Chinese Journal of Clinical Oncology Feb. 2007, Vol. 4, No. 1 P36~41 Li Sun et al.38

Aberrant Expression of Notch1 in Cervical Cancer

Li Sun1Qimin Zhan2Wenhua Zhang1Yongmei Song2Tong Tong2

1 Department of Gynecological Oncology, Cancer Hospital and Institute, Chinese Academy of Medical Sciences, Beij ing 100021,China.2 State Key Laboratory of Molecular Oncol-ogy, Cancer Hospital and Institute, Chinese Academy of Medical Sciences, Beij ing 100021,China.

Correspondence to: Li SunFax: 86-10-87702203E-mail: xjsunli0331@hotmail.com

Received December 19, 2006; accepted January 18, 2007.

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 signifi-cantly 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 immu-nohistochemistry 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 cervi-cal tumorigenesis. Determination of Notch1 expression may be helpful for preoperative diagnosis and accuracy of staging. But its clinical use for cervi-cal cancer requires further investigation.

KEYWORDS: cervical cancer, tumour suppressor, Notch1.

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 treat-ment 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 di-agnosis. 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 affect-ing 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 differ-entiation of normal cells, but as an oncogene or tumor suppressor

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[SpringerLink] DOI 10.1007/s11805-007-0038-3

39Chinese Journal of Clinical Oncology Feb. 2007, Vol. 4, No. 1 P36~41 Li Sun et al.

gene is also associated with the initiation and devel-opment 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 pro-vide molecular evidence for the development of indi-vidualized, targeted therapeutics for clinical diagnosis and treatment.

MATERIALS AND METHODS

MaterialsThe 40 cervical squamous carcinomas and 30 normal tissues were obtained from Chinese patients in the Cancer Hospital and Institute of the Chinese Acad-emy of Medical Sciences, Beijing. The specimens were frozen in liquid nitrogen within 1 h after resec-tion and then transferred to -80 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 g/ml aprotenin, 20 g/ml leupeptin and 1 mM PMSF). The protein concentrations were determined using the Bradford method (BIO-RAD, Hercules, CA). About 20 g of protein were separated on 10% of SDS-PAGE gels and transferred to PVDF membranes. After being blocked with 10% non-fat milk, the mem-branes were incubated with anti-Rabbit clonal anti-body (Santa Cruz Bitechnology Inc, Santa Cruz, CA) at 4 overnight. After six 3-min washes the mem-branes were incubated with goat anti-mouse IgG for 1 h at room temperature. The signals were developed with the ECL kit (Amersham Pharmacia Biotechnol-ogy Inc, Milpitas, CA), using anti-mouse antibody (Santa Cruz) as an internal control.

RNA isolation and semi-quantitative RT-PCRTotal RNA was isolated from CC tissues with TRIzol reagent (Invitrogen) according to the manufacturers 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 in-cluded with the Superscript II-reverse transcriptase kit (Invitrogen). Five g of total RNA were used for each reaction and 1 l 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 stainingSurgical samples were fixed in formalin and para-formaldehyde and embedded in paraffin. Five-um sections were deparaffinized with xylene, and endog-enous peroxidase was quenched with H2O2. The spec-imens were incubated for 10 min at room temperature with a protein-blocking solution and then incubated overnight at 4 with antibodies (1:200). Bound anti-bodies were detected using a kit from the Zhongshan Biochemistry Co (REF).The sections were counter-stained with hematoxylin.

Statistical analysisThe correlations among the Notch1 expression and clinicopathological parameters were analyzed using Fishers Exact Test. Chi-square tests were performed to determine the differences between normal epithe-lium and tumor tissue in Notch1 expression. Differ-ences were considered statistically significant at the P

Chinese Journal of Clinical Oncology Feb. 2007, Vol. 4, No. 1 P36~41 Li Sun et al.40

M C1 C2 C3 N1 N2 N3

Fig.2. 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 cer-vical tissuesUsing immunohistochemical staining assay, the Notch1 protein was shown to localize predominantly in the cytoplasm and nucleus of the normal cervi-cal squamous epithelium. Unlike normal cells that showed staining, no staining was observed in the CC cells. This result was similar to that of Western blot-ting and RT-PCR (Fig.3).

Fig.3. IHC analysis of Notch1 protein expression in cervical tissues, 200. A, B: H&E and IHC staining of the normal cer-vical tissue; C, D: H&E and IHC staining of the CC tissues.

Notch1 protein expression and clini-copathological correlationsThe results of statistical analysis correlating Notch1 protein expression in tumor tissues with clinicopatho-logical data showed that there were no significant correlations with age, tumor size and lymph node in-volvement However, a significant correlation between Notch1 protein expression and the clinical stage was observed, suggesting that the loss of its expres-sion 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).

Table 1. Notch1 protein expression and clinico-pathological characteristics.

Characteristics NumberNotch1 ProteinNegative Positive P

Age 0.689

41Chinese Journal of Clinical Oncology Feb. 2007, Vol. 4, No. 1 P36~41 Li Sun et al.

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 pre-cancerous 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 progres-sion 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 inva-sive 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 lev-els of Notch1 protein expression were significantly higher in the early stage compared to those in an ad-vanced stage, which may be helpful in identifing the tumor stages. In conclusion, Notch1 plays a role as a tumor sup-pressor in cervical tumorigenesis. Whether Notch1 can be used as a tumor marker and therapeutic target in cervical cancer requires further investigation.

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