Research Note Journal of International Medical Research 41(6) 1838–1847 ! The Author(s) 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0300060513498021 imr.sagepub.com
Expression and clinical significance of the programmed cell death 5 gene and protein in laryngeal squamous cell carcinoma Fenglei Xu1, Kai Wu2, Miaoqing Zhao3, Yejun Qin3 and Ming Xia4
Abstract Objective: To determine the expression of the gene programmed cell death 5 (PDCD5) and its protein PDCD5 in laryngeal squamous cell carcinoma and to analyse possible correlations with clinicopathological parameters. Methods: PDCD5 mRNA expression was assessed using reverse transcription–polymerase chain reaction and expression of PDCD5 protein was studied using Western blot analysis and immunohistochemistry in laryngeal squamous cell carcinoma and morphologically normal paracarcinoma tissue. Results: A total of 41 laryngeal squamous cell carcinoma and 29 normal para-carcinoma tissue specimens were examined. Expression of both PDCD5 mRNA and PDCD5 protein was significantly reduced in laryngeal squamous cell carcinoma compared with normal tissue. Expression was correlated with clinical stage and histological grade, but was not associated with age, sex, location of primary tumour or the presence of lymph node metastases. Conclusion: The expression of PDCD5 and its protein were shown to be reduced in laryngeal squamous cell carcinoma. The functional importance of PDCD5 as a regulating agent in cell apoptosis suggests that it may play a key role in tumour pathogenesis and development.
4 1
Department of Otorhinolaryngology – Head and Neck Surgery, Jinling Hospital of Nanjing University Medical School, Jiangsu, China 2 Department of Pathology, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan, China 3 Department of Pathology, Provincial Hospital Affiliated to Shandong University, Shandong, China
Department of Otorhinolaryngology, Qilu Hospital, Shandong University, Shandong, China Corresponding author: Dr Fenglei Xu, Department of Otorhinolaryngology – Head and Neck Surgery, Jinling Hospital of Nanjing University Medical School, 305 East Zhongshan Road, Nanjing 210002, Jiangsu, China. Email: [email protected]
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Keywords Programmed cell death 5, PCDC5, TFAR19, expression, laryngeal squamous cell carcinoma, clinical significance Date received: 17 March 2013; accepted: 30 March 2013
Introduction Laryngeal squamous cell carcinoma is one of most frequent cancers in elderly men and its incidence has increased markedly during recent years.1 The pathogenesis and development of laryngeal squamous cell carcinoma involves multiple factors, multiple genes and multiple stages,2 and the exact mechanisms are unknown. The gene programmed cell death 5 (PDCD5), also known as TF-1 cell apoptosis-related gene 19 (TFAR19), was first identified in the leukaemic cell clone TF-1 at the Human Disease Gene Center of Peking University and was shown to participate in the process of cell apoptosis.3 Subsequent studies have confirmed the functional importance of PDCD5 in regulating cell apoptosis and its downregulation in a number of cancers, suggesting its clinical potential in the early diagnosis and treatment of malignancies.4–7 The present study aimed to determine the expression of PDCD5 and its protein PDCD5 in laryngeal squamous cell carcinoma and to analyse possible correlations with clinicopathological parameters.
Patients and methods Patients Patients undergoing first-line surgical treatment for laryngeal squamous cell carcinoma in the Department of Otorhinolaryngology, Qilu Hospital, Shandong University, Shandong, China, between January 2010 and October 2012 who had not received preoperative radiotherapy, chemotherapy or biological therapy (defined as immunotherapy, biotherapy or biological response modifier treatment) were recruited to the
study. Imaging studies, histological grading using the American Joint Committee on Cancer cancer staging system8 and clinical staging using the TNM criteria9 were performed before surgery. The study was approved by the ethics committees of the Qilu Hospital of Shandong University, Shandong, China, and the Jinling Hospital of Nanjing University Medical School, Jiangsu, China. Written informed consent was obtained from all study participants.
Specimens Specimens of tumour tissue and morphologically normal tissue adjacent to the tumour were harvested during open surgery: tumour tissue and normal tissue were identified visually and tumour tissue was harvested directly from the centre of the tumour. Normal tissue samples were obtained 5 mm from the margin of the tumour. Each fresh tissue specimen was divided into two: one half was fixed with ice-cold 10% paraformaldehyde for immunohistochemical analysis and other half was stored at 80 C for reverse transcription– polymerase chain reaction (RT–PCR) and Western blot analysis.
RT–PCR Expression of PDCD5 mRNA was measured using RT–PCR. Total RNA was extracted from thawed samples weighing approximately 1 g using TRIzolÕ reagent (Invitrogen, Shanghai, China) according to the manufacturer’s instructions. Reverse transcription was performed on 1 mg RNA using Moloney murine leukaemia virus
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reverse transcriptase (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The resulting cDNA was then amplified by PCR using GoTaqÕ PCR Core Systems (Promega) according to the manufacturer’s instructions, with primers for human PDCD5 (forward primer 50 -CAA CAGGAAGCAAAGCAC-30 , reverse primer 50 -GATCTTAACTTCTGCCTAG AC-30 ; 296 bp) and the internal control gene b-actin (forward primer 50 -ATCAT GTTTGAGACCTTCAACA-30 , reverse primer 50 - CATCTCTTGCTCGAAGTC CA-30 ; 318 bp) (Invitrogen, Shanghai, China). The PCR protocol consisted of preliminary denaturation at 95 C for 4 min, followed by 35 cycles of denaturation at 95 C for 30 s, annealing at 56 C for 30 s and elongation at 72 C for 30 s, with a final elongation step at 72 C for 5 min. The PCR products were electrophoresed on 1.5% agarose gel and stained with ethidium bromide. Expression levels were calculated after adjustment for the expression levels of the housekeeping gene b-actin using Kodak 1D Image Analysis software (Eastman Kodak, Rochester, NY, USA) and given as the ratio of PDCD5 to b-actin. Positive specimens were defined as those with a ratio of PDCD5 to b-actin >0.25.
Western blot analysis Expression of PDCD5 protein was measured using Western blotting. Total protein was extracted from thawed tissue homogenates using TRIzolÕ reagent (Invitrogen) according to the manufacturer’s instructions. After quantification by the Bradford method,10 equal amounts of protein (30 mg) were separated using a sodium dodecyl sulphate–polyacrylamide gel electrophoresis kit with a 15% acrylamide gel (Pierce, Rockford, IL, USA) and transferred to nitrocellulose membranes using the TransBlotÕ system (Bio-Rad, Hercules, CA, USA) (2 h at 450 mA in 25 mM Tris-buffered
saline, pH 6.8, 192 mM glycine and 20% methanol). The membranes were then blocked with 50 g/l skimmed milk powder overnight at 4 C and incubated at 37 C for 1 h with rabbit polyclonal antibody to human PDCD5 (Abcam, Cambridge, MA, USA) at a dilution of 1:150. After washing with Tris-buffered saline and Tween 20 (TBST) buffer (pH 7.6), the membranes were incubated with horseradish peroxidase-labelled goat anti-rabbit secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA) at a dilution of 1:500 with gentle agitation for 1 h at room temperature. The membranes were then rinsed thoroughly with TBST buffer and the bands were developed using 3,30 -diaminobenzidine (DAB) for 10 min. The bands were detected using the AlphaImagerÕ HP system (ProteinSimple, Santa Clara, CA, USA); positive expression of PDCD5 was defined as the appearance of a clear band. Expression was quantified by densitometry using Quantity OneÕ analysis software (BioRad). The PDCD5 protein level was normalized to that of b-actin, which was used as an internal control.
Immunohistochemistry Expression of PDCD5 protein was also analysed by immunohistochemistry using a streptavidin–biotin–peroxidase complex kit (MaiXin Bio, Fuzhou, China) according to the manufacturer’s instructions. Tissues were embedded in paraffin and cut into 4 mm sections. The sections were deparaffinized and incubated with rabbit anti-human PDCD5 antibody (Abcam) at a dilution of 1:600 overnight at 4 C and then washed three times in phosphate-buffered saline (pH 7.4). The specimens were then incubated with biotinized goat anti-rabbit immunoglobulin G secondary antibody (Abcam) at 37 C for 30 min. After a further three washes in phosphate-buffered saline, antibody binding was visualized using a DAB
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kit (Jingmei, Shenzhen, China) according to the manufacturer’s instructions and counterstained with haematoxylin. Negative control sections were incubated with secondary antibody alone. The protein PDCD5 was identified as brown staining in the cytoplasm under light microscopy and areas with the most and well-distributed stained cells under low power were selected. The number of stained cells was counted in at least five randomly chosen high-power (400 magnification) fields for each specimen. PDCD5 expression was quantified according to the percentage of cells stained and the intensity of staining. A score of 0 was assigned to specimens with no stained cells, 50% stained cells had a score of 4. The intensity of staining was scored as 0 for no staining, 1 for weak staining, 2 for moderate staining and 3 for strong staining.11 The product of these two scores was termed the staining score and was used to quantify PDCD5 expression. Each section was analysed by two experienced technicians who were blinded to the study group and the final result was obtained by consensus in cases of discrepancies.12 Specimens with a staining score 1 were defined as being positive for PDCD5 protein expression.
Statistical analyses Results were expressed as the mean SD. Intergroup differences were analysed using one-way analysis of variance. Non-parametric statistics were used for non-normally distributed results. The percentages of positive specimens in the two groups were compared using the Mann– Whitney U-test. Semiquantitative expression of PDCD5 mRNA and PDCD5 protein was compared in matched specimens from the two groups using the paired Student’s ttest. Correlation analysis was performed
using the Pearson test or 2 test. A Pvalue < 0.05 was considered to be statistically significant. All statistical analyses were performed using SPSS software version 11.0 (SPSS Inc., Chicago, IL, USA).
Results A total of 41 laryngeal squamous cell carcinoma specimens were collected from 41 patients (mean SD 56.35 12.31 years); in addition, 29 normal para-carcinoma tissue specimens were collected from 29 of the 41 patients who consented to this.
Expression of PDCD5 Examples of expression of PDCD5 mRNA as detected by RT–PCR are shown in Figure 1. Expression was seen in a significantly lower proportion of carcinoma tissue specimens than in normal tissue specimens (P ¼ 0.0038) (Table 1). Semiquantitative analysis of expression in the 29 paired samples produced similar results, with expression of PDCD5 mRNA being significantly higher in normal tissue than in carcinoma tissue (P ¼ 0.013) (Table 2).
Expression of PDCD5 protein A high proportion of normal tissue specimens were shown to express PDCD5 protein on Western blotting, whereas most tumour tissue specimens had lost the ability to express PDCD5 or showed only low expression (P ¼ 0.0012) (Table 1; Figure 2). Semiquantitative analysis of PDCD5 protein expression in the 29 paired samples produced similar results, with expression being significantly higher in normal tissue than in carcinoma tissue (P ¼ 0.027) (Table 2). These findings were confirmed on immunohistochemistry. PDCD5 protein was
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Figure 1. Expression of programmed cell death 5 gene (PDCD5) mRNA in representative normal para-carcinoma tissue and laryngeal squamous cell carcinoma as detected by reverse transcription– polymerase chain reaction; data were normalized to b-actin.
Table 1. Proportions of normal para-carcinoma and laryngeal squamous cell carcinoma specimens positive for expression of programmed cell death 5 (PDCD5) mRNA and PDCD5 protein. PDCD5 protein PDCD5 mRNA
Western blot
Immunohistochemistry
Tissue
n
Positive specimens
Statistical significancea
Positive specimens
Statistical significancea
Positive specimens
Statistical significancea
Normal Carcinoma
29 41
24 (82.8) 20 (48.8)
P ¼ 0.0038
29 (100) 18 (43.9)
P ¼ 0.0012
23 (79.3) 20 (48.8)
P ¼ 0.0097
Data presented as n (%) of specimens. a Using Mann–Whitney U-test.
located in the cytoplasm but not in the nucleus. The proportion of cells expressing PDCD5 protein was significantly lower in carcinoma tissue than in normal tissue (P ¼ 0.0097) (Table 1; Figure 3). Comparison of staining scores in the 29 paired samples showed that PDCD5 protein expression was significantly higher in normal tissue
than in carcinoma tissue (P ¼ 0.0079) (Table 2).
Correlation between PDCD5 mRNA and PDCD5 protein expression A significant association was identified between PDCD5 mRNA expression and
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Table 2. Semiquantitative expression of programmed cell death 5 (PDCD5) mRNA and PDCD5 protein in 29 matched normal para-carcinoma and laryngeal squamous cell carcinoma specimens. PDCD5 protein PDCD5 mRNA
Western blot
Immunohistochemistry
Tissue
Expression ratioa
Statistical significanceb
Expression ratioa
Statistical significanceb
Staining score
Statistical significanceb
Normal Carcinoma
0.72 0.39 0.81 0.47
P ¼ 0.013
0.57 0.24 0.42 0.19
P ¼ 0.027
6.21 3.72 4.03 1.67
P ¼ 0.0079
Data presented as mean SD. a Relative to b-actin. b Using paired Student’s t-test.
correlated with TNM stage and histological grade (Table 3). Moreover, PDCD5 mRNA expression was also associated with TNM stage and histological grade (Table 3).
Discussion Figure 2. Expression of programmed cell death 5 (PDCD5) protein in representative normal para-carcinoma tissue and laryngeal squamous cell carcinoma as detected by Western blot; data were normalized to b-actin.
PDCD5 protein expression in laryngeal squamous cell carcinoma using the Pearson test (r ¼ 0.695, P < 0.01).
Correlation between PDCD5 mRNA or PDCD5 protein expression and clinicopathological features Associations between PDCD5 mRNA expression or PDCD5 protein expression and clinicopathological features in laryngeal squamous cell carcinoma were analysed using the 2 test. PDCD5 protein expression determined by Western blot or immunohistochemistry was not significantly associated with age, sex, location of primary or lymph node metastasis, but was significantly
Loss of signal transduction pathways is a frequent occurrence in cancer cells and several key components in cell apoptosis were first identified through their effects on pro-cancer transformation or mutation in cancer tissues.13–15 Several studies have shown that PDCD5 promotes cell apoptosis mediated by various factors and inhibits cell proliferation.3,5,16 The use of a variety of technologies has confirmed that PDCD5 is downregulated in many diseases, especially cancers. Spectrum analysis of gene expression in hereditary breast cancer tissues has shown that PDCD5 expression is increased in BRCA1-mutation cancers and decreased in BRCA2-mutation cancers.17 In addition, a study of differential gene expression using DNA chip technology demonstrated that some cell apoptosis-related genes, including PDCD5, were downregulated in hepatocellular carcinoma.18 It has also been reported that expression of PDCD5 in ovarian epithelial cancer tissue is lower than in normal ovary or benign ovarian tumour; moreover,
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Figure 3. Expression of programmed cell death 5 (PDCD5) protein in representative (A) normal para-carcinoma tissue and (B) laryngeal squamous cell carcinoma as detected by immunohistochemistry. Magnification 400. Table 3. Expression of programmed cell death 5 (PDCD5) mRNA and PDCD5 protein according to clinicopathological features in laryngeal squamous cell carcinoma specimens. PDCD5 protein
Parameter Age, years 60 >60 Sex Male Female Location of primary tumour Supraglottic Glottis Subglottic Histological grade I II III TNM stage I þ II III þ IV Metastases in nodes No Yes
PDCD5 mRNA
Western blot
Immunohistochemistry
n
Expression ratioa
Statistical significanceb
Expression ratioa
Statistical significanceb
Staining score
Statistical significanceb
19 22
0.61 0.32 0.53 0.33
NS
0.28 0.17 0.32 0.19
NS
4.19 2.50 3.72 2.28
NS
34 7
0.41 0.26 0.54 0.29
NS
0.31 0.21 0.38 0.17
NS
3.85 2.40 4.07 2.35
NS
10 25 6
0.41 0.26 0.54 0.29 0.41 0.26
NS
0.37 0.16 0.43 0.18 0.34 0.11
NS
4.15 2.72 3.62 2.08 3.43 2.13
NS
14 18 9
0.63 0.34 0.49 0.29 0.24 0.21
P ¼ 0.032
0.61 0.32 0.43 0.25 0.31 0.12
P ¼ 0.027
4.65 2.69 3.52 2.76 2.12 1.73
P ¼ 0.014
23 18
0.51 0.27 0.34 0.19
P ¼ 0.021
0.42 0.22 0.29 0.17
P ¼ 0.016
4.69 2.578 2.93 1.37
P ¼ 0.021
20 21
0.41 0.26 0.54 0.29
NS
0.43 0.21 0.47 0.26
NS
4.39 2.23 3.52 2.43
NS
Data presented as mean SD. a Relative to b-actin. b Using 2 test. NS, no statistically significant differences (P 0.05).
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PDCD5 expression decreased with increasing histological grade.4 In the present study, expression of both PDCD5 mRNA and PDCD5 protein in laryngeal squamous cell carcinoma was significantly lower than that in normal tissue (P ¼ 0.0038 and P ¼ 0.0012, respectively). These results were confirmed by immunohistochemical analysis. Further evaluation of associations between PDCD5 protein expression on Western blot and the pathological features of laryngeal squamous cell carcinoma showed that the reduction in expression was not associated with age, sex, location of the primary or the presence of lymph node metastases, but was associated with histological grade and TNM stage (P ¼ 0.027 and P ¼ 0.016, respectively). Moreover, the reduction in PDCD5 mRNA expression was also associated with histological grade and TNM stage (P ¼ 0.032 and P ¼ 0.021, respectively). There are currently few studies on the apoptosis-related gene PDCD5 and the exact mechanisms of its role in apoptosis regulation are unknown. Fernandez et al.19 reported that when normal human breast epithelial cells were exposed in vitro to bisphenol A, an endocrine disruptor, expression of many genes associated with DNA repair were increased but expression of PDCD5 and BCL2L11, both of which are involved in apoptosis, were downregulated. In the osteosarcoma cell line MG-63, PDCD5 has been shown to induce apoptosis and G2 phase arrest.20 In addition, expression of PDCD5 inhibited the Ras/Raf/MEK/ERK signalling pathway, leading to inhibition of cyclin B and cyclin-dependent kinase 1, with downregulation of ERK resulting in activation of caspase 3 and 9.20 Furthermore, caspase 3 regulates Bax translocation from cytoplasm to mitochondria, which plays an essential role in apoptosis.21 In another study, PCDC5 was identified as a substrate for casein kinase 2 (CK2); PDCD5 was
phosphorylated in vitro by both the CK2a subunit and the CK2 holoenzyme at residue S118.22 Transfection of the nonphosphorylatable mutant (S118A) impaired PDCD5 acceleration of either doxorubimicin- or ultraviolet-induced apoptosis in U2OS cells.22 On the other hand, when PDCD5 was transfected into gastric cancer cell line BG823, the stable transfection resulted in G2/M cell cycle arrest, increased apoptosis and nuclear translocation of PDCD5 and p53 after cisplatin treatment, indicating that regulation of PDCD5 may be a novel strategy for improving chemotherapeutic effects in cancer.23 Expression of PDCD5 protein has been shown to be significantly associated with expression of p53.24 As the pathway mediated by p53 is important in cancer-cell apoptosis, this association is consistent with PDCD5 having a role in apoptosis. It has been shown that PDCD5 can bind with p53 within residues 41–56 of the p53 TAD2 subdomain, while p53 binds preferentially to the positively charged surface region around the C-terminals of helices a3 and a5 and the N-terminal of helix a4 of PDCD5.25 In conclusion, the present study found that expression of both PDCD5 mRNA and PDCD5 protein was significantly decreased in laryngeal squamous cell carcinoma, indicating that PDCD5 may have an important role in the pathogenesis and development of this cancer as a regulating agent in cell apoptosis. However, the mechanism of loss of expression of this pro-apoptosis gene in laryngeal squamous cell carcinoma is still unknown. In addition, the pathways via which PDCD5 exerts its functions remain to be elucidated. Declaration of conflicting interest The authors declare that there are no conflicts of interest.
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Funding This research received no specific grant from any funding agency in the public, commercial or notfor-profit sectors.
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22. Salvi M, Xu D, Chen Y, et al. Programmed cell death protein 5 (PDCD5) is phosphorylated by CK2 in vitro and in 293T cells. Biochem Biophys Res Commun 2009; 387: 606–610. 23. Xu HY, Chen ZW, Pan YM, et al. Transfection of PDCD5 effect on the biological behavior of tumor cells and sensitized gastric cancer cells to
cisplatin-induced apoptosis. Dig Dis Sci 2012; 57: 1847–1856. 24. Wang Y, Li D, Fan H, et al. Cellular uptake of exogenous human PDCD5 protein. J Biol Chem 2006; 281: 24803–24817. 25. Yao H, Feng Y, Zhou T, et al. NMR studies of the interaction between human programmed cell death 5 and human p53. Biochemistry 2012; 51: 2684–2693.
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Expression and clinical significance of the programmed cell death 5 gene and protein in laryngeal squamous cell carcinoma Fenglei Xu1, Kai Wu2, Miaoqing Zhao3, Yejun Qin3 and Ming Xia4
Abstract Objective: To determine the expression of the gene programmed cell death 5 (PDCD5) and its protein PDCD5 in laryngeal squamous cell carcinoma and to analyse possible correlations with clinicopathological parameters. Methods: PDCD5 mRNA expression was assessed using reverse transcription–polymerase chain reaction and expression of PDCD5 protein was studied using Western blot analysis and immunohistochemistry in laryngeal squamous cell carcinoma and morphologically normal paracarcinoma tissue. Results: A total of 41 laryngeal squamous cell carcinoma and 29 normal para-carcinoma tissue specimens were examined. Expression of both PDCD5 mRNA and PDCD5 protein was significantly reduced in laryngeal squamous cell carcinoma compared with normal tissue. Expression was correlated with clinical stage and histological grade, but was not associated with age, sex, location of primary tumour or the presence of lymph node metastases. Conclusion: The expression of PDCD5 and its protein were shown to be reduced in laryngeal squamous cell carcinoma. The functional importance of PDCD5 as a regulating agent in cell apoptosis suggests that it may play a key role in tumour pathogenesis and development.
4 1
Department of Otorhinolaryngology – Head and Neck Surgery, Jinling Hospital of Nanjing University Medical School, Jiangsu, China 2 Department of Pathology, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan, China 3 Department of Pathology, Provincial Hospital Affiliated to Shandong University, Shandong, China
Department of Otorhinolaryngology, Qilu Hospital, Shandong University, Shandong, China Corresponding author: Dr Fenglei Xu, Department of Otorhinolaryngology – Head and Neck Surgery, Jinling Hospital of Nanjing University Medical School, 305 East Zhongshan Road, Nanjing 210002, Jiangsu, China. Email: [email protected]
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Keywords Programmed cell death 5, PCDC5, TFAR19, expression, laryngeal squamous cell carcinoma, clinical significance Date received: 17 March 2013; accepted: 30 March 2013
Introduction Laryngeal squamous cell carcinoma is one of most frequent cancers in elderly men and its incidence has increased markedly during recent years.1 The pathogenesis and development of laryngeal squamous cell carcinoma involves multiple factors, multiple genes and multiple stages,2 and the exact mechanisms are unknown. The gene programmed cell death 5 (PDCD5), also known as TF-1 cell apoptosis-related gene 19 (TFAR19), was first identified in the leukaemic cell clone TF-1 at the Human Disease Gene Center of Peking University and was shown to participate in the process of cell apoptosis.3 Subsequent studies have confirmed the functional importance of PDCD5 in regulating cell apoptosis and its downregulation in a number of cancers, suggesting its clinical potential in the early diagnosis and treatment of malignancies.4–7 The present study aimed to determine the expression of PDCD5 and its protein PDCD5 in laryngeal squamous cell carcinoma and to analyse possible correlations with clinicopathological parameters.
Patients and methods Patients Patients undergoing first-line surgical treatment for laryngeal squamous cell carcinoma in the Department of Otorhinolaryngology, Qilu Hospital, Shandong University, Shandong, China, between January 2010 and October 2012 who had not received preoperative radiotherapy, chemotherapy or biological therapy (defined as immunotherapy, biotherapy or biological response modifier treatment) were recruited to the
study. Imaging studies, histological grading using the American Joint Committee on Cancer cancer staging system8 and clinical staging using the TNM criteria9 were performed before surgery. The study was approved by the ethics committees of the Qilu Hospital of Shandong University, Shandong, China, and the Jinling Hospital of Nanjing University Medical School, Jiangsu, China. Written informed consent was obtained from all study participants.
Specimens Specimens of tumour tissue and morphologically normal tissue adjacent to the tumour were harvested during open surgery: tumour tissue and normal tissue were identified visually and tumour tissue was harvested directly from the centre of the tumour. Normal tissue samples were obtained 5 mm from the margin of the tumour. Each fresh tissue specimen was divided into two: one half was fixed with ice-cold 10% paraformaldehyde for immunohistochemical analysis and other half was stored at 80 C for reverse transcription– polymerase chain reaction (RT–PCR) and Western blot analysis.
RT–PCR Expression of PDCD5 mRNA was measured using RT–PCR. Total RNA was extracted from thawed samples weighing approximately 1 g using TRIzolÕ reagent (Invitrogen, Shanghai, China) according to the manufacturer’s instructions. Reverse transcription was performed on 1 mg RNA using Moloney murine leukaemia virus
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reverse transcriptase (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The resulting cDNA was then amplified by PCR using GoTaqÕ PCR Core Systems (Promega) according to the manufacturer’s instructions, with primers for human PDCD5 (forward primer 50 -CAA CAGGAAGCAAAGCAC-30 , reverse primer 50 -GATCTTAACTTCTGCCTAG AC-30 ; 296 bp) and the internal control gene b-actin (forward primer 50 -ATCAT GTTTGAGACCTTCAACA-30 , reverse primer 50 - CATCTCTTGCTCGAAGTC CA-30 ; 318 bp) (Invitrogen, Shanghai, China). The PCR protocol consisted of preliminary denaturation at 95 C for 4 min, followed by 35 cycles of denaturation at 95 C for 30 s, annealing at 56 C for 30 s and elongation at 72 C for 30 s, with a final elongation step at 72 C for 5 min. The PCR products were electrophoresed on 1.5% agarose gel and stained with ethidium bromide. Expression levels were calculated after adjustment for the expression levels of the housekeeping gene b-actin using Kodak 1D Image Analysis software (Eastman Kodak, Rochester, NY, USA) and given as the ratio of PDCD5 to b-actin. Positive specimens were defined as those with a ratio of PDCD5 to b-actin >0.25.
Western blot analysis Expression of PDCD5 protein was measured using Western blotting. Total protein was extracted from thawed tissue homogenates using TRIzolÕ reagent (Invitrogen) according to the manufacturer’s instructions. After quantification by the Bradford method,10 equal amounts of protein (30 mg) were separated using a sodium dodecyl sulphate–polyacrylamide gel electrophoresis kit with a 15% acrylamide gel (Pierce, Rockford, IL, USA) and transferred to nitrocellulose membranes using the TransBlotÕ system (Bio-Rad, Hercules, CA, USA) (2 h at 450 mA in 25 mM Tris-buffered
saline, pH 6.8, 192 mM glycine and 20% methanol). The membranes were then blocked with 50 g/l skimmed milk powder overnight at 4 C and incubated at 37 C for 1 h with rabbit polyclonal antibody to human PDCD5 (Abcam, Cambridge, MA, USA) at a dilution of 1:150. After washing with Tris-buffered saline and Tween 20 (TBST) buffer (pH 7.6), the membranes were incubated with horseradish peroxidase-labelled goat anti-rabbit secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA) at a dilution of 1:500 with gentle agitation for 1 h at room temperature. The membranes were then rinsed thoroughly with TBST buffer and the bands were developed using 3,30 -diaminobenzidine (DAB) for 10 min. The bands were detected using the AlphaImagerÕ HP system (ProteinSimple, Santa Clara, CA, USA); positive expression of PDCD5 was defined as the appearance of a clear band. Expression was quantified by densitometry using Quantity OneÕ analysis software (BioRad). The PDCD5 protein level was normalized to that of b-actin, which was used as an internal control.
Immunohistochemistry Expression of PDCD5 protein was also analysed by immunohistochemistry using a streptavidin–biotin–peroxidase complex kit (MaiXin Bio, Fuzhou, China) according to the manufacturer’s instructions. Tissues were embedded in paraffin and cut into 4 mm sections. The sections were deparaffinized and incubated with rabbit anti-human PDCD5 antibody (Abcam) at a dilution of 1:600 overnight at 4 C and then washed three times in phosphate-buffered saline (pH 7.4). The specimens were then incubated with biotinized goat anti-rabbit immunoglobulin G secondary antibody (Abcam) at 37 C for 30 min. After a further three washes in phosphate-buffered saline, antibody binding was visualized using a DAB
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kit (Jingmei, Shenzhen, China) according to the manufacturer’s instructions and counterstained with haematoxylin. Negative control sections were incubated with secondary antibody alone. The protein PDCD5 was identified as brown staining in the cytoplasm under light microscopy and areas with the most and well-distributed stained cells under low power were selected. The number of stained cells was counted in at least five randomly chosen high-power (400 magnification) fields for each specimen. PDCD5 expression was quantified according to the percentage of cells stained and the intensity of staining. A score of 0 was assigned to specimens with no stained cells, 50% stained cells had a score of 4. The intensity of staining was scored as 0 for no staining, 1 for weak staining, 2 for moderate staining and 3 for strong staining.11 The product of these two scores was termed the staining score and was used to quantify PDCD5 expression. Each section was analysed by two experienced technicians who were blinded to the study group and the final result was obtained by consensus in cases of discrepancies.12 Specimens with a staining score 1 were defined as being positive for PDCD5 protein expression.
Statistical analyses Results were expressed as the mean SD. Intergroup differences were analysed using one-way analysis of variance. Non-parametric statistics were used for non-normally distributed results. The percentages of positive specimens in the two groups were compared using the Mann– Whitney U-test. Semiquantitative expression of PDCD5 mRNA and PDCD5 protein was compared in matched specimens from the two groups using the paired Student’s ttest. Correlation analysis was performed
using the Pearson test or 2 test. A Pvalue < 0.05 was considered to be statistically significant. All statistical analyses were performed using SPSS software version 11.0 (SPSS Inc., Chicago, IL, USA).
Results A total of 41 laryngeal squamous cell carcinoma specimens were collected from 41 patients (mean SD 56.35 12.31 years); in addition, 29 normal para-carcinoma tissue specimens were collected from 29 of the 41 patients who consented to this.
Expression of PDCD5 Examples of expression of PDCD5 mRNA as detected by RT–PCR are shown in Figure 1. Expression was seen in a significantly lower proportion of carcinoma tissue specimens than in normal tissue specimens (P ¼ 0.0038) (Table 1). Semiquantitative analysis of expression in the 29 paired samples produced similar results, with expression of PDCD5 mRNA being significantly higher in normal tissue than in carcinoma tissue (P ¼ 0.013) (Table 2).
Expression of PDCD5 protein A high proportion of normal tissue specimens were shown to express PDCD5 protein on Western blotting, whereas most tumour tissue specimens had lost the ability to express PDCD5 or showed only low expression (P ¼ 0.0012) (Table 1; Figure 2). Semiquantitative analysis of PDCD5 protein expression in the 29 paired samples produced similar results, with expression being significantly higher in normal tissue than in carcinoma tissue (P ¼ 0.027) (Table 2). These findings were confirmed on immunohistochemistry. PDCD5 protein was
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Figure 1. Expression of programmed cell death 5 gene (PDCD5) mRNA in representative normal para-carcinoma tissue and laryngeal squamous cell carcinoma as detected by reverse transcription– polymerase chain reaction; data were normalized to b-actin.
Table 1. Proportions of normal para-carcinoma and laryngeal squamous cell carcinoma specimens positive for expression of programmed cell death 5 (PDCD5) mRNA and PDCD5 protein. PDCD5 protein PDCD5 mRNA
Western blot
Immunohistochemistry
Tissue
n
Positive specimens
Statistical significancea
Positive specimens
Statistical significancea
Positive specimens
Statistical significancea
Normal Carcinoma
29 41
24 (82.8) 20 (48.8)
P ¼ 0.0038
29 (100) 18 (43.9)
P ¼ 0.0012
23 (79.3) 20 (48.8)
P ¼ 0.0097
Data presented as n (%) of specimens. a Using Mann–Whitney U-test.
located in the cytoplasm but not in the nucleus. The proportion of cells expressing PDCD5 protein was significantly lower in carcinoma tissue than in normal tissue (P ¼ 0.0097) (Table 1; Figure 3). Comparison of staining scores in the 29 paired samples showed that PDCD5 protein expression was significantly higher in normal tissue
than in carcinoma tissue (P ¼ 0.0079) (Table 2).
Correlation between PDCD5 mRNA and PDCD5 protein expression A significant association was identified between PDCD5 mRNA expression and
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Table 2. Semiquantitative expression of programmed cell death 5 (PDCD5) mRNA and PDCD5 protein in 29 matched normal para-carcinoma and laryngeal squamous cell carcinoma specimens. PDCD5 protein PDCD5 mRNA
Western blot
Immunohistochemistry
Tissue
Expression ratioa
Statistical significanceb
Expression ratioa
Statistical significanceb
Staining score
Statistical significanceb
Normal Carcinoma
0.72 0.39 0.81 0.47
P ¼ 0.013
0.57 0.24 0.42 0.19
P ¼ 0.027
6.21 3.72 4.03 1.67
P ¼ 0.0079
Data presented as mean SD. a Relative to b-actin. b Using paired Student’s t-test.
correlated with TNM stage and histological grade (Table 3). Moreover, PDCD5 mRNA expression was also associated with TNM stage and histological grade (Table 3).
Discussion Figure 2. Expression of programmed cell death 5 (PDCD5) protein in representative normal para-carcinoma tissue and laryngeal squamous cell carcinoma as detected by Western blot; data were normalized to b-actin.
PDCD5 protein expression in laryngeal squamous cell carcinoma using the Pearson test (r ¼ 0.695, P < 0.01).
Correlation between PDCD5 mRNA or PDCD5 protein expression and clinicopathological features Associations between PDCD5 mRNA expression or PDCD5 protein expression and clinicopathological features in laryngeal squamous cell carcinoma were analysed using the 2 test. PDCD5 protein expression determined by Western blot or immunohistochemistry was not significantly associated with age, sex, location of primary or lymph node metastasis, but was significantly
Loss of signal transduction pathways is a frequent occurrence in cancer cells and several key components in cell apoptosis were first identified through their effects on pro-cancer transformation or mutation in cancer tissues.13–15 Several studies have shown that PDCD5 promotes cell apoptosis mediated by various factors and inhibits cell proliferation.3,5,16 The use of a variety of technologies has confirmed that PDCD5 is downregulated in many diseases, especially cancers. Spectrum analysis of gene expression in hereditary breast cancer tissues has shown that PDCD5 expression is increased in BRCA1-mutation cancers and decreased in BRCA2-mutation cancers.17 In addition, a study of differential gene expression using DNA chip technology demonstrated that some cell apoptosis-related genes, including PDCD5, were downregulated in hepatocellular carcinoma.18 It has also been reported that expression of PDCD5 in ovarian epithelial cancer tissue is lower than in normal ovary or benign ovarian tumour; moreover,
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Figure 3. Expression of programmed cell death 5 (PDCD5) protein in representative (A) normal para-carcinoma tissue and (B) laryngeal squamous cell carcinoma as detected by immunohistochemistry. Magnification 400. Table 3. Expression of programmed cell death 5 (PDCD5) mRNA and PDCD5 protein according to clinicopathological features in laryngeal squamous cell carcinoma specimens. PDCD5 protein
Parameter Age, years 60 >60 Sex Male Female Location of primary tumour Supraglottic Glottis Subglottic Histological grade I II III TNM stage I þ II III þ IV Metastases in nodes No Yes
PDCD5 mRNA
Western blot
Immunohistochemistry
n
Expression ratioa
Statistical significanceb
Expression ratioa
Statistical significanceb
Staining score
Statistical significanceb
19 22
0.61 0.32 0.53 0.33
NS
0.28 0.17 0.32 0.19
NS
4.19 2.50 3.72 2.28
NS
34 7
0.41 0.26 0.54 0.29
NS
0.31 0.21 0.38 0.17
NS
3.85 2.40 4.07 2.35
NS
10 25 6
0.41 0.26 0.54 0.29 0.41 0.26
NS
0.37 0.16 0.43 0.18 0.34 0.11
NS
4.15 2.72 3.62 2.08 3.43 2.13
NS
14 18 9
0.63 0.34 0.49 0.29 0.24 0.21
P ¼ 0.032
0.61 0.32 0.43 0.25 0.31 0.12
P ¼ 0.027
4.65 2.69 3.52 2.76 2.12 1.73
P ¼ 0.014
23 18
0.51 0.27 0.34 0.19
P ¼ 0.021
0.42 0.22 0.29 0.17
P ¼ 0.016
4.69 2.578 2.93 1.37
P ¼ 0.021
20 21
0.41 0.26 0.54 0.29
NS
0.43 0.21 0.47 0.26
NS
4.39 2.23 3.52 2.43
NS
Data presented as mean SD. a Relative to b-actin. b Using 2 test. NS, no statistically significant differences (P 0.05).
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PDCD5 expression decreased with increasing histological grade.4 In the present study, expression of both PDCD5 mRNA and PDCD5 protein in laryngeal squamous cell carcinoma was significantly lower than that in normal tissue (P ¼ 0.0038 and P ¼ 0.0012, respectively). These results were confirmed by immunohistochemical analysis. Further evaluation of associations between PDCD5 protein expression on Western blot and the pathological features of laryngeal squamous cell carcinoma showed that the reduction in expression was not associated with age, sex, location of the primary or the presence of lymph node metastases, but was associated with histological grade and TNM stage (P ¼ 0.027 and P ¼ 0.016, respectively). Moreover, the reduction in PDCD5 mRNA expression was also associated with histological grade and TNM stage (P ¼ 0.032 and P ¼ 0.021, respectively). There are currently few studies on the apoptosis-related gene PDCD5 and the exact mechanisms of its role in apoptosis regulation are unknown. Fernandez et al.19 reported that when normal human breast epithelial cells were exposed in vitro to bisphenol A, an endocrine disruptor, expression of many genes associated with DNA repair were increased but expression of PDCD5 and BCL2L11, both of which are involved in apoptosis, were downregulated. In the osteosarcoma cell line MG-63, PDCD5 has been shown to induce apoptosis and G2 phase arrest.20 In addition, expression of PDCD5 inhibited the Ras/Raf/MEK/ERK signalling pathway, leading to inhibition of cyclin B and cyclin-dependent kinase 1, with downregulation of ERK resulting in activation of caspase 3 and 9.20 Furthermore, caspase 3 regulates Bax translocation from cytoplasm to mitochondria, which plays an essential role in apoptosis.21 In another study, PCDC5 was identified as a substrate for casein kinase 2 (CK2); PDCD5 was
phosphorylated in vitro by both the CK2a subunit and the CK2 holoenzyme at residue S118.22 Transfection of the nonphosphorylatable mutant (S118A) impaired PDCD5 acceleration of either doxorubimicin- or ultraviolet-induced apoptosis in U2OS cells.22 On the other hand, when PDCD5 was transfected into gastric cancer cell line BG823, the stable transfection resulted in G2/M cell cycle arrest, increased apoptosis and nuclear translocation of PDCD5 and p53 after cisplatin treatment, indicating that regulation of PDCD5 may be a novel strategy for improving chemotherapeutic effects in cancer.23 Expression of PDCD5 protein has been shown to be significantly associated with expression of p53.24 As the pathway mediated by p53 is important in cancer-cell apoptosis, this association is consistent with PDCD5 having a role in apoptosis. It has been shown that PDCD5 can bind with p53 within residues 41–56 of the p53 TAD2 subdomain, while p53 binds preferentially to the positively charged surface region around the C-terminals of helices a3 and a5 and the N-terminal of helix a4 of PDCD5.25 In conclusion, the present study found that expression of both PDCD5 mRNA and PDCD5 protein was significantly decreased in laryngeal squamous cell carcinoma, indicating that PDCD5 may have an important role in the pathogenesis and development of this cancer as a regulating agent in cell apoptosis. However, the mechanism of loss of expression of this pro-apoptosis gene in laryngeal squamous cell carcinoma is still unknown. In addition, the pathways via which PDCD5 exerts its functions remain to be elucidated. Declaration of conflicting interest The authors declare that there are no conflicts of interest.
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Funding This research received no specific grant from any funding agency in the public, commercial or notfor-profit sectors.
11.
12.
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