| BACKGROUND AND OBJECIVENasopharyngeal carcinoma (NPC) is the most common cancer of the head and neck, and it is highly prevalent and endemic in Southern China and Southeast Asia. NPC originates in the epithelial lining of the nasopharynx and is caused by the synergetic effect of many factors, including Epstein-Barr virus (EBV) infection, genetic susceptibility, environmental and dietary factors and so on. Currently, NPC patients are mainly treated by radiation or chemo-radiation, and many early stage patients can be cured. However, there are still a substantial proportion of NPC patients (approximately30%-40%) who fail to achieve long-term disease control and die from local recurrence and metastasis. Recently, molecular target therapeutic reagents, such as the anti-epidermal growth factor receptor (EGFR) antibody cetuximab and the anti-vascular endothelial growth factor (VEGF) antibody bevacizumab, have been employed against NPC. The combination of molecular target reagents and radiation may provide a more effective therapeutic strategy for NPC.The phosphatidylinositol3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is an intracellular signaling pathway that plays a critical role in tumor formation, and these proteins are central to the regulation of tumor cell proliferation, metabolism, cell cycle progression, apoptosis and survival. The possible mechanisms by which this pathway regulates NPC proliferation include mutations in PI3K, phosphatase and tensin homologue deleted from chromosome10(PTEN) and Ras oncogene, as well as EGFR amplification. Previous studies showed that PI3K/Akt/mTOR signaling is frequently activated in many cancers, such as lung, gastric, renal cell and ovarian cancers, and inhibition of this pathway can increase radiosensitivity by inducing tumor cell death. Nasopharyngeal carcinoma cell lines and tissues also expressed phosphorylated Akt (p-Akt). Recently, we reported that Akt expression in NPC cells and biopsies from NPC patients can be increased by irradiation, which is associated with metastasis, suggesting that the PI3K/mTOR pathway might be an ideal therapeutic target in NPC. Preclinical studies have demonstrated that targeting the PI3K/mTOR pathway by a dual PI3K/mTOR inhibitor, NVP-BEZ235, sensitized the antitumor effect of cisplatin in NPC. However, there is little data about the effect of inhibition of PI3K/Akt/mTOR signaling on tumor progression and radiosensitivity in NPC.GSK2126458has been identified as a highly potent, orally bioavailable inhibitor of p110α, p110β, p110γ, p110δ, mTORCl and mTORC2. It can induce a significant reduction in the levels of p-Akt (S473), inhibit growth and induce G1phase arrest in breast cancer. Further, GSK2126458is currently in a phase Ⅰ clinical trial (NCT00972686) in patients with solid tumors. PKI-587is a highly potent dual inhibitor of PI3Kα, PI3Kγ and mTOR. Preclinical studies have demonstrated potent inhibitory effects for PKI-587on a variety of human cancer cell lines, such as breast, glioma, lung, melanoma, colon and liver cancer. Because PKI-587has strong antitumor activity in vitro and in xenograft models, it is currently being evaluated in a phase I clinical trial (NCT00940498).In this study, we investigated the antitumor and radiation-sensitizing effect of dual PI3K/mTOR inhibitors in NPC both in vitro and in vivo. To provide better insight into the effect of dual PI3K/mTOR inhibitors and test whether comparable results would be obtained, we investigated two dual PI3K/mTOR inhibitors, GSK2126458and PKI-587. We propose that the combination of dual PI3K/mTOR inhibitors and radiation may lead to important clinical benefits in NPC and provide the basis for further development of a targeted therapeutic strategy for NPC.MATERIALS AND METHODS1. Cell proliferation assayCell proliferation measured using the3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) dye reduction method. In brief, tumor cells (2×103/100ul/well) were plated into each well of96-well plates in RMPI-1640with10%FBS and incubated for24h. Several concentrations of GSK2126458or PKI-587and/or culture medium were added to each well, and the cells were incubated for a further72h. Next,50μl of MTT (2mg/ml; Sigma) were added to each well, and cells were incubated for2h at37℃. The media containing MTT solution were removed, and the dark blue crystals were dissolved by adding100μl DMSO. The absorbance was measured with a microplate reader at test and reference wavelengths of550and630nm, respectively. The percentage growth was determined relative to untreated controls. Each experiment was performed at least three times, each with triplicate samples.2. Antibodies and western blottingCells were lysed in cell lysis buffer with phosphatase and proteinase inhibitor cocktails (Cell Signaling Technology). Protein concentrations were determined by BCA Assay Reagent (KeyGen Biotechnology). Total protein (40μg) was resolved by SDS-PAGE, and the proteins were transferred onto polyvinylidenedifluoride membranes (Bio-Rad). After four washes, the membranes were blocked in5%BSA buffer for1h at room temperature, followed by overnight incubation at4℃with primary antibodies. After three washes, the membranes were incubated for1h at room temperature with secondary antibodies (species-specific horseradish peroxidase-conjugated). Immunoreactive bands were measured with the Immobilon western chemiluminescent HRP substrate (Millipore). Each experiment was performed at least three times independently.3. Wound-healing assayCells were seeded on6-well plates at80%-90%confluence. After24h, cells were wounded by using a0.2-ml pipette tip to scratch the subconfluent cell monolayer. Then, the detached cells were removed by washing with PBS before GSK2126458or PKI-587was added to the plates. Cells were maintained in serum-free medium and allowed to migrate for24h. An inverted microscope was used to photograph the cell migration images at different time points after wounding (0,12and24h), and the speed of migration was acquired by dividing the length of the gap at different time points. Motic Images Advanced3.2software was used to analyze the relative surface distance traveled by the leading edge. Three replicates each of two independent experiments were performed.4. Cell migration and invasion assayCell migration and invasion assays were evaluated in transwell chambers (Corning), and cell invasion was evaluated by adding Matrigel (BD Bioscience) to the chambers. Before the assay, cells were serum-starved for24h. The top and bottom transwell chambers were separated by an8-μm-pore filter that had been coated with collagen (30μg/filter). For the migration assay,1×105tumor cells were plated in the top chamber with0.1%BSA. For the invasion assay, cells were seeded on filters coated with20-50μg/cm2of reconstituted Matrigel basement membranes (BD Biosciences). Culture medium containing GSK2126548or PKI-587was added to the bottom chamber. After incubation for24h at37℃, cotton swabs were used to remove the cells that had not migrated or invaded from the top surface of the filters. The cells that had migrated or invaded to the bottom surface were fixed in100%methanol and stained with0.5%crystal violet. Migration or invasion cells were quantitated by counting cells in six randomly selected fields on each filter under a microscope at x200magnification and graphed as the mean of three independent experiments.5. Clonogenic survival assaysThe four human nasopharyngeal carcinoma cells were seeded onto60-mm dishes at specific cell densities. GSK2126458or PKI-587was added1h before cells exposed to different doses of radiation (0,2,4,6,8Gy) and maintained for another24h. After14days, cells were fixed in100%methanol and stained with0.5%crystal violet. Then, colonies of at least50normal-appearing cells were counted, and the surviving fraction was graphed against the dose of IR. Three independent experiments were performed, each in triplicate. The data were analyzed using single-hit multi-target model. Using GraphPad Prism5.0software (GraphPad, La Jolla, CA), the survival fractions (SF) were fitted to the following single-hit multi-target formula: SF=1-(1-e-D/D0)N.6. γ-H2AX assayDouble-stranded DNA breaks lead to the formation of γ-H2AX. Therefore, we measured residual y-H2AX foci to assess residual DNA damage in irradiated nasopharyngeal carcinoma cells. We used a y-H2AX antibody (Abcam) to visualize dsDNA breaks, as previously described. In brief, cells were plated in chamber slides, incubated for24h and pretreated with either GSK2126458(0.003μM) or PKI-587(0.03μM)1h before radiation (4Gy), and the number of residual foci was determined at24h after irradiation. Cells were fixed in4%paraformaldehyde and incubated with a primary antibody against y-H2AX. Then, the primary antibody was washed off, and a secondary antibody conjugated to fluorescein isothiocyanate (FITC) was applied to the slides. DNA damage was visualized with a fluorescence microscope (Olympus). For each group, the y-H2AX foci were counted in at least50cells.7. Cell cycle and apoptosisCells were plated in6-well plates and treated with GSK2126458or PKI-587for1h before irradiation with a single dose of4Gy and were harvested at24h after irradiation. DNA content was analyzed by propidium iodide (PI) staining and flow cytometry. After24h of radiation, cells were collected, gently washed with cold PBS containing2%fetal bovine serum, fixed in70%cold ethanol and stored at-20℃overnight. Then, cells were pelleted, washed and stained with P1/ribonuclease staining buffer (BD Biosciences) for15min at room temperature. Apoptosis was measured with the annexin V-FITC apoptosis detection kit (BD Biosciences) according to the manufacturer’s protocols. Analysis was performed on the FACS Calibur using Cell Quest software (Becton Dickinson). All experiments were performed at least three times.8. Xenograft studiesAll animal experiments were carried out in strict accordance with the principles and procedures approved by the Committee on the Ethics of Animal Experiments of Southern Medical University. Suspensions of5×106/0.2ml5-8F cells were injected subcutaneously into the right hindlimbs of5-to7-week-old female BALB/c-nu/nu nude mice. When tumor volumes reached200mm3, mice were randomly assigned to control and treated groups (5mice per group). The treated groups received300μg/kg GSK2126458,25mg/kg PKI-587, IR,300μg/kg GSK2126458combined with IR, or25mg/kg PKI-587combined with IR. GSK2126458was administered by intragastric administration once daily for five consecutive days each week. PKI-587was administered by intravenous injection via tail vein once per five days. Mice in the IR groups were irradiated with2Gy every other day for four treatments with a linear accelerator (Varian2300EX, USA)(Precision X-ray) with a dose rate of500cGy/min, with copper shielding. The source-to-target distance was100cm. In the combination groups, GSK2126458or PKI-587was administered2h before IR exposure. Tumor sizes were calculated every two days using the formula (lengthxwidth2)/2. The mice were killed in day12after treatments since the tumor volumes were beyond3000mm3. Tumor tissues were collected for immunohistochemistry (IHC) and TUNEL assays. All animal experiments complied with the guidelines for the Institute for Experimental Animals, Southern Medical University, Guangzhou, China.9. Immunohistochemistry and TUNEL assaysImmunostaining was performed using tissues (5mm thick) harvested from the5-8F xenografts. Tissue sections were fixed overnight in4%formaldehyde and then embedded in paraffin for IHC. After deparaffinization and hydration, paraffin-embedded sections were pretreated for20minutes with10mM sodium citrate buffer in a microwave for antigen retrieval. Next, the tissue sections were incubated overnight at4℃with primary antibodies. The sections were then incubated with HRP-conjugated secondary antibodies (SP-9000; Zhongshan Gold Bridge, Beijing, China) for1h. Finally, slides were counterstained with hematoxylin and eosin and observed and photographed with a microscope-mounted camera system. At least three random fields were examined in each section.The TUNEL staining was performed using TdT-mediated TUNEL method, performed according to manufacturer’s instructions of Apoptosis Detection System (Promega, Madison, WI).10. Statistical analysisAll data are expressed as the mean±standard deviations (SD) of experiments repeated at least three times. Significant differences between the means were measured by two-tailed unpaired student’s t-test or one-way ANOVA. For measurement of synergistic antitumor effect in vivo, factorial analysis was used. P<0.05was considered statistically significant.RESULTS:1. GSK2126458and PKI-587inhibit NPC proliferation by blocking PI3K/mTOR signalingWestern blotting showed strong expression of phosphorylated mTOR, Akt, S6, and4EBP1in NPC cells, while only weak expression of these proteins was detected in NP69, indicating that a high level of activation of the PI3K/mTOR pathway is characteristic of NPC cells. Next, we evaluated the effect of GSK2126458and PKI-587on the proliferation of NPC cells in vitro with the MTT assay. GSK2126458and PKI-587decreased the proliferation of four NPC cell lines at low nanomolar concentrations and in a dose-dependent manner. All four NPC cell lines were sensitive to GSK2126458and PKI-587, especially CNE-2and5-8F. Furthermore, we determined whether GSK2126458and PKI-587inhibited proliferation of NPC cell lines by modulating the PI3K/Akt signaling pathways. GSK2126458and PKI-587inhibited phosphorylation of mTOR, Akt, S6and4EBP1, leading to increased cleaved-PARP in NPC cell lines in a dose-and time-dependent manner. These results demonstrated that PI3K/mTOR inhibitors, both GSK2126458and PKI-587, effectively inhibited proliferation of NPC cells by blocking the PI3K/mTOR/S6/4EBP1signaling pathway.2. GSK2126458and PKI-587inhibit migration and invasion of NPC cells We performed wound healing and migration assays with GSK2126458and PKI-587, these compounds significantly decreased the speed of wound healing. Moreover, we found that, in migration assays, the number of cells migrating through the transwell chamber polycarbonate membrane in the GSK2126458and PKI-587groups was markedly less than that in the control group. Furthermore, treatment with GSK2126458or PKI-587also substantially impaired ability of cells to invade the filters. Further investigation indicated that for epithelial-mesenchymal transition (EMT) markers, the level of E-cadherin was increased and the level of Vimentin was reduced in CNE-2and5-8F cells24h after treatment with GSK2126458or PKI-587. Collectively, these results suggest that GSK2126458and PKI-587inhibited the migration and invasion ability of NPC cells which may associate with reduced EMT phenotypic expression.3. Targeting the PI3K/mTOR pathway with GSK2126458or PKI-587increases the radiosensitivity of NPC cells.We exposed NPC cells to increasing doses of IR (2-8Gy) in the presence of0.003μM GSK2126458or0.03μM PKI-587, which was the lowest dose that efficiently inhibited colony formation. Our result showed, GSK2126458and PKI-587groups developed significantly fewer colonies than control groups. More importantly, the combination of IR with either of these two compounds further reduced clonogenic survival in all NPC cell. The SER10(sensitizer enhancement ratio at10%survival) in GSK2126458or PKI-587-treated cells compared with IR alone were shown as blows: CNE-1:GSK21264581.31, PKI-5871.17; CNE-2:GSK21264581.56, PKI-5871.49;5-8F:GSK21264581.51, PKI-5871.46;6-10B:GSK21264581.42, PKI-5871.35We further performed western blotting to investigate the mechanism of this effect. Our data showed that GSK2126458or PKI-587significantly decreased the activation of PI3K/mTOR signaling proteins, whereas irradiation alone (4Gy) trends to slightly enhanced the phosphorylation of Akt, mTOR, and S6. However, combination of IR with either of these two reagents further inhibited phosphorylation of PI3K and mTOR, causing reduced phosphorylation of S6and4EBP1in NPC cell lines. These data strongly suggest that GSK2126458and PKI-587can increase the radiosensitivity of NPC cell lines by inhibiting the PI3K/mTOR pathway.4. Radiosensitization induced by the dual PI3K/mTOR inhibitors is accompanied by persistence of y-H2AX fociWe investigated the effects of GSK2126458and PKI-587on the DNA damage response by measuring the number of y-H2AX foci after irradiation, which is a well-known marker of DNA double strand breakage and repair. We found that treatment with GSK2126458(0.003μM) or PKI-587(0.03μM) in combination with IR (4Gy) led to a dramatic persistence of y-H2AX foci at24h post-IR administration compared with exposure to IR alone. The median number of y-H2AX foci per cell in the GSK2126458-or PKI-587-treated groups was significantly greater than that in the IR-alone group of NPC cells, indicating that radiosensitization of these reagents is accompanied by the persistence of y-H2AX foci.5. The combination of IR and dual PI3K/mTOR inhibitors induces cell cycle arrest and apoptosis in NPC cells.To investigate whether these PI3K/mTOR inhibitors radiosensitized NPC cells via redistribution of cell cycle, we performed cell cycle assay on NPC cells exposed to IR and/or the inhibitors. Treatment with GSK2126458or PKI-587alone led to an increased percentage of cells in G1phase, indicating a Gl block, whereas IR alone led to a G2/M block. Strikingly, the combination of IR with GSK2126458or PKI-587induced a further arrest in the G2/M phase, which highlights the potential radiosensitizing capability of these reagents. Further investigation indicated that the level of cyclin D1, which is a vital protein required for the Gl/S transition, was decreased24h after treatment with GSK2126458or PKI-587, which supports the observations on cell cycle changes. Meanwhile the expression of p21and p27, both of which are associated with suppressing cell cycle progression, were increased24h after IR. The combination of IR with GSK2126458or PKI-587further increased the level of p21and p27, accompanied with dramatic decrease of cyclin D1.To investigate the effect of GSK2126458and PKI-587in combination with IR on apoptosis of NPC cells, we performed an apoptosis assay by flow cytometry after24h of treatment. Treatment withGSK2126458or PKI-587alone was slightly induced apoptosis in5-8F cells when compared with untreated cells (CNE-2:PGSK=0.002, PPKI=0.030;5-8F:PGSK=0.007, PPKI=0.030). Strikingly, the combination of either GSK2126458or PKI-587with IR induced apoptosis was much more than IR alone. These data indicated that GSK2126458-or PKI-587-mediated radiosensitization is associated with increased cell apoptosis.Taken together, these results suggest that the radiosensitization effects of the dual PI3K/mTOR inhibitors GSK1216458and PKI-587were primarily due to inhibition of DNA repair or augmentation of damage by cycle arrest and induction of apoptosis.6. Dual inhibition of PI3K/mTOR leads to a remarkable radiosensitization in vivo.To assess whether the combination of dual PI3K/mTOR inhibitors and IR could exert a synergistic antitumor effect on NPC cell lines in vivo, we used the5-8F xenografted model, which is a well-established model of NPC using one of the NPC cell lines from our in vitro experiments. We found that GSK2126458or PKI-587alone had a modest antitumor activity that was not statistically significant and that the IR showed better inhibition of tumor progression. In contrast, the combination of IR with GSK2126458or PKI-587showed much stronger suppression of tumor growth than IR alone. Factorial analysis revealed an interaction between IR with GSK2126458(P<0.05) or PKI-587(P<0.05), indicating that the combination of GSK2126458or PKI-587with IR has a synergistic antitumor effect.To confirm that PI3K/mTOR inhibitor inhibits PI3K/Akt signaling in vivo, we assessed expression of phosphorylated Akt, mTOR and4EBP1in the xenograft tumors. Pathologic examination of sections stained with H&E revealed no significant morphologic abnormalities in any group. However, GSK2126458or PKI-587inhibited phosphorylated Akt, mTOR and4EBP1. Consistent with in vitro findings, we observed dramatically lower levels of phosphorylated mTOR, Akt and4EBP1in tumors from the combination groups. Interestingly, IR alone slightly reduced the phosphorylation of these proteins in vivo. Western blotting revealed that high levels of phosphorylated Akt and4EBP1were detected in control tumors and IR-treated tumors, but lower levels were found in tumors treated with either GSK2126458or PKI-587. The lowest levels were detected in both groups of combination-treated tumors, although the inhibition by PKI-587alone seems weaker than inhibition by GSK2126458alone. These data showed the efficacy of GSK2126458and PKI-587as dual PI3K/mTOR inhibitors in vivo.Consistent with our in vitro findings, we detected significantly fewer proliferating cells (Ki-67-positive) and more apoptotic cells (TUNEL-positive) in the combination-treated tumors when compared with either treatment alone. Taken together, our in vivo results demonstrated that treatment with GSK2126458or PKI-587alone did not significantly inhibit tumor growth, whereas the combination of dual PI3K/mTOR inhibition and IR markedly suppressed tumor progression. This synergistic antitumor effect is associated with PI3K/Akt pathway inhibition and apoptosis.CONCLUSIONS1. Dual PI3K/mTOR inhibitor, GSK2126458and PKI-587could block PI3K/mTOR signaling and inhibit NPC proliferation, migration and invasion.2. PI3K/mTOR inhibition sensitized NPC cells to irradiation by inducing apoptosis, DNA damage and cell-cycle arrest.3. In vivo, the dual PI3K/mTOR inhibitor and irradiation interact synergistically against NPC. |
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