br Available online November br Table br
Available online 16 November 2018
The correlation between RAD51 expression and clinicopathological character-istics of included patients.
Variables RAD51 expression
Female 7 5
I and II
Lymph node metastasis 17 6
cells to radiotherapy by inducing Methoctramine arrest and apoptosis . In addition, AIF has also shown anti-neoplastic activity in lung cancer , renal cell carcinoma , and melanoma .
The aim of this study was to determine the potential anti-cancer effect of AIF on CRC cells. We found that AIF inhibited CRC cell growth and triggered apoptosis in a dose-dependent manner by inducing DNA DSBs and blocking DNA damage repair, as evidenced by RAD51 down-regulation and increased γH2AX foci in the CRC cells. Furthermore, inhibition of RAD51 in vitro augmented the anti-cancer effects of AIF. Taken together, AIF suppressed CRC cell growth by modulating RAD51.
2. Materials and methods
2.1. CRC tissue samples
In present study, all investigation and experiments have obtained patients' consent and been approved by the Ethic Committee of Puyang Oilfield General Hospital (Henan, China). Resected 47 pairs CRC tumors tissues and adjacent non-tumor tissues were sampled from a total of 47 patients in Puyang Oilfield General Hospital from Jan 2011 to March 2013. Diagnosis and staging were carried out by 2 independent senior oncologists blinded to the data. The level of RAD51 in tissue samples was determined by immunohistochemical assay. According to the per-centage of positively stained cells, the sections were graded by five levels: 0 (≤5%), 1 positive cells and the staining intensity were mul-tiplied to generate an immune-reactive score for each specimen (0−12). Based on the total score, scores of 0–3 were considered as the low expression group, and scores of 4–12 were defined as the high expression group. All the patients were followed up by telephone up to April 30, 2018, to obtain the survival data. The correlation between RAD51 expression and clinicopathological characteristics of included patients was listed in Table 1. Life Sciences 216 (2019) 259–270
2.2. Antibodies and reagents
2.3. Cell lines and culture
Two well-established CRC cell lines, HCT-116 (#CCL-247) and SW480 (#CCL-228) cells were purchased from the American Type Culture Collection. Cells were cultured in McCoy's 5a medium (HCT-
2.4. Cell proliferation assay
The viability of CRC cells treated with AIF was analyzed using the CCK-8 kit (#C0038, Beyotime Biotechnology, Shanghai, China) ac-cording to the manufacturer's instructions. Briefly, 5 × 103 CRC cells were seeded in 96-well plates and stimulated with different con-centrations of AIF for the indicated durations. The culture medium was replaced with 100 μl fresh medium containing 10 μl CCK-8 reagent per well, and the cells were incubated further for 2 h. The absorbance at 450 mm was measured using a microplate reader (Multiskan FC, ThermoFisher, Waltham, MA). r> 2.5. Colony formation assay
HCT-116 and SW480 cells were seeded into 6-well plates at the density of 300 cells/well and cultured with different concentrations of AIF or the vehicle for 14 days. The ensuing colonies were fixed with 4% paraformaldehyde (#P0099, Beyotime Biotechnology, Shanghai, China) for 15 min and stained with 1% crystal violet (#C0121, Beyotime Biotechnology, Shanghai, China) for 10 min as previously described . The number of colonies were counted in five randomly chosen fields under an inverted microscope (40×), and the colony-forming efficiency was calculated as the number of colonies/ 500 × 100%.
Chromatin condensation in HCT-116 and SW480 cells was de-termined using Hoechst 33258 staining. Briefly, 1 × 105 cells were seeded per well in 6-well plates and cultured with AIF or the vehicle for 48 h. The cells were fixed with 4% paraformaldehyde for 15 min at room temperature, and stained with Hoechst 33258 (#C1017, Beyotime Biotechnology, Shanghai, China) according to the manufac-turer's instructions. The cells were observed under a fluorescence mi-croscope (Olympus Corporation, Tokyo, Japan).
(caption on next page)
Fig. 1. AIF induces apoptosis in CRC cells. A. HCT-116 and SW480 cells were treated with AIF (5 and 10 μM) for 24 h and 48 h, and viability was determined by CCK-8 assay. B. Colony formation assay of HCT-116 and SW480 cells treated with AIF (5 and 10 μM) for the indicated durations. (C, D). AIF-treated cells were stained with Hoechst 33258 and percentage of apoptotic cells was measured using flow cytometry. E. Western blot showing the relative levels of the apoptosis-associated proteins pro-caspase-3, caspase-3, Bcl-2 and Bax normalized to GAPDH. Data are presented as mean ± SD from three independent experiments, **p < 0.01.