Zhaoyang Zhong

Multifunctional magnetic bead-based electrochemical immunoassay for the detection of aflatoxin B1 in food

A sensitive and reusable electrochemical immunoassay for aflatoxin B(1) (AFB(1)) in food has been developed. A multifunctional magnetic bead (MMB) was initially synthesized using magnetic CoFe(2)O(4) nanoparticle as the core and Prussian blue nanoparticle (PBNP)-doped silica as the shell, and then the prepared MMB was used as an affinity support for the immobilization of the AFB(1)-bovine serum albumin conjugate (AFB(1)-BSA). With the aid of an external magnet, the AFB(1)-BSA-conjugated MMBs were attached on the surface of an indium tin oxide (ITO) electrode. Gold nanoparticles, labeled with horseradish peroxidase (HRP)-bound anti-AFB(1) antibodies (HRP-anti-AFB(1)), were employed as detection antibodies. With a competitive immunoassay format, the concentrations of AFB(1) in samples were measured in PBS (pH 7.0) by using PBNP-doped MMBs as the mediator, HRP-anti-AFB(1) as the tracer and hydrogen peroxide (H(2)O(2)) as the enzyme substrate, and the linear range was 0.05-12 ng/mL with a detection limit of 6.0 pg/mL AFB(1) (at 3sigma). Intra- and inter-assay coefficients of variation were less than 7.5%. In addition, the content of AFB(1) in red paprika specimens has been assayed by the developed immunoassay and a commercially available enzyme-linked immunosorbent assay (ELISA) method, respectively, and consistent results were obtained. The as-prepared immunoassay provides a promising approach for the screening of organic pollutants because it is simple, rapid, highly sensitive, specific, and without the need of sample pre-concentration.

Identification and characterization of mitochondrial targeting sequence of human apurinic/apyrimidinic endonuclease 1.

Dually targeted mitochondrial proteins usually possess an unconventional mitochondrial targeting sequence (MTS), which makes them difficult to predict by current bioinformatics approaches. Human apurinic/apyrimidinic endonuclease (APE1) plays a central role in the cellular response to oxidative stress. It is a dually targeted protein preferentially residing in the nucleus with conditional distribution in the mitochondria. However, the mitochondrial translocation mechanism of APE1 is not well characterized because it harbors an unconventional MTS that is difficult to predict by bioinformatics analysis. Two experimental approaches were combined in this study to identify the MTS of APE1. First, the interactions between the peptides from APE1 and the three purified translocase receptors of the outer mitochondrial membrane (Tom) were evaluated using a peptide array screen. Consequently, the intracellular distribution of green fluorescent protein-tagged, truncated, or mutated APE1 proteins was traced by tag detection. The results demonstrated that the only MTS of APE1 is harbored within residues 289–318 in the C terminus, which is normally masked by the intact N-terminal structure. As a dually targeted mitochondrial protein, APE1 possesses a special distribution pattern of different subcellular targeting signals, the identification of which sheds light on future prediction of MTSs.

Vector-based Ape1 small interfering RNA enhances the sensitivity of human osteosarcoma cells to endostatin in vivo

Osteosarcoma is a highly vascular and extremely destructive malignancy, and the survival of patients with osteosarcoma has not improved significantly in recent years. Antiangiogenic therapy currently holds great potential in conjunction with conventional treatment modalities for osteosarcoma. However, there are examples of gradual loss of response, and perhaps acquired resistance to antiangiogenic drugs. The acquired resistance of antiangiogenesis may be associated with a lot of hypoxia‐response genes. The human apurinic/apyrimidinic endonuclease (Ape1) protein, a bifunctional redox factor and apurinic/apyrimidinic (AP) endonuclease, plays a crucial role in protecting against cell death due to hypoxia. We therefore hypothesized that Ape1 may contribute to the resistance of antiangiogenic therapy. To investigate the effect of Ape1 on the sensitivity of human osteosarcoma cells to endostatin, we constructed an Ape1 small interfering RNA expression vector, pSilenceApe1. Transfection of human osteosarcoma 9901 and HOS cells with pSilenceApe1 resulted in a dose‐dependent loss of Ape1 protein. pSilenceApe1 also significantly suppressed the expression of vascular endothelial growth factor (VEGF) protein in the 9901 cells. Combined treatment with pSilenceApe1 and recombinant human endostatin (rhES) showed potent antiangiogenic effects in the transwell chamber invasion assay. Then, 20 nude mice bearing 9901 xenografts were divided into four groups: the phosphate‐buffered saline treatment control group; the rhES treatment group (1.5 mg/kg, daily); the pSilenceApe1 treatment group (20 µg, once every 3 days); and the combination of rhES and pSilenceApe1 treatment group. pSilenceApe1 significantly suppressed the expression of Ape1 and VEGF protein in the 9901 xenografts. The tumor‐inhibition rate of the pSilenceApe1, rhES, and combination of rhES and pSilenceApe1 treatment groups was 38.23, 35.29, and 62.18%, respectively. Furthermore, a significant decrease in microvessel density with an increase in apoptosis was observed following combined treatment with pSilenceApe1 and rhES, compared with control and either agent alone in 9901 xenografts. These results indicate that Ape1 small interfering RNA could enhance the sensitivity of osteosarcoma cells to endostatin. (Cancer Sci 2007; 98: 1993–2001)

Human apurinic/apyrimidinic endonuclease siRNA inhibits the angiogenesis induced by X-ray irradiation in lung cancer cells.

Objective: Radiotherapy is an important and effective treatment method for non-small cell lung cancer (NSCLC). Nonetheless, radiotherapy can alter the expression of proangiogenic molecules and induce angiogenesis. Human apurinic/apyrimidinic endonuclease (APE1) is a multifunctional protein, which has DNA repair and redox function. Our previous studies indicated APE1 is also a crucial angiogenic regulator. Thus, we investigated the effect of APE1 on radiation-induced angiogenesis in lung cancer and its underlying mechanism. Methods: Tumor specimens of 136 patients with NSCLC were obtained from 2003 to 2008. The APE1 and vascular endothelial growth factor (VEGF) expression, as well as microvessel density (MVD) were observed with immunohistochemistry in tumor samples. Human lung adenocarcinoma A549 cells were treated with Ad5/F35-APE1 siRNA and/or irradiation, and then the cells were used for APE1 analysis by Western blot and VEGF analysis by RT-PCR and ELISA. To elucidate the underline mechanism of APE1 on VEGF expression, HIF-1α protein level was determined by Western blot, and the DNA binding activity of HIF-1α was detected by EMSA. Transwell migration assay and capillary-like structure assay were used to observe the migration and capillary-like structure formation ability of human umbilical veins endothelial cells (HUVECs) that were co-cultured with Ad5/F35-APE1 siRNA and (or) irradiation treated A549 cells culture medium. Results: The high expression rates of APE1 and VEGF in NSCLC were 77.94% and 66.18%, respectively. The expressions of APE1 was significantly correlated with VEGF and MVD (r=0.369, r=0.387). APE1 and VEGF high expression were significantly associated with reduced disease free survival (DFS) time. The high expressions of APE1 and VEGF on A549 cells were concurrently induced by X-ray irradiation in a dose-dependent manner. Silencing of APE1 by Ad5/F35-APE1 siRNA significantly decreased DNA binding activity of HIF-1α and suppressed the expression of VEGF in A549 cells, moreover, significantly inhibited the endothelial cells immigration and capillary-like structure formation induced by irradiated A549 cells. Conclusion: Our results indicate that APE1 may play a crucial role in angiogenesis induced by irradiation. Administration of Ad5/F35-APE1 siRNA during radiotherapy could be a potent adjuvant therapeutic approach to enhance the radiotherapy response, effectively eliminate metastasis and improve the efficacy of radiotherapy for NSCLC.

Apurinic/apyrimidinic endonuclease 1 regulates angiogenesis in a transforming growth factor β-dependent manner in human osteosarcoma

Angiogenesis plays an important role in tumor growth and metastasis and has been reported to be inversely correlated with overall survival of osteosarcoma patients. It has been shown that apurinic/apyrimidinic endonuclease 1 (APE1), a dually functional protein possessing both base excision repair and redox activities, is involved in tumor angiogenesis, although these mechanisms are not fully understood. Our previous study showed that the expression of transforming growth factor β (TGFβ) was significantly reduced in APE1‐deficient osteosarcoma cells. Transforming growth factor β promotes cancer metastasis through various mechanisms including immunosuppression, angiogenesis, and invasion. In the current study, we initially revealed that APE1, TGFβ, and microvessel density (MVD) have pairwise correlation in osteosarcoma tissue samples, whereas TGFβ, tumor size, and MVD were inversely related to the prognosis of the cohort. We found that knocking down APE1 in osteosarcoma cells resulted in TGFβ downregulation. In addition, APE1‐siRNA led to suppression of angiogenesis in vitro based on HUVECs in Transwell and Matrigel tube formation assays. Reduced secretory protein level of TGFβ of culture medium also resulted in decreased phosphorylation of Smad3 of HUVECs. In a mouse xenograft model, siRNA‐mediated silencing of APE1 downregulated TGFβ expression, tumor size, and MVD. Collectively, the current evidence indicates that APE1 regulates angiogenesis in osteosarcoma by controlling the TGFβ pathway, suggesting a novel target for anti‐angiogenesis therapy in human osteosarcoma.

Serum APE1 as a predictive marker for platinum-based chemotherapy of non-small cell lung cancer patients

Purpose To define the role of the DNA repair protein apurinic/apyrimidinic endonuclease 1 (APE1) in predicting the prognosis and chemotherapeutic response of non-small cell lung cancer patients receiving platinum-containing chemotherapy. Results Our investigations found that serum APE1 level was significantly elevated in 229 of 412 NSCLC patients and correlated with its level in tissue (r2 = 0.639, p < 0.001). The elevated APE1 level in both tissue and serum of patients prior to chemotherapy was associated with worse progression-free survival (HR: 2.165, p < 0.001, HR: 1.421, p = 0.012), but not with overall survival. After 6 cycles of chemotherapy, a low APE1 serum level was associated with better overall survival (HR: 0.497, p = 0.010). Experimental Design We measured APE1 protein levels in biopsy tissue from 172 NSCLC patients and sera of 412 NSCLC patients receiving platinum-based chemotherapy by immunohistochemistry and a newly established sensitive and specific enzyme-linked immunosorbent assay, respectively. APE1 levels in sera of 523 healthy donors were also determined as control. Conclusions Our studies indicate that APE1 is a biomarker for predicting prognosis and therapeutic efficacy in NSCLC. The chemotherapy-naïve serum APE1 level, which correlated with its tissue level inversely associated with progression-free survival of platinum-containing doublet chemotherapy, whereas post-treatment serum APE1 level was inversely associated with overall survival.

MicroRNA-765 Enhances the Anti-Angiogenic Effect of CDDP via APE1 in Osteosarcoma

Human osteosarcoma (HOS) is the most common malignancy in children and adolescents and has a heterogeneous presentation and high mortality. Previous studies have shown that microRNAs contribute to RNA silencing and post-transcriptional regulation of gene expression. Here, we showed that significantly increased expression of miR-765 with or without CDDP (Cisplatin) down-regulates APE1 expression and angiogenesis-related markers (VEGF, FGF2, TGFβ, and CD34). Further investigation showed that miR-765 modulates osteosarcoma cell migration and angiogenesis following treatment with cisplatin in vitro and in vivo. MiR-765 increases the anti-angiogenic effect of CDDP in human osteosarcoma. Elucidation of the mechanism of the miR-765-APE1 axis in tumor progression of HOS will be beneficial in identifying biomarkers and therapeutic target of osteosarcoma.

miR-513a-5p regulates radiosensitivity of osteosarcoma by targeting human apurinic/apyrimidinic endonuclease

Radiotherapy in osteosarcoma patients is problematic due to radioresistance; therefore, understanding the mechanism of radioresistance is integral to providing effective radiotherapeutic regimens for osteosarcoma. We now report the activity of an miRNA, miR-513a-5p, in stimulating radiosensitivity of osteosarcoma cells in vitro and in vivo. MiR-513a-5p expression is decreased in osteosarcoma tissue from patients and cultured osteosarcoma cell lines. However, exogenous re-expression of this miRNA in osteosarcoma cell lines, including HOS, U2OS and 9901, can induce sensitization to ionizing radiation. We also confirm that miR-513a-5p suppresses APE1 expression, and that both the redox and DNA repair activity of APE1 were decreased in miR-513a-5p expressing cell lines. By suppressing APE1, miR-513a-5p induces the DNA damage response which stimulates apoptosis after irradiation. Our report establishes miR-513a-5p as a radiosensitizing miRNA and identifies its activity in the suppression of APE1, which could directly lead to radiosensitization.