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Expression of P33ING1b Protein in Colorectal Cancer

BACKGROUND Colorectal cancer (CRC) is the second most common malignancy in the world. However, its mortality rate can be reduced if diagnosed early. P33ING1b is a tumor suppressor protein, which plays a role in growth control and apoptosis. Suppression of p33ING1b is associated with the loss of cellular growth control. However, p33 ING1b expression in CRC and its correlations with clinicopathological factors have been less studied. The aim of this study was to examine p33ING1b expression in patients with CRC and evaluate its potential correlations with clinicopathological factors. METHODS P33ING1b protein expression was examined in 70 cases of CRC tissue samples and their corresponding neighboring normal tissues by immunhistochemistry. Moreover, p33ING1b expression in CRC and its correlations with clinicopathological variables including patients’ sex and age, tumor type, location, stage, and differentiation grade were examined. RESULTS P33ING1b expression was significantly lower in tumor samples compared with the normal adjacent samples (p<0.002). CONCLUSION Low expression of P33ING1b in patients with colorectal cancer, may be an important molecular event in the pathogenesis of colorectal cancer. Our data suggest that reduced expression of p33ING1b may be contribute to tumor genesis and accompanied by the loss of cellular growth control. In fact cell growth is out of control in lower expression of P33 and dysfunctional program cell death. P33 expression might explain the etiology of CRC for reducing the expression of tumor suppressor proteins.


INTRODUCTION
Colorectal cancer (CRC) is the second most prevalent malignant neoplasm in the world after the breast cancer. 1,2 It is the third most common cancer in men and the second in women; accounting for 8% (n=608,700) of all cancer deaths worldwide. The highest incidence rate of colon cancer is in the Eastern Europe and Asia. CRC is also the third most common cancer in Iranians in except of the skin cancers. It occurs at younger ages with an increasing trend similar to the Asia-Pacific countries. 3 Often, the first line of treatment is surgical resection followed by chemotherapy because most patients are diagnosed at an advanced stage. 4,5 About one quarter of the sufferers have Dukes' C disease and a 5-year survival rate of 30-40%. 4 Several risk factors may play a role in CRC development. The incidence is higher in men than women. 6 Prognosis for CRC is affected by a variety of features presented at the time of initial diagnosis including age, sex, duration of symptoms, and tumor location. 7 There are two major classes of tumor associated genes that have been implicated in tumorgenesis: oncogenes and tumor suppressor genes. Inactivation (by loss or mutation) of tumor suppressor genes plays an essential role in the genesis of many tumors. 8 Inhibitor of Growth 1 (ING1) is a recently cloned novel growth inhibitor and a candidate tumor suppressor gene detected through a method of cDNA subtractive hybridization from normal and cancer cells followed by an in vitro selection assay. ING1 mapped to the human chromosome 13q33-34, a region that has been linked to the progression of various tumors. 9 Several mechanisms of malfunction for ING1 gene have been proposed including: gene malfunctions (mutations, rearrangements, lack of heterozygosity, homozygous loss and DNA CpG island hypermethylation), decreased mRNA expression, reduced protein expression, and protein malformations. 8 ING1 encodes four protein isoforms: p47 ING1a ,  p33 ING1b , p24 ING1c , and p27 ING1d , 4 among which p33 ING1b is the most widely expressed isoform 10 that is involved in growth control and apoptosis as well a role in senescence. 4 The p33 ING1b protein plays a fundamental role in multiple cellular activities such as growth regulation, apoptosis, senescence, and DNA repair upon UV-induced DNA damage, which are all typical characteristics of tumor suppressors. 11 while suppression of p33 ING1b is accompanied by the loss of cellular growth control and immortalization, its overexpression arrests cells in the G0/G1 phase of the cell cycle, 10 which inhibits cell growth and induces apoptosis. 4 By contrast, a reduced p33 ING1b expression has been detected in lymphoid malignancy, and esophageal, gastric, brain, colon, liver, lung, astrocytoma, and breast cancers, suggesting that the reduced expression of p33 ING1b might contribute to tumorigenesis. 10 P33 ING1b expression in CRC is low and seems to play an important role in carcinogenesis or progression. 12 Patients with p33 ING1b protein under expression exhibit an overall shorter survival compared with those with normal p33 ING1b expression levels. 4 Chen and colleagues have shown that p33 ING1b expression reduces in patients with CRC, and that the average mRNA expression levels in the cancerous tissues of Dukes' stages C and D were significantly lower than in stages A and B. 12 However, the correlation between p33 ING1b expression in CRC and clinicopathological factors has not been studied. In a study by Ahmed and colleagues, it was found that in patients with Dukes' C stage of CRC, p33 ING1b protein was under expressed in 32% of samples. It was also reported that patients with p33 ING1b under expression had a shorter overall survival in comparison with those with normal p33 ING1b expression. 4 Studies on CRC show that patients with mucinous adenocarcinoma and high stage, with metastasis to the lymph nodes, have a poorer prognosis. 13,14 Here, we aimed to evaluate p33 ING1b expression in CRC tumor samples in comparison with normal tissues and further analyze the potential correlation between p33 ING1b expression and different clinicopathological variables including sex, age, tumor location, type, stage, and grade of differentiation.

Human Tissue Samples
In this retrospective study, a total of 70 formalinfixed paraffin-embedded tissue samples of CRC and their matched normal colorectal tissues (at least 10 cm away from the tumor margins) were obtained from patients by surgical colectomy (Department of Pathology at Al-Zahra Hospital in Isfahan, Iran). The information regarding patients' sex, age, tumor location, stage, and grade of differentiation were obtained from hospital records. All diagnoses were verified by an independent pathologist who did not have any information about stained slides. This project was approved by the Ethics Committee of Isfahan University of Medical Sciences (No:388207).

Immunohistological staining and evaluation
The expression intensity of p33 ING1b protein in the paraffin-embedded histological sections was determined using immunohistochemical analysis. One section block was hematoxylin & eosinstained and another section was prepared and examined for p33 ING1b expression. First, sections (4-5 μm thick) were dewaxed in xylene and then rehydrated by immersion in different percentages of ethanol. Endogenous peroxides were blocked by pretreatment of the sections with a 0.5% solution of hydrogen peroxide in methanol for 10 minutes. The slides were then washed under running tap water. A one-minute pressure cooking step in citrate buffer (200 mM citric acid, 500 mM NaOH, pH = 6.0) was used for antigen retrieval. Treated sections were transferred into Tris-buffered saline (TBS) for five minutes and blocked with 10% normal goat serum in TBS for 5 minutes. Excess serum was removed and the sections were incubated at room temperature with the primary anti-ING1 monoclonal antibody (wH0003621M1; Sigma.Aldrich. co) at 1:200 dilutions for 60 minutes. The sections were washed twice with TBS for five minutes each, and then incubated with the secondary biotinylated rabbit anti-mouse antibody at a 1500 dilution for 45 minutes at room temperature. After rinsing the samples twice with TBS, the peroxidase activity was developed with 3,3′-diaminobenzidine tetrahydrochloride (DAB), in sterile H 2 O 2 solution for five minutes. Sections were then washed with tap water, counterstained with hematoxylin, dehydrated in different percentages of ethanol, and mounted in DPX [A mixture of distyrene (a polystyrene), a plasticizer (tricresyl phosphate), and xylene]. Sections of normal colon tissue samples were used as positive controls for p33 ING1b expression. Sections incubated with PBS instead of the corresponding primary antibody were used as negative controls.

Scoring System
The proportions of cellular staining for p33 ING1b were estimated under light microscopy at 100× and 400× magnifications by using Motic Image Advanced Plus Software, version 3.2. At least 1,000 cells per slide from 10 different fields were counted. The staining intensity was assessed on a fourpoint scale as follows: 0= negative, 1= weak, 2=intermediate, and 3=strong. The staining intensities were also verified by an independent pathologist. The percentages of stained cells were assessed on a three-point scale as follows: 1=0-15%, 2=16-50%, and 3=51-100%.

Statistical analysis
The SPSS software version 20 (SPSS Inc., Chicago, IL) was used for all statistical analyses. The paired t test was used to test the relationship between p33 ING1b expression in normal and tumor samples, as well as the relationship between p33 ING1b expression and clinicopathological variables. Data were represented as mean ± SD and P values less than 0.05 were considered as statistically significant.

General
Positive p33 ING1b staining was observed as a nuclear pattern with various staining intensities (figures 1,2). Overall, a significant reduction in p33 ING1b protein expression was noted in the tumor samples in comparison with the normal tissues

Expression of P33 ING1b
(p< 0.000) Table 1 shows the results of the paired t test comparing the mean tumor suppressor protein expression in tumor specimens and adjacent normal tissue samples. There was a significant difference (p< 0.000) between the first group (T.exp3-C.exp3) and group IV (T.exp0-C.exp0). But in the second group (T.exp.2-C.exp.2) and group III (T.exp.1-C.

exp.1) this difference was not significant.
The overall correlation between the mean percentages of p33 ING1b expressing cells with demographic and clinical characteristics of patients with CRC Out of the 70 samples analyzed, 41 (58.57%) came from the male patients and 52 (74.3%) were from patients above 50 years old. Among these patients whose ages ranged from 30 to 87 years (with an average age of 61.95 ± 13 years). Most of the patients with CRC (67.15%) were in stage B. The tumors were mainly non-mucinous (85.71%) and well differentiated (88.34%). And about two third of them (68.57%) were in the colon.
As table 2 shows the comparison of the mean tumor suppressor protein expression in tumor specimens with sex in the four-point scale, the second group (T.exp.2) and group III (T.exp.1) showed a significant difference (p=0.037 and p=0.029, respectively).
To compare the mean tumor suppressor protein 47 Torabizadeh et al.  (Table 3). Results showed that different levels vary with each other. In fact T.exp.2 between different levels of tumor stage was significant (p=0.045). Also in this scale, stage A was significantly different from stage B and C (p=0.014).
Comparing the mean tumor suppressor protein expression in tumor specimens with the other variables showed no statistically significant difference (Tables 4-7).

DISCUSSION
Several studies have shown that the levels of p33 ING1 expression are reduced in some cancers including head and neck, squamous cell, stomach, liv-       15 However, the low levels of p33 ING1b expression have been reported with variable frequencies. [16][17][18][19][20][21][22] For instance, in breast cancer, the low levels of p33 ING1b expression were observed in 58-80% of cases. 16,[20][21] This study was done on Iranian people and the expression intensity of p33 ING1b protein in the paraffin-embedded histological sections was determined using immunohistochemical analysis. Also the correlation between p33 ING1b expression in CRC and clinicopathological factors was examined.
Here, we showed that the mean intensity of nuclear p33 ING1b expression as well as the number of positive p33 ING1b expressing cells were low in all CRC cases. This is in support of the findings by Jian-guang and colleagues who reported that the p33 ING1b expression in CRC was reduced. 23 Also a study by Chen and colleagues on mRNA expression in sporadic CRC tissue samples by Real-Time PCR, showed that p33 ING1b mRNA levels were significantly lower in cancerous tissue. The authors further suggested that low levels expression of p33 ING1b might play an important role in CRC carcinogenesis and progression. 12 The study by Jian-Guang and co-workers did not show a meaningful correlation between low levels of p33 ING1b expression and patients' sex, age, and tumor differentiation grades and site. 23 Jian-Guang and colleagues have also reported that the low lev-els of p33 ING1b expression markedly related with the Dukes classification. This is in agreement with an earlier report by Liang and colleagues indicating that the rates of p33 ING1b expression in cancerous tissues at Dukes' A and B stages were significantly higher than that at stages C and D. 24 In conclusion, low expression of p33 ING1b may be an important molecular event in the pathogenesis of CRC. Our data suggest that reduced expression of p33 ING1b may be contribute to tumorigenesis and accompanied by the loss of cellular growth control. In fact cell growth is out of control in lower expression of p33 and dysfunctional program cell death. P33 expression might explain the etiology of CRC for reducing the expression of tumor suppressor proteins.

ACKNOwLEDGMENTS
This study has been supported by grant No. 388207 from Isfahan University of Medical Sciences, and also in part by the Mohajeri and Mehzad laboratories at Sadoghi and Kashani Hospitals in Isfahan. The authors would like to thank Dr. Mitra Heidarpour for help in reviewing the slides. we also thank the Department of Pathology at Al-Zahra Hospital in Isfahan.