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Suppression of the notch signaling pathway by γ-secretase inhibitor GSI inhibits human nasopharyngeal carcinoma cell proliferation
Cancer Letters 306 (2011) 76–84
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Cancer Letters journal homepage: www.elsevier.com/locate/canlet
Suppression of the notch signaling pathway by c-secretase inhibitor GSI inhibits human nasopharyngeal carcinoma cell proliferation Shi-Ming Chen a,b, Jun-Ping Liu b, Jun-Xu Zhou a, Chen Chen a, Yu-Qin Deng a, Yan Wang a, Ze-Zhang Tao a,⇑ a b
Department of Otolaryngology-Head and Neck Surgery, Renmin Hospital of Wuhan University, Wuhan 430060, China Molecular Signaling Laboratory, Department of Immunology, Central Clinical School, Monash University, Melbourne, Victoria 3004, Australia
a r t i c l e
i n f o
Article history: Received 18 January 2011 Received in revised form 18 February 2011 Accepted 21 February 2011
Keywords: Notch Nasopharyngeal carcinoma cells GSI Apoptosis AKT
a b s t r a c t Notch signaling has been suggested to be required for many human cancers. However, the role of Notch signaling in human nasopharyngeal carcinoma cells (NPC) remains unknown. Here, we report that Notch-1, Notch-2, Notch-3 and Notch-4 are all detected in NPC cells. Notch inhibitor, GSI, suppresses the levels of Notch-1, Notch-2 and Notch-4, but not Notch3. In addition, GSI inhibits NPC cell proliferation by inducing the cell cycle arrest and apoptosis. Furthermore, GSI inhibits the AKT and MEK signaling, without affecting P38 and JNK1/2. Thus, NPC cells may up-regulate Notch signaling to maintain cell proliferation and targeting the Notch signaling pathway may offer a potential alternative strategy for the treatment of NPC. Ó 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Overexpression of the Notch receptors and/or their ligands has been identified in multiple cancers of the breast [1,2], ovarian [3,4], prostate [5], brain [6] and sarcoma [7–10]. The overexpression of Notch elements in cancers has been related to a poor clinical outcome. For example, overexpression of Notch-1 is associated with the rapid recurrence in breast cancers [11], and increased expression of JAG-1 is associated with a high rate of recurrence in prostate cancers [5]. Consequently, many laboratories have studied the effects of blocking the Notch signaling pathway by targeting the c-secretase protein complex to inhibit the activation of Notch receptors [12]. It has been shown that inhibition of the c-secretase complex prevents the growth of T cell ALL lines leading to apoptosis [13–15]. Notch signaling inhibitors (GSIs) suppress the growth of multiple tumors,
⇑ Corresponding author. Tel.: +86 13907141892; fax: +86 27 88043958. E-mail address: [email protected] (Z.-Z. Tao). 0304-3835/$ - see front matter Ó 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2011.02.034
including sarcoma [7,16], medulloblastoma [6], ovarian cancer [17], prostate cancer [18] and breast cancer [2,19]. However, Notch signaling is also involved in suppressing tumorigenesis in some cancers, such as human papillomavirus-positive cervical cancer cells [20]. Although the Notch signaling may participate in the development of human cervical carcinoma, overexpressed active Notch1 to activate Notch signaling inhibits tumor growth through induction of cell cycle arrest and apoptosis [21,22]. Among the various forms of human lung cancers, small cell lung cancer (SCLC) exhibits a characteristic neuroendocrine (NE) phenotype with the cell cycle arrest by Notch signaling [22]. In the treatment of nasopharyngeal carcinoma, there have been significant research advances including the introduction of novel radiotherapy, chemotherapies and gene targeting agents, but the overall survival rate remains low and many nasopharyngeal carcinomas eventually develop resistance to radiotherapy and chemotherapy. Research has demonstrated that a number of prosurvival pathways play important roles during the development of therapeutic resistance [12,23]. To date, to our knowl-
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edge, activation of Notch signaling in nasopharyngeal carcinoma (NPC) cells has not been examined. In this work, we show the expression and prosurvival role of the Notch signaling pathway in NPC. 2. Materials and methods 2.1. Cell lines The human nasopharyngeal carcinoma cell lines CNE1 and 5–8F (China Center for Type Culture Collection, CCTCC) were maintained in tissue culture plates containing RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Gibco-BRL, Carlsbad, CA, USA), 100 units/ ml penicillin, and 100 lg/ml streptomycin at 37 °C and 5% CO2 gas phase. CNE1 is well differentiated, and 5–8F is poorly differentiated. They represent different type cells of NPC. All cell lines were tested to rule out mycoplasma contamination. 2.2. Drugs Notch signaling inhibitor, c-secretase inhibitor (GSI), provided by Merck Company (KGaA, Darmstadt, Germany), was dissolved in DMSO, stored at 20 °C, and diluted in media. The final concentration of DMSO was 0.1% or less in all experiments. 2.3. Western blotting Cell lines were treated with GSI (0–15 lmol/L) or 0.1% DMSO (as a control) for 24–48 h. Total protein lysates were prepared. Treated or untreated CNE1 and 5–8F cells were lysed in cell lysis buffer (25 mM HEPES-KOH, pH 7.5, 150 mM NaCl, 5 mM EDTA, 2 mM Na3VO4, leupeptin, Pefa-block, 1% Triton X-100, and 0.5% Nonidet P-40). The protein concentrations were determined using the BCA protein assay reagent from Pierce Biotechnology Inc. Equal amounts of protein from the cells were loaded and electrophoresed on a sodium dodecyl sulfate–polyacrylamide gel. Proteins were then transferred to an Immobilon-P membrane (Millipore). The membrane was incubated with primary antibodies at 4 °C for 12 h and washed with TBS-T (10 mM Tris–HCl, pH 8.0, 150 mM NaCl, and 0.1% Tween20). Proteins were probed with the following primary antibodies: Notch intracellular domain (NICD), Notch-1,2,3,4, Jagged1, c-Myc, HEY1, HEY2, cyclin D, cyclin E1, cyclin E2, pRb, P21, P27, P53, AKT, GSK3b, ERK1/2, P38, JNK1/2, caspase3/6/8/9, PARP, t-Bid, Cyto C, Smac, and GAPDH (all from Santa Cruz or Cell Signaling). It was then incubated with anti-mouse or rat second antibodies conjugated with horseradish peroxidase (Santa Cruz Biotechnology) at 37 °C for 2 h and the immune complex was visualized with the ECL system (Santa Cruz Biotechnology). 2.4. c-secretase activity assay The activity of the c-secretase was measured using the R&D Systems (R&D, Minneapolis, MN, USA) c-secretase Activity Kit and following the manufacturer’s instructions.
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In brief, cell lines were treated with GSI or with DMSO control for 48 h. Cells were then washed twice with ice-cold phosphate-buffered saline (PBS), harvested in the cell extraction buffer, and incubated on ice for at least 10 min. Lysates were centrifuged at 10,000g for 1 min and supernatants were collected with a total protein yield of 0.5–1.0 mg/ml. Pierce BCA Protein Assay (Pierce, Rockford, IL, USA) was used to determine the protein concentration in each sample. Protein (200 lg) was incubated with the c-secretase fluorogenic substrate for 2 h at 37 °C and fluorescence was measured at 355/460 nm.
2.5. Viability assays CNE1 and 5–8F cells were plated in 96-well plates and treated with GSI (0–20 lmol/L), or DMSO only for 24– 72 h. Viability was assessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and referred previously by Li et al. [24]. The dye MTT is taken up and metabolized to a colored product by viable mitochondria. Thus, the measurement of this metabolic reduction reaction was used as a marker of mitochondrial viability. Briefly, after the removal of medium, 100 ll of MTT (5 mg/10 ml of medium) was added to each well, and the tissue culture plate was incubated for 4 h at 37 °C. The MTT solution was removed, and 100 ll of dimethyl sulfoxide was added to each well, the color intensity was assessed with an enzyme-linked immunosorbent assay (ELISA) plate reader at a wavelength of 570 nm. Each experiment was repeated three times.
2.6. Hoechst 33258 staining Morphological changes resulting from apoptosis were determined by Hoechst 33258 staining. All the procedures were performed according to the manufacturer’s instructions and our previous work [25] (Apoptosis detection kit; Beyotime Biotech Co., Shanghai, China.). Briefly, cancer cells suspended in PBS were stained with 2 mg/L Hoechst 33258 and observed under fluorescence microscope using a blue filter. Cells showing nuclear shrinkage and chromatin condensation or fragmentation were defined as apoptotic cells.
2.7. Flow cytometry for DNA content CNE1 and 5–8F cells were initially seeded at 1 106 cells in 6-well dishes. Cells were then treated with GSI or DMSO only for 24 h. Cells were then centrifuged, washed once in PBS, and resuspended in 200 ml of PBS. Cells were then added dropwise to 5 ml of ice-cold 70% ethanol with vortexing and stored at 20 °C until analysis. Fixed cells were collected by centrifugation, washed once in PBS, and incubated in 300 ml of PI staining buffer [10 lg/ml PI and 250 lg/ml RNase A] for 30 min at room temperature. Samples were then acquired using a FACSCalibur flow cytometer, and the sub-G0-G1 peak and cell cycles were quantized using CELLQuest software.
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2.8. Measurement of mitochondrial membrane potential (Dwm) The Dwm was quantified by using a cationic fluorescent probe, rhodamine 123 (Molecular probes), which accumulates in functional mitochondria having high Dwm. CNE1 and 5–8F cells were incubated with or without GSI for different time, were then recovered, and 5 ll of 1 mg/ml rhodamine 123 was added to 1 106 cells per milliliter and incubated 10 min at 37 °C. Cells were then analyzed by FACS analysis. All the procedures were performed according to the manufacturer’s instructions and as described by Li et al. [24].
concentrations were determined by using the BCA protein assay reagent from Pierce Biotechnology Inc. A total of 75 lg of the S-100 fraction was used for Western blotting analysis of cyto c and Smac. 2.10. Statistical analysis All values are expressed as the mean ± SD. Statistical analyses were carried out by one-way ANOVA performed using the SPSS statistical software (SPSS Inc., Chicago, IL, USA). Probability values of P < 0.05 were considered statistically significant. 3. Results
2.9. Preparation of S-100 and Western blotting analysis of cytosolic cyto c and Smac Treated and untreated cells were harvested and centrifuged at 1000g for 10 min at 4 °C. The cell pellets were washed once with ice-cold PBS and resuspended with five volumes of buffer [20 mM HEPES-KOH (pH 7.5), 10 mM KCl, 1.5 mM MgCl2, 1 mM sodium EDTA, 1 mM sodium EGTA, 1 mM DTT, and 0.1 mM phenylmethyl sulfonylfluoride] containing 250 mM sucrose. The cells were homogenized with a 22-gauge needle, and the homogenates were centrifuged at 10,000g for 10 min at 4 °C. The supernatants were further centrifuged at 10,000g for 30 min. The resulting supernatants (S-100) were collected, and the protein
3.1. GSI suppresses the c-secretase activity, NICD and c-Myc in CNE1 and 5– 8F cells To test our hypothesis that Notch signaling is overexpressed and thereby important for nasopharyngeal carcinoma cell proliferation, we analyzed the levels of Notch-1, Notch-2, Notch-3 and Notch-4 in NPC CNE1 and 5–8F cells. We found that Notch-1, Notch-2, Notch-3 and Notch-4 were all positive in these cancer cells. After treated with GSI for 48 h, Notch-1, Notch-2 and Notch-4 were significantly reduced in both cell lines. Notch-3 showed no change in these cells (Fig. 1A). Next, we assessed the NICD expression and c-secretase activity. As shown in Fig. 1B and C, the NICD expression and the relative c-secretase activity were significantly inhibited after treating the cells with GSI for 48 h, an effect that was dose-dependent. When the cells were treated with GSI at 0.5 lM, the inhibitory effect was apparent (compared with the DMSO control, P < 0.05), and when the dose was 15 lM, the inhibitory
Fig. 1. The effects of GSI on the levels of Notch and c-secretase activity. (A) Suppression of Notch-1, Notch-2 and Notch-4 by GSI in 48 h. (B) and (C) Inhibition of NICD and the c-secretase activity after GSI treatment at different doses for 48 h. DP < 0.05 compared with that of DMSO control; P < 0.01 compared with that of DMSO control; #P > 0.05 compared with that of 10 lM. (D) Inhibition of the expression of Jagged1, c-Myc and HEY1 in association with inhibited NICD.
S.-M. Chen et al. / Cancer Letters 306 (2011) 76–84 effect was maximal (compared with the DMSO control, P < 0.01). However, no statistical difference was observed between the dose 10 lM and 15 lM (compared with the 10 lM, P > 0.05). Previous work has demonstrated that Jagged1 [26,27], c-Myc [28–30], HEY1, HEY2 [31] are the down-stream targeted gene of Notch signaling. In NPC, we found that the expression of these genes was also detected. As shown in Fig. 1D, the expression of Jagged1, c-Myc and HEY1 was significantly suppressed consistent with the reduced expression of NICD, although no change in HEY2 gene expression was observed under the conditions where NICD was inhibited in these cancer cells. 3.2. GSI prevents the proliferation and triggers apoptosis of CNE1 and 5–8F cells When the c-secretase activity and NICD expression were suppressed by GSI, the proliferations of CNE1 and 5–8F cells were inhibited in both time- and dose-dependent manners. As shown in Fig. 2A, after the cells were treated with GSI for 48–72 h, the inhibition of cell proliferation was observed at all doses (5 lM, 10 lM, 20 lM). Significant inhibitory effect was noted between the doses of 5 lM and 10 lM (P < 0.01). No statistical difference of inhibitory effect was observed between the doses of 10 lM and 20 lM (P > 0.05), reflective of a maximal inhibitory dose at 10–20 lM. In CNE1 cells, treatment with 20 lM for 72 h resulted in the highest inhibitory effect of 89.1%. In 5–8F cells, the maximal inhibition was 87.9%. When we examined the apoptotic cell death by flow cytometry, as shown in Fig. 2B and C, we found that GSI triggered apoptosis in both CNE1 and 5–8F cells at the doses of 5–15 lM. The apoptotic ratio of CNE1 cells was 5.4%, 13.2%, 25.4% and 76.7% in control, 5 lM, 10 lM and 15 lM dose, respectively. And the apoptotic ratio of 5–8F cells was 4.6%, 9.0%, 18% and 66.5% in control, 5 lM, 10 lM and 15 lM dose, respectively. There existed significant differences in the cell apoptosis ratio between the control and the doses at 5 lM, 10 lM and 15 lM (P < 0.05). A similar pattern was found with the apoptotic effects of GSI on these cancer cells when apoptosis was examined by Hoechst 33258 staining to visualize the presence of apoptotic nuclei (Fig. 2D). There presented many apoptotic nuclei in the two cancer cell lines after GSI treatment for 24– 48 h. 3.3. GSI induces the G2/M cell cycle arrest through regulating the cell cycle proteins cyclin D, cyclin E1, cyclin E2, pRb, P21, P27 and P53 The effect of GSI on the cell cycle was assessed by flow cytometry. Fig. 3A presents the results of the DNA flow cytometric analyses of CNE1 and 5–8F cells after incubation with GSI at 0–15 lM for 24 h. Both cell lines responded to the treatment with a dose–response accumulation of the G2/M phase cells. As shown in Fig. 3A, CNE1 cells showed a dosedependent accumulation in the G2/M phase from 5.2% (0 lM GSI) to 62.2% (15 lM GSI) after 24 h of incubation with GSI. As to 5–8F cells, the percentage of the G2/M phase cells increased from 9.6% (0 lM GSI) to 70.7% (15 lM GSI). As shown in Fig. 3B, the expressions of cyclin D, cyclin E1, cyclin E2 and pRb were decreased significantly in both CNE1 and 5–8F cells after GSI treatment for 48 h. In contrast, the expressions of P21, P27 and P53 were increased significantly in these cells treated with the same dose of GSI for 48 h. 3.4. GSI induces caspase 3/6/9 activation through mitochondrial pathway The percentage of the G2/M phase cells and the apoptotic cells increased significantly when CNE1 and 5–8F cells were treated with GSI. To determine whether mitochondrial disruption may account for the apoptotic effect of GSI, we tested the effect of GSI on mitochondrial permeability. Compared with the DMSO control, we found that the mitochondrial membrane polarization increased in a dose-dependent manner in the cells treated with GSI (Fig. 4A). CNE1 cells showed a dose-dependent accumulation with depolarized mitochondria from 10.1% (0 lM GSI) to 90.2% (15 lM GSI) after 48 h of GSI treatment. As to 5–8F cells, it increased from 10.3% (0 lM GSI) to 78.2% (15 lM GSI). Disruption of mitochondrial membrane function results in the release of several pro-apoptotic proteins, such as cyto c and Smac from mitochondria to the cytosol. Fig. 4B demonstrated that the levels of cyto
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c and Smac were increased significantly in CNE1 and 5–8F cells when the cells were treated with GSI. These effects were in a dose-dependent manner. As shown in Fig. 4C, the expression of cleaved caspase 3/6/9 and cleaved PARP were increased in a dose-dependent manner in CNE1 and 5–8F cells when the cells were treated with GSI for 48 h. 3.5. GSI also induces caspase 8 and Bid activation To further demonstrate the apoptotic process, the activation of caspase 8 and Bid was assessed. We found that caspase 8 and t-Bid were significantly increased when CNE1 and 5–8F cells were treated with GSI. As shown in Fig. 5, caspase 8 and t-Bid responded to GSI treatment with a dose-dependent increase in both CNE1 and 5–8F cell lines. 3.6. GSI suppresses AKT/mTOR/GSK3b and MEK/ERK1/2 signaling pathways, without affecting P38 and JNK1/2 signaling It has been reported that Akt activation induces NICD production through induction of c-secretase [32]. Here, the relationships between Notch signals, AKT, ERK1/2, P38 and JNK1/2 signaling pathway were examined. As shown in Fig. 6, in both NCE1 and 5–8F cell lines, the levels of phosphorylated AKT (P-AKT), P-GSK3b and P-ERK1/2 were decreased in a dose-dependent manner after the cells were treated with GSI for 60 min. However, no change in the total protein kinases (T-AKT, T-GSK3b and T-ERK1/2) was observed. By contrast, no change in total and phosphorylated p38 and JNK kinases was observed in the same cells treated with GSI.
4. Discussion The activation of the Notch pathway occurs when specific ligands like Delta-like-3 (DLL3) and Jagged-1 (JAG-1) bind to the four transmembrane receptors, Notch-1, Notch-2, Notch-3 and Notch-4. The binding activates the c-secretase protein complex. Gamma-secretase that cleaves the Notch receptor in the transmembrane domain, to release the cytoplasmic portion known as the Notch intracellular domain (NICD). After translocating into the nucleus, the NICD binds three cofactors to form a complex that acts as a transcriptional coactivator [33]. Notch signaling induces the expression of a variety of genes involved in cellular proliferation, such as c-Myc [28–30] and cyclin D1 [34,35], and in cellular survival [33]. In this study, we provided evidence for the first time that the Notch pathway is overexpressed in nasopharyngeal carcinoma cells (NPC cells) and that when Notch-1, Notch-2 and Notch-4 are inhibited, NPC cells undergo the cell cycle arrest and apoptosis. Previously studies reported that Jagged1, c-Myc, HEY1 and HEY2 are the downstream targets of Notch signaling [26–31]. Consistently, we found that the expression of Jagged1, c-Myc and HEY1 are significantly suppressed when NPC cells treated by Notch signaling inhibitor. However, in contrast to previous studies in other cancers, the expression of HEY2 in NPC cells is not suppressed following the inhibition of the Notch signaling by GSI. We cannot exclude the possibility that HEY2 is transcriptionally regulated by Notch-3 which is not inhibited by GSI in NPC cells. The inability of GSI inhibition of Notch-3 is not understood, although it implicated a specificity control of the selective inhibitions of other Notches and a need in future studies to elucidate the role of Notch-3 and its specific downstream targets. We demonstrate that the inhibition of NPC cell proliferation by GSI in a time- and dose-dependent manner, and similarly GSI induces apoptotic cell death in a dose- and
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Fig. 2. GSI prevents cell proliferation and triggers apoptosis in CNE1 and 5–8F cell cultures. (A) After treated with GSI for 48–72 h, the entire experiment dose presented significant growth inhibition in these cells. (B) Graphs represent combined data from three experiments of flow cytometry. GSI triggered apoptosis in both CNE1 and 5–8F cells at the doses of 5–15 lM. DP < 0.05 compared with that of DMSO control; #P < 0.01 compared with that of DMSO control. (C) Representative flow cytometry results. (D) There presented many apoptotic nuclei in the two cancer cell lines after GSI treatment for 24–48 h. Cells showing nuclear shrinkage and chromatin condensation or fragmentation were apoptotic cells.
time-dependent manner. These data suggest that GSI inhibition-induced cellular phenotype is likely to be specific,
mediated by inhibition of the Notch receptors. Our findings that GSI induced the G2/M cell cycle arrest are also consis-
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Fig. 3. The effect of GSI on the cell cycle and related proteins. (A) Both cell lines responded to the treatment with a dose–response accumulation of percentage of the G2/M phase cells. #P < 0.01 compared with that of DMSO control; DP > 0.05 compared with that of DMSO control. (B) The expression of cyclin D, cyclin E1, cyclin E2 and pRb were decreased significantly in both CNE1 and 5–8F cells after GSI treatment for 48 h. In contrast, the expression of P21, P27 and P53 were increased significantly in these cells treated with the same dose of GSI for 48 h.
Fig. 4. The effect of GSI on mitochondrial permeability and effector caspase. (A) The mitochondrial membrane polarization increased in a dose-dependent manner in cancer cells treated with GSI. P < 0.05 compared with that of DMSO control; #P < 0.01 compared with that of DMSO control. (B) The expression of cyto c and Smac was increased significantly after mitochondrial membrane function disrupted. (C) The expression of cleaved caspase 3/6/9 and cleaved PARP were increased in a dose-dependent response in CNE1 and 5–8F cells after the cells were treated with GSI for 48 h.
tent with the effects on the inhibition of cell proliferation and induction of cell death. Moreover, we substantiated the changes of molecular profiles underlying the cell cycle
arrest. We demonstrate that GSI treatment results in a significant suppression of the cell cycle proteins, cyclin D, cyclin E1, cyclin E2 and pRb in parallel with the mainte-
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Fig. 5. The effect of GSI on the activation of caspase 8 and Bid. The expression of caspase 8 and t-Bid in both cell lines responded to GSI treatment with a dose-dependent increase.
Fig. 6. The effect of GSI on AKT/mTOR/GSK3b, MEK/ERK1/2, P38 and JNK1/2 signaling pathways. Both in NCE1 and 5–8F cell lines, the levels of P-AKT, PGSK3b and P-ERK1/2 were decreased in a dose-dependent manner after the cells were treated with GSI. However, no change in the total protein kinases (TAKT, T-GSK3b and T-ERK1/2) was observed. By contrast, no change in total and phosphorylated p38 and JNK kinases was observed in the same cells treated with GSI.
nance of the expression of P21, P27 and P53. Furthermore, we demonstrate that GSI-induced compromise of mitochondrial membrane Dwm to release cyto c and Smac into the cytosol may be responsible in triggering caspase-9 and downstream effector caspase cascades. Consistently, GSI treatment of NPC cells results in a caspase-8-mediated Bid cleavage. Thus, GSI-induced proliferation inhibition, cell cycle arrest and apoptosis of NPC cells involve multiple molecular elements concurred in the NPC cells within the time window of GSI treatment. In addition to the effector molecules controlling the cell cycle and apoptosis, AKT protein kinase has been impli-
cated in transducing Notch signaling. AKT activation induces NICD through induction of c-secretase [36]. Consistently, we found that phosphorylated AKT is significantly reduced in GSI treated NPC cells, suggesting that AKT inhibition contributes to GSI-inhibition of the Notch signaling. More interestingly, while little is known of the relationship between Notch signaling and MAP kinase signaling pathways, examination of ERK1/2, P38 and JNK1/2 in GSI treated NPC cells revealed that P-GSK3b and PERK1/2 are decreased in a dose-dependent manner in response to GSI, whereas the expression of total GSK3b and ERK1/2 showed no change. These data suggest for the first
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time that GSI treatment results in inhibition of the MAP kinase signaling pathway with specificity on MEK/ERK1/2 signaling (but not P38 and JNK1/2 signaling). Previous studies have shown that Notch signaling can serve as a tumor promoter in most cancers, whereas it can also serve as a tumor suppressor in some cancers. Our data support that Notch signaling serves as a tumor promoter in NPC cells. Previous studies also show that blocking the Notch signaling with GSI sensitizes cells to chemotherapy. For example, inhibition of Notch signaling prevented bone marrow-mediated drug resistance and sensitized myeloma cells to chemotherapy [37]. The chemosensitization has been shown to be mediated by Notch-1, as inhibition of Notch-1 by gene targeting enhances chemosensitivity whereas overexpression of NICD increases chemoresistance [33]. Moreover, Notch has been shown to promote radioresistance in glioma stem cells [38]. Since radiotherapy and chemotherapy are the two main standard treatments in NPC at present, future studies will need to investigate if there exists an enhanced effect of GSI on the chemotherapeutic and radiotherapy in NPC cells. In summary, our data show for the first time that inhibition of Notch signaling with c-secretase inhibitor GSI results in inhibition of NPC cell proliferation in association with increased arrest of the cell cycle and apoptosis. We demonstrate for the first time that in addition the AKT pathway, ERK signaling is suppressed which participates in GSI-induced suppressions of various effector proteins important in the cell cycle progress and survival. Our data suggest that blocking Notch pathway in combination with other therapies may represent a novel approach for the treatment of nasopharyngeal carcinoma. Conflict of interest There is no any conflict of interest about this paper. Acknowledgements The authors would like to thank Prof. Hong-Liang Li for technical assistance to this study. This work was supported by the Grants from the National Natural Science Foundation of China (Nos. 30901662 and 30872851), the Science and Technology Program of Hubei Province of China (No. 2007AA302B08), and the Science and Technology Program of Wuhan City (Nos. 200951199455 and 200950431168). References [1] M. Reedijk, S. Odorcic, L. Chang, H. Zhang, N. Miller, D.R. McCready, G. Lockwood, S.E. Egan, High-level coexpression of JAG1 and NOTCH1 is observed in human breast cancer and is associated with poor overall survival, Cancer Res. 65 (2005) 8530–8537. [2] C.L. Efferson, C.T. Winkelmann, C. Ware, T. Sullivan, S. Giampaoli, J. Tammam, S. Patel, G. Mesiti, J.F. Reilly, R.E. Gibson, C. Buser, T. Yeatman, D. Coppola, C. Winter, E.A. Clark, G.F. Draetta, P.R. Strack, P.K. Majumder, Downregulation of notch pathway by a gammasecretase inhibitor attenuates AKT/mammalian target of rapamycin signaling and glucose uptake in an ERBB2 transgenic breast cancer model, Cancer Res. 70 (2010) 2476–2484. [3] O. Hopfer, D. Zwahlen, M.F. Fey, S. Aebi, The notch pathway in ovarian carcinomas and adenomas, Br. J. Cancer 93 (2005) 709–718.
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Contents lists available at ScienceDirect
Cancer Letters journal homepage: www.elsevier.com/locate/canlet
Suppression of the notch signaling pathway by c-secretase inhibitor GSI inhibits human nasopharyngeal carcinoma cell proliferation Shi-Ming Chen a,b, Jun-Ping Liu b, Jun-Xu Zhou a, Chen Chen a, Yu-Qin Deng a, Yan Wang a, Ze-Zhang Tao a,⇑ a b
Department of Otolaryngology-Head and Neck Surgery, Renmin Hospital of Wuhan University, Wuhan 430060, China Molecular Signaling Laboratory, Department of Immunology, Central Clinical School, Monash University, Melbourne, Victoria 3004, Australia
a r t i c l e
i n f o
Article history: Received 18 January 2011 Received in revised form 18 February 2011 Accepted 21 February 2011
Keywords: Notch Nasopharyngeal carcinoma cells GSI Apoptosis AKT
a b s t r a c t Notch signaling has been suggested to be required for many human cancers. However, the role of Notch signaling in human nasopharyngeal carcinoma cells (NPC) remains unknown. Here, we report that Notch-1, Notch-2, Notch-3 and Notch-4 are all detected in NPC cells. Notch inhibitor, GSI, suppresses the levels of Notch-1, Notch-2 and Notch-4, but not Notch3. In addition, GSI inhibits NPC cell proliferation by inducing the cell cycle arrest and apoptosis. Furthermore, GSI inhibits the AKT and MEK signaling, without affecting P38 and JNK1/2. Thus, NPC cells may up-regulate Notch signaling to maintain cell proliferation and targeting the Notch signaling pathway may offer a potential alternative strategy for the treatment of NPC. Ó 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Overexpression of the Notch receptors and/or their ligands has been identified in multiple cancers of the breast [1,2], ovarian [3,4], prostate [5], brain [6] and sarcoma [7–10]. The overexpression of Notch elements in cancers has been related to a poor clinical outcome. For example, overexpression of Notch-1 is associated with the rapid recurrence in breast cancers [11], and increased expression of JAG-1 is associated with a high rate of recurrence in prostate cancers [5]. Consequently, many laboratories have studied the effects of blocking the Notch signaling pathway by targeting the c-secretase protein complex to inhibit the activation of Notch receptors [12]. It has been shown that inhibition of the c-secretase complex prevents the growth of T cell ALL lines leading to apoptosis [13–15]. Notch signaling inhibitors (GSIs) suppress the growth of multiple tumors,
⇑ Corresponding author. Tel.: +86 13907141892; fax: +86 27 88043958. E-mail address: [email protected] (Z.-Z. Tao). 0304-3835/$ - see front matter Ó 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2011.02.034
including sarcoma [7,16], medulloblastoma [6], ovarian cancer [17], prostate cancer [18] and breast cancer [2,19]. However, Notch signaling is also involved in suppressing tumorigenesis in some cancers, such as human papillomavirus-positive cervical cancer cells [20]. Although the Notch signaling may participate in the development of human cervical carcinoma, overexpressed active Notch1 to activate Notch signaling inhibits tumor growth through induction of cell cycle arrest and apoptosis [21,22]. Among the various forms of human lung cancers, small cell lung cancer (SCLC) exhibits a characteristic neuroendocrine (NE) phenotype with the cell cycle arrest by Notch signaling [22]. In the treatment of nasopharyngeal carcinoma, there have been significant research advances including the introduction of novel radiotherapy, chemotherapies and gene targeting agents, but the overall survival rate remains low and many nasopharyngeal carcinomas eventually develop resistance to radiotherapy and chemotherapy. Research has demonstrated that a number of prosurvival pathways play important roles during the development of therapeutic resistance [12,23]. To date, to our knowl-
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edge, activation of Notch signaling in nasopharyngeal carcinoma (NPC) cells has not been examined. In this work, we show the expression and prosurvival role of the Notch signaling pathway in NPC. 2. Materials and methods 2.1. Cell lines The human nasopharyngeal carcinoma cell lines CNE1 and 5–8F (China Center for Type Culture Collection, CCTCC) were maintained in tissue culture plates containing RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Gibco-BRL, Carlsbad, CA, USA), 100 units/ ml penicillin, and 100 lg/ml streptomycin at 37 °C and 5% CO2 gas phase. CNE1 is well differentiated, and 5–8F is poorly differentiated. They represent different type cells of NPC. All cell lines were tested to rule out mycoplasma contamination. 2.2. Drugs Notch signaling inhibitor, c-secretase inhibitor (GSI), provided by Merck Company (KGaA, Darmstadt, Germany), was dissolved in DMSO, stored at 20 °C, and diluted in media. The final concentration of DMSO was 0.1% or less in all experiments. 2.3. Western blotting Cell lines were treated with GSI (0–15 lmol/L) or 0.1% DMSO (as a control) for 24–48 h. Total protein lysates were prepared. Treated or untreated CNE1 and 5–8F cells were lysed in cell lysis buffer (25 mM HEPES-KOH, pH 7.5, 150 mM NaCl, 5 mM EDTA, 2 mM Na3VO4, leupeptin, Pefa-block, 1% Triton X-100, and 0.5% Nonidet P-40). The protein concentrations were determined using the BCA protein assay reagent from Pierce Biotechnology Inc. Equal amounts of protein from the cells were loaded and electrophoresed on a sodium dodecyl sulfate–polyacrylamide gel. Proteins were then transferred to an Immobilon-P membrane (Millipore). The membrane was incubated with primary antibodies at 4 °C for 12 h and washed with TBS-T (10 mM Tris–HCl, pH 8.0, 150 mM NaCl, and 0.1% Tween20). Proteins were probed with the following primary antibodies: Notch intracellular domain (NICD), Notch-1,2,3,4, Jagged1, c-Myc, HEY1, HEY2, cyclin D, cyclin E1, cyclin E2, pRb, P21, P27, P53, AKT, GSK3b, ERK1/2, P38, JNK1/2, caspase3/6/8/9, PARP, t-Bid, Cyto C, Smac, and GAPDH (all from Santa Cruz or Cell Signaling). It was then incubated with anti-mouse or rat second antibodies conjugated with horseradish peroxidase (Santa Cruz Biotechnology) at 37 °C for 2 h and the immune complex was visualized with the ECL system (Santa Cruz Biotechnology). 2.4. c-secretase activity assay The activity of the c-secretase was measured using the R&D Systems (R&D, Minneapolis, MN, USA) c-secretase Activity Kit and following the manufacturer’s instructions.
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In brief, cell lines were treated with GSI or with DMSO control for 48 h. Cells were then washed twice with ice-cold phosphate-buffered saline (PBS), harvested in the cell extraction buffer, and incubated on ice for at least 10 min. Lysates were centrifuged at 10,000g for 1 min and supernatants were collected with a total protein yield of 0.5–1.0 mg/ml. Pierce BCA Protein Assay (Pierce, Rockford, IL, USA) was used to determine the protein concentration in each sample. Protein (200 lg) was incubated with the c-secretase fluorogenic substrate for 2 h at 37 °C and fluorescence was measured at 355/460 nm.
2.5. Viability assays CNE1 and 5–8F cells were plated in 96-well plates and treated with GSI (0–20 lmol/L), or DMSO only for 24– 72 h. Viability was assessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and referred previously by Li et al. [24]. The dye MTT is taken up and metabolized to a colored product by viable mitochondria. Thus, the measurement of this metabolic reduction reaction was used as a marker of mitochondrial viability. Briefly, after the removal of medium, 100 ll of MTT (5 mg/10 ml of medium) was added to each well, and the tissue culture plate was incubated for 4 h at 37 °C. The MTT solution was removed, and 100 ll of dimethyl sulfoxide was added to each well, the color intensity was assessed with an enzyme-linked immunosorbent assay (ELISA) plate reader at a wavelength of 570 nm. Each experiment was repeated three times.
2.6. Hoechst 33258 staining Morphological changes resulting from apoptosis were determined by Hoechst 33258 staining. All the procedures were performed according to the manufacturer’s instructions and our previous work [25] (Apoptosis detection kit; Beyotime Biotech Co., Shanghai, China.). Briefly, cancer cells suspended in PBS were stained with 2 mg/L Hoechst 33258 and observed under fluorescence microscope using a blue filter. Cells showing nuclear shrinkage and chromatin condensation or fragmentation were defined as apoptotic cells.
2.7. Flow cytometry for DNA content CNE1 and 5–8F cells were initially seeded at 1 106 cells in 6-well dishes. Cells were then treated with GSI or DMSO only for 24 h. Cells were then centrifuged, washed once in PBS, and resuspended in 200 ml of PBS. Cells were then added dropwise to 5 ml of ice-cold 70% ethanol with vortexing and stored at 20 °C until analysis. Fixed cells were collected by centrifugation, washed once in PBS, and incubated in 300 ml of PI staining buffer [10 lg/ml PI and 250 lg/ml RNase A] for 30 min at room temperature. Samples were then acquired using a FACSCalibur flow cytometer, and the sub-G0-G1 peak and cell cycles were quantized using CELLQuest software.
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2.8. Measurement of mitochondrial membrane potential (Dwm) The Dwm was quantified by using a cationic fluorescent probe, rhodamine 123 (Molecular probes), which accumulates in functional mitochondria having high Dwm. CNE1 and 5–8F cells were incubated with or without GSI for different time, were then recovered, and 5 ll of 1 mg/ml rhodamine 123 was added to 1 106 cells per milliliter and incubated 10 min at 37 °C. Cells were then analyzed by FACS analysis. All the procedures were performed according to the manufacturer’s instructions and as described by Li et al. [24].
concentrations were determined by using the BCA protein assay reagent from Pierce Biotechnology Inc. A total of 75 lg of the S-100 fraction was used for Western blotting analysis of cyto c and Smac. 2.10. Statistical analysis All values are expressed as the mean ± SD. Statistical analyses were carried out by one-way ANOVA performed using the SPSS statistical software (SPSS Inc., Chicago, IL, USA). Probability values of P < 0.05 were considered statistically significant. 3. Results
2.9. Preparation of S-100 and Western blotting analysis of cytosolic cyto c and Smac Treated and untreated cells were harvested and centrifuged at 1000g for 10 min at 4 °C. The cell pellets were washed once with ice-cold PBS and resuspended with five volumes of buffer [20 mM HEPES-KOH (pH 7.5), 10 mM KCl, 1.5 mM MgCl2, 1 mM sodium EDTA, 1 mM sodium EGTA, 1 mM DTT, and 0.1 mM phenylmethyl sulfonylfluoride] containing 250 mM sucrose. The cells were homogenized with a 22-gauge needle, and the homogenates were centrifuged at 10,000g for 10 min at 4 °C. The supernatants were further centrifuged at 10,000g for 30 min. The resulting supernatants (S-100) were collected, and the protein
3.1. GSI suppresses the c-secretase activity, NICD and c-Myc in CNE1 and 5– 8F cells To test our hypothesis that Notch signaling is overexpressed and thereby important for nasopharyngeal carcinoma cell proliferation, we analyzed the levels of Notch-1, Notch-2, Notch-3 and Notch-4 in NPC CNE1 and 5–8F cells. We found that Notch-1, Notch-2, Notch-3 and Notch-4 were all positive in these cancer cells. After treated with GSI for 48 h, Notch-1, Notch-2 and Notch-4 were significantly reduced in both cell lines. Notch-3 showed no change in these cells (Fig. 1A). Next, we assessed the NICD expression and c-secretase activity. As shown in Fig. 1B and C, the NICD expression and the relative c-secretase activity were significantly inhibited after treating the cells with GSI for 48 h, an effect that was dose-dependent. When the cells were treated with GSI at 0.5 lM, the inhibitory effect was apparent (compared with the DMSO control, P < 0.05), and when the dose was 15 lM, the inhibitory
Fig. 1. The effects of GSI on the levels of Notch and c-secretase activity. (A) Suppression of Notch-1, Notch-2 and Notch-4 by GSI in 48 h. (B) and (C) Inhibition of NICD and the c-secretase activity after GSI treatment at different doses for 48 h. DP < 0.05 compared with that of DMSO control; P < 0.01 compared with that of DMSO control; #P > 0.05 compared with that of 10 lM. (D) Inhibition of the expression of Jagged1, c-Myc and HEY1 in association with inhibited NICD.
S.-M. Chen et al. / Cancer Letters 306 (2011) 76–84 effect was maximal (compared with the DMSO control, P < 0.01). However, no statistical difference was observed between the dose 10 lM and 15 lM (compared with the 10 lM, P > 0.05). Previous work has demonstrated that Jagged1 [26,27], c-Myc [28–30], HEY1, HEY2 [31] are the down-stream targeted gene of Notch signaling. In NPC, we found that the expression of these genes was also detected. As shown in Fig. 1D, the expression of Jagged1, c-Myc and HEY1 was significantly suppressed consistent with the reduced expression of NICD, although no change in HEY2 gene expression was observed under the conditions where NICD was inhibited in these cancer cells. 3.2. GSI prevents the proliferation and triggers apoptosis of CNE1 and 5–8F cells When the c-secretase activity and NICD expression were suppressed by GSI, the proliferations of CNE1 and 5–8F cells were inhibited in both time- and dose-dependent manners. As shown in Fig. 2A, after the cells were treated with GSI for 48–72 h, the inhibition of cell proliferation was observed at all doses (5 lM, 10 lM, 20 lM). Significant inhibitory effect was noted between the doses of 5 lM and 10 lM (P < 0.01). No statistical difference of inhibitory effect was observed between the doses of 10 lM and 20 lM (P > 0.05), reflective of a maximal inhibitory dose at 10–20 lM. In CNE1 cells, treatment with 20 lM for 72 h resulted in the highest inhibitory effect of 89.1%. In 5–8F cells, the maximal inhibition was 87.9%. When we examined the apoptotic cell death by flow cytometry, as shown in Fig. 2B and C, we found that GSI triggered apoptosis in both CNE1 and 5–8F cells at the doses of 5–15 lM. The apoptotic ratio of CNE1 cells was 5.4%, 13.2%, 25.4% and 76.7% in control, 5 lM, 10 lM and 15 lM dose, respectively. And the apoptotic ratio of 5–8F cells was 4.6%, 9.0%, 18% and 66.5% in control, 5 lM, 10 lM and 15 lM dose, respectively. There existed significant differences in the cell apoptosis ratio between the control and the doses at 5 lM, 10 lM and 15 lM (P < 0.05). A similar pattern was found with the apoptotic effects of GSI on these cancer cells when apoptosis was examined by Hoechst 33258 staining to visualize the presence of apoptotic nuclei (Fig. 2D). There presented many apoptotic nuclei in the two cancer cell lines after GSI treatment for 24– 48 h. 3.3. GSI induces the G2/M cell cycle arrest through regulating the cell cycle proteins cyclin D, cyclin E1, cyclin E2, pRb, P21, P27 and P53 The effect of GSI on the cell cycle was assessed by flow cytometry. Fig. 3A presents the results of the DNA flow cytometric analyses of CNE1 and 5–8F cells after incubation with GSI at 0–15 lM for 24 h. Both cell lines responded to the treatment with a dose–response accumulation of the G2/M phase cells. As shown in Fig. 3A, CNE1 cells showed a dosedependent accumulation in the G2/M phase from 5.2% (0 lM GSI) to 62.2% (15 lM GSI) after 24 h of incubation with GSI. As to 5–8F cells, the percentage of the G2/M phase cells increased from 9.6% (0 lM GSI) to 70.7% (15 lM GSI). As shown in Fig. 3B, the expressions of cyclin D, cyclin E1, cyclin E2 and pRb were decreased significantly in both CNE1 and 5–8F cells after GSI treatment for 48 h. In contrast, the expressions of P21, P27 and P53 were increased significantly in these cells treated with the same dose of GSI for 48 h. 3.4. GSI induces caspase 3/6/9 activation through mitochondrial pathway The percentage of the G2/M phase cells and the apoptotic cells increased significantly when CNE1 and 5–8F cells were treated with GSI. To determine whether mitochondrial disruption may account for the apoptotic effect of GSI, we tested the effect of GSI on mitochondrial permeability. Compared with the DMSO control, we found that the mitochondrial membrane polarization increased in a dose-dependent manner in the cells treated with GSI (Fig. 4A). CNE1 cells showed a dose-dependent accumulation with depolarized mitochondria from 10.1% (0 lM GSI) to 90.2% (15 lM GSI) after 48 h of GSI treatment. As to 5–8F cells, it increased from 10.3% (0 lM GSI) to 78.2% (15 lM GSI). Disruption of mitochondrial membrane function results in the release of several pro-apoptotic proteins, such as cyto c and Smac from mitochondria to the cytosol. Fig. 4B demonstrated that the levels of cyto
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c and Smac were increased significantly in CNE1 and 5–8F cells when the cells were treated with GSI. These effects were in a dose-dependent manner. As shown in Fig. 4C, the expression of cleaved caspase 3/6/9 and cleaved PARP were increased in a dose-dependent manner in CNE1 and 5–8F cells when the cells were treated with GSI for 48 h. 3.5. GSI also induces caspase 8 and Bid activation To further demonstrate the apoptotic process, the activation of caspase 8 and Bid was assessed. We found that caspase 8 and t-Bid were significantly increased when CNE1 and 5–8F cells were treated with GSI. As shown in Fig. 5, caspase 8 and t-Bid responded to GSI treatment with a dose-dependent increase in both CNE1 and 5–8F cell lines. 3.6. GSI suppresses AKT/mTOR/GSK3b and MEK/ERK1/2 signaling pathways, without affecting P38 and JNK1/2 signaling It has been reported that Akt activation induces NICD production through induction of c-secretase [32]. Here, the relationships between Notch signals, AKT, ERK1/2, P38 and JNK1/2 signaling pathway were examined. As shown in Fig. 6, in both NCE1 and 5–8F cell lines, the levels of phosphorylated AKT (P-AKT), P-GSK3b and P-ERK1/2 were decreased in a dose-dependent manner after the cells were treated with GSI for 60 min. However, no change in the total protein kinases (T-AKT, T-GSK3b and T-ERK1/2) was observed. By contrast, no change in total and phosphorylated p38 and JNK kinases was observed in the same cells treated with GSI.
4. Discussion The activation of the Notch pathway occurs when specific ligands like Delta-like-3 (DLL3) and Jagged-1 (JAG-1) bind to the four transmembrane receptors, Notch-1, Notch-2, Notch-3 and Notch-4. The binding activates the c-secretase protein complex. Gamma-secretase that cleaves the Notch receptor in the transmembrane domain, to release the cytoplasmic portion known as the Notch intracellular domain (NICD). After translocating into the nucleus, the NICD binds three cofactors to form a complex that acts as a transcriptional coactivator [33]. Notch signaling induces the expression of a variety of genes involved in cellular proliferation, such as c-Myc [28–30] and cyclin D1 [34,35], and in cellular survival [33]. In this study, we provided evidence for the first time that the Notch pathway is overexpressed in nasopharyngeal carcinoma cells (NPC cells) and that when Notch-1, Notch-2 and Notch-4 are inhibited, NPC cells undergo the cell cycle arrest and apoptosis. Previously studies reported that Jagged1, c-Myc, HEY1 and HEY2 are the downstream targets of Notch signaling [26–31]. Consistently, we found that the expression of Jagged1, c-Myc and HEY1 are significantly suppressed when NPC cells treated by Notch signaling inhibitor. However, in contrast to previous studies in other cancers, the expression of HEY2 in NPC cells is not suppressed following the inhibition of the Notch signaling by GSI. We cannot exclude the possibility that HEY2 is transcriptionally regulated by Notch-3 which is not inhibited by GSI in NPC cells. The inability of GSI inhibition of Notch-3 is not understood, although it implicated a specificity control of the selective inhibitions of other Notches and a need in future studies to elucidate the role of Notch-3 and its specific downstream targets. We demonstrate that the inhibition of NPC cell proliferation by GSI in a time- and dose-dependent manner, and similarly GSI induces apoptotic cell death in a dose- and
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Fig. 2. GSI prevents cell proliferation and triggers apoptosis in CNE1 and 5–8F cell cultures. (A) After treated with GSI for 48–72 h, the entire experiment dose presented significant growth inhibition in these cells. (B) Graphs represent combined data from three experiments of flow cytometry. GSI triggered apoptosis in both CNE1 and 5–8F cells at the doses of 5–15 lM. DP < 0.05 compared with that of DMSO control; #P < 0.01 compared with that of DMSO control. (C) Representative flow cytometry results. (D) There presented many apoptotic nuclei in the two cancer cell lines after GSI treatment for 24–48 h. Cells showing nuclear shrinkage and chromatin condensation or fragmentation were apoptotic cells.
time-dependent manner. These data suggest that GSI inhibition-induced cellular phenotype is likely to be specific,
mediated by inhibition of the Notch receptors. Our findings that GSI induced the G2/M cell cycle arrest are also consis-
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Fig. 3. The effect of GSI on the cell cycle and related proteins. (A) Both cell lines responded to the treatment with a dose–response accumulation of percentage of the G2/M phase cells. #P < 0.01 compared with that of DMSO control; DP > 0.05 compared with that of DMSO control. (B) The expression of cyclin D, cyclin E1, cyclin E2 and pRb were decreased significantly in both CNE1 and 5–8F cells after GSI treatment for 48 h. In contrast, the expression of P21, P27 and P53 were increased significantly in these cells treated with the same dose of GSI for 48 h.
Fig. 4. The effect of GSI on mitochondrial permeability and effector caspase. (A) The mitochondrial membrane polarization increased in a dose-dependent manner in cancer cells treated with GSI. P < 0.05 compared with that of DMSO control; #P < 0.01 compared with that of DMSO control. (B) The expression of cyto c and Smac was increased significantly after mitochondrial membrane function disrupted. (C) The expression of cleaved caspase 3/6/9 and cleaved PARP were increased in a dose-dependent response in CNE1 and 5–8F cells after the cells were treated with GSI for 48 h.
tent with the effects on the inhibition of cell proliferation and induction of cell death. Moreover, we substantiated the changes of molecular profiles underlying the cell cycle
arrest. We demonstrate that GSI treatment results in a significant suppression of the cell cycle proteins, cyclin D, cyclin E1, cyclin E2 and pRb in parallel with the mainte-
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Fig. 5. The effect of GSI on the activation of caspase 8 and Bid. The expression of caspase 8 and t-Bid in both cell lines responded to GSI treatment with a dose-dependent increase.
Fig. 6. The effect of GSI on AKT/mTOR/GSK3b, MEK/ERK1/2, P38 and JNK1/2 signaling pathways. Both in NCE1 and 5–8F cell lines, the levels of P-AKT, PGSK3b and P-ERK1/2 were decreased in a dose-dependent manner after the cells were treated with GSI. However, no change in the total protein kinases (TAKT, T-GSK3b and T-ERK1/2) was observed. By contrast, no change in total and phosphorylated p38 and JNK kinases was observed in the same cells treated with GSI.
nance of the expression of P21, P27 and P53. Furthermore, we demonstrate that GSI-induced compromise of mitochondrial membrane Dwm to release cyto c and Smac into the cytosol may be responsible in triggering caspase-9 and downstream effector caspase cascades. Consistently, GSI treatment of NPC cells results in a caspase-8-mediated Bid cleavage. Thus, GSI-induced proliferation inhibition, cell cycle arrest and apoptosis of NPC cells involve multiple molecular elements concurred in the NPC cells within the time window of GSI treatment. In addition to the effector molecules controlling the cell cycle and apoptosis, AKT protein kinase has been impli-
cated in transducing Notch signaling. AKT activation induces NICD through induction of c-secretase [36]. Consistently, we found that phosphorylated AKT is significantly reduced in GSI treated NPC cells, suggesting that AKT inhibition contributes to GSI-inhibition of the Notch signaling. More interestingly, while little is known of the relationship between Notch signaling and MAP kinase signaling pathways, examination of ERK1/2, P38 and JNK1/2 in GSI treated NPC cells revealed that P-GSK3b and PERK1/2 are decreased in a dose-dependent manner in response to GSI, whereas the expression of total GSK3b and ERK1/2 showed no change. These data suggest for the first
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time that GSI treatment results in inhibition of the MAP kinase signaling pathway with specificity on MEK/ERK1/2 signaling (but not P38 and JNK1/2 signaling). Previous studies have shown that Notch signaling can serve as a tumor promoter in most cancers, whereas it can also serve as a tumor suppressor in some cancers. Our data support that Notch signaling serves as a tumor promoter in NPC cells. Previous studies also show that blocking the Notch signaling with GSI sensitizes cells to chemotherapy. For example, inhibition of Notch signaling prevented bone marrow-mediated drug resistance and sensitized myeloma cells to chemotherapy [37]. The chemosensitization has been shown to be mediated by Notch-1, as inhibition of Notch-1 by gene targeting enhances chemosensitivity whereas overexpression of NICD increases chemoresistance [33]. Moreover, Notch has been shown to promote radioresistance in glioma stem cells [38]. Since radiotherapy and chemotherapy are the two main standard treatments in NPC at present, future studies will need to investigate if there exists an enhanced effect of GSI on the chemotherapeutic and radiotherapy in NPC cells. In summary, our data show for the first time that inhibition of Notch signaling with c-secretase inhibitor GSI results in inhibition of NPC cell proliferation in association with increased arrest of the cell cycle and apoptosis. We demonstrate for the first time that in addition the AKT pathway, ERK signaling is suppressed which participates in GSI-induced suppressions of various effector proteins important in the cell cycle progress and survival. Our data suggest that blocking Notch pathway in combination with other therapies may represent a novel approach for the treatment of nasopharyngeal carcinoma. Conflict of interest There is no any conflict of interest about this paper. Acknowledgements The authors would like to thank Prof. Hong-Liang Li for technical assistance to this study. This work was supported by the Grants from the National Natural Science Foundation of China (Nos. 30901662 and 30872851), the Science and Technology Program of Hubei Province of China (No. 2007AA302B08), and the Science and Technology Program of Wuhan City (Nos. 200951199455 and 200950431168). References [1] M. Reedijk, S. Odorcic, L. Chang, H. Zhang, N. Miller, D.R. McCready, G. Lockwood, S.E. Egan, High-level coexpression of JAG1 and NOTCH1 is observed in human breast cancer and is associated with poor overall survival, Cancer Res. 65 (2005) 8530–8537. [2] C.L. Efferson, C.T. Winkelmann, C. Ware, T. Sullivan, S. Giampaoli, J. Tammam, S. Patel, G. Mesiti, J.F. Reilly, R.E. Gibson, C. Buser, T. Yeatman, D. Coppola, C. Winter, E.A. Clark, G.F. Draetta, P.R. Strack, P.K. Majumder, Downregulation of notch pathway by a gammasecretase inhibitor attenuates AKT/mammalian target of rapamycin signaling and glucose uptake in an ERBB2 transgenic breast cancer model, Cancer Res. 70 (2010) 2476–2484. [3] O. Hopfer, D. Zwahlen, M.F. Fey, S. Aebi, The notch pathway in ovarian carcinomas and adenomas, Br. J. Cancer 93 (2005) 709–718.
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