Abstract
OBJECTIVE To investigate the putative role of the Notch1 receptor in cervical cancer carcinogenesis and progression.
METHODS The expression of the Notch1 protein was analyzed by a Western-blotting approach in 40 cervical cancer and 30 normal cervical tissues. Some tissues were examined using RT-PCR to determine mRNA levels. Celluar localization of the Notch1 protein in the paraffin-embedded cervical tissues was also analyzed by immunohistochemistry.
RESULTS The Notch1 protein was detected in all 30 normal cervical tissues. In contrast, only 6 samples of 40 cervical cancer tissues showed Notch1 expression. The level of the Notch1 protein expression was significantly lower in cervical cancer tissues than that in normal tissue samples. In agreement with these observations, levels of Notch1 mRNA were found to be substantially down-regulated in cervical cancer tissues. In the immunohistochemistry staining assay, the Notch1 protein was shown to localize predominantly in the cytoplasm and nucleoli of the normal cervical squamous epithelium of the cervix, but no staining was observed in the cervical cancer cells. Notch1 expression was observed to correlate with the clinical disease stage, but there were no correlations with age, tumor size, grade or lymph node metastasis (P>0.05). The levels of Notch1 protein expression were significantly higher in early stages (I~IIa, 66.7%) compared to those in the advanced stages (IIb~IV,12.6%)(P=0.001).
CONCLUSION Notch1 may play a role as a tumor suppressor in cervical tumorigenesis. Determination of Notch1 expression may be helpful for preoperative diagnosis and accuracy of staging. But its clinical use for cervical cancer requires further investigation.
keywords
INTRODUCTION
Cervical cancer (CC) is the one of the most common tumors among women. Every year more than 130 million new cases are diagnosed worldwide. Little progress has been made in the treatment of late-stage patients over the past 30 years, with a 5-year survival of about 60%[1,2]. The incidence of CC is estimated to be 5 times higher in China compared to developed countries, and the majority of Chinese patients are first diagnosed in the late stages of disease. To date, there is no credible tumor marker for CC diagnosis. To identify tumor markers for diagnosis, individualized, targeted therapeutics must be developed[3,4].
The Notch gene, which was first identified in 1919, is broadly involved in cell specification, proliferation, and apoptosis affecting the development and the functions of many organs. Mammals such as mice and humans possess four Notch proteins (Notch1-4). Among them, Notch1 not only plays an important role in differentiation of normal cells, but as an oncogene or tumor suppressor gene is also associated with the initiation and development of many tumors[5].
To elucidate the molecular mechanisms involved in CC, we analyzed the relationships between Notch1 and cervical cancer. The goal of the study was to provide molecular evidence for the development of individualized, targeted therapeutics for clinical diagnosis and treatment.
MATERIALS AND METHODS
Materials
The 40 cervical squamous carcinomas and 30 normal tissues were obtained from Chinese patients in the Cancer Hospital and Institute of the Chinese Academy of Medical Sciences, Beijing. The specimens were frozen in liquid nitrogen within 1 h after resection and then transferred to -80°C freezers for storage until use.
Western blot
Total cell lysates were prepared by lysing cells with the buffer (1% SDS, 10 mM Tris-Cl, pH 7.6, 20 μg/ml aprotenin, 20 μg/ml leupeptin and 1 mM PMSF). The protein concentrations were determined using the Bradford method (BIO-RAD, Hercules, CA). About 20 μg of protein were separated on 10% of SDS-PAGE gels and transferred to PVDF membranes. After being blocked with 10% non-fat milk, the membranes were incubated with anti-Rabbit clonal antibody (Santa Cruz Bitechnology Inc, Santa Cruz, CA) at 4°C overnight. After six 3-min washes the membranes were incubated with goat anti-mouse IgG for 1 h at room temperature. The signals were developed with the ECL kit (Amersham Pharmacia Biotechnology Inc, Milpitas, CA), using anti-mouse antibody (Santa Cruz) as an internal control.
RNA isolation and semi-quantitative RT-PCR
Total RNA was isolated from CC tissues with TRIzol reagent (Invitrogen) according to the manufacturer’ s instructions. Agarose gel electrophoresis at 1% and spectrophotometric analysis (A260:280 nm ratio) were used to assess the RNA quality. First strand cDNA was synthesized by using a random primer included with the Superscript II-reverse transcriptase kit (Invitrogen). Five μg of total RNA were used for each reaction and 1 μl aliquots of the cDNA then subjected to RT-PCR. The primers used for the amplification of Notch1 were as follows: sense 5’-GGC CAC CTG GGC CGG AGG TTA-3’; antisense 5’-GCG ATC TGG GAC TGC ATG CTG-3’. As an internal control, GAPDH was amplified to ensure cDNA quality and quantity for each RT-PCR.
Immunohistochemical staining
Surgical samples were fixed in formalin and paraformaldehyde and embedded in paraffin. Five-um sections were deparaffinized with xylene, and endogenous peroxidase was quenched with H2O2. The specimens were incubated for 10 min at room temperature with a protein-blocking solution and then incubated overnight at 4°C with antibodies (1:200). Bound antibodies were detected using a kit from the Zhongshan Biochemistry Co (REF).The sections were counterstained with hematoxylin.
Statistical analysis
The correlations among the Notch1 expression and clinicopathological parameters were analyzed using Fisher’s Exact Test. Chi-square tests were performed to determine the differences between normal epithelium and tumor tissue in Notch1 expression. Differences were considered statistically significant at the P<0.01 level.
RESULTS
Expression of Notch1 in CC tissue samples
The results from Western blotting for 40 CC tissue samples and 30 normal cervical epithelia showed that the expression of the Notch1 protein was lower in the CC tissue compared to the normal cervical epithelia (Fig.1). Notch1 protein was expressed in a significantly lower percentage of CC tissues (15%) than normal tissues (100%) (Chi Square Test, P=0.001).
Representative examples of results of Western blot analysis of Notch1 protein expression, using beta-actin as the internal control. N: normal tissues; C: cancer tissues.
By use of RT-PCR, we found that 3 CC tissue samples in which the Notch1 protein was absent also lacked detectable levels of Notch1 mRNA. The three normal cervical epithelia showed high levels of Notch1 expression (Fig.2). The expression patterns of Notch1 were similar in the levels of protein and in mRNA, suggesting that the expression of Notch1 was down-regulated at its mRNA level.
RT-PCR analysis of Notch1 expression. GAPDH was used as internal control. M: marker; C: cancer tissues; N: normal tissues.
The localization of Notch1 in the cervical tissues
Using immunohistochemical staining assay, the Notch1 protein was shown to localize predominantly in the cytoplasm and nucleus of the normal cervical squamous epithelium. Unlike normal cells that showed staining, no staining was observed in the CC cells. This result was similar to that of Western blotting and RT-PCR (Fig.3).
IHC analysis of Notch1 protein expression in cervical tissues, ×200. A, B: H&E and IHC staining of the normal cervical tissue; C, D: H&E and IHC staining of the CC tissues.
Notch1 protein expression and clinicopathological correlations
The results of statistical analysis correlating Notch1 protein expression in tumor tissues with clinicopathological data showed that there were no significant correlations with age, tumor size and lymph node involvement However, a significant correlation between Notch1 protein expression and the clinical stage was observed, suggesting that the loss of its expression was associated with the development of the CC (P=0.001) (Table 1). The levels of Notch1 protein expression are significantly higher in the early Stages (I~IIa, 66.7%) than in advanced Stages (IIb~IV, 12.6%) (P=0.001).
Notch1 protein expression and clinicopathological characteristics.
DISCUSSION
Our results suggest that the CC tissues lose expression of the Notch1 protein. The Notch gene family encodes transmembrane receptors of 300k Da that are involved in cell fate choices in vertebrates and invertebrates. Struhl et al.[6] has proposed a proteolysis model of Notch signaling. The model states that ligand binding results in proteolytic cleavage of Notch and consequent release of the Notch intracellular domain (NICD), which enters the nucleus and activates transcription of the target gene. The molecular active form of Notch was 120 kDa but not full-length 300 kDa Notch[7,8].
A number of reports suggest that Notch signaling may be involved in neoplastic transformation. Deletion of most of the extracellular domain as a consequence of a (9:7) chromosomal translocation results in generation of a truncated Notch, which is associated with a subgroup of human T-cell acute lymphoblastic leukemias. Many studies have demonstrated that loss of the extracellular subunit of Notch receptors can mediate transformation. Aberrant expression of Notch receptors has been found in lung, renal and breast cancers[9,10]. However, there is mounting evidence that deviant Notch signaling is not the only relationship with oncogensis, as it also can function as a tumor suppressor[11,12].
In our study, a 120 kDa Notch protein was detected by use of Western blots. Our results showed that the expression of the Notch1 protein was lost in the CC tissues compared to the normal cervical epithelia. Using RT-PCR the loss of Notch1 expression was found in at level of mRNA. Previous studies showed that increased Notch1 expression existed in the precancerous lesions caused by HPV infection[13]. Since our results showed a high level of Notch1 expression in normal cervical epithelium, but decreased Notch1 expression in invasive CC, we believe that in progression of normal cervical epithelium to precancerous lesions to CC, Notch1 expression decreases more and more. That is to say that Notch1 might function as a tumor suppressor in the final development of CC. Our results were similar to those reported by Talora et al.[14,15].
The effeciency of therapy for CC mostly depends on the clinical stage. Carcinoma in situ can be totally cured, while the 5-year survival of early stage invasive carcinomas has showed to be 67%[16]. Thus, the diferential diagnosis of in situ and invasive carcinoma has an important clinical significance. A significant correlation between Notch1 protein expression and the clinical stage was observed in our study. The levels of Notch1 protein expression were significantly higher in the early stage compared to those in an advanced stage, which may be helpful in identifing the tumor stages.
In conclusion, Notch1 plays a role as a tumor suppressor in cervical tumorigenesis. Whether Notch1 can be used as a tumor marker and therapeutic target in cervical cancer requires further investigation.
- Received December 19, 2006.
- Accepted January 18, 2007.
- Copyright © 2007 by Tianjin Medical University Cancer Institute & Hospital and Springer
REFERENCES
In this issue
Jump to section
Related Articles
Cited By...
- No citing articles found.