Iodine-125 induces apoptosis via regulating p53, microvessel density, and vascular endothelial growth factor in colorect
- PDF / 630,279 Bytes
- 7 Pages / 595.276 x 793.701 pts Page_size
- 25 Downloads / 195 Views
RESEARCH
WORLD JOURNAL OF SURGICAL ONCOLOGY
Open Access
Iodine-125 induces apoptosis via regulating p53, microvessel density, and vascular endothelial growth factor in colorectal cancer Zhenhuan Ma1,2, Yong Yang1,2*, Guokai Yang1,2, Jia Wan1,2, Guojian Li1,2, Ping Lu1,2 and Lingjuan Du1,2
Abstract Background: Iodine interstitial brachytherapy has been widely reported for treating colorectal cancer (CRC). However, the inhibitory molecular mechanism of iodine-125 (I-125) on CRC has not been reported. Methods: To illustrate the inhibitory mechanism of iodine-125 (I-125) on CRC, we established the animal models of CRC via the injection of HCT-8 cells into nude mice. Subsequently, the I-125 granules were implanted into the tumor of the animal model at different dosages. Proliferating cell nuclear antigen and terminal transferase dUTP nick end labeling were used to detect the apoptosis of the tumor cells. Immunohistochemistry SP staining was used to measure the expression of p53 protein. The protein levels were examined with western blot and ELISA. Meanwhile, microvessel density (MVD) was counted by endothelial cells immunostained by anti-CD34 antibody. Results: The results showed that I-125 protests against CRC via increasing the protein level of p53 and decreasing the level of vascular endothelial growth factor (VEGF), leading to the decrease of MVD in CRC (P 50% nuclei). The numerical scoring was confirmed by a second independent examination, blinded to the initial score. Proliferating cell nuclear antigen (PCNA) and terminal transferase dUTP nick end labeling (TUNEL) assay for apoptosis
PCNA was examined to investigate cell proliferation, which can reflect the degree of the cellular apoptosis. All cells were fixed in PBS formalin, embedded in paraffin and stained. PCNA was performed using a labeled streptavidin-biotin. Anti-PCNA monoclonal antibody (DAKO, Carpenteria, CA, USA) reacted exclusively with nuclei. TUNEL was also performed to examine the apoptosis according to a previous report [20]. All cells were suspended in PBS buffer by gently vortexing the vials and aliquoted to approximately 1 × 106 cells/mL per test. The cells were collected via centrifuge for 5 min (300 g) and resuspended in 1 mL of the wash buffer in each tube. After washing three times, 50 μL of the TdT enzyme mixture were added to the sample and incubated for 60 min at 37°C in a water bath. Subsequently, the cells were washed three times and resuspended in 0.1 mL of the antibody labeling mix. The tubes were incubated in the dark for 30 min at room temperature. The cells were washed three times and suspended in 0.9 mL PBS. One hundred μL propidium iodide/RNase were added to each tube. After 3 hours of staining, the samples were analyzed by flow cytometry.
Ma et al. World Journal of Surgical Oncology 2014, 12:222 http://www.wjso.com/content/12/1/222
Detection for the expression of vascular endothelial growth factor (VEGF) and quantitation of microvessel density (MVD)
The expression level of VEGF was determined according to Takahashi stan
Data Loading...
