Expression of CD133 and Extracellular Matrix Molecules in Malignant Brain Tumors ()
1. Introduction
A small population of cancer stem cells (CSCs) within neoplasm is supposed to be responsible for long-term tumor proliferation [1]. Some groups have also subsequently showed that CSCs exist in brain tumor, [2], and CD133 could be characterized as a marker of CSCs subpopulation and an invasive tumor phenotype [3,4].
The malignant brain tumors contain CSCs are also thought to show higher NOTCH activity [5]. The NOTCH pathway may be also related to many remodeling factors such as matrix metalloproteinases (MMP) in the brain tumors. It is reported that the NOTCH pathway represents a possible target in stem-like malignant brain tumor cells [6]. Furthermore, it has recently been reported that common chemotherapeutic drugs, as well as traditional radiation therapy, predominantly targeted the CD133- negative population [7,8]. The NOTCH pathway represents a possible target in stem-like malignant brain tumor cells [9,10]. In this study, we investigate the relationship of CD133 and other remodeling factors such as MMPs concerning with the NOTCH pathway in the malignant brain tumors [11,12].
2. Patients and Methods
Tumors from 15 patients with malinant brain tumors (Table 1) were studied to investigate the expressionpatterns of CD133, EGFR, MT1-MMP, MMP7, and CD44 using the immunostaining and RT-PCR analysis.
3. Immunostaining
All these drugs are reported to be unable to penetrate the blood-brain-barrier. Sections of paraffin-embedded tissues were studied by the immunohistochemistry(IHC) using monoclonal antibodies to CD133, EGFR, MT1- MMP, MMP7, and CD44 (Oncogene Research Labs). Using the methods described previously [13], 8-micro mm sections were treated using the avidin-biotin-peroxidase complex method (Vectastatin Elite ABC kit; Vector Labs). Endogenous peroxidase activity was blocked with 3% H2O2 in methanol for 30 min. Sections were blocked with 20% horse serum for 1 h. Primary antibody at 1:100 dilution was applied overnight at 40˚C. Secondary biotinylated antibody at 1:200 dilution was applied for 1 h to visualize the bound antibody. ABC reaction was performed for 1 h at room temperature. The peroxi
Table 1. Characteristics of patients.
dase activity was developed by incubation in 0.05% 3,3-diaminobenzidine for 3 min. Control sections were obtained from the surrounding brain tissues where no cancer cells were detected.
4. Reverse Transcription/Polymerase Reaction (RT-PCR)
The obtained brain tumor tissues were frozen in liquid nitrogen and stored at –80˚C. Total RNA was prepared using the RNase Mini Kit according to the manufacture directions(Qiagen, Chatsworth, CA). The RNA samples were reverse-transcribed using Superscript reverse transcriptase (Life Technologies Inc., Gaithersburg, MD.), random hexamers, and dNTPs. cDNA synthesized were amplified by PCR( 30 cycles) with Taq DNA polymerase (Qiagen, Chatsworth, CA) in the presence of both dNTP and an appropriate pair of primers. The following sense and anti sense primer was used:
CD133 forward primer 5’-CAG AGT ACA ACG CCA AAC CA-3’
CD133 reverse primer 5’-AAA TCA CGA TGA GGG TCA GC-3’.
EGFR sense; 5-AGCGGATAACAATTTCACACAGG-3
anti-sense; 5-GTCGTCTTTCCAGACGTTAGT-3
MT1-MMP sense; 5-ACAGTCTGCGGAACGGAGC AG-3
anti-sense; 5-GTCAATTGTGTTTCTGTCCAC-3
MMP7 sense; 5-GTGGTCACCTACAGGATCGTA-3
anti-sense; 5-CTGAAGTTTCTATTTCTTTTTGA-3
CD44 sense; 5-GTACGTCTTCAAATACCA-3
anti-sense; 5-GTGGTTGAAATGGTGC-3
β-actin forward primer 5’-GTC TTC CCC TCC ATC GTG-3’β-actin reverse primer 5’-AGG TGT GGT GCC AGA TTT TC-3’Results were expressed as band intensity in each lane relative to GAPDH and compared statistically using Student’s t test.
5. Statistical Analysis
The immunohistochemical and PCR activities measured in this study were analyses using the Student t-test. A probability value less than 0.05 was considered statistically significant.
6. Results
6.1. Immunohistochemistry
EGFR immunostaining was detected in 75% (6/8) and 67% (1/3) of brain metastasis from lung adenocarcinoma and breast cancer, respectively. MT1-MMP immunostaining was also detected in 73% (8/11) of these brain metastasis. CD133 was not detected in these 13 patients. EGFR immunostaining was detected in 75% (6/8) and 67% (1/3) of brain metastasis from lung adenocarcinoma and breast cancer, respectively (Figure 1). MT1-MMP immunostaining was also detected in 73% (8/11) of these brain metastasis. CD133 was not detected in these 13 patients (Table 2).
6.2. Identification of Gene Expression
Figure 2 shows the relative gene expression levels of brain metastasis from lung cancer for EGFR, MT1-MMP, MMP7, and CD44. Table 3 summarizes the RT-PCR results. EGFR mRNA was detected in 83.3% (25/30) and 70% (7/10) of brain metastasis from lung cancer and breast cancer, respectively. MT1-MMP mRNA was detected in 73.3% (22/30) specimens of brain metastasis from lung cancer, and only 13.3% (4/30) specimens of these patients expressed MMP7 mRNA. MT1-MMP mRNA was also detected in 50% (5/10) of brain metastasis from breast cancer. As for the correlation between EGFR and other gene expression, gene expression of