Yi Qing

Nanogold-enwrapped graphene nanocomposites as trace labels for sensitivity enhancement of electrochemical immunosensors in clinical immunoassays: Carcinoembryonic antigen as a model.

A new, highly sensitive electrochemical immunosensor with a sandwich-type immunoassay format was designed to quantify carcinoembryonic antigen (CEA), as a model tumor marker, using nanogold-enwrapped graphene nanocomposites (NGGNs) as trace labels in clinical immunoassays. The device consisted of a glassy carbon electrode coated with Prussian Blue (PB) on whose surface gold nanoparticles were electrochemically deposited to the further modified with the specific analyte-capturing molecule, anti-CEA antibodies. The immunoassay was performed using horseradish peroxidase (HRP)-conjugated anti-CEA as secondary antibodies attached on the NGGN surface (HRP-anti-CEA-NGGN). The method using HRP-anti-CEA-NGGNs as detection antibodies shows high signal amplification, and exhibits a dynamic working range of 0.05-350 ng/mL with a low detection limit of 0.01 ng/mL CEA (at 3s). The assayed results of serum samples with the sensor received an acceptable agreement with the reference values. Importantly, the methodology provides a promising ultrasensitive assay strategy for clinical applications.

Upregulation of PD-L1 and APE1 is associated with tumorigenesis and poor prognosis of gastric cancer

Introduction Gastric cancer is a fatal malignancy with a rising incidence rate. Effective methods for early diagnosis, monitoring metastasis, and prognosis are currently unavailable for gastric cancer. In this study, we examined the association of programmed death ligand-1 (PD-L1) and apurinic/apyrimidinic endonuclease 1 (APE1) expression with the prognosis of gastric cancer. Methods The expressions of PD-L1 and APE1 were detected by immunohistochemistry in 107 cases of human gastric carcinoma. The correlation of PD-L1 and APE1 expression with the clinicopathologic features of gastric carcinoma was analyzed by SPSS version 19.0. Results The positive expression rates of PD-L1 and APE1 in gastric cancer tissues were 50.5% (54/107) and 86.9% (93/107), respectively. PD-L1 and APE1 positive expressions were significantly associated with depth of invasion, lymph node metastasis, pathological type, overall survival, and higher T stage. Furthermore, the expression of PD-L1 in highly differentiated gastric cancers was higher than that in poorly differentiated cancers (P=0.008). Moreover, the expression of APE1 and PD-L1 in gastric cancers was positively correlated (r=0.336, P<0.01). Multivariate analysis showed that the depth of invasion was a significant prognostic factor (risk ratio 19.91; P=0.000), but there was no significant relationship with PD-L1, APE1, prognosis, and other characteristics. Conclusion The deregulation of PD-L1 and APE1 might contribute to the development and the poor prognosis of gastric cancer. Our findings suggest that high expression of PD-L1 and APE1 is a risk factor of gastric cancer and a new biomarker to predict the prognosis of gastric cancer. Furthermore, our findings suggest that targeting the PD-L1 and APE1 signaling pathways may be a new strategy for cancer immune therapy and targeted therapy for gastric cancer, especially in patients with deep invasion and lymph node metastasis.

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.

Targeting truncated APE1 in mitochondria enhances cell survival after oxidative stress

The high steady-state level of mitochondrial DNA (mtDNA) oxidative lesions is assumed to be the result of high susceptibility to DNA damage attack and limited DNA repair capacity in mitochondria. As a key enzyme of base excision repair (BER), human apurinic/apyrimidinic endonuclease (APE1) is often scarce in mitochondria, and mitochondria-targeted APE1 with robust repair activity represents a promising therapeutic candidate. In this study, overexpression vectors of mitochondria-targeted truncated APE1 (mtAPE1) and that of full-length APE1 (flAPE1) were constructed and transfected to human umbilical vein endothelial cells to test their protective effects after hydrogen peroxide-induced oxidative stress. The overexpression of truncated APE1 was achieved at protein and enzyme activity levels in mitochondria of mtAPE1-transfected cells. In parallel, enhanced mtDNA repair capacity and increased cell survival were observed. MtAPE1 transfection also prevented apoptosis by blocking mitochondria-dependent pathways. In contrast, flAPE1 transfection rendered slight elevation of nuclear APE1 protein level and nuclear APE activity but no benefits for cell resistance to oxidative stress. The present results suggest that overexpression of the truncated APE1 in mitochondria appears to be a viable approach to protecting healthy cells from some deleterious effects of oxidative stress.

Serum APE1 Autoantibodies: A Novel Potential Tumor Marker and Predictor of Chemotherapeutic Efficacy in Non-Small Cell Lung Cancer

Apurinic/apyrimidinic endonuclease 1 (APE1), which has the dual functions of both DNA repair and redox activity, has been reported to be highly expressed in non-small cell lung cancer (NSCLC), and this appears to be a characteristic related to chemotherapy resistance. In this study, we identified serum APE1 autoantibodies (APE1-AAbs) in NSCLC patients and healthy controls by immunoblotting and investigated the expression of APE1-AAbs by indirect ELISA from the serum of 292 NSCLC patients and 300 healthy controls. In addition, serum APE1-AAbs level alterations of 91 patients were monitored before and after chemotherapy. Our results showed that serum APE1-AAbs can be detected in both NSCLC patients and healthy controls. Serum APE1-AAbs were significantly higher than those of healthy controls and closely related to APE1 antigen levels both in tumor tissues and the peripheral blood. Moreover, the change in levels of serum APE1-AAbs in NSCLC is closely associated with the response to chemotherapy. These results suggest that APE1-AAbs is a potential tumor marker and predictor of therapeutic efficacy in NSCLC.

Human AP endonuclease/redox factor APE1/ref-1modulates mitochondrial function after oxidative stress by regulating the transcriptional activity of NRF1

Maintenance of mitochondrial functionality largely depends on nuclear transcription because most mitochondrial proteins are encoded by the nuclear genome and transported to the mitochondria. Nuclear respiration factor 1 (NRF1) plays a crucial role in regulating the expression of a broad range of mitochondrial genes in the nucleus in response to cellular oxidative stress. However, little is known about the redox regulatory mechanism of the transcriptional activity of NRF1. In this study, we show that the human apurinic/apyrimidinic endonuclease/redox factor (APE1/Ref-1) is involved in mitochondrial function regulation by modulating the DNA-binding activity of NRF1. Our results show that both APE1 expression level and its redox activity are essential for maintenance of the mitochondrial function after tert-butylhydroperoxide-induced oxidative stress. Upon knocking down or redox mutation of APE1, NRF1 DNA-binding activity was impaired and, consequently, the expression of its downstream genes, including Tfam, Cox6c, and Tomm22, was significantly reduced. NRF1 knockdown blocked the restoration of mitochondrial function by APE1 overexpression, which further suggests APE1 regulates mitochondrial function through an NRF1-dependent pathway. Taken together, our results reveal APE1 as a new coactivator of NRF1, which highlights an additional regulatory role of APE1 in maintenance of mitochondrial functionality.

CALIFORNIUM-252 BRACHYTHERAPY COMBINED WITH EXTERNAL-BEAM RADIOTHERAPY FOR CERVICAL CANCER: LONG-TERM TREATMENT RESULTS

PURPOSE To observe, by retrospective analysis, the curative effects and complications due to californium-252 (252Cf) neutron intracavitary brachytherapy (ICBT) combined with external-beam radiotherapy (EBRT) in the treatment of cervical cancer. METHODS AND MATERIALS From February 1999 to December 2007, 696 patients with cervical cancer (Stages IB to IIIB) were treated with 252Cf-ICBT in combination of EBRT. Of all, 31 patients were at Stage IB, 104 at IIA, 363 at IIB, 64 at IIIA, and 134 at IIIB. Californium-252 ICBT was delivered at 7-12 Gy per insertion per week, with a total dose of 29-45 Gy to reference point A in three to five insertions. The whole pelvic cavity was treated with 8-MV X-ray external irradiation at 2 Gy per fraction, four times per week. After 16-38 Gy of external irradiation, the center of the whole pelvic field was blocked with a 4-cm-wide lead shield, with a total external irradiation dose of 44-56 Gy. The total treatment course was 5 to 6 weeks. RESULTS Overall survival rate at 3 and 5 years for all patients was 76.0% and 64.9%, respectively. Disease-free 3- and 5-year survival rates of patients were 71.2% and 58.4%, respectively. Late complications included vaginal contracture and adhesion, radiation proctitis, radiation cystitis, and inflammatory bowel, which accounted for 5.8%, 7.1%, 6.2%, and 4.9%, respectively. Univariate analysis results showed significant correlation of stage, age, histopathologic grade, and lymph node status with overall survival. Cox multiple regression analysis showed that the independent variables were stage, histopathologic grade, tumor size, and lymphatic metastasis in all patients. CONCLUSION Results of this series suggest that the combined use of 252Cf-ICBT with EBRT is an effective method for treatment of cervical cancer.

Elevated Expression of APE1/Ref-1 and its Regulation on IL-6 and IL-8 in Bone Marrow Stromal Cells of Multiple Myeloma

A number of growth factors secreted by bone marrow stromal cells (BMSCs), including interleukin-6 and -8 (IL-6/8), are important for the initiation and progression of multiple myeloma (MM). However, the mechanisms that regulate the production of IL-6/8 by BMSC have not yet been well characterized. Human dual functional protein apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE1/Ref-1) is essential for cell survival and proliferation. Previous studies showed that APE1/Ref-1 was overexpressed in tumor cells, but few studies showed its expression in supportive cells in the tumor microenvironment. We first detected APE1/Ref-1 expression in BMSCs of normal, initial, and recurrent MM patients, and then explore the correlation between APE1/Ref-1 level and IL-6/8 secretion of BMSCs. A marked increase of APE1/Ref-1 expression and abnormal subcellular distribution were observed in MM BMSCs. APE1/Ref-1 overexpression was related to higher secretary level of IL-6/8 by MM BMSCs and the IL-6/8 secretion was blocked significantly by adenovirus-mediated APE1/Ref-1-specific (small interfering RNA) siRNA. Our results also demonstrated that APE1/Ref-1-specific siRNA significantly inhibited DNA binding activity of AP-1 and nuclear factor-κB (NF-κB), 2 important transcription factors in the regulation IL-6/8 secretion in MM BMSCs. The results provided by the present study indicate APE1/Ref-1, which plays a regulatory role in IL-6/8 production by BMSCs, may be a potential therapeutic target of MM.

Single Nucleotide Polymorphisms of DNA Base-excision Repair Genes (APE1, OGG1 and XRCC1) Associated with Breast Cancer Risk in a Chinese Population

Altered DNA repair capacity can result in increased susceptibility to cancer. The base excision repair (BER) pathway effectively removes DNA damage caused by ionizing radiation and reactive oxidative species (ROS). In the current study, we analyzed the possible relation of polymorphisms in BER genes, including 8-oxoguanine DNA glycosylase (OGG1), apurinic/apyrimidinic endonuclease 1 (APE1), and X-ray repair cross-complementing group 1 protein (XRCC1), with breast cancer risk in Chinese Han women. This case-control study examined 194 patients with breast cancer and 245 cancer-free hospitalized control subjects. Single nucleotide polymorphisms (SNPs) of OGG1 (Ser326Cys), XRCC1 (Arg399Gln), and APE1 (Asp148Glu and -141T/G) were genotyped and analyzed for their association with breast cancer risk using multivariate logistic regression models. We found that XRCC1 Arg399Gln was significantly associated with an increased risk of breast cancer. Similarly, the XRCC1 Gln allele was significantly associated with an elevated risk in postmenopausal women and women with a high BMI (≥ 24 kg/m2). The OGG1 Cys allele provided a significant protective effect against developing cancer in women with a low BMI (< 24 kg/m2). When analyzing the combined effects of these alleles on the risk of breast cancer, we found that individuals with ≥ 2 adverse genotypes (XRCC1 399Gln, APE1 148Asp, and OGG1 326Ser) were at a 2.18-fold increased risk of breast cancer (P = 0.027). In conclusion, our data indicate that Chinese women with the 399Gln allele of XRCC1 have an increased risk of breast cancer, and the combined effects of polymorphisms of BER genes may contribute to tumorigenesis.

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.