Radiosensitivity enhancement by combined treatment of nimotuzumab and celecoxib on nasopharyngeal carcinoma cells.

INTRODUCTION: In this study, the radiation-enhancing effects of combined treatment with nimotuzumab, a humanized EGFR-blocking antibody, and celecoxib, a COX-2 selective inhibitor, in human nasopharyngeal carcinoma (NPC) cells were investigated.
MATERIALS AND METHODS: 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide and clonogenic survival assays were done to evaluate the combined cytotoxic and radiosensitizing effects of nimotuzumab or celecoxib or the combination on CNE1 and CNE2 cells. Western blot analysis was performed to identify the effect of nimotuzumab and/or celecoxib with or without irradiation on the cytoplasmic and nuclear EGFR signaling pathways in CNE2 cells.
RESULTS: Our results demonstrated that concurrent administration of nimotuzumab and celecoxib cooperatively enhanced the cytotoxicity and radiosensitivity of CNE2 cells but not CNE1 cells. The combination of both drugs with or without irradiation also cooperatively inhibited cytoplasmic and nuclear EGFR signaling pathways in CNE2 cells.
CONCLUSION: Our results suggest a promising approach for the treatment of poorly differentiated NPC.

Introduction

Nasopharyngeal carcinoma (NPC) is an endemic malignancy in Asia, especially in Southern China, and most patients are diagnosed with locally advanced disease (LA-NPC). Standard treatment for NPC is radiotherapy for patients in early stage and concurrent chemoradiation for those in advanced stage. However, up to 30% of LA-NPC patients still die of distant metastasis, and 10%–20% develop locoregional relapse.1–3 Moreover, the addition of concurrent chemotherapy results in significantly increased toxicities, which in turn impairs the delivery of radiation, and consequently, the benefit of concurrent chemotherapy on survival may be dimmed.4,5 Hence, novel systemic strategies are needed for the treatment of NPC.

Overexpression of EGFR has been documented in a large body of literature on NPC, and also its involvement in chemo-/radioresistance and poor survival.6,7 Nimotuzumab, a humanized monoclonal antibody against EGFR, has been shown to yield promising clinical outcomes when combined with radiotherapy in patients with LA-NPC.8,9 Nevertheless, no evidence of locoregional control or overall survival benefit was observed in the nimotuzumab-based studies as compared to the conventional regime of concurrent chemoradiation.

In addition to EGFR, COX-2 is another potential target in NPC. Likewise, COX-2 has been found to be frequently overexpressed and plays an important role in carcinogenesis and aggressiveness of NPC.10,11 Celecoxib, the first US FDA-approved selective COX-2 inhibitor, has been shown to have the capacity to radiosensitize tumor cells in vitro.12 Interestingly, it was reported that COX-2 was intimately related with EGFR in tumor cells. Activation of EGFR signaling could enhance the transcriptional activity of COX-2 and increase the production of prostaglandin E2, which subsequently lead to further augmentation of EGFR activity.13,14 Moreover, a linear association was observed between the intensity of expression of COX-2 and EGFR proteins in NPC patients.11

Given the positive feedback loop between EGFR and COX-2 signaling pathways, the concurrent administration of EGFR and COX-2 inhibitors appears an attractive strategy for the treatment of NPC. Therefore, in this study, we assessed the effects of combined nimotuzumab and celecoxib on cytotoxicity and radiosensitivity of NPC cells to determine whether this drug combination may be beneficial for NPC patients.

Materials and methods

Chemicals

Roswell Park Memorial Institute 1640 (RPMI-1640) medium and fetal bovine serum (FBS) were obtained from Hyclone (Logan, UT, USA). Nimotuzumab was purchased from Biotech Pharmaceutical Co. Ltd. (Beijing, People’s Republic of China). Celecoxib was kindly provided by Pfizer Pharmaceuticals LLC (New York, NY, USA) and dissolved in dimethyl sulfoxide to obtain a stock concentration of 5 × 105 μmol/L, stored at −20°C and diluted to the desired concentration in fresh medium immediately before use.

Cell culture and radiation administration

Human NPC cell lines CNE1 and CNE2 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). CNE1 cell line was established from well-differentiated squamous NPC, while CNE2 cell line was established from poorly differentiated NPC. Both cell lines were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FBS, 100 units/mL penicillin and 100 μg/mL streptomycin in 5% CO2 humidified atmosphere at 37°C. Cells were irradiated with 160 kV X-ray at a dose rate of 1.15 Gy/min by a biological research irradiator (Rad Source Technologies, Suwanee, GA, USA).

Cell viability assay

The effects of nimotuzumab and/or celecoxib on cell viability were evaluated by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich, St. Louis, MO, USA) assay. Briefly, cells were seeded in 96-well plates at a density of 3 × 104 cells per 100 μL/well. Six wells were assigned to each experimental treatment. After allowing 24 hours for attachment, cells were treated with various concentrations of nimotuzumab or celecoxib or the combination of both drugs at indicated concentrations for 24, 48 and 72 hours. Thereafter, 20 μL of MTT solution was added to each well and incubated for 4 hours. Absorbance at 570 nm was recorded using a microplate reader. All experiments were repeated three times.

Clonogenic survival assay

Cells were seeded into six-well plates in triplicate. To determine cytotoxicity, cells were exposed to nimotuzumab and/or celecoxib at indicated concentrations for 48 hours. To calculate radiosensitizing effects, cells were preincubated with nimotuzumab or celecoxib or the combination for 24 hours and then exposed to graded doses of X-ray radiation and further incubated for 24 hours. Subsequently, cells were incubated with drug-free medium for 14 days to form visible colonies and then fixed with methanol and stained with 1% crystal violet. The cell colonies were counted under a microscope using the standard definition of a colony as one containing >50 cells. All experiments were performed three times.

Subcellular fractionation and Western blot analysis

Cytoplasmic and nuclear extracts were prepared according to the instructions of the nuclear and cytoplasmic extraction kit (Beyotime Biotechnology, Haimen, People’s Republic of China). Western blotting was performed as described previously.15 Briefly, cell lysate containing equal amount of protein was subjected to 10% SDS-PAGE (8% for EGFR and p-EGFR [Y1068]), and electrically transferred to PVDF membranes (Bio-Rad Laboratories, Hercules, CA, USA). Nonspecific binding was blocked with Tris-buffered saline containing 5% (w/v) skim milk for 2 hours at room temperature. The membranes were then incubated with primary antibodies for p-EGFR (Y1068), EGFR, COX-2 (Cell Signaling Technology, Danvers, MA, USA), p-ERK1/2 (Thr202/Tyr204), p-DNA-PK (Ser2056) (Abcam, Cambridge, UK), p-STAT3 (Y705) (Epitomics, Burlingame, CA, USA), p-AKT (Ser473), PCNA (Abgent, San Diego, CA, USA), α-tubulin and Lamin B1 (Beyotime Biotechnology) and then incubated with horseradish peroxidase-conjugated second antibody. Immunoblotting signals were detected using an enhanced chemiluminescence method. All experiments were carried out three times.

Statistical analysis

Data were recorded as mean ± standard deviation in independent experiments. Analysis was performed by one-way analysis of variance (ANOVA) using SPSS 18.0 software (SPSS Inc., Chicago, IL, USA). Results with a p-value <0.05 were considered statistically significant. Factorial ANOVA was applied to quantitatively evaluate the cooperative cytotoxic and radiosensitizing effects of nimotuzumab and celecoxib. If a statistically significant interaction between the two factors was found, a combination index (CI) of <1 indicated synergistic effect, and a CI of >1 indicated antagonistic effect.

Results

Effect of nimotuzumab and/or celecoxib on cell viability of NPC cells

Firstly, the effects of nimotuzumab or celecoxib on cell viability of each cell line were detected by MTT assay. The results showed that celecoxib (0–200 μmol/L) significantly decreased proliferation of both cell lines in a dose- and time-dependent manner (Figure 1A and B), whereas nimotuzumab (0–200 μg/mL) was not toxic to either cell line, even when the cells were treated at the concentration of 200 μg/mL for 72 hours (Figure 1C and D). Subsequently, the antineoplastic effects of nimotuzumab plus celecoxib were investigated. The concentrations of nimotuzumab and celecoxib selected were 50 μg/mL and 25 μmol/L, respectively. CNE1 and CNE2 cells were incubated with nimotuzumab (50 μg/mL, N50) or celecoxib (25 μmol/L, C25) or both (N50 + C25), and a supra-additive cell viability inhibition was observed in the combinational setting after 48 and 72 hours of incubation (Figure 2A and B).

Figure 1

Effect of nimotuzumab or celecoxib on the cell viability of nasopharyngeal carcinoma cells. (A and B) CNE1 and CNE2 cells were treated with various concentrations of celecoxib for 24, 48 and 72 hours. (C and D) CNE1 and CNE2 cells were treated with various concentrations of nimotuzumab for 24, 48 and 72 hours. Cell viability was evaluated by MTT assay. *p < 0.05 vs 0 μmol/L celecoxib at indicated time points. Δp < 0.05 vs 24-hour incubation at indicated drug concentrations. #p < 0.05 vs 48-hour incubation at indicated drug concentrations.

Abbreviation: MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide.

Figure 2

Antineoplastic effects of nimotuzumab plus celecoxib on nasopharyngeal carcinoma cells. (A and B) CNE1 and CNE2 cells were treated with N50 (nimotuzumab, 50 μg/mL) or C25 (celecoxib, 25 μmol/L) or both for 24, 48 and 72 hours. Cell viability was evaluated by MTT assay. *p < 0.05, C25 + N50 vs control, C25 or N50 group at indicated time points.

Abbreviation: MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide.

Combination of celecoxib and nimotuzumab sensitized CNE2 cells but not CNE1 cells

To further confirm the cytotoxicity of combined nimotuzumab and celecoxib on NPC, CNE1 and CNE2 cells were exposed to N50 or C25 or the combination for 48 hours and then permitted to form colonies in drug-free medium. As shown in Figure 3A–D, the results of factorial ANOVA indicated that N50 or C25 alone showed no significant decrease in the colony formation and surviving fractions in both CNE1 and CNE2 cell lines (p > 0.05), while the combination showed a synergistic effect in CNE2 cell line (CI = 0.80, p = 0.03). On the contrary, N50 plus C25 could not decrease the surviving fraction of CNE1 cell line, and no statistically significant interaction between the two factors was found (p > 0.05).

Figure 3

Combination of nimotuzumab and celecoxib could sensitize CNE2 cells but not CNE1 cells. CNE1 and CNE2 cells were exposed to N50 and/or C25 for 48 hours, and clonogenic survival assay was performed. (A and B) Surviving fraction and colony formation of CNE1 cells. (C and D) Surviving fraction and colony formation of CNE2 cells. *p < 0.05, C25 + N50 vs control, C25 or N50 group.

Radiosensitizing effects of nimotuzumab and/or celecoxib on NPC cells

According to the cell viability assay, 25 μmol/L celecoxib and a clinically relevant dose of 50 μg/mL nimotuzumab were selected.16 To evaluate whether interaction between N50 and C25 is effective at decreasing clonogenic survival at different doses of X-ray irradiation, a dose × N50 × C25-factor repeated measure factorial design was applied. CNE1 and CNE2 cells were exposed to graded doses of X-ray radiation (0, 2, 4, 6 and 8 Gy) with drug-free medium, N50 or C25 or the combination for 48 hours. Radiation was administered 24 hours after the start of drug treatment. The radiosensitizing effects conferred by the two-drug combination treatment are shown in Figure 4A and B. The results demonstrated that N50 or C25 alone showed slight radiosensitizing effect in CNE2 cell line, while the combination of both drugs cooperatively enhanced the radiosensitivity. Clonogenic analysis indicated that treatment with N50 plus C25 significantly decreased clonogenic survival of CNE2 cells in combination with 4, 6 and 8 Gy of X-ray irradiation in a synergistic manner; the CI values were 0.75, 0.83 and 0.92, respectively (p < 0.05). In contrast, no radiosensitivity enhancement was found in CNE1 cell line when treated with either one drug or the combination of both drugs.

Figure 4

Radiosensitizing effects of nimotuzumab and/or celecoxib on nasopharyngeal carcinoma cells. CNE1 and CNE2 cells were preincubated with N50 or C25 or the combination for 24 hours, and then exposed to graded doses of X-ray radiation and further incubated for 24 hours. (A) Clonogenic survival assay of CNE1 cells was performed. (B) Clonogenic survival assay of CNE2 cells was performed. *p < 0.05, C25 + N50 vs C25, N50 group; #p < 0.05, N50 vs control group; Δp < 0.05, C25 vs control group.

Combined nimotuzumab and celecoxib cooperatively inhibited cytoplasmic and nuclear EGFR signaling pathways in CNE2 cell line

To elucidate the molecular mechanisms of the cytotoxicity enhancement by nimotuzumab and celecoxib on CNE2 cell line, the cells were incubated with N50 or C25 or the combination for 48 hours. Cytoplasmic and nuclear fractions were prepared, and protein levels of EGFR and the downstream signaling cascades were determined. Results of these experiments indicated that combined nimotuzumab and celecoxib was significantly more effective at reducing cytoplasmic levels of EGFR, p-EGFR and p-AKT (Figure 5A), as well as nuclear levels of EGFR and PCNA, than either drug alone (Figure 5B).

Figure 5

Combined nimotuzumab and celecoxib cooperatively inhibits cytoplasmic and nuclear EGFR signaling pathways in CNE2 cell line. CNE2 cells were incubated with N50 or C25 or the combination for 48 hours. Cytoplasmic and nuclear extracts were harvested for Western blot analysis. (A) Cytoplasmic levels of EGFR, p-EGFR, p-ERK1/2, p-STAT3, p-AKT and COX-2. (B) Nuclear levels of EGFR and PCNA.

Combined nimotuzumab and celecoxib cooperatively blocked radiation-induced EGFR activation and internalization in CNE2 cell line

To find an explanation for the radiosensitizing effects of nimotuzumab plus celecoxib on CNE2 cell line, we screened for alteration of cytoplasmic and nuclear proteins after irradiation plus N50 or C25 or both. Cells were preincubated with N50 or C25 or the combination for 24 hours and then exposed to 4 Gy of X-ray radiation and further incubated for 2 hours. Thereafter, cytoplasmic and nuclear extracts were harvested for Western blot analysis.

It has been previously shown that radiation treatment of tumor cells could lead to EGFR activation and internalization, and nuclear-localized EGFR could enhance DNA-PK activity and DNA damage repair, which plays a critical role in resistance to radiation.17 In this study, increased cytoplasmic levels of p-EGFR, p-ERK1/2, p-STAT3 and p-AKT (Figure 6A), as well as nuclear levels of EGFR and p-DNA-PK, were also found upon exposure to 4 Gy of X-ray radiation (Figure 6B).

Figure 6

Combined nimotuzumab and celecoxib cooperatively blocks radiation-induced EGFR activation and internalization in CNE2 cell line. CNE2 cells were preincubated with N50 or C25 or the combination for 24 hours, and then exposed to 4 Gy of X-ray radiation and further incubated for 2 hours. Cytoplasmic and nuclear extracts were harvested for Western blot analysis. (A) Cytoplasmic levels of EGFR, p-EGFR, p-ERK1/2, p-STAT3, p-AKT and COX-2. (B) Nuclear levels of EGFR and p-DNA-PK.

Abbreviation: IR, irradiation.

Results of these experiments also indicated that nimotuzumab or celecoxib alone has been shown to decrease cytoplasmic levels of p-EGFR, p-ERK1/2, p-STAT3 and p-AKT, as well as nuclear levels of EGFR and p-DNA-PK, when combined with radiation compared with radiation alone, and the combination of both drugs was significantly more effective than either drug alone in CNE2 cell line.

Discussion

The EGFR has been one of the most extensively researched targets over the last decade for the treatment of NPC. However, the observed benefit from EGFR inhibitors occurs only in a minority of patients, suggesting that a more comprehensive understanding of EGFR biology is needed. Growing number of evidence reveals an intimate cross talk between EGFR signaling and COX-2 activity, both of which contribute to carcinogenesis and development of NPC.11,13,14 As a result, concurrent inhibition of EGFR and COX-2 seems an attractive approach for the treatment of NPC.

In this study, it was demonstrated that combined nimotuzumab and celecoxib cooperatively enhanced the cytotoxicity and radiosensitivity of CNE2 cells but not CNE1 cells, which may be attributed to the intrinsic differences of biological behaviors in these cell lines as poorly differentiated NPC differs greatly from the well-differentiated tumors with respect to epidemiology, etiology, pathology, clinical presentation and response to treatment.18 In other words, poorly differentiated subtype constitutes most cases of patients and is associated with Epstein–Barr virus infection, high invasiveness and high radiation sensitivity. In fact, similar additive or synergistic antineoplastic effects of COX-2 inhibitors and EGFR inhibitors were also observed in many other kinds of cancers, including lung cancer, hepatocellular carcinoma and non-NPC head and neck cancer,19–22 suggesting a promising prospect of this drug combination.

As is well known, EGFR is a transmembrane receptor that contains an extracellular ligand-binding domain, a single membrane-spanning region and an intracellular tyrosine kinase domain. Various ligands, such as EGF and TGF-α, bind to the extracellular domain, resulting in EGFR dimerization and ATP-dependent phosphorylation of specific tyrosine residues in the intracellular domain and, thereby, causing phosphorylated activation of three primary growth-promoting downstream signaling cascades including ERK1/2, AKT and STAT3.23 In addition, activation of the plasma membrane’s EGFR can also result in enhanced expression of COX-2 and increased synthesis of prostaglandins E2, which, in turn, lead to further activation of the cytoplasmic EGFR signaling.13,14 In our study, concurrent administration of nimotuzumab and celecoxib with or without irradiation was shown to be more effective than either drug alone at inhibiting cytoplasmic EGFR signaling pathways in CNE2 cells, which might partly contribute to the increased cytotoxicity and radiosensitivity. The cooperative inhibition of cytoplasmic EGFR signaling was also observed in non-NPC head and neck cancer cells with tyrosine kinase inhibitor erlotinib and celecoxib.24

It is noteworthy that, besides classic cytoplasmic signaling, a new function mode of EGFR is now being unraveled. EGFR has been also detected in the nuclei of cancer cells, which functions in ways distinct from its counterpart bound to the plasma membrane. In the nucleus, EGFR has been shown to act as a co-transcription factor to enhance the transcription of COX-2.25 Nuclear EGFR can also activate and stabilize PCNA and DNA-PK to enhance DNA replication and DNA repair.26,27 Interestingly, it has been pointed out that nuclear EGFR is translocated from plasma membrane EGFR, and its function may be independent of kinase activity.28,29 Previous studies have demonstrated that pretreatment with celecoxib or nimotuzumab alone could enhance the radiosensitivity of cancer cells by blocking radiation-induced nuclear localization of EGFR.30,31 In NPC, it was reported that nuclear EGFR could directly bind to cyclin D1 promoter in an LMP1-dependent manner and ultimately promote cell proliferation and cell cycle progression.32 In this study, we firstly reported that combined nimotuzumab and celecoxib could cooperatively inhibit nuclear levels of EGFR and PCNA in CNE2 cell line. We also found that upon exposure to 4 Gy of X-ray radiation, elevated levels of EGFR and p-DNA-PK were detected in the nucleus, and nimotuzumab or celecoxib alone was shown to block the radiation-induced nuclear localization of EGFR and DNA-PK activity, whereas the combined treatment was significantly more effective than either drug alone in CNE2 cells. Results of these experiments indicated that nuclear EGFR might play a crucial role in the progression and radiation resistance of NPC cells.

Conclusion

Collectively, in this work, we suggest that combined treatment of nimotuzumab and celecoxib could cooperatively enhance the cytotoxicity and radiosensitivity of CNE2 cells by inhibition of not only cytoplasmic EGFR signaling pathways but also nuclear EGFR signaling. Our results suggest a promising approach for the treatment of poorly differentiated NPC. However, further clinical trials are needed to confirm these findings and to evaluate the potential toxicities in NPC patients.