br apoptosis evasion in the pre malignant
apoptosis evasion in the pre-malignant cells [5,6]. Following the DSB stimuli, the ataxia telangiectasia mutated (ATM) kinase phosphorylates the hydroxyl terminal of H2AX at the serine-139 residue, which sub-sequently recruits DNA-repair related proteins and cyclins to the da-maged site. Studies show that down-regulating H2AX levels in tumor cells enhances their sensitivity to chemo- and radiotherapy [7,8]. Taken together, targeting RAD51 and H2AX can potentially sensitize tumor cells to radio- and chemotherapy, and induce apoptosis.
High-throughput screening of small-molecule libraries has helped discover numerous novel chemotherapeutic agents, including pacli-taxel, camptothecin, Vinca alkaloids and their derivatives etc. . In recent decades, the focus of anti-cancer drug discovery has shifted to screening for the bioactive ingredients in natural products because of their lower toxicity and higher efficacy . Alpinumisoflavone (AIF), the principal bioactive component isolated from D. eriocarpa, a tradi-tional Chinese medicine herb, has potent pharmacological properties, including atheroprotective , molluscicidal , estrogenic , and anti-bacterial activities . Zhang et al. reported that AIF en-hanced the sensitivity of esophageal squamous cell carcinoma (ESCC)
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 Ferrozine 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).