doi: 10.1111/jop.12229
J Oral Pathol Med (2015) 44: 193–200 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd wileyonlinelibrary.com/journal/jop
Decreased expression of glutathione S-transferase pi correlates with poorly differentiated grade in patients with oral squamous cell carcinoma Hai-long Ma1, Cong Yu1, Ying Liu1, Yi-ran Tan1, Jin-ke Qiao1, Xi Yang1, Li-zhen Wang2, Jiang Li2, Qiong Chen3, Fu-xiang Chen3, Zhi-yuan Zhang1, Lai-ping Zhong1 1
Department of Oral & Maxillofacial-Head & Neck Oncology, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; 2Department of Oral Pathology, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; 3Department of Clinical Laboratories, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
BACKGROUND: Glutathione S transferase pi (GSTP1) is a member of phase II detoxification enzymes as a major regulator of cell signaling in response to stress, hypoxia, growth factors, and other stimuli. The clinical role of GSTP1 in cancer is still unclear. The aim of this study was to investigate the serum GSTP1 level in patients with oral squamous cell carcinoma (OSCC) and the GSTP1 expression in tissue samples from patients with OSCC and OSCC lines. METHODS: One hundred and sixty-six patients with OSCC and 120 normal persons were used to screen potential serum peptide biomarkers using matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Serum GSTP1 concentration was detected in 18 patients with OSCC and 18 normal persons using ELISA. Immunohistochemistry was used to detect GSTP1 expression in tissue samples from twenty-eight OSCC patients. Western blot and real-time PCR were used to detect GSTP1 expression in nine OSCC lines. RESULTS: Decreased GSTP1 concentration was found in the patients with OSCC compared with the normal persons by MALDI-TOF-MS, which was then confirmed by ELISA (P = 0.019). Decreased GSTP1 mRNA level and protein expression were also found in the OSCC lines. Decreased GSTP1 expression was found correlating with pathological differentiation grade in the tissue samples from OSCC patients, a lower GSTP1 expression indicating a poorer pathological differentiation grade (P = 0.041).
Correspondence: Lai-ping Zhong, MD, PhD, Department of Oral & Maxillofacial-Head & Neck Oncology, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine; No.639 Zhizaoju Road, Shanghai 200011, China. Tel: +86 21 23271699 5160, Fax: +86 21 63136856, E-mail: [email protected] Hai-long Ma and Cong Yu contributed equally to this paper. Accepted for publication June 13, 2014
CONCLUSIONS: These results suggest that decreased GSTP1 expression in patients with OSCC and a lower GSTP1 expression indicating a poorer pathological differentiation grade in OSCC tissue samples. J Oral Pathol Med (2015) 44: 193–200 Keywords: glutathione S-transferase pi; oral squamous cell carcinoma; pathological differentiation grade
Introduction Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cancer by incidence worldwide (1). Oral squamous cell carcinoma (OSCC), a subset of this disease, has a poor clinical outcome with a 5-year survival rate of only 50–60% and an even lower survival rate in patients with locally advanced OSCC (1, 2). Currently, the treatment strategy for patients with locally advanced and resectable OSCC is radical surgery followed by post-operative radiation or chemoradiation, depending on the presence of highrisk features in the surgical specimen (3, 4). Tumor biomarkers that can distinguish differences in tumor behavior in vitro or in vivo are expected to be useful for diagnosis and prognostic monitoring. Proteomic analysis is a powerful tool for global evaluation of protein expression. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been widely used to identify potential biomarkers for diagnosis and prognosis in many types of cancers, such as breast cancer, pancreatic cancer, colon cancer, lung cancer, head and neck cancer, ovarian cancer, hepatocellular cancer, leukemia, etc. (5–9). Glutathione S transferases (GSTs), are a family of phase II detoxification enzymes that catalyses the conjugation of nonpolar compounds that contain an electrophilic carbon,
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nitrogen or sulfur atom to reduced glutathione (10). Mammalian GST isozymes can be classified into seven types, and GSTP1 is the major regulator of cell signaling in response to stress, hypoxia, growth factors, and other stimuli (11). It exists mainly in cytosol, and will be released into serum when cells are injured. The inconsistent results of GSTP1 expression suggest potential different carcinogenesis mechanisms in different cancers. In OSCC, there are still debates on the relationships between GSTP1 expression and carcinogenesis, both positive and negative relationships have been reported in the literatures (12, 13). In this study, we aimed to investigate the expression level of GSTP1 and its potential clinical significance in patients with OSCC. We applied the MALDI-TOF-MS technique with magnetic nano-beads to compare the serum peptide profile between patients with OSCC and normal persons, and validated the GSTP1 expression using ELISA, PCR, Western blot, and immunohistochemistry in serum samples, tissue samples and cell lines. We hypothesized reduction of GSTP1 expression in patients with OSCC comparing with normal persons.
Materials and methods Materials From March 2008 to June 2009, 166 primary patients with OSCC (103 males and 63 females, age ranging from 26 to 87 years old with a mean of 56.3 years old) and 120 normal persons (75 males and 45 females, age ranging from 19 to 70 years old with a mean of 47.2 years old) were enrolled in this study to screen potential serum peptide biomarkers using MALDI-TOF-MS technique with magnetic nanobeads. Another 18 primary patients with OSCC and 18 normal persons were enrolled to validate serum GSTP1 concentrations using ELISA. Twenty eight primary patients with OSCC were enrolled to validate tissue GSTP1 protein expression using immunohistochemistry. All subjects came from the Department of Oral & Maxillofacial-Head & Neck Oncology, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine. The diagnostic confirmation of OSCC was according to the WHO histological criteria (14). The clinical stage of patients with OSCC was according the TNM staging system (UICC, 2002). After signing the informed consent forms approved by the Institutional Ethical Committee of Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine (with the approval number of 2008 [12]), pretreatment venous blood samples and surgical tissue samples were collected. The baseline demographic and lifestyle information of all subjects were also collected, and the constituent ratio of baseline demographic and lifestyle between patients with OSCC and normal persons was similar without significant difference. The clinical characteristics of patients with OSCC were reviewed and collected from the hospital medical records. Cell culture Nine cell lines were used in this study, including HIOECs (human immortalized oral epithelial cells), HB, HN4, HN6, HN12, HN13, CAL27, HN30, and SCC25. HIOEC and HB were from our previously established in vitro cellular J Oral Pathol Med
carcinogenesis model of OSCC (15); HN4, HN6, HN12, HN13 were OSCC lines, HN30 was a pharyngeal squamous cell carcinoma line, as gifts from the University of Maryland Dental School, USA; CAL27 and SCC25 were OSCC lines, which were purchased from ATCC (Manassas, VA, USA). HIOECs were cultured in defined keratinocyte-SFM (Invitrogen, Carlsbad, CA, USA). HB, HN4, HN6, HN12, HN13, CAL27, and HN30 cells were cultured in DMEM (Gibico, Grand Island, NY, USA) supplemented with 10% fetal bovine serum, 1% glutamine, and 1% penicillin–streptomycin. SCC25 cells were cultured in DMEM/F12 (Gibico) supplemented with 10% fetal bovine serum, 1% glutamine, and 1% penicillin–streptomycin. All cells were cultured in a humidified atmosphere of 5% CO2 at 37°C. Serum collection, preparation and MALDI-TOF MS 2.5 ml pretreatment fasting venous blood samples from the patients with OSCC and normal persons was collected. After inverting gently, the samples were kept at room temperature (25°C) for 1 h and then centrifuged at 1200 g per minute at 25°C for 10 min. The cell-free supernatant of serum was transferred to an EP tube carefully and stored at 80°C. Serum samples were assayed in combination with weak cationic exchange magnetic beads (WCX-MB; Bruker Daltonics, Billerica, Germany) according to the kit instructions. Duplicate serum samples (5 ll each) were mixed thoroughly with 10 ll MB and bead buffer, and incubated for 5 min. Then, the samples were put on magnet separator for 1 min and after seven washings; 5 ll of bead elution buffer was added into the affinity bead-purified serum protein to pre-fractionate the surrogate markers. Candidate marker solution was stored with bead stable buffer, analyzed by mass spectrometry directly or frozen at 20°C before analysis within 24 h. In the procession, human serum (Sigma, Santa Clara, CA, USA) was applied as calibration criterion to assure repeatability and reproducibility. The samples and spectras were analyzed statistically with the aid of Autoflex II (Bruker Daltonics) and Clinprot tandem mass bioinformatics software. MALDI-TOF MS was performed to identify the peptides profile ranging from 1000 to 20 000 Da. Peptides solutions were processed automatically with robotic instrumentation. Aliquots of the eluted peptides (0.8–1.2 ll) were mixed with 0.4 mg/ml 5 ll HCCA matrix solutions (absolute ethyl alcohol and 1.2 mg/ll a-Cyano-4-hydroxycinnamic acid at 1:2 stored at 20°C for 2 days). The mixture was spotted on the 600 lm anchor chip target and dried at 25°C. Then, samples were transferred to MALDI-TOF MS instrumentation for analysis, and the peptide calibration standard samples were detected with guidance of Clinprot standard methodology, with mean molecule variation
J Oral Pathol Med (2015) 44: 193–200 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd wileyonlinelibrary.com/journal/jop
Decreased expression of glutathione S-transferase pi correlates with poorly differentiated grade in patients with oral squamous cell carcinoma Hai-long Ma1, Cong Yu1, Ying Liu1, Yi-ran Tan1, Jin-ke Qiao1, Xi Yang1, Li-zhen Wang2, Jiang Li2, Qiong Chen3, Fu-xiang Chen3, Zhi-yuan Zhang1, Lai-ping Zhong1 1
Department of Oral & Maxillofacial-Head & Neck Oncology, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; 2Department of Oral Pathology, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; 3Department of Clinical Laboratories, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
BACKGROUND: Glutathione S transferase pi (GSTP1) is a member of phase II detoxification enzymes as a major regulator of cell signaling in response to stress, hypoxia, growth factors, and other stimuli. The clinical role of GSTP1 in cancer is still unclear. The aim of this study was to investigate the serum GSTP1 level in patients with oral squamous cell carcinoma (OSCC) and the GSTP1 expression in tissue samples from patients with OSCC and OSCC lines. METHODS: One hundred and sixty-six patients with OSCC and 120 normal persons were used to screen potential serum peptide biomarkers using matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Serum GSTP1 concentration was detected in 18 patients with OSCC and 18 normal persons using ELISA. Immunohistochemistry was used to detect GSTP1 expression in tissue samples from twenty-eight OSCC patients. Western blot and real-time PCR were used to detect GSTP1 expression in nine OSCC lines. RESULTS: Decreased GSTP1 concentration was found in the patients with OSCC compared with the normal persons by MALDI-TOF-MS, which was then confirmed by ELISA (P = 0.019). Decreased GSTP1 mRNA level and protein expression were also found in the OSCC lines. Decreased GSTP1 expression was found correlating with pathological differentiation grade in the tissue samples from OSCC patients, a lower GSTP1 expression indicating a poorer pathological differentiation grade (P = 0.041).
Correspondence: Lai-ping Zhong, MD, PhD, Department of Oral & Maxillofacial-Head & Neck Oncology, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine; No.639 Zhizaoju Road, Shanghai 200011, China. Tel: +86 21 23271699 5160, Fax: +86 21 63136856, E-mail: [email protected] Hai-long Ma and Cong Yu contributed equally to this paper. Accepted for publication June 13, 2014
CONCLUSIONS: These results suggest that decreased GSTP1 expression in patients with OSCC and a lower GSTP1 expression indicating a poorer pathological differentiation grade in OSCC tissue samples. J Oral Pathol Med (2015) 44: 193–200 Keywords: glutathione S-transferase pi; oral squamous cell carcinoma; pathological differentiation grade
Introduction Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cancer by incidence worldwide (1). Oral squamous cell carcinoma (OSCC), a subset of this disease, has a poor clinical outcome with a 5-year survival rate of only 50–60% and an even lower survival rate in patients with locally advanced OSCC (1, 2). Currently, the treatment strategy for patients with locally advanced and resectable OSCC is radical surgery followed by post-operative radiation or chemoradiation, depending on the presence of highrisk features in the surgical specimen (3, 4). Tumor biomarkers that can distinguish differences in tumor behavior in vitro or in vivo are expected to be useful for diagnosis and prognostic monitoring. Proteomic analysis is a powerful tool for global evaluation of protein expression. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been widely used to identify potential biomarkers for diagnosis and prognosis in many types of cancers, such as breast cancer, pancreatic cancer, colon cancer, lung cancer, head and neck cancer, ovarian cancer, hepatocellular cancer, leukemia, etc. (5–9). Glutathione S transferases (GSTs), are a family of phase II detoxification enzymes that catalyses the conjugation of nonpolar compounds that contain an electrophilic carbon,
GSTP1 in OSCC Ma et al.
194
nitrogen or sulfur atom to reduced glutathione (10). Mammalian GST isozymes can be classified into seven types, and GSTP1 is the major regulator of cell signaling in response to stress, hypoxia, growth factors, and other stimuli (11). It exists mainly in cytosol, and will be released into serum when cells are injured. The inconsistent results of GSTP1 expression suggest potential different carcinogenesis mechanisms in different cancers. In OSCC, there are still debates on the relationships between GSTP1 expression and carcinogenesis, both positive and negative relationships have been reported in the literatures (12, 13). In this study, we aimed to investigate the expression level of GSTP1 and its potential clinical significance in patients with OSCC. We applied the MALDI-TOF-MS technique with magnetic nano-beads to compare the serum peptide profile between patients with OSCC and normal persons, and validated the GSTP1 expression using ELISA, PCR, Western blot, and immunohistochemistry in serum samples, tissue samples and cell lines. We hypothesized reduction of GSTP1 expression in patients with OSCC comparing with normal persons.
Materials and methods Materials From March 2008 to June 2009, 166 primary patients with OSCC (103 males and 63 females, age ranging from 26 to 87 years old with a mean of 56.3 years old) and 120 normal persons (75 males and 45 females, age ranging from 19 to 70 years old with a mean of 47.2 years old) were enrolled in this study to screen potential serum peptide biomarkers using MALDI-TOF-MS technique with magnetic nanobeads. Another 18 primary patients with OSCC and 18 normal persons were enrolled to validate serum GSTP1 concentrations using ELISA. Twenty eight primary patients with OSCC were enrolled to validate tissue GSTP1 protein expression using immunohistochemistry. All subjects came from the Department of Oral & Maxillofacial-Head & Neck Oncology, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine. The diagnostic confirmation of OSCC was according to the WHO histological criteria (14). The clinical stage of patients with OSCC was according the TNM staging system (UICC, 2002). After signing the informed consent forms approved by the Institutional Ethical Committee of Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine (with the approval number of 2008 [12]), pretreatment venous blood samples and surgical tissue samples were collected. The baseline demographic and lifestyle information of all subjects were also collected, and the constituent ratio of baseline demographic and lifestyle between patients with OSCC and normal persons was similar without significant difference. The clinical characteristics of patients with OSCC were reviewed and collected from the hospital medical records. Cell culture Nine cell lines were used in this study, including HIOECs (human immortalized oral epithelial cells), HB, HN4, HN6, HN12, HN13, CAL27, HN30, and SCC25. HIOEC and HB were from our previously established in vitro cellular J Oral Pathol Med
carcinogenesis model of OSCC (15); HN4, HN6, HN12, HN13 were OSCC lines, HN30 was a pharyngeal squamous cell carcinoma line, as gifts from the University of Maryland Dental School, USA; CAL27 and SCC25 were OSCC lines, which were purchased from ATCC (Manassas, VA, USA). HIOECs were cultured in defined keratinocyte-SFM (Invitrogen, Carlsbad, CA, USA). HB, HN4, HN6, HN12, HN13, CAL27, and HN30 cells were cultured in DMEM (Gibico, Grand Island, NY, USA) supplemented with 10% fetal bovine serum, 1% glutamine, and 1% penicillin–streptomycin. SCC25 cells were cultured in DMEM/F12 (Gibico) supplemented with 10% fetal bovine serum, 1% glutamine, and 1% penicillin–streptomycin. All cells were cultured in a humidified atmosphere of 5% CO2 at 37°C. Serum collection, preparation and MALDI-TOF MS 2.5 ml pretreatment fasting venous blood samples from the patients with OSCC and normal persons was collected. After inverting gently, the samples were kept at room temperature (25°C) for 1 h and then centrifuged at 1200 g per minute at 25°C for 10 min. The cell-free supernatant of serum was transferred to an EP tube carefully and stored at 80°C. Serum samples were assayed in combination with weak cationic exchange magnetic beads (WCX-MB; Bruker Daltonics, Billerica, Germany) according to the kit instructions. Duplicate serum samples (5 ll each) were mixed thoroughly with 10 ll MB and bead buffer, and incubated for 5 min. Then, the samples were put on magnet separator for 1 min and after seven washings; 5 ll of bead elution buffer was added into the affinity bead-purified serum protein to pre-fractionate the surrogate markers. Candidate marker solution was stored with bead stable buffer, analyzed by mass spectrometry directly or frozen at 20°C before analysis within 24 h. In the procession, human serum (Sigma, Santa Clara, CA, USA) was applied as calibration criterion to assure repeatability and reproducibility. The samples and spectras were analyzed statistically with the aid of Autoflex II (Bruker Daltonics) and Clinprot tandem mass bioinformatics software. MALDI-TOF MS was performed to identify the peptides profile ranging from 1000 to 20 000 Da. Peptides solutions were processed automatically with robotic instrumentation. Aliquots of the eluted peptides (0.8–1.2 ll) were mixed with 0.4 mg/ml 5 ll HCCA matrix solutions (absolute ethyl alcohol and 1.2 mg/ll a-Cyano-4-hydroxycinnamic acid at 1:2 stored at 20°C for 2 days). The mixture was spotted on the 600 lm anchor chip target and dried at 25°C. Then, samples were transferred to MALDI-TOF MS instrumentation for analysis, and the peptide calibration standard samples were detected with guidance of Clinprot standard methodology, with mean molecule variation
