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Effect of nimesulide on the growth of human laryngeal squamous cell carcinoma☆ Zhuoping Liang, MM a, 1 , Jinbo Liu, MD b, 1 , Leiji Li, MM a , Haiyang Wang, MM a , Chong Zhao, MM a , Liang Jiang, MM a , Gang Qin, MD a,⁎ a Department of Otolaryngology Head and Neck Surgery, Affiliated Hospital of Luzhou Medical College, Luzhou 646000, Sichuan Province, China b Department of Clinical Laboratory, Affiliated Hospital of Luzhou Medical College, Luzhou 646000, Sichuan Province, China
ARTI CLE I NFO
A BS TRACT
Article history:
Purpose: To investigate the effect of nimesulide on the growth of human laryngeal
Received 27 August 2013
squamous cell carcinoma. Materials and methods: The effect of NIM on Hep-2 cell proliferation was measured by the MTT assay. Flow cytometry was used to evaluate the cell cycle and apoptosis in Hep-2 cells. A Western blot analysis was used to detect changes in the protein expression levels of COX-2, Survivin and proliferating cell nuclear antigen (PCNA) in Hep-2 cells. A Hep-2 tumor xenograft model was established in nude mice to observe tumor growth. The changes in the xenograft tumors were observed after hematoxylin/eosin staining. The expression levels of COX-2, Survivin and PCNA proteins and mRNA were measured by immunohistochemical analysis and RT-PCR, respectively. Results: NIM had time- and dose-dependent inhibitory effect on the proliferation of Hep-2 cells. NIM could prevent the progression of the cell cycle. After NIM treatment, COX-2, Survivin and PCNA protein levels were reduced in the Hep-2 cells. The volume and weight of the xenograft tumors in the NIM treatment group were significantly reduced. The NIM treatment group also exhibited significantly reduced expression levels of COX-2, Survivin and PCNA at both the protein and mRNA levels. Conclusions: Our results suggested that NIM has significant inhibitory effects on the growth of Hep-2 cells and xenograft tumors in nude mice. Selective COX-2 inhibitors could potentially become part of a comprehensive treatment for laryngeal squamous cell carcinoma. Additional research and development will provide new and broader prospects for the prevention and treatment of laryngeal squamous cell carcinoma. © 2014 Elsevier Inc. All rights reserved.
1.
Introduction
Recent studies have demonstrated that non-steroidal antiinflammatory drugs (NSAIDs) can prevent and treat various tumors. Selective cyclooxygenase-2 (COX-2) inhibitors exhibit high preferential inhibition of COX-2 and only minor effects on
cyclooxygenase-1(COX-1). With their lower side effects and high specificity, selective COX-2 inhibitors have become a focus for cancer prevention and treatment, gradually replacing traditional NSAIDs in clinical applications [1]. Nimesulide (NIM), which only weakly inhibits the protective COX-1, was a highly specific COX-2 selective inhibitor. Because the ratio of its
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None of the authors has any conflict of interest, financial or otherwise. ⁎ Corresponding author. Department of Otolaryngology Head and Neck Surgery, Affiliated Hospital of Luzhou Medical College, 25 Taiping Street, Luzhou 646000, Sichuan Province, China. Tel.: +86 830 3165641; fax: +86 830 2392753. E-mail address: [email protected] (G. Qin). 1 Zhuoping Liang and Jinbo Liu contributed equally to this work and are to be regarded as equivalent authors. 0196-0709/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.amjoto.2013.10.009
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IC50 values for COX-2 and COX-1 is 139, NIM can exert effective anti-inflammatory effects with fewer side effects, such as peptic ulcers, gastrointestinal bleeding and renal prostaglandin inhibition, than other NSAIDs (e.g., aspirin, indomethacin, naproxen, piroxicam, and ibuprofen) [2,3]. In vivo and in vitro experiments demonstrated that NIM could inhibit the growth of colon, pancreatic, breast and lung cancers and multiple other tumors. NIM is frequently used in cancer prevention and treatment studies [4–7]. Selective COX-2 inhibitors have been increasingly applied to head and neck cancers that have high expression levels of COX-2. However, there have been no reports on the application and the mechanism of action of NIM in the treatment of laryngeal squamous cell carcinoma. COX-2 is strongly implicated in tumorigenesis. Its functions include stimulating tumor cell proliferation, inhibiting apoptosis, promoting tumor angiogenesis, enhancing tumor cell invasion, decreasing tumor-mediated immune suppression, inducing DNA damage and promoting carcinogenesis [8]. Recent studies have indicated that COX-2 expression is significantly increased in head and neck tumor tissues [9], and COX-2 over-expression can lead to unrestricted cell proliferation by affecting the expression levels of certain genes involved in cell proliferation and apoptosis, thus promoting the development of head and neck tumors [10]. Survivin is a member of the apoptosis inhibitor proteins and is normally expressed only in embryonic tissues and not in most terminally differentiated adult tissues. However, Survivin is selectively expressed in most tumor tissues, and overexpression is involved in the prognosis of certain tumors [11,12]. It can inhibit cytochrome C or caspase-8-induced caspase activation and can also prevent spontaneous activation of caspase-3 and caspase-7. Survivin prevents apoptosis via its regulation of the cell cycle at the G2/M phase [13–15]. In normal proliferating cells and transformed cells, proliferating cell nuclear antigen(PCNA) levels display a clear cyclical pattern. The levels of PCNA begin to increase in the DNA synthesis phase, reach their peak in the G1 and S phases of the cell cycle, begin to decline in the G2 and M phases, and reach their lowest levels in the G0 phase. The changes in the expression levels of PCNA are correlated with the DNA synthesis process. Due to this pattern of expression and its important function in cell proliferation, PCNA can be used as an indicator to evaluate cell proliferation and cell proliferation kinetics [16]. In this study, we investigated the effects of NIM on laryngeal squamous cell carcinoma Hep-2 cells by examining in vitro cell growth, cell cycle phase distribution, the growth of Hep-2 tumor xenografts in nude mice and the protein and mRNA expression levels of COX-2, Survivin and PCNA in tumors. This study was designed to explore the anti-tumor mechanisms of NIM and provide experimental evidence for the application of NIM in the prevention and treatment of laryngeal squamous cell carcinoma.
2.
Materials and methods
2.1.
Cell culture
The frozen Hep-2 cells (a human laryngeal squamous cell line from the Center for Biotherapy of Cancer of the
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National Key Laboratory of Biotherapy in the West China Hospital at Sichuan University) were recovered and seeded in 25 cm2 tissue culture flasks. After adding RPMI 1640 tissue culture medium, the cells were placed in a CO2 incubator at 37 °C and allowed to grow for 3–4 days. The cells were passaged after growing to confluence. Trypsin (0.25%) was added to digest the cell attachments for 1–2 min at room temperature. Once most of the cells were detached, as confirmed by inverted microscopy, the trypsin solution was removed. The culture medium was added, and the cell suspension was repeatedly pipetted to mix. After counting, the cell concentration was adjusted to 5 × 108 cells/l before seeding them in new tissue culture flasks. The cell growth was observed daily.
2.2.
Cell proliferation assay
Hep-2 cells in the logarithmic growth phase were digested with 0.25% trypsin and re-suspended to a single-cell suspension with RPMI 1640 cell culture medium. After adjusting the concentration of the cell suspension to 105 cells/ml, the cells were plated in 96-well culture plates at a density of 200 μl/well and then cultured in a CO2 incubator for 24 h. The tissue culture plates were removed the next day, and various concentrations of NIM (50 μM, 100 μM and 200 μM) (SigmaAldrich, St. Louis, MO) dissolved in culture medium at a final concentration of 0.1% DMSO were added to the cells for subsequent analyses. For each concentration, the experiment was performed in triplicate. In addition, a control group was treated with PBS alone, DMSO was as the solvent control. The cells were subsequently cultured for an additional 24, 48, 72 and 96 h. The plates were then removed, and 5 μl of 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5 mg/ml) solution was added to each well. The culture supernatant was discarded after 4 h of incubation in the dark at 37 °C. The OD492 value of each well was measured by a microplate reader. The rate of inhibition was calculated as (1 − [OD492 value of the experimental drug group]/[OD492 value of the blank control group]) × 100%.
2.3.
Flow cytometry (FCM)
The cells were treated with different concentrations of NIM (50 μM, 100 μM or 200 μM) and cultured for 48 h. The control group was treated with only culture medium. The single-cell suspensions were prepared by spinning down the cells at 800– 1000 rpm for 5 min, discarding the supernatants, washing twice with PBS and then re-suspending the cell pellets in PBS. The cells were fixed with cold 70% ethanol overnight at 4 °C, washed with PBS two times, stained with propidium iodide (PI) staining solution for 30 min at 4 °C and then filtered through a 400 μm mesh sieve. The cells were then collected, and the cellular DNA content in each phase of the cell cycle was measured by FCM in accordance with the instructions from the apoptosis detection kit. A DNA content histogram was generated, the proportion of cells in each phase was automatically simulated and the rate of apoptosis was calculated. For each concentration of NIM, the experiment was performed in triplicate.
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2.4. Measurement of COX-2, Survivin and PCNA by western blotting analysis Hep-2 cells in the logarithmic growth phase were seeded in 6well tissue culture plates at a density of 1 × 104 cells/well and cultured for 24 h. The cells were subsequently treated with 50 μM, 100 μM or 200 μM NIM, whereas the control group was treated with PBS. The culture medium was discarded after 48 h of incubation. The cells were washed with pre-chilled PBS twice and lysed with RIPA cell lysis buffer. The lysates were incubated on ice for 30 min, and the supernatant was collected after centrifuging at 10,000 rpm for 10 min at 4 °C. The supernatants were quantified using a Coomassie brilliant blue kit, and the aliquots were stored at − 70 °C. The total cellular protein (10 μg) was separated by a 12% (COX-2, PCNA) or a 15% (Survivin) denatured protein SDS-PAGE and then transferred to a PVDF membrane. After blocking the membrane in 5% skim milk for 2 h at 37 °C, the primary antibodies for a sheep anti-human COX-2 polyclonal antibody (Santa Cruz), a rabbit anti-human Survivin polyclonal antibody (Santa Cruz) and a rabbit anti-human PCNA polyclonal antibody (Wuhan Boster Biological Engineering Co) (1:200 dilution) were incubated overnight at 4 °C. The secondary antibodies were incubated for 2 h at 37 °C. After washing the membrane, the ECL substrate was added, and the membrane was exposed to film. The films were processed by developing and fixing.
2.5. Establishment and treatment of Hep-2 xenograft tumors in nude mice Six- to eight- week-old Balb/c nu/nu nude mice were acquired from the Experimental Animal Center at Sichuan University and housed in specific pathogen-free conditions. Hep-2 cells in the logarithmic growth phase were suspended in PBS at a concentration of 108 cells/ml. The cell suspensions (100 μl) were injected subcutaneously into the right sides of the backs of nude mice. When the tumor diameters became greater than 5 mm approximately 2 weeks later, the nude mice were randomly divided into the experimental and the control groups, with six mice in each group. The following procedures were performed. The experimental group (NIM treatment group) was treated with 0.2 ml of NIM injected intraperitoneally at a dosage of 50 mg/kg/2 d for a total of 4 weeks. The control group (saline group) was injected with normal saline intraperitoneally at 0.2 ml/2 d for a total of 4 weeks. The subcutaneous tumor long diameters (a) and short diameters (b) were measured every other day to calculate the tumor volumes (V = a × b2/2) for the tumor growth curves. The body weights of the nude mice were also measured once every other day. The mice were sacrificed after 4 weeks to dissect the orthotopic tumors. The tumor and body weights of nude mice were measured and recorded. The tumor inhibition rate was calculated as follows: (tumor weight of the control group − tumor weight of the experimental group)/(tumor weight of the control group) × 100%. The tumor tissue from each nude mouse was divided into two halves. One half was immediately cryopreserved in liquid nitrogen for RNA extraction, while the other half was fixed and paraffin embedded for sectioning and staining according to routine procedures. All animal experi-
ments were performed in accordance with a guideline from the Administration of Animal Experiments for Medical Research Purposes issued by the Ministry of Health of China. The protocol was approved by the Animal Experiment Administration Committee of Sichuan University. All surgery was performed under sodium pentobarbital anesthesia and accomplished in a clean surgery room with sterilized instruments. All efforts were made to minimize the suffering of the mice during the experiments.
2.6.
Pathological observations
The tumor tissue was processed with routine hematoxylin/ eosin staining. The pathological changes, such as necrosis and changes in cell morphology, in the two xenograft tumor groups were observed and compared by light microscopy.
2.7. Test of COX-2, Survivin and PCNA by immunohistochemistry analysis A goat anti-human COX-2 polyclonal antibody, a rabbit antihuman Survivin antibody and a rabbit anti-human PCNA polyclonal antibody were the primary antibodies used. PBS replaced the primary antibody in the negative control group. The positive control was the positive biopsy provided in the staining kit. Immunohistochemical staining was performed according to the manufacturer’s instructions. Brown staining in the cytoplasm or nucleus (PCNA was mainly in the nucleus) was classified as positive expression. The slides were examined under double-blind conditions. For each slide, representative areas (10 fields) with clear, positive staining, no nonspecific background color and a characteristic staining intensity were observed under 400 × magnification to detect positive cells.
2.8.
Detection of COX-2, Survivin and PCNA by RT-PCR
Portions of excised xenograft tumors from each group were immediately stored in liquid nitrogen for total RNA extraction. A two-step method was used for RT-PCR. The β-actin PCR reaction (upstream primer: TAGAAGCATTTGCGGTGG, downstream primer: GCTACGAGCTGCCTGACG, amplified product of 412 bp) consisted of the following: an initial denaturation for 4 min at 94 °C; 30 cycles of amplification with denaturation for 30 s at 94 °C, annealing for 30 s at 61 °C, and extension for 30 s at 72 °C; and a final extension for 7 min at 72 °C. The reaction for COX-2 (upstream primer: ACGCTGTCTAGCCAGAGTTT, downstream primer: TATAAGTGCGATTGTACCCG, amplified product of 567 bp) consisted of the following: an initial denaturation for 4 min at 94 °C; 35 cycles of amplification with denaturation for 30 s at 94 °C, annealing for 30 s at 56 °C, and extension for 30 s at 72 °C; and a final extension for 7 min at 72 °C. The reaction for Survivin (upstream primer: ATTGCTAAGGGGCCCACAGG, downstream primer: CTCAAGGACCACCGCATCTC, amplified product of 368 bp) consisted of the following: an initial denaturation for 4 min at 94 °C; 35 cycles of amplification with denaturation for 30 s at 94 °C, annealing for 30 s at 59 °C, and extension for 30 s at 72 °C; and a final extension for 7 min at 72 °C. The reaction for PCNA (upstream primer: AGTGTCCCATATCCGCAATT,
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Table 1 – The cell viability after NIM treatment for 24, 48, 72 or 96 h. Processing method Control DMSO 50 μM NIM 100 μM NIM 200 μM NIM a,b
Cell absorbance value 24 h 315.00 301.17 249.83 210.67 180.67
± ± ± ± ±
10.06 5.04 10.17a 12.47a 15.04a
48 h 324.50 302.17 226.00 201.83 72.17
± ± ± ± ±
7.31 4.40 6.96a 14.22a 11.43a
72 h 762.17 708.83 407.67 331.5 82.00
± ± ± ± ±
1160.17 1074.17 481.83 427.67 86.33
± ± ± ± ±
34.18 25.85 13.36a 46.34a 6.09a
24 h
48 h
72 h
96 h
0.00 4.39 20.86b 33.11b 42.63b
0.00 6.89 30.35b 37.81b 77.75b
0.00 7.00 46.51b 56.51b 89.24b
0.00 7.41 58.48b 63.14b 92.56b
P < 0.05, compared with control and DMSO group.
Statistical analysis
The SPSS15.0 statistical analysis software was used for the statistical analysis of the results. All values are shown as the mean ± standard error. The groups were compared using t-tests. The results with α = 0.05 and P < 0.05 were considered statistically significant.
3.
Results
3.1.
MTT test results
Compared with the control and DMSO groups, NIM treatment significantly inhibited Hep-2 cell growth in a time- and dosedependent manner (P < 0.05) (Table 1 and Fig. 1). The time at which this effect could first be observed varied according to the concentration of NIM in the tissue culture medium. Then, there was a not significant inhibition of cell growth using PBS compared with DMSO (P > 0.05), it indicated that DMSO had no obviously toxic properties.
3.2.
96 h
8.98 6.27 16.11a 5.79a 24.36a
downstream primer: CTCAAGGACCTCATCAACGA, amplified product of 685 bp) consisted of the following: an initial denaturation for 4 min at 94 °C; 30 cycles of amplification with denaturation for 30 s at 94 °C, annealing for 40 s at 52 °C, and extension for 30 s at 72 °C; and a final extension for 7 min at 72 °C. The final products (5 μl each) of the reactions were analyzed on 1.0% agarose gels.
2.9.
Inhibition of cell proliferation rate(%)
Analysis of apoptosis and the cell cycle by FCM
As shown in Fig. 2 and Table 2, the FCM results demonstrated that NIM could induce apoptosis in the Hep-2 cells in a dosedependent manner. When comparing cell phase distribution, the percentage of cells in the G0/G1 phase of the experimental group dropped from 74.90% to 56.40%, the percentage of cells in the S phase gradually increased from 16.3% to 41.90%, and the percentage of cells in the G2/M phase decreased from 8.83% to 1.67%. The differences were significant compared with the control group (P < 0.05) (Table 2 and Fig. 2). This indicated that NIM mostly caused an accumulation of cells in the S phase, thus blocking cell cycle progression. However, compared with control, treatment of Hep-2 cell with DMSO showed that the rate of apoptosis was not markedly increased (P > 0.05), indicating that DMSO was not obviously toxic to Hep-2 cells once again.
3.3. The effects of NIM on COX-2, Survivin, and PCNA protein expression in Hep-2 cells The Western blot analysis results showed that the Hep-2 cells had basal levels of COX-2, Survivin and PCNA protein expression. After treatment with NIM, the expression levels of COX-2, Survivin and PCNA were significantly lower compared with the control group. Moreover, NIM in a dose dependent manner inhibited both the COX-2 and Survivin protein expression level, but there were no obvious changes in the PCNA protein expression among different NIM concentrations (Fig. 3).
3.4.
Growth of xenograft tumors in nude mice
Subcutaneous tumor formation was observed 5 days after the xenograft in nude mice. The size of the tumors gradually increased with time with 100% tumor formation. The xenograft tumors were round and oval-nodular shaped. The tumor surfaces were smooth initially, but as the tumor sizes increased, the surfaces of some of the tumors showed ulceration, necrosis and scabbing. In the control group, the tumors in the nude mice grew continuously and significantly increased in volume. The NIM treatment group displayed slow tumor growth, with tumor volumes that were significantly smaller than the control group; the difference was statistically significant (P < 0.05) (Fig. 4). After 4 weeks of treatment, there was no significant difference in the average weight of nude mice between the two groups (P > 0.05), while there was a statistically significant difference in tumor nodule weight (P < 0.05) (Table 3). The growth of tumors was inhibited by 51.81% with NIM treatment.
Fig. 1 – The cell growth was measured by MTT assay at designed time points. A time- and dose-dependent growth inhibition was induced by NIM treatment .1 control, 2 DMSO, 3 50 μM NIM, 4 100 μM NIM, 5 200 μM NIM.
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control group. After NIM treatment, the expression levels of COX-2, Survivin and PCNA mRNA were significantly reduced (Fig. 7).
4.
The loss of regulatory control of cell proliferation and apoptosis is an important cause of tumorigenesis. Interventions against the dysfunctions of cell proliferation and apoptosis have become a well-studied area in cancer research. In this study, we found that NIM could inhibit Hep-2 cell proliferation, as determined by the MTT assay. NIM also significantly inhibited cell viability and induced cell apoptosis. Consistent with our findings, numerous studies using the MTT assay and FCM have also confirmed that selective COX-2 inhibitors can inhibit cell proliferation and induce apoptosis of head and neck cancer and esophageal cancer cells in a timeand dose-dependent manner [17–20]. One of the basic characteristics of tumor cells is uncontrolled cell growth. The mechanism underlying uncontrolled proliferation is the destruction of the cell cycle control machinery. However, regulation of cell cycle progression can potentially trigger cellular apoptosis to achieve anti-tumor effects. It has previously been reported that after treating esophageal cancer cells with 50 μM NIM, the cell cycle phase distribution displayed a gradual increase in G1/S phase cells and a reduction in G2/M phase cells the rate of apoptosis was dose-dependent [21]. This suggests that the effect of NIM on apoptosis was achieved by altering cell cycle progression. Our study did not identify cell cycle arrest in the G1 phase. In contrast, the percentage of cells in G1 phase decreased significantly compared with the control group, and the cells were arrested in S phase. Because the apoptotic cells in the experimental group accounted for a large population of the total cells and those apoptotic cells were mostly derived from cells in G1 phase, we believe that the G1 phase arrest might still occur in laryngeal squamous cell carcinoma Hep-2 cells before apoptosis. The mechanism by which the COX-2 inhibitors affect the cell cycle remains unclear. Han et al. [22] found that selective COX-2 inhibitors, such as NS-398, NIM and sulindac, can inhibit the expression of cyclins. The COX-2 inhibitors mainly act through inhibition of the positive cell cycle-regulated factor cyclin D1, thus inhibiting the activation of CDK4/6 and reducing the level of dephosphorylated Rb protein. This significantly decreases DNA synthesis and cell proliferation and increases the levels of cyclin-dependent
Fig. 2 – The effect of NIM on cell cycle distribution. Hep-2 cell was stained with propidium iodide and analyzed by flow cytometry. 1 control, 2 DMSO, 3 50 μM NIM, 4 100 μM NIM, 5 200 μM NIM.
3.5.
Discussion
Pathological observations
Hematoxylin/eosin (H&E) staining revealed that the tumor tissue from the NIM treatment group exhibited significant apoptosis and large areas of necrosis, with necrotic zones displaying homogeneous pink staining. We also observed a loss of cellular structure, decreased tumor cell density and reduced pathological mitosis. Fibrosis and inflammatory cell infiltration were also observed. In contrast, the tumor tissues from the control group showed no obvious necrosis, normal cell structure and significant tumor cell density and pathological mitosis (Fig. 5).
3.6. COX-2, Survivin and PCNA protein levels in xenograft tumor tissue The immunohistochemical analysis showed that COX-2, Survivin and PCNA proteins were expressed in the cytoplasm or nuclei (PCNA) of the xenograft tumor cells, with pale yellow to brown diffuse distributions. The protein expression levels of COX-2, Survivin and PCNA were all significantly reduced in the treated group (Fig. 6).
3.7. Expression of COX-2, Survivin and PCNA mRNA in xenograft tumor tissue The RT-PCR results showed that there were significant differences in the absorbance ratios of COX-2, Survivin and PCNA to β-actin between the NIM treatment group and the
Table 2 – Effects of NIM on apoptosis and the cell cycle in Hep-2 cells. Processing
Control DMSO 50 μM NIM 100 μm NIM7 200 μm NIM a,b
The rate of apoptosis (%) 2.63 4.00 40.17 0.10 87.53
± ± ± ± ±
0.33 0.56 1.72a 3.21a 1.96a
P < 0.05, compared with control and DMSO group.
Cell cycle distribution G0/G1(%) 74.90 72.97 58.50 56.63 56.40
± ± ± ± ±
0.26 0.55 0.44b 0.45b 0.36b
S(%) 16.30 18.10 37.90 39.93 41.90
± ± ± ± ±
0.36 0.46 0.36b 0.76b 0.36b
G2/M(%) 8.83 8.83 3.63 3.40 1.67
± ± ± ± ±
0.15 0.25 0.21b 0.26b 0.21b
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Fig. 3 – Suppression of COX-2, Survivin and PCNA expression of Hep-2 cells by NIM. Samples were from cultures treated with PBS (lane 1), with NIM 50 μΜ (lane 2), with NIM 100 μΜ (lane 3), or 200 μΜ (lane 4).
kinase inhibitors, such as p21 and p27. The inhibition of cell proliferation and the pro-apoptotic effects are achieved because cells cannot pass the G1/S checkpoint and are arrested in G0/G1 phase. Moreover, other reports have
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shown that the effects of NS-398 on esophageal squamous cell carcinoma TE-12 cell viability and arrest of the cell cycle in G1/S phase are mainly due to an increased level of p27Kip1 and a reduced level of cyclin B1 [23]. There are many studies that have focused on the NIM inhibition of the growth of head and neck cancer xenograft tumors in animal models. However, few studies of the effects of NIM on laryngeal squamous cell carcinoma have been reported. Our study successfully established a xenograft model of human laryngeal squamous cell carcinoma Hep-2 cells in nude mice with a 100% tumor formation rate. The results showed that the tumor growth of the NIM treatment group was significantly slower than the control group. Compared with the control group, the tumor volumes and the weights of the xenograft tumors were significantly reduced. These results indicate that NIM can significantly inhibit the growth of laryngeal squamous cell carcinoma xenografts in nude mice. Shaik et al. [24] also confirmed the role of NIM in inhibiting tumor growth by applying NIM to the lung adenocarcinoma A549 cell xenografts in nude mice. Likewise, other studies reported that 50 mg/2 d of NIM can inhibit the growth of poorly differentiated colon cancer
Fig. 4 – Effect of NIM on inhibition of Hep-2 cell xenograft tumors in nude mice. (A) The tumor growth curve. (B) Photographs of killed nude mice at 28 days after inoculation with Hep-2 cells. (C) The image of individual tumors.
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Table 3 – Effect of NIM on the growth of Hep-2 xenograft tumors in nude mice. Groups Experimental Control a,b
Sample numbers (n) 6 6
Volume of tumor (mm3) a
172.67 ± 115.25 478.69 ± 341.84
Weight of nude mice (g)
Weight of tumor (g)
24.05 ± 1.79 23.22 ± 1.78
0.40 ± 0.33b 0.83 ± 0.27
P < 0.05, compared with control group.
C-26 cells in Balb/c mice without significant side effects and that NIM can sooth radiotherapy-induced pain [25]. Moreover, this study found that NIM had no significant side effects, indicating that the dosage was within safety limits. In addition, the pathological and morphological evaluation of the xenograft tumor tissue revealed that the tumor tissue from the NIM group displayed more significant tumor necrosis, reduced tumor cell density and pathological mitosis than the control group, as well as substantial tumor cell apoptosis. These observations demonstrated, from a morphological standpoint, that NIM could inhibit tumor growth by inducing apoptosis and necrosis. Oyama et al. [26] found that COX-2 plays an important role in the processes of inflammation, dysplasia and adenocarcinoma. COX-2 and PGE2 expression and proliferation and the frequency of esophagitis and Barrett's esophagus were much lower in the NIM treatment group than in the control group. This suggests that NIM can inhibit COX-2 expression, thus preventing Barrett's esophagus and esophageal adenocarcinoma. Another study utilizing Western blot analysis to examine COX-2 protein expression showed that NIM had no effect on COX-2 protein expression in lung adenocarcinoma xenograft tumors [27]. In addition, celecoxib can inhibit the growth of head and neck squamous cell carcinoma xenograft tumors in nude mice without a significant impact on COX-2 protein expression [10], while PGE2 levels in tumor tissues were significantly reduced. Although the selective COX-2 inhibitors did not affect COX-2 expression levels in the two tumor xenograft groups, COX-2 activity was inhibited in both groups, thereby reducing the level of COX-2-induced PGE2 and subsequently inducing apoptosis and tumor growth inhibition. These studies indicate that the COX-2 activity-dependent pathways may be one of the mechanisms by which COX2 inhibitors repress tumors. Most scholars believe that NSAIDs play an anti-tumor role mainly in a COX-2 activity- or
expression level-dependent manner as discussed above [28–33]. Our study examined the expression level of COX-2 protein in laryngeal squamous cell carcinoma Hep-2 cells and the levels of COX-2 protein and mRNA in xenograft tumor tissues. The results showed that NIM effectively inhibits the levels of COX-2 protein and mRNA in the Hep-2 xenograft tumors. We thus concluded that NIM inhibition of the growth of laryngeal squamous cell carcinoma Hep-2 cells and xenograft tumors in nude mice might depend on the COX-2 expression level. Survivin is an important inhibitor of apoptosis. Scheper et al. [10] utilized in vivo and in vitro experiments to demonstrate that sulindac downregulates Survivin expression by acting on the Stat3 signaling pathway, which inhibits proliferation of the laryngeal squamous cell carcinoma Hep-2 cells and induces cell apoptosis. Apoptosis consists of a complex network of regulatory processes. The specific molecular mechanisms underlying apoptosis remain unclear. Previous studies have reported that celecoxib can significantly reduce the expression of Survivin in cells. The fact that Survivin expression depends on the expression of cellular COX-2 suggests that COX-2 and Survivin might share a common activation mechanism and that COX-2 might act as an upstream regulatory factor, the high expression of which upregulates Survivin expression [34,35]. The level of tumor cell proliferation reflects the degree of malignancy. The loss of tumor suppressor genes or point mutations disrupts normal cell proliferation and differentiation, which can be manifested through malignant transformation of cells and the development of cancer caused by overexpression of the cell proliferation marker PCNA. This is consistent with the biological behavior of laryngeal squamous cell carcinomas.. During a study of the effects of baicalin on tumor cell proliferation and PGE2 synthesis, Zhang et al. [36] observed that both celecoxib and NIM could reduce the expression of PCNA by inhibiting
Fig. 5 – Histopathology(H&E) staining in Hep-2 cell xenograft tumor masses of each sample. Necrosis and changes of cell morphology in the two xenograft tumor groups were observed and compared by light microscopy. (A) Experimental group ( × 100). (B) Control group( × 100).
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Fig. 6 – Immunocytochemical staining for COX-2, Survivin and PCNA proteins in xenograft tumor tissues. (A), (C), (E) refer to experimental group, respectively( × 400). (B), (D), (F) refer to control group, respectively( × 400).
PGE2 synthesis. A preliminary conclusion may be that COX-2 affects PCNA expression via PGE2, which subsequently influences cell proliferation and promotes tumorigenesis. This is consistent with the fact that cell proliferation was increased
in COX-2-induced tumorigenesis and suggests that selective COX-2 inhibitors may inhibit PCNA by affecting the expression or activity of the COX-2 product PGE2, thus inhibiting the growth of laryngeal squamous cell carcinomas. The present
Fig. 7 – Expression of COX-2, Survivin and PCNA mRNA in xenograft tumor tissues in nude mice by RT-PCR analysis.
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study examined the expression levels of Survivin and PCNA in Hep-2 cells in vitro and xenograft tumors in vivo. Our results showed that the protein levels of Survivin and PCNA were significantly reduced compared with the control group after treating Hep-2 cells with NIM. Similarly, after treating xenograft tumors with NIM, the protein and mRNA expression levels of Survivin and PCNA were significantly reduced, suggesting that the effects of NIM, including inducing Hep-2 cell apoptosis and inhibiting cell proliferation and xenograft tumors growth in nude mice, may be related to the downregulation of Survivin and PCNA. NIM, a new type of NSAID, has strong anti-tumor effect and low side effect. The application of NIM in the clinical treatment of colon cancer has been reported, but its application in head and neck cancer is still in the experimental stage, and NIM has not yet been used clinically on a large scale. NIM demonstrated excellent anti-tumor properties in laryngeal squamous cell carcinoma Hep-2 cells and in vivo experiments. We suggest that its function in inducing apoptosis and inhibiting cell proliferation and the growth of laryngeal squamous cell carcinoma xenografts in nude mice is the combined result of its effects on the cell cycle, the downregulation of COX-2, Survivin and PCNA, and a variety of other factors. However, the specific mechanism may be much more complicated and diverse and requires further study. We believe that additional research on the relationship between COX-2, tumors, including laryngeal squamous cell carcinoma, and selective COX-2 inhibitors, would lead to the development of selective COX-2 inhibitors with higher specificity, better efficacy and fewer side effects.
5.
Conclusion
Selective COX-2 inhibitor NIM inhibited the growth of laryngeal squamous cell carcinoma Hep-2 cell and xenografts in nude mice. NIM has no obvious toxic side effect. Selective COX-2 inhibitor is expected to become a part of comprehensive treatment for laryngeal squamous cell carcinoma in the future, it may provide a new prospect for the prevention and treatment of laryngeal squamous cell carcinoma.
Acknowledgments The authors sincerely acknowledge Ling Yu M.D of ophthalmology, Affiliated Hospital of Luzhou Medical College and Yongsheng Wang M.D, Honggang Lin M.M and Huashan Shi M. D of the Center for Biotherapy of Cancer of the National Key Laboratory of Biotherapy in the West China Hospital at Sichuan University, for their generous technical support and help. Then the authors thank the grant support of Sichuan Province applied basic research project (05JY029-104).
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Effect of nimesulide on the growth of human laryngeal squamous cell carcinoma☆ Zhuoping Liang, MM a, 1 , Jinbo Liu, MD b, 1 , Leiji Li, MM a , Haiyang Wang, MM a , Chong Zhao, MM a , Liang Jiang, MM a , Gang Qin, MD a,⁎ a Department of Otolaryngology Head and Neck Surgery, Affiliated Hospital of Luzhou Medical College, Luzhou 646000, Sichuan Province, China b Department of Clinical Laboratory, Affiliated Hospital of Luzhou Medical College, Luzhou 646000, Sichuan Province, China
ARTI CLE I NFO
A BS TRACT
Article history:
Purpose: To investigate the effect of nimesulide on the growth of human laryngeal
Received 27 August 2013
squamous cell carcinoma. Materials and methods: The effect of NIM on Hep-2 cell proliferation was measured by the MTT assay. Flow cytometry was used to evaluate the cell cycle and apoptosis in Hep-2 cells. A Western blot analysis was used to detect changes in the protein expression levels of COX-2, Survivin and proliferating cell nuclear antigen (PCNA) in Hep-2 cells. A Hep-2 tumor xenograft model was established in nude mice to observe tumor growth. The changes in the xenograft tumors were observed after hematoxylin/eosin staining. The expression levels of COX-2, Survivin and PCNA proteins and mRNA were measured by immunohistochemical analysis and RT-PCR, respectively. Results: NIM had time- and dose-dependent inhibitory effect on the proliferation of Hep-2 cells. NIM could prevent the progression of the cell cycle. After NIM treatment, COX-2, Survivin and PCNA protein levels were reduced in the Hep-2 cells. The volume and weight of the xenograft tumors in the NIM treatment group were significantly reduced. The NIM treatment group also exhibited significantly reduced expression levels of COX-2, Survivin and PCNA at both the protein and mRNA levels. Conclusions: Our results suggested that NIM has significant inhibitory effects on the growth of Hep-2 cells and xenograft tumors in nude mice. Selective COX-2 inhibitors could potentially become part of a comprehensive treatment for laryngeal squamous cell carcinoma. Additional research and development will provide new and broader prospects for the prevention and treatment of laryngeal squamous cell carcinoma. © 2014 Elsevier Inc. All rights reserved.
1.
Introduction
Recent studies have demonstrated that non-steroidal antiinflammatory drugs (NSAIDs) can prevent and treat various tumors. Selective cyclooxygenase-2 (COX-2) inhibitors exhibit high preferential inhibition of COX-2 and only minor effects on
cyclooxygenase-1(COX-1). With their lower side effects and high specificity, selective COX-2 inhibitors have become a focus for cancer prevention and treatment, gradually replacing traditional NSAIDs in clinical applications [1]. Nimesulide (NIM), which only weakly inhibits the protective COX-1, was a highly specific COX-2 selective inhibitor. Because the ratio of its
☆
None of the authors has any conflict of interest, financial or otherwise. ⁎ Corresponding author. Department of Otolaryngology Head and Neck Surgery, Affiliated Hospital of Luzhou Medical College, 25 Taiping Street, Luzhou 646000, Sichuan Province, China. Tel.: +86 830 3165641; fax: +86 830 2392753. E-mail address: [email protected] (G. Qin). 1 Zhuoping Liang and Jinbo Liu contributed equally to this work and are to be regarded as equivalent authors. 0196-0709/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.amjoto.2013.10.009
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IC50 values for COX-2 and COX-1 is 139, NIM can exert effective anti-inflammatory effects with fewer side effects, such as peptic ulcers, gastrointestinal bleeding and renal prostaglandin inhibition, than other NSAIDs (e.g., aspirin, indomethacin, naproxen, piroxicam, and ibuprofen) [2,3]. In vivo and in vitro experiments demonstrated that NIM could inhibit the growth of colon, pancreatic, breast and lung cancers and multiple other tumors. NIM is frequently used in cancer prevention and treatment studies [4–7]. Selective COX-2 inhibitors have been increasingly applied to head and neck cancers that have high expression levels of COX-2. However, there have been no reports on the application and the mechanism of action of NIM in the treatment of laryngeal squamous cell carcinoma. COX-2 is strongly implicated in tumorigenesis. Its functions include stimulating tumor cell proliferation, inhibiting apoptosis, promoting tumor angiogenesis, enhancing tumor cell invasion, decreasing tumor-mediated immune suppression, inducing DNA damage and promoting carcinogenesis [8]. Recent studies have indicated that COX-2 expression is significantly increased in head and neck tumor tissues [9], and COX-2 over-expression can lead to unrestricted cell proliferation by affecting the expression levels of certain genes involved in cell proliferation and apoptosis, thus promoting the development of head and neck tumors [10]. Survivin is a member of the apoptosis inhibitor proteins and is normally expressed only in embryonic tissues and not in most terminally differentiated adult tissues. However, Survivin is selectively expressed in most tumor tissues, and overexpression is involved in the prognosis of certain tumors [11,12]. It can inhibit cytochrome C or caspase-8-induced caspase activation and can also prevent spontaneous activation of caspase-3 and caspase-7. Survivin prevents apoptosis via its regulation of the cell cycle at the G2/M phase [13–15]. In normal proliferating cells and transformed cells, proliferating cell nuclear antigen(PCNA) levels display a clear cyclical pattern. The levels of PCNA begin to increase in the DNA synthesis phase, reach their peak in the G1 and S phases of the cell cycle, begin to decline in the G2 and M phases, and reach their lowest levels in the G0 phase. The changes in the expression levels of PCNA are correlated with the DNA synthesis process. Due to this pattern of expression and its important function in cell proliferation, PCNA can be used as an indicator to evaluate cell proliferation and cell proliferation kinetics [16]. In this study, we investigated the effects of NIM on laryngeal squamous cell carcinoma Hep-2 cells by examining in vitro cell growth, cell cycle phase distribution, the growth of Hep-2 tumor xenografts in nude mice and the protein and mRNA expression levels of COX-2, Survivin and PCNA in tumors. This study was designed to explore the anti-tumor mechanisms of NIM and provide experimental evidence for the application of NIM in the prevention and treatment of laryngeal squamous cell carcinoma.
2.
Materials and methods
2.1.
Cell culture
The frozen Hep-2 cells (a human laryngeal squamous cell line from the Center for Biotherapy of Cancer of the
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National Key Laboratory of Biotherapy in the West China Hospital at Sichuan University) were recovered and seeded in 25 cm2 tissue culture flasks. After adding RPMI 1640 tissue culture medium, the cells were placed in a CO2 incubator at 37 °C and allowed to grow for 3–4 days. The cells were passaged after growing to confluence. Trypsin (0.25%) was added to digest the cell attachments for 1–2 min at room temperature. Once most of the cells were detached, as confirmed by inverted microscopy, the trypsin solution was removed. The culture medium was added, and the cell suspension was repeatedly pipetted to mix. After counting, the cell concentration was adjusted to 5 × 108 cells/l before seeding them in new tissue culture flasks. The cell growth was observed daily.
2.2.
Cell proliferation assay
Hep-2 cells in the logarithmic growth phase were digested with 0.25% trypsin and re-suspended to a single-cell suspension with RPMI 1640 cell culture medium. After adjusting the concentration of the cell suspension to 105 cells/ml, the cells were plated in 96-well culture plates at a density of 200 μl/well and then cultured in a CO2 incubator for 24 h. The tissue culture plates were removed the next day, and various concentrations of NIM (50 μM, 100 μM and 200 μM) (SigmaAldrich, St. Louis, MO) dissolved in culture medium at a final concentration of 0.1% DMSO were added to the cells for subsequent analyses. For each concentration, the experiment was performed in triplicate. In addition, a control group was treated with PBS alone, DMSO was as the solvent control. The cells were subsequently cultured for an additional 24, 48, 72 and 96 h. The plates were then removed, and 5 μl of 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5 mg/ml) solution was added to each well. The culture supernatant was discarded after 4 h of incubation in the dark at 37 °C. The OD492 value of each well was measured by a microplate reader. The rate of inhibition was calculated as (1 − [OD492 value of the experimental drug group]/[OD492 value of the blank control group]) × 100%.
2.3.
Flow cytometry (FCM)
The cells were treated with different concentrations of NIM (50 μM, 100 μM or 200 μM) and cultured for 48 h. The control group was treated with only culture medium. The single-cell suspensions were prepared by spinning down the cells at 800– 1000 rpm for 5 min, discarding the supernatants, washing twice with PBS and then re-suspending the cell pellets in PBS. The cells were fixed with cold 70% ethanol overnight at 4 °C, washed with PBS two times, stained with propidium iodide (PI) staining solution for 30 min at 4 °C and then filtered through a 400 μm mesh sieve. The cells were then collected, and the cellular DNA content in each phase of the cell cycle was measured by FCM in accordance with the instructions from the apoptosis detection kit. A DNA content histogram was generated, the proportion of cells in each phase was automatically simulated and the rate of apoptosis was calculated. For each concentration of NIM, the experiment was performed in triplicate.
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2.4. Measurement of COX-2, Survivin and PCNA by western blotting analysis Hep-2 cells in the logarithmic growth phase were seeded in 6well tissue culture plates at a density of 1 × 104 cells/well and cultured for 24 h. The cells were subsequently treated with 50 μM, 100 μM or 200 μM NIM, whereas the control group was treated with PBS. The culture medium was discarded after 48 h of incubation. The cells were washed with pre-chilled PBS twice and lysed with RIPA cell lysis buffer. The lysates were incubated on ice for 30 min, and the supernatant was collected after centrifuging at 10,000 rpm for 10 min at 4 °C. The supernatants were quantified using a Coomassie brilliant blue kit, and the aliquots were stored at − 70 °C. The total cellular protein (10 μg) was separated by a 12% (COX-2, PCNA) or a 15% (Survivin) denatured protein SDS-PAGE and then transferred to a PVDF membrane. After blocking the membrane in 5% skim milk for 2 h at 37 °C, the primary antibodies for a sheep anti-human COX-2 polyclonal antibody (Santa Cruz), a rabbit anti-human Survivin polyclonal antibody (Santa Cruz) and a rabbit anti-human PCNA polyclonal antibody (Wuhan Boster Biological Engineering Co) (1:200 dilution) were incubated overnight at 4 °C. The secondary antibodies were incubated for 2 h at 37 °C. After washing the membrane, the ECL substrate was added, and the membrane was exposed to film. The films were processed by developing and fixing.
2.5. Establishment and treatment of Hep-2 xenograft tumors in nude mice Six- to eight- week-old Balb/c nu/nu nude mice were acquired from the Experimental Animal Center at Sichuan University and housed in specific pathogen-free conditions. Hep-2 cells in the logarithmic growth phase were suspended in PBS at a concentration of 108 cells/ml. The cell suspensions (100 μl) were injected subcutaneously into the right sides of the backs of nude mice. When the tumor diameters became greater than 5 mm approximately 2 weeks later, the nude mice were randomly divided into the experimental and the control groups, with six mice in each group. The following procedures were performed. The experimental group (NIM treatment group) was treated with 0.2 ml of NIM injected intraperitoneally at a dosage of 50 mg/kg/2 d for a total of 4 weeks. The control group (saline group) was injected with normal saline intraperitoneally at 0.2 ml/2 d for a total of 4 weeks. The subcutaneous tumor long diameters (a) and short diameters (b) were measured every other day to calculate the tumor volumes (V = a × b2/2) for the tumor growth curves. The body weights of the nude mice were also measured once every other day. The mice were sacrificed after 4 weeks to dissect the orthotopic tumors. The tumor and body weights of nude mice were measured and recorded. The tumor inhibition rate was calculated as follows: (tumor weight of the control group − tumor weight of the experimental group)/(tumor weight of the control group) × 100%. The tumor tissue from each nude mouse was divided into two halves. One half was immediately cryopreserved in liquid nitrogen for RNA extraction, while the other half was fixed and paraffin embedded for sectioning and staining according to routine procedures. All animal experi-
ments were performed in accordance with a guideline from the Administration of Animal Experiments for Medical Research Purposes issued by the Ministry of Health of China. The protocol was approved by the Animal Experiment Administration Committee of Sichuan University. All surgery was performed under sodium pentobarbital anesthesia and accomplished in a clean surgery room with sterilized instruments. All efforts were made to minimize the suffering of the mice during the experiments.
2.6.
Pathological observations
The tumor tissue was processed with routine hematoxylin/ eosin staining. The pathological changes, such as necrosis and changes in cell morphology, in the two xenograft tumor groups were observed and compared by light microscopy.
2.7. Test of COX-2, Survivin and PCNA by immunohistochemistry analysis A goat anti-human COX-2 polyclonal antibody, a rabbit antihuman Survivin antibody and a rabbit anti-human PCNA polyclonal antibody were the primary antibodies used. PBS replaced the primary antibody in the negative control group. The positive control was the positive biopsy provided in the staining kit. Immunohistochemical staining was performed according to the manufacturer’s instructions. Brown staining in the cytoplasm or nucleus (PCNA was mainly in the nucleus) was classified as positive expression. The slides were examined under double-blind conditions. For each slide, representative areas (10 fields) with clear, positive staining, no nonspecific background color and a characteristic staining intensity were observed under 400 × magnification to detect positive cells.
2.8.
Detection of COX-2, Survivin and PCNA by RT-PCR
Portions of excised xenograft tumors from each group were immediately stored in liquid nitrogen for total RNA extraction. A two-step method was used for RT-PCR. The β-actin PCR reaction (upstream primer: TAGAAGCATTTGCGGTGG, downstream primer: GCTACGAGCTGCCTGACG, amplified product of 412 bp) consisted of the following: an initial denaturation for 4 min at 94 °C; 30 cycles of amplification with denaturation for 30 s at 94 °C, annealing for 30 s at 61 °C, and extension for 30 s at 72 °C; and a final extension for 7 min at 72 °C. The reaction for COX-2 (upstream primer: ACGCTGTCTAGCCAGAGTTT, downstream primer: TATAAGTGCGATTGTACCCG, amplified product of 567 bp) consisted of the following: an initial denaturation for 4 min at 94 °C; 35 cycles of amplification with denaturation for 30 s at 94 °C, annealing for 30 s at 56 °C, and extension for 30 s at 72 °C; and a final extension for 7 min at 72 °C. The reaction for Survivin (upstream primer: ATTGCTAAGGGGCCCACAGG, downstream primer: CTCAAGGACCACCGCATCTC, amplified product of 368 bp) consisted of the following: an initial denaturation for 4 min at 94 °C; 35 cycles of amplification with denaturation for 30 s at 94 °C, annealing for 30 s at 59 °C, and extension for 30 s at 72 °C; and a final extension for 7 min at 72 °C. The reaction for PCNA (upstream primer: AGTGTCCCATATCCGCAATT,
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Table 1 – The cell viability after NIM treatment for 24, 48, 72 or 96 h. Processing method Control DMSO 50 μM NIM 100 μM NIM 200 μM NIM a,b
Cell absorbance value 24 h 315.00 301.17 249.83 210.67 180.67
± ± ± ± ±
10.06 5.04 10.17a 12.47a 15.04a
48 h 324.50 302.17 226.00 201.83 72.17
± ± ± ± ±
7.31 4.40 6.96a 14.22a 11.43a
72 h 762.17 708.83 407.67 331.5 82.00
± ± ± ± ±
1160.17 1074.17 481.83 427.67 86.33
± ± ± ± ±
34.18 25.85 13.36a 46.34a 6.09a
24 h
48 h
72 h
96 h
0.00 4.39 20.86b 33.11b 42.63b
0.00 6.89 30.35b 37.81b 77.75b
0.00 7.00 46.51b 56.51b 89.24b
0.00 7.41 58.48b 63.14b 92.56b
P < 0.05, compared with control and DMSO group.
Statistical analysis
The SPSS15.0 statistical analysis software was used for the statistical analysis of the results. All values are shown as the mean ± standard error. The groups were compared using t-tests. The results with α = 0.05 and P < 0.05 were considered statistically significant.
3.
Results
3.1.
MTT test results
Compared with the control and DMSO groups, NIM treatment significantly inhibited Hep-2 cell growth in a time- and dosedependent manner (P < 0.05) (Table 1 and Fig. 1). The time at which this effect could first be observed varied according to the concentration of NIM in the tissue culture medium. Then, there was a not significant inhibition of cell growth using PBS compared with DMSO (P > 0.05), it indicated that DMSO had no obviously toxic properties.
3.2.
96 h
8.98 6.27 16.11a 5.79a 24.36a
downstream primer: CTCAAGGACCTCATCAACGA, amplified product of 685 bp) consisted of the following: an initial denaturation for 4 min at 94 °C; 30 cycles of amplification with denaturation for 30 s at 94 °C, annealing for 40 s at 52 °C, and extension for 30 s at 72 °C; and a final extension for 7 min at 72 °C. The final products (5 μl each) of the reactions were analyzed on 1.0% agarose gels.
2.9.
Inhibition of cell proliferation rate(%)
Analysis of apoptosis and the cell cycle by FCM
As shown in Fig. 2 and Table 2, the FCM results demonstrated that NIM could induce apoptosis in the Hep-2 cells in a dosedependent manner. When comparing cell phase distribution, the percentage of cells in the G0/G1 phase of the experimental group dropped from 74.90% to 56.40%, the percentage of cells in the S phase gradually increased from 16.3% to 41.90%, and the percentage of cells in the G2/M phase decreased from 8.83% to 1.67%. The differences were significant compared with the control group (P < 0.05) (Table 2 and Fig. 2). This indicated that NIM mostly caused an accumulation of cells in the S phase, thus blocking cell cycle progression. However, compared with control, treatment of Hep-2 cell with DMSO showed that the rate of apoptosis was not markedly increased (P > 0.05), indicating that DMSO was not obviously toxic to Hep-2 cells once again.
3.3. The effects of NIM on COX-2, Survivin, and PCNA protein expression in Hep-2 cells The Western blot analysis results showed that the Hep-2 cells had basal levels of COX-2, Survivin and PCNA protein expression. After treatment with NIM, the expression levels of COX-2, Survivin and PCNA were significantly lower compared with the control group. Moreover, NIM in a dose dependent manner inhibited both the COX-2 and Survivin protein expression level, but there were no obvious changes in the PCNA protein expression among different NIM concentrations (Fig. 3).
3.4.
Growth of xenograft tumors in nude mice
Subcutaneous tumor formation was observed 5 days after the xenograft in nude mice. The size of the tumors gradually increased with time with 100% tumor formation. The xenograft tumors were round and oval-nodular shaped. The tumor surfaces were smooth initially, but as the tumor sizes increased, the surfaces of some of the tumors showed ulceration, necrosis and scabbing. In the control group, the tumors in the nude mice grew continuously and significantly increased in volume. The NIM treatment group displayed slow tumor growth, with tumor volumes that were significantly smaller than the control group; the difference was statistically significant (P < 0.05) (Fig. 4). After 4 weeks of treatment, there was no significant difference in the average weight of nude mice between the two groups (P > 0.05), while there was a statistically significant difference in tumor nodule weight (P < 0.05) (Table 3). The growth of tumors was inhibited by 51.81% with NIM treatment.
Fig. 1 – The cell growth was measured by MTT assay at designed time points. A time- and dose-dependent growth inhibition was induced by NIM treatment .1 control, 2 DMSO, 3 50 μM NIM, 4 100 μM NIM, 5 200 μM NIM.
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control group. After NIM treatment, the expression levels of COX-2, Survivin and PCNA mRNA were significantly reduced (Fig. 7).
4.
The loss of regulatory control of cell proliferation and apoptosis is an important cause of tumorigenesis. Interventions against the dysfunctions of cell proliferation and apoptosis have become a well-studied area in cancer research. In this study, we found that NIM could inhibit Hep-2 cell proliferation, as determined by the MTT assay. NIM also significantly inhibited cell viability and induced cell apoptosis. Consistent with our findings, numerous studies using the MTT assay and FCM have also confirmed that selective COX-2 inhibitors can inhibit cell proliferation and induce apoptosis of head and neck cancer and esophageal cancer cells in a timeand dose-dependent manner [17–20]. One of the basic characteristics of tumor cells is uncontrolled cell growth. The mechanism underlying uncontrolled proliferation is the destruction of the cell cycle control machinery. However, regulation of cell cycle progression can potentially trigger cellular apoptosis to achieve anti-tumor effects. It has previously been reported that after treating esophageal cancer cells with 50 μM NIM, the cell cycle phase distribution displayed a gradual increase in G1/S phase cells and a reduction in G2/M phase cells the rate of apoptosis was dose-dependent [21]. This suggests that the effect of NIM on apoptosis was achieved by altering cell cycle progression. Our study did not identify cell cycle arrest in the G1 phase. In contrast, the percentage of cells in G1 phase decreased significantly compared with the control group, and the cells were arrested in S phase. Because the apoptotic cells in the experimental group accounted for a large population of the total cells and those apoptotic cells were mostly derived from cells in G1 phase, we believe that the G1 phase arrest might still occur in laryngeal squamous cell carcinoma Hep-2 cells before apoptosis. The mechanism by which the COX-2 inhibitors affect the cell cycle remains unclear. Han et al. [22] found that selective COX-2 inhibitors, such as NS-398, NIM and sulindac, can inhibit the expression of cyclins. The COX-2 inhibitors mainly act through inhibition of the positive cell cycle-regulated factor cyclin D1, thus inhibiting the activation of CDK4/6 and reducing the level of dephosphorylated Rb protein. This significantly decreases DNA synthesis and cell proliferation and increases the levels of cyclin-dependent
Fig. 2 – The effect of NIM on cell cycle distribution. Hep-2 cell was stained with propidium iodide and analyzed by flow cytometry. 1 control, 2 DMSO, 3 50 μM NIM, 4 100 μM NIM, 5 200 μM NIM.
3.5.
Discussion
Pathological observations
Hematoxylin/eosin (H&E) staining revealed that the tumor tissue from the NIM treatment group exhibited significant apoptosis and large areas of necrosis, with necrotic zones displaying homogeneous pink staining. We also observed a loss of cellular structure, decreased tumor cell density and reduced pathological mitosis. Fibrosis and inflammatory cell infiltration were also observed. In contrast, the tumor tissues from the control group showed no obvious necrosis, normal cell structure and significant tumor cell density and pathological mitosis (Fig. 5).
3.6. COX-2, Survivin and PCNA protein levels in xenograft tumor tissue The immunohistochemical analysis showed that COX-2, Survivin and PCNA proteins were expressed in the cytoplasm or nuclei (PCNA) of the xenograft tumor cells, with pale yellow to brown diffuse distributions. The protein expression levels of COX-2, Survivin and PCNA were all significantly reduced in the treated group (Fig. 6).
3.7. Expression of COX-2, Survivin and PCNA mRNA in xenograft tumor tissue The RT-PCR results showed that there were significant differences in the absorbance ratios of COX-2, Survivin and PCNA to β-actin between the NIM treatment group and the
Table 2 – Effects of NIM on apoptosis and the cell cycle in Hep-2 cells. Processing
Control DMSO 50 μM NIM 100 μm NIM7 200 μm NIM a,b
The rate of apoptosis (%) 2.63 4.00 40.17 0.10 87.53
± ± ± ± ±
0.33 0.56 1.72a 3.21a 1.96a
P < 0.05, compared with control and DMSO group.
Cell cycle distribution G0/G1(%) 74.90 72.97 58.50 56.63 56.40
± ± ± ± ±
0.26 0.55 0.44b 0.45b 0.36b
S(%) 16.30 18.10 37.90 39.93 41.90
± ± ± ± ±
0.36 0.46 0.36b 0.76b 0.36b
G2/M(%) 8.83 8.83 3.63 3.40 1.67
± ± ± ± ±
0.15 0.25 0.21b 0.26b 0.21b
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Fig. 3 – Suppression of COX-2, Survivin and PCNA expression of Hep-2 cells by NIM. Samples were from cultures treated with PBS (lane 1), with NIM 50 μΜ (lane 2), with NIM 100 μΜ (lane 3), or 200 μΜ (lane 4).
kinase inhibitors, such as p21 and p27. The inhibition of cell proliferation and the pro-apoptotic effects are achieved because cells cannot pass the G1/S checkpoint and are arrested in G0/G1 phase. Moreover, other reports have
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shown that the effects of NS-398 on esophageal squamous cell carcinoma TE-12 cell viability and arrest of the cell cycle in G1/S phase are mainly due to an increased level of p27Kip1 and a reduced level of cyclin B1 [23]. There are many studies that have focused on the NIM inhibition of the growth of head and neck cancer xenograft tumors in animal models. However, few studies of the effects of NIM on laryngeal squamous cell carcinoma have been reported. Our study successfully established a xenograft model of human laryngeal squamous cell carcinoma Hep-2 cells in nude mice with a 100% tumor formation rate. The results showed that the tumor growth of the NIM treatment group was significantly slower than the control group. Compared with the control group, the tumor volumes and the weights of the xenograft tumors were significantly reduced. These results indicate that NIM can significantly inhibit the growth of laryngeal squamous cell carcinoma xenografts in nude mice. Shaik et al. [24] also confirmed the role of NIM in inhibiting tumor growth by applying NIM to the lung adenocarcinoma A549 cell xenografts in nude mice. Likewise, other studies reported that 50 mg/2 d of NIM can inhibit the growth of poorly differentiated colon cancer
Fig. 4 – Effect of NIM on inhibition of Hep-2 cell xenograft tumors in nude mice. (A) The tumor growth curve. (B) Photographs of killed nude mice at 28 days after inoculation with Hep-2 cells. (C) The image of individual tumors.
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Table 3 – Effect of NIM on the growth of Hep-2 xenograft tumors in nude mice. Groups Experimental Control a,b
Sample numbers (n) 6 6
Volume of tumor (mm3) a
172.67 ± 115.25 478.69 ± 341.84
Weight of nude mice (g)
Weight of tumor (g)
24.05 ± 1.79 23.22 ± 1.78
0.40 ± 0.33b 0.83 ± 0.27
P < 0.05, compared with control group.
C-26 cells in Balb/c mice without significant side effects and that NIM can sooth radiotherapy-induced pain [25]. Moreover, this study found that NIM had no significant side effects, indicating that the dosage was within safety limits. In addition, the pathological and morphological evaluation of the xenograft tumor tissue revealed that the tumor tissue from the NIM group displayed more significant tumor necrosis, reduced tumor cell density and pathological mitosis than the control group, as well as substantial tumor cell apoptosis. These observations demonstrated, from a morphological standpoint, that NIM could inhibit tumor growth by inducing apoptosis and necrosis. Oyama et al. [26] found that COX-2 plays an important role in the processes of inflammation, dysplasia and adenocarcinoma. COX-2 and PGE2 expression and proliferation and the frequency of esophagitis and Barrett's esophagus were much lower in the NIM treatment group than in the control group. This suggests that NIM can inhibit COX-2 expression, thus preventing Barrett's esophagus and esophageal adenocarcinoma. Another study utilizing Western blot analysis to examine COX-2 protein expression showed that NIM had no effect on COX-2 protein expression in lung adenocarcinoma xenograft tumors [27]. In addition, celecoxib can inhibit the growth of head and neck squamous cell carcinoma xenograft tumors in nude mice without a significant impact on COX-2 protein expression [10], while PGE2 levels in tumor tissues were significantly reduced. Although the selective COX-2 inhibitors did not affect COX-2 expression levels in the two tumor xenograft groups, COX-2 activity was inhibited in both groups, thereby reducing the level of COX-2-induced PGE2 and subsequently inducing apoptosis and tumor growth inhibition. These studies indicate that the COX-2 activity-dependent pathways may be one of the mechanisms by which COX2 inhibitors repress tumors. Most scholars believe that NSAIDs play an anti-tumor role mainly in a COX-2 activity- or
expression level-dependent manner as discussed above [28–33]. Our study examined the expression level of COX-2 protein in laryngeal squamous cell carcinoma Hep-2 cells and the levels of COX-2 protein and mRNA in xenograft tumor tissues. The results showed that NIM effectively inhibits the levels of COX-2 protein and mRNA in the Hep-2 xenograft tumors. We thus concluded that NIM inhibition of the growth of laryngeal squamous cell carcinoma Hep-2 cells and xenograft tumors in nude mice might depend on the COX-2 expression level. Survivin is an important inhibitor of apoptosis. Scheper et al. [10] utilized in vivo and in vitro experiments to demonstrate that sulindac downregulates Survivin expression by acting on the Stat3 signaling pathway, which inhibits proliferation of the laryngeal squamous cell carcinoma Hep-2 cells and induces cell apoptosis. Apoptosis consists of a complex network of regulatory processes. The specific molecular mechanisms underlying apoptosis remain unclear. Previous studies have reported that celecoxib can significantly reduce the expression of Survivin in cells. The fact that Survivin expression depends on the expression of cellular COX-2 suggests that COX-2 and Survivin might share a common activation mechanism and that COX-2 might act as an upstream regulatory factor, the high expression of which upregulates Survivin expression [34,35]. The level of tumor cell proliferation reflects the degree of malignancy. The loss of tumor suppressor genes or point mutations disrupts normal cell proliferation and differentiation, which can be manifested through malignant transformation of cells and the development of cancer caused by overexpression of the cell proliferation marker PCNA. This is consistent with the biological behavior of laryngeal squamous cell carcinomas.. During a study of the effects of baicalin on tumor cell proliferation and PGE2 synthesis, Zhang et al. [36] observed that both celecoxib and NIM could reduce the expression of PCNA by inhibiting
Fig. 5 – Histopathology(H&E) staining in Hep-2 cell xenograft tumor masses of each sample. Necrosis and changes of cell morphology in the two xenograft tumor groups were observed and compared by light microscopy. (A) Experimental group ( × 100). (B) Control group( × 100).
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Fig. 6 – Immunocytochemical staining for COX-2, Survivin and PCNA proteins in xenograft tumor tissues. (A), (C), (E) refer to experimental group, respectively( × 400). (B), (D), (F) refer to control group, respectively( × 400).
PGE2 synthesis. A preliminary conclusion may be that COX-2 affects PCNA expression via PGE2, which subsequently influences cell proliferation and promotes tumorigenesis. This is consistent with the fact that cell proliferation was increased
in COX-2-induced tumorigenesis and suggests that selective COX-2 inhibitors may inhibit PCNA by affecting the expression or activity of the COX-2 product PGE2, thus inhibiting the growth of laryngeal squamous cell carcinomas. The present
Fig. 7 – Expression of COX-2, Survivin and PCNA mRNA in xenograft tumor tissues in nude mice by RT-PCR analysis.
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study examined the expression levels of Survivin and PCNA in Hep-2 cells in vitro and xenograft tumors in vivo. Our results showed that the protein levels of Survivin and PCNA were significantly reduced compared with the control group after treating Hep-2 cells with NIM. Similarly, after treating xenograft tumors with NIM, the protein and mRNA expression levels of Survivin and PCNA were significantly reduced, suggesting that the effects of NIM, including inducing Hep-2 cell apoptosis and inhibiting cell proliferation and xenograft tumors growth in nude mice, may be related to the downregulation of Survivin and PCNA. NIM, a new type of NSAID, has strong anti-tumor effect and low side effect. The application of NIM in the clinical treatment of colon cancer has been reported, but its application in head and neck cancer is still in the experimental stage, and NIM has not yet been used clinically on a large scale. NIM demonstrated excellent anti-tumor properties in laryngeal squamous cell carcinoma Hep-2 cells and in vivo experiments. We suggest that its function in inducing apoptosis and inhibiting cell proliferation and the growth of laryngeal squamous cell carcinoma xenografts in nude mice is the combined result of its effects on the cell cycle, the downregulation of COX-2, Survivin and PCNA, and a variety of other factors. However, the specific mechanism may be much more complicated and diverse and requires further study. We believe that additional research on the relationship between COX-2, tumors, including laryngeal squamous cell carcinoma, and selective COX-2 inhibitors, would lead to the development of selective COX-2 inhibitors with higher specificity, better efficacy and fewer side effects.
5.
Conclusion
Selective COX-2 inhibitor NIM inhibited the growth of laryngeal squamous cell carcinoma Hep-2 cell and xenografts in nude mice. NIM has no obvious toxic side effect. Selective COX-2 inhibitor is expected to become a part of comprehensive treatment for laryngeal squamous cell carcinoma in the future, it may provide a new prospect for the prevention and treatment of laryngeal squamous cell carcinoma.
Acknowledgments The authors sincerely acknowledge Ling Yu M.D of ophthalmology, Affiliated Hospital of Luzhou Medical College and Yongsheng Wang M.D, Honggang Lin M.M and Huashan Shi M. D of the Center for Biotherapy of Cancer of the National Key Laboratory of Biotherapy in the West China Hospital at Sichuan University, for their generous technical support and help. Then the authors thank the grant support of Sichuan Province applied basic research project (05JY029-104).
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