoptosis. We have shown previously that depleting 14-3-3η, a 14-3-3 isoform, enhances mitotic cell death, and that combining it with microtubule agents is more effective for anticancer therapeutics. In this study, we investigated whether depleting 14-3-3η can be combined with radiotherapy to enhance its therapeutic efficacy. We found that depleting 14-3-3η resulted in a synergistic radiosensitizing effect when combined with radiotherapy in several glioblastoma cell lines, where its specific expression and correlation of its expression level with malignancy have been reported. The radiosensitizing effect was associated with enhanced mitotic cell death by 14-3-3η depletion but not with mitotic catastrophe, which is one of the major cell death mechanisms observed in response to irradiation of most solid tumors. These results suggest that 14-3-3η may be a therapeutic target to overcome radioresistance in glioblastoma.
14-3-3 proteins are found in all eukaryotic organisms, particularly in the mammalian brain. The 14-3-3 family consists of seven isotypes (β, γ, η, τ, σ, ɛ and ζ), which are highly homologous in mammals.1 These proteins are intimately involved in a wide variety of cellular processes including the cell cycle, the DNA damage checkpoint and programmed cell death. 14-3-3 proteins interact with a variety of partners mainly by binding to the phosphorylated consensus motifs of their target proteins and regulating subcellular localization and enzymatic activity. In particular, the regulatory roles of 14-3-3 proteins during the G1/S and G2/M transitions have been studied extensively, and it is understood that they inhibit premature entry into S phase and mitosis.1, 2, 3
Radiotherapy is a typical approach for cancer treatment, and inherent and acquired resistance of cancer cells is a major hurdle of radiotherapy.4, 5 To overcome radioresistance, surgery, chemotherapy, and other anti-cancer therapeutics have been combined with radiotherapy. As regulation of gene expression is closely related with tumorigenesis and resistance to several anti-cancer therapies, it may be reasonable to target genes responsible for tumorigenesis or radioresistance to enhance cancer cell radiosensitivity.6, 7
Glioblastoma multiforme is the most frequent and malignant primary central nervous system tumor.8 However, little information about glioblastoma multiforme is available, including its cause, genetic factors that regulate its course and prognosis markers, despite decades of research.7, 9 Glioblastoma multiforme treatments include neurosurgery, polychemotherapy and ionizing irradiation. However, the survival rate is very low due to treatment resistance. Thus, more efforts are required to develop effective therapeutics and overcome resistance to current treatments.
We have shown previously that depleting 14-3-3η enhances mitotic cell death when combined with microtubule damaging agents, which is independent of mitotic catastrophe.10 Because mitotic catastrophe is one of the major cell death mechanisms observed in response to irradiation of most solid tumors, we speculated that depleting 14-3-3η could be synergistically combined with radiation for cancer therapy.11 In this study, we investigated whether depleting 14-3-3η sensitizes cancer cells to radiotherapy. We found that depleting 14-3-3η radiosensitized several glioblastoma cell lines, which are relatively resistant to irradiation. The radiosensitizing effect was due to the enhanced mitotic cell death caused by depleting 14-3-3η but not due to mitotic catastrophe. These results suggest that 14-3-3η may be a therapeutic target to overcome radioresistance in glioblastoma.
Materials and methods
Plasmid construction, cell culture and transfection
Short hairpin RNAs (shRNAs) targeting 14-3-3η were described previously.10 HeLa, T98G, U251 and U87MG cells were purchased from the American Type Culture Collection (Manassas, VA, USA). Cell culture and transfection were performed as described previously.10, 12
Gamma irradiation was performed using a BIOBEAM 8000 instrument (Gamma Service Medical GmbH, Leipzig, Germany). Gamma irradiation was delivered at room temperature using a 77.33-TBq 137Cs source at a dose rate of 2.52 Gy min−1.
Flow cytometry analysis
To estimate the sub-G1 fractions, cells were harvested at the indicated time points after radiation, fixed in 70% ethanol and stained with 40 μg ml−1 propidium iodide in the presence of 50 μg ml−1 RNase A. At least 10 000 cells were obtained to analyze DNA content on a Becton Dickinson FACS Aria flow cytometer (Franklin Lakes, NJ, USA) using FACSDiva software. To measure the mitotic index, cells were harvested at the indicated time points after radiation, fixed in 70% ethanol, stained with anti-phospho-MPM2 (Upstate, Temecula, CA, USA) for 1 h and incubated for 1 h with allophycocyanin-labeled secondary antibody. Cells were further stained with 40 μg ml−1 propidium iodide in the presence of 50 μg ml−1 RNase A and analyzed on the flow cytometer.
Western blot analysis
Protein samples were prepared in TNN lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.5% nonidet P-40, 10 mM NaF, 1 mM Na3VO4, 1 mM diothiothreitol, 1 mM phenylmethanesulfonyl floride and protease inhibitors) and subjected to western blot analysis with anti-poly(ADP-ribose) polymerase (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-caspase-3 (Cell Signaling Technology, Danvers, MA, USA), anti-14-3-3η (Serotec, Oxford, UK), anti-γ-H2AX (Phospho S139) (Abcam, Cambridge, MA, USA) and anti-actin (Sigma, St Louis, MO, USA) antibodies.
Cell proliferation assay
Cell proliferation was measured using the CellTiter96 Non-Radioactive Cell Proliferation Assay (MTT) kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions.
Cells were transfected with the indicated shRNAs, and 2 × 103 cells were spread in 60-mm dishes. The cells were irradiated 24 h later and then incubated for 10–14 days to allow colonies to form. Colonies were stained with crystal violet. The number of colonies was counted, and images were taken.
Terminal transferase dUTP nick end labeling (TUNEL) assay immunofluorescence staining
Cells were irradiated at 8 Gy 36 h after transfection. The cells were fixed in 4% paraformaldehyde 24 h later, and the TUNEL assay was performed using an In Situ Cell Death Detection Kit, TMR red according to the manufacturer's instructions (Roche, Indianapolis, IN, USA). After the TUNEL assay, the cells were further stained with anti-phospho-MPM2 antibody (Millipore, Milford, MA, USA), fluorescein-conjugated secondary antibody (Vector Laboratories, Burlingame, CA, USA) and DAPI. Images were acquired and analyzed with ECLIPSE 80i (Nikon, Tokyo, Japan) and Image-Pro Plus (MediaCybernetics, Bethesda, MD, USA).
Depletion of 14-3-3η radiosensitizes HeLa cells
We have shown previously that depleting 14-3-3η results in enhanced mitotic cell death and that 14-3-3η-depleted cells become sensitized to microtubule damage agents.10 The enhanced mitotic cell death by 14-3-3η depletion was not due to mitotic catastrophe, which is a main cell death mechanism during solid tumor radiotherapy. A synergistic effect may be expected when two different cell death mechanisms are combined to kill tumors. Thus, we investigated whether enhanced mitotic cell death by depleting 14-3-3η could be synergistically combined with irradiation. HeLa cells were transfected with control luciferase shRNA (shLuc) or shRNAs targeting 14-3-3η (sh14-3-3η-179 and -616) and then irradiated at several doses. The number of viable cells in each control group that was not irradiated (shLuc- or sh14-3-3 eta-0 Gy) was set to 100%, and relative viability was compared between the groups. As shown in Figure 1a, lower viabilities were observed in the 14-3-3η-depleted groups at all radiation doses tested compared with that of the control group, suggesting that the combination of 14-3-3η depletion and irradiation may have a synergistic effect (radiosensitizing effect). The enhanced cell death due to this combination therapy was further confirmed by enhanced DNA-damage and cell-death markers. The combination therapy resulted in enhanced phospho-γ-H2AX, cleaved poly (ADP-ribose) polymerase, and cleaved caspase 3, as shown by western blot analysis (Figure 1b).
Depletion of 14-3-3η radiosensitizes glioblastoma cell lines
14-3-3σ is known to be induced by DNA damages in a p53-dependent manner.13 Thus, we have investigated whether expression of 14-3-3η is also regulated by DNA damages and also in a p53-dependent manner. HeLa and glioblastoma cell lines (T98G, U251: mutant p53, U87MG: wild-type p53) were treated with doxorubicin or irradiated and then expression of 14-3-3η was analyzed. Unlike 14-3-3σ, expression of 14-3-3η is not changed by doxorubicin- or radiation-induced DNA damages and not differentially regulated between the mutant p53 cell lines (HeLa, T98G and U251) and the wild-type p53 cell line (U87MG) (Figure 2a).
14-3-3η is expressed specifically in astrocytoma tissues and glioblastoma cell lines, and its expression level is well correlated with astrocytoma malignancy grade.14 This result suggests that 14-3-3η is a possible therapeutic target for astrocytoma. Astrocytoma, including glioblastoma, is a main target of radiotherapy but is relatively resistant to radiotherapy. Thus, we investigated whether 14-3-3η depletion could radiosensitize glioblastoma cells. As shown in Hela cells, 14-3-3η was depleted by both shRNAs in glioblastoma cell lines (Figure 2b). U251 cells were relatively resistant to radiotherapy, as only 8–9% of the cells died at a dose of 8 Gy in the control group. However, depleting 14-3-3η induced 33–70% cell death when combined with 8 Gy radiation (Figure 3a). This suggested very effective therapeutic efficacy, considering the radioresistance of the glioblastoma cell line. We further investigated whether the effect of a combination of these two approaches was additive or synergistic. As shown in Figure 3b, depleting 14-3-3η clearly radiosensitized U251 cells compared with that in the control group. Similar results were obtained from the clonogenic assay (Figure 3c). The radiosensitizing effect may not be due to off-target effects because two different shRNAs targeting different regions of the 14-3-3η gene showed similar results. We have used two more glioblastoma cell lines, T98G and U87MG, to confirm the radiosensitizing effect of 14-3-3η depletion. As observed in assays using U251 cells, 14-3-3η depletion induced a radiosensitizing effect in the T98G and U87MG cell lines (Figure 3d). Taken together, these results suggest that combination therapy may be very effective for astrocytoma/glioblastoma, which is relatively resistant to radiotherapy.
Radiosensitizing effect due to 14-3-3η depletion is not dependent on mitotic catastrophe
We investigated the molecular mechanisms of the radiosensitizing effect induced by depleting 14-3-3η. It has been established that mitotic catastrophe is the major cell death mechanism following irradiation for solid tumor radiotherapy.11 To determine if mitotic catastrophe is involved in the radiosensitizing effect due to 14-3-3η depletion, U251 cells were transfected with shLuc or sh14-3-3η RNAs in combination with shRNA targeting BubR1, a mitotic checkpoint regulator, as mitotic catastrophe is dependent on activating the mitotic checkpoint. As shown in Figure 4a, a similar radiosensitizing effect was induced by depleting 14-3-3η in both the mitotic checkpoint-active and -inactivated conditions. Thus, mitotic catastrophe may not be involved in the radiosensitizing effect due to 14-3-3η depletion.
Radiosensitizing effect due to 14-3-3η depletion is correlated with enhanced mitotic cell death
Because depleting 14-3-3η enhanced mitotic cell death alone and in combination with microtubule-damaging agents, we investigated whether the radiosensitizing effect due to depleting 14-3-3η correlated with the extent of mitotic cell death. U251 cells transfected with the indicated shRNAs were irradiated (8 Gy), and mitotic cell death was compared by evaluating the number of TUNEL-positive cells among the mitotic cells (MPM2-positive). As shown in Figure 4b, >60% of the mitotic cells (MPM2-positive cells) in 14-3-3η-depleted groups were TUNEL-positive upon irradiation, suggesting enhanced mitotic cell death by depleting 14-3-3η upon irradiation.
We further investigated the relationship between the radiosensitizing effect and mitotic cell death. U251 cells transfected with the indicated shRNAs were irradiated and then the mitotic population (phospho-MPM2-positive) and cell death (Sub-G1) were analyzed (Figure 5a). Control and 14-3-3η-depleted U251 cells were irradiated, and cell cycle progression was analyzed using MPM2, a mitotic marker, and propidium iodide staining. Both cell types were arrested at the G2/M checkpoint, possibly due to DNA damage from irradiation (Figure 5a, 9 h). Control cells resumed the cell cycle, and the number of mitotic cells increased (Figure 5b). However, the percentage of mitotic cells among the 14-3-3η-depleted cells was lower than that of the control throughout the assay. The sub-G1 fraction of 14-3-3η-depleted cells increased concomitantly, but that of the control cells remained unchanged. These results suggest that enhanced mitotic cell death in the 14-3-3η-depleted cells contributed to enhanced cancer cell death when combined with irradiation. Thus, the 14-3-3η depletion-mediated radiosensitizing effect was accompanied by enhanced DNA damage and mitotic cell death.
Radiotherapy is one of the most popular cancer therapy modalities. However, some cancers are resistant owing to various mechanisms, such as the presence of cancer stem cells, expression of antiapoptotic proteins and increased ability to repair DNA damage.15 Cancer cell resistance is a common problem with current anticancer therapies.5 Several approaches are currently under development to overcome the resistance of cancer cells to radiotherapy. One of these approaches could be a combination of two different anticancer strategies. In this study, we combined irradiation with 14-3-3η depletion, which induced mitotic cell death, and observed that the combination enhanced the anticancer effect of irradiation in glioblastoma cell lines, which are relatively resistant to irradiation.
It is generally believed that synergy can be achieved when two different pathways are involved in the final outcome. The major cell death mechanism in solid tumors induced by irradiation is mitotic catastrophe.11 We showed previously that mitotic cell death in cancer cells induced by depleting 14-3-3η is different from mitotic catastrophe.10 Because the cell death mechanisms by irradiation and 14-3-3η depletion are different, the combination of the two approaches can be synergistic. Consistent with this hypothesis, we showed that depleting 14-3-3η induced radiosensitization in glioblastoma cell lines.
The cell death mechanisms following 14-3-3η depletion are different from mitotic catastrophe and other known mechanisms and have not been fully characterized. Both the control and 14-3-3η-depleted cells were initially arrested at the G2/M checkpoint and then entered mitosis. Although cells have checkpoints to maintain chromosome stability by preventing entry into the S phase or mitosis with unrepaired DNA damage, there are still some limitations in the checkpoints. The G2/M checkpoint arrests cells only when DNA damage includes ⩾10–20 double-strand breaks.16 Furthermore, cancer cells contain some cell cycle regulation defects.17 Thus, irradiated cells enter mitosis with unrepaired DNA damage. Some may die during mitosis by mitotic catastrophe, which is the major cell death mechanism during radiotherapy, whereas others may survive, leave mitosis and enter the next cell cycle by several mechanisms11, 18 However, 14-3-3η-depleted cells may accelerate mitotic cell death due to defects in mitotic progression. Thus, these two cell death mechanisms were synergistically combined and resulted in a radiosensitizing effect in the glioblastoma cell lines. Taken together, 14-3-3η could function as a good therapeutic target by either depleting its expression or interrupting its interaction with cellular binding partners to overcome the radioresistance of glioblastoma.
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This study was supported by a National Research Foundation grant funded by the National R&D Program through the Dongnam Institute of Radiological & Medical Sciences (DIRAMS) funded by the Ministry of Science, ICT and Future Planning (50597-2013). We thank Dr Jung Ki Kim, Min Young Kim, and Min Su Ju for administrative support.
The authors declare no conflict of interest.
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