Abstract
Background/Aim: We lack reports on the clinicopathological characteristics and prognostic value of serum sirtuin 1 (SIRT1) levels and their association with SIRT1 expression in tissues of patients with gastric cancer (GC). Thus, we investigated the pathological characteristics and prognostic values of SIRT1 tissue expression and its serum concentration in GC. Moreover, we investigated the correlation between these two factors. Materials and Methods: A total of 78 patients with GC who underwent curative gastrectomy were evaluated in this study. The expression of SIRT1 in the surgical specimens was assessed using immunohistochemistry. Serum levels of SIRT1 were measured using an enzyme-linked immunosorbent assay. The association of tissue and serum SIRT1 with the clinicopathological features and prognosis were evaluated. Results: Positive SIRT1 tissue expression was significantly related to an advanced cancer stage (p=0.017). Furthermore, a significant relationship existed between a positive SIRT1 tissue expression and poorer overall survival (OS) and relapse-free survival (RFS) (p=0.033 and p=0.033, respectively). In contrast, serum SIRT1 levels showed no significant association with clinicopathological characteristics besides age. In addition, no significant correlation was observed between tissue SIRT1 expression and serum SIRT1 concentration. Conclusion: Tissue SIRT1 expression may be a valuable novel prognostic biomarker; nonetheless, further studies are required to clarify the relationship between tissue SIRT1 expression and serum SIRT1 levels in GC.
Gastric cancer (GC) is one of the most prevalent and deadly malignancies worldwide, with the fifth and fourth highest incidence and mortality rates, respectively (1). To date, several issues regarding the genetics of GC remain unresolved. Therefore, identifying prognostic and specific molecular markers as therapeutic targets for GC is expected to increase the effectiveness of multidisciplinary treatment and thus contribute to the advancement of medical care.
Sirtuins (SIRTs) are a highly conserved family of proteins that exist in a wide range of prokaryotic and eukaryotic organisms. Their functional activity depends on the cofactor nicotinamide adenine dinucleotide (NAD+). The mammalian SIRT family is a homolog of the yeast silent information regulator 2 (Sir2) protein and consists of seven members: SIRT1-7 (2). Sirtuin1 is involved in many biological functions, including ageing, DNA repair, metabolic regulation, apoptosis, and inflammation, and is reportedly associated with diverse diseases (3). SIRT1 reportedly acts as either a tumor promoter or suppressor in various cancer types (4). Similarly, there are conflicting reports regarding the role of SIRT1 in GC, and its exact function remains unclear (5). In most previous studies, SIRT1 expression was determined in tumor tissue using immunohistochemistry, and high tissue SIRT1 expression has been demonstrated to correlate with poor prognosis (6). However, tissue expression is heterogeneous and requires the excision of samples via biopsy or surgery, which are relatively highly invasive procedures. Therefore, more accurate and less-invasive methods are required.
Serum SIRT3 levels have been reported to be a useful biomarker in esophageal and lung cancer (7, 8), suggesting that serum SIRTs are worth exploring as minimally invasive biomarkers. However, we lack reports on the clinicopathological characteristics and prognostic value of serum SIRT1 levels and their association with SIRT1 tissue expression in patients with GC.
Thus, in this study, we investigated the pathological characteristics and prognostic value of tissue SIRT1 expression and serum SIRT1 concentration in patients with GC and the correlation between these two factors.
Materials and Methods
Patients and materials. Primary GC specimens were obtained from 78 patients with histologically confirmed GC at Chiba University Hospital between 2015 and 2016. The blood samples evaluated in this study were collected during the patients’ first visit to our department. All patients underwent radical gastrectomy with lymph node dissection. Patients who had undergone any other prior treatment or whose histological diagnosis was not GC were excluded from the study. The staging classification was assessed using the 8th American Joint Committee on Cancer (AJCC) guidelines (9), and clinical data were collected from the clinical database. The study was conducted in accordance with the Declaration of Helsinki. The institutional ethics committee approved this study (No. M10369), and informed consent was obtained from all patients.
Immunohistochemistry. SIRT1 expression was investigated via immunohistochemistry using the peroxidase-anti-peroxidase complex method, as previously reported (10). Anti-SIRT1 monoclonal antibody (1:200; ab110304; Abcam, Cambridge, UK) was used as the primary antibody, and anti-mouse antibody (EnVision+System-HRP Labelled Polymer, anti-mouse, K4001; DAKO Japan, Tokyo, Japan) was used as the secondary antibody.
Two observers independently assessed the staining results. We divided patients into two groups according to their SIRT1 expression. The expression was considered positive if over 30% of tumor cells were stained and negative if less than 30% were stained (11-13).
Measurement of SIRT1 serum concentration. Peripheral blood samples collected from patients were separated into serum and blood cells. In this study, the serum fraction was used for analysis. Serum concentrations of SIRT1 were measured using enzyme-linked immunosorbent assay (ELISA) with a commercial kit for human SIRT1 (MBS2601311; MyBiosource, Inc., San Diego, CA, USA) according to the manufacturer’s protocol. All samples were analyzed in duplicates, and the average values were calculated.
Approximately 100 μl of samples or human SIRT1 standards of varying concentrations were dispensed into the corresponding wells. The blank wells (0 ng/ml) were filled with the standard diluent. The ELISA plate was sealed with adhesive tape and incubated at 37°C for 90 min. The plate was washed twice with wash buffer, and 100 μl of biotinylated human SIRT1 antibody solution was added to each well. The plate was sealed with adhesive tape and incubated at 37°C for 60 min. Then, the plate was washed thrice with wash buffer, and 100 μl of each enzyme-conjugate liquid was added to each well. The plate was sealed with adhesive tape and kept at 37°C for 30 min. Subsequently, the plate was washed five times with wash buffer, and 100 μl of color reagent solution was added to each well and incubated in the dark at 37°C. The color change was monitored, and the reaction was stopped when the standard curve showed a color gradient, and 100 μl of color reagent C was added to each well. The optical density was measured using a microplate spectrophotometer (xMark; Bio-Rad Laboratories Inc., Hercules, CA, USA) set at 450 nm. Protein concentrations were calculated using Microplate Manager version 6 (Bio-Rad Laboratories Inc). All samples were analyzed in duplicates, and the average values were calculated. Standard curves were prepared with samples of defined concentrations and used to measure SIRT1 concentrations in the serum samples.
Statistical analyses. The correlation between tissue SIRT1 expression and serum SIRT1 concentration and the clinicopathological features of patients was evaluated using the chi-squared test and Wilcoxon rank-sum test, respectively. The association between tissue SIRT1 expression and serum SIRT1 concentration was assessed using the Wilcoxon rank-sum test. Patients were divided into two groups using the median serum SIRT1 concentration as the cut-off for survival analyses. Survival rates were calculated using the Kaplan–Meier method, and the log-rank test was used to assess differences. The JMP data analysis software version 15 (SAS Institute Inc., Cary, NC, USA) was used for all statistical analyses, and statistical significance was defined as a p-value <0.05.
Results
Patient characteristics. Patient characteristics are listed in Table I. The median patient age was 73 years. The male-to-female ratio was 50 to 28. Most patients did not have positive lymph nodes (57/78, 73%). The tumor depth varied from T1a to T4a, and the stages ranged from stage IA to stage IIIC. The median serum SIRT1 concentration was 11.85 pg/ml.
Patient characteristics (n=78).
Relationship between SIRT1 tissue expression and clinicopathological features. Among the 78 cases, 35 (44.9%) had tumors with positive SIRT1 expression, and 43 (55.1%) had tumors with negative SIRT1 expression. Representative images of SIRT1 expression are shown in Figure 1A.
Tissue SIRT1 expression. (A) Images of low expression (a and b) and high expression (c and d) are shown (100× in the upper section, 400× in the lower section). (B) Correlation between serum sirtuin 1 (SIRT1) concentration and SIRT1 tissue expression in resected specimens.
The positive SIRT1 tissue expression was significantly associated with the advanced cancer stage (p=0.017). No significant relationship was observed between SIRT1 tissue expression and other clinicopathological factors, including sex (p=0.836), age (p=0.561), tumor depth (p=0.114), lymph node (LN) status (p=0.066), lymphovascular invasion (p=0.058), and tumor differentiation (p=0.15). However, the positive expression group tended to have a higher proportion of advanced tumor depth and LN metastases (Table II).
Relationship between SIRT1 tissue expression and clinicopathological features.
Relationship between serum SIRT1 levels and clinicopathological features. The serum levels of SIRT1 for each clinicopathological feature are shown in Table III. Serum SIRT1 levels did not differ according to sex (p=0.085), tumor depth (p=0.906), LN metastasis (p=0.411), lymphovascular invasion (p=0.93), stage (p=0.253), or tumor differentiation (p=0.158). However, the mean of serum SIRT1 levels was significantly higher in the group aged 80 years or less than in patients over 80 years (p=0.047).
Relationship between serum SIRT1 levels and clinicopathological features.
Correlation between serum SIRT1 levels and the expression of SIRT1 in resected specimens. A comparison of SIRT1 expression in tumor tissue and serum SIRT1 concentration is shown in Figure 1B. The average serum SIRT1 levels were 12.27 pg/ml (2.31-31.76) and 18.26 pg/ml (3.06-176.05) in patients with SIRT1-positive and SIRT1-negative tumors, respectively. However, this difference was not statistically significant (p=0.419).
Survival analyses. Survival analyses using the Kaplan–Meier method and log-rank test are shown in Figure 2.
Kaplan–Meier survival curves of 78 patients with gastric cancer (GC). (A) Overall survival (OS) for sirtuin 1 (SIRT1) tissue expression. (B) Relapse-free survival (RFS) for SIRT1 tissue expression. (C) OS for serum SIRT1 level. (D) RFS for serum SIRT1 level. (E) OS for serum SIRT1 level in the 80 years or less group. (F) The OS for serum SIRT1 level in the group over 80 years of age.
The positive SIRT1 tissue expression was associated with significantly poorer overall survival (OS) and relapse-free survival (RFS) than that of lower expression (five-year OS rate: positive/negative=62.8%/83.1%, p=0.033, 5-year RFS rate: positive/negative=58.3%/78.3%, p=0.033, respectively). In contrast, there were no significant differences in the OS and RFS between the serum SIRT1-high and -low groups (5-year OS rate: high/low=75.6%/72.9%, p=0.458; 5-year RFS rate: high/low=67.6%/71.3%, p=0.574).
Subgroup analysis of serum SIRT1 concentration based on patient age. As the mean serum SIRT1 concentration significantly differed between patients below and above 80 years of age, we divided the patients into two subgroups with a cut-off age of 80. We performed survival analyses according to serum SIRT1 levels.
There was no statistically significant difference in OS between serum SIRT1-high and SIRT1-low levels in the group aged 80 years or less (five-year OS rate: high/low=74.3%/78.6%, p=0.962) (Figure 2E). Similarly, there was no statistically significant difference in OS according to serum SIRT1 levels in the group aged over 80 years. However, patients over 80 years old with a high SIRT1 concentration tended to have a better prognosis than those with a low SIRT1 concentration (five-year OS rate: high/low=100%/53.3%, p=0.283) (Figure 2F).
Discussion
GC is one of the most aggressive cancers with a poor prognosis. Therefore, identifying effective prognostic biomarkers and therapeutic targets is desirable for the management of patients with GC. SIRT1 is extensively involved in vital functions, and its relationship with GC has been increasingly examined in recent years. However, there are unresolved issues, such as the relationship between SIRT1 tissue expression and its serum concentration.
In this study, positive SIRT1 tissue expression was significantly associated with the advanced stage (p=0.017). Furthermore, there was a significant relationship between the positive tissue expression of SIRT1 and poorer OS and RFS (p=0.033 and p=0.033, respectively). No significant differences were observed between serum SIRT1 levels and the clinicopathological characteristics besides age. In addition, no significant correlation was observed between tissue SIRT1 expression and serum SIRT1 concentration. These results suggest that tissue expression of SIRT1 may be a valuable candidate prognostic biomarker for patients with GC. Notably, the findings suggest that serum SIRT1 levels are not a substitute for tissue expression of SIRT1. To the best of our knowledge, this is the first study to determine the relationship between SIRT1 tissue expression and its serum levels.
Several reports have described SIRT1 expression in GC tissues as a tumor-promoting and unfavorable prognostic factor (11, 12, 14). These results are consistent with our findings, which suggest that tissue SIRT1 expression is a valuable prognostic biomarker in patients with GC.
In the current study, serum SIRT1 levels were not associated with its tissue expression. As SIRT1 is located almost exclusively in the nucleus of tissue cells, serum SIRT1 may represent the overall SIRT1 expression in many tissues of the body rather than its expression levels in a specific tissue. Serum SIRT1 is reportedly associated with various diseases, such as asthma, rheumatoid arthritis, type 2 diabetes, and Alzheimer’s disease (15-18) and can be considered a valuable biomarker. In the present study, serum SIRT1 levels were significantly lower in patients aged over 80 years. Among the older patients, those with higher serum SIRT1 levels tended to have a relatively better prognosis. Zhong et al. reported that SIRT1 levels decrease with age (19), which is consistent with our findings. The relatively better prognosis of the elderly group, in whom serum SIRT1 levels are maintained, may be because SIRT1 as a longevity gene becomes more critical in the elderly than in the young. High tissue expression is also a poor prognostic factor in lung cancer (20, 21). Hosseninia et al. reported that serum SIRT1 was lower in patients with lung cancer than in healthy individuals (22). Therefore, further studies on the relationship between tissue SIRT1 expression and its serum levels in patients with cancer are warranted.
The strength of this study lies in the rarity of measuring tissue and serum SIRT1 expression in patients with GC, in addition to exploring their characteristics and their association. This study was possible because of our department’s integrated surgical, patient management, and laboratory procedures. However, this work has several limitations. First, this study has a retrospective design; thus, potential selection bias could not be ruled out. Second, this was a relatively small, single-center study conducted in East Asia, and a more extensive studies in other regions are desirable to generalize its results.
In conclusion, we demonstrated that the positive SIRT1 tissue expression correlates with advanced cancer stage and poor prognosis in patients with GC. However, we did not find a significant association between SIRT1 tissue expression and serum levels. These findings suggest that SIRT1 tissue expression may be a valuable novel prognostic biomarker. However, further studies are required to clarify the relationship between tissue SIRT1 expression and its serum levels in GC.
Acknowledgements
This study was supported by a Grant-in-Aid for Scientific Research (grant nos. 22K16481) from the Japan Society for the Promotion of Science.
Footnotes
Authors’ Contributions
Ryota Otsuka conceived and designed the study. Keiko Iida and Ryota Otsuka conducted the experiments. Hiroki Morishita and Ryota Otsuka performed the data analysis. Ryota Otsuka prepared the manuscript under the supervision of Kentaro Murakami, Koichi Hayano, Satoshi Endo, Takeshi Toyozumi, Yasunori Matsumoto, Yoshihiro Kurata, Kazuya Kinoshita, Takuma Sasaki, Shinichiro Iida, Yuri Nishioka, and Hisahiro Matsubara. All Authors read and approved the final manuscript.
Conflicts of Interest
The Authors declare that they have no conflicts of interest in relation to this study.
- Received January 18, 2023.
- Revision received January 31, 2023.
- Accepted February 1, 2023.
- Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
