Mengxia Li

Genetic Polymorphism of DNA Base-excision Repair Genes (APE1, OGG1 and XRCC1) and Their Correlation with Risk of Lung Cancer in a Chinese Population

BACKGROUND AND AIMS Reactive oxygen species (ROS) and numerous carcinogens may cause DNA damage including oxidative base lesions that contribute to the risk of lung cancer. The base excision repair (BER) pathway could effectively remove oxidative lesions in which 8-oxoguanine glycosylase-1 (OGG1), x-ray repair cross-complementing 1 (XRCC1), and apurinic/apyimidinic endonuclease 1 (APE1) play key roles. The aim of this study was to analyze the polymorphisms of DNA BER genes (OOG1, XRCC1 and APE1) and explore their associations, and the combined effects of these variants, with risk of lung cancer. METHODS In a hospital-based, case-control study of 455 lung cancer cases and 443 cancer-free hospital controls, the SNPs of OGG1 (Ser326Cys), XRCC1 (Arg399Gln), APE1 (Asp148Glu and -141T/G) were genotyped and analyzed for their correlation with the risk of lung cancer in multivariate logistic regression models. RESULTS Individuals homozygous for the variants APE1 -141GG showed a protective effect for lung cancer overall (OR=0.62; 95% CI: 0.42-0.91; p=0.02) and for lung adenocarcinoma (OR=0.65; 95% CI, 0.44-0.96; p=0.03). When analyzing the combined effects of variant alleles, 84 patients and controls were identified who were homozygous for two or three of the potential protective alleles (i.e., OGG1 326Cys, XRCC1 399Gln and APE1 -141G). ORs were significantly reduced when all patients were analyzed (OR=0.62; 95% CI: 0.38-0.99; p=0.05). CONCLUSIONS The combined effects of polymorphisms within BER genes may contribute to the tumorigenesis of lung cancer.

Knock-in reconstitution studies reveal an unexpected role of Cys-65 in regulating APE1/Ref-1 subcellular trafficking and function

The multifunctional APE1 protein is required for tumor progression and is associated with cancer resistance. It is shown that APE1 presents structural elements that function in distinct cellular roles, highlighting the molecular determinants of the multifunctional nature of this protein and providing the basis for a new role of the C65 residue.

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.

Association of DNA base excision repair genes (OGG1, APE1 and XRCC1) polymorphisms with outcome to platinum-based chemotherapy in advanced nonsmall-cell lung cancer patients.

Polymorphism of DNA base excision repair (BER) genes affects DNA repair capacity and may alter sensitivity to platinum‐based chemotherapy regimens. This study investigated polymorphisms of OGG1 Ser326Cys, APE1 Asp148Glu APE1‐141T/G and XRCC1 Arg399Gln for association with clinical outcome in 235 advanced inoperable nonsmall‐cell lung cancer (NSCLC) patients after treatment with platinum‐based chemotherapy. The multivariate analysis showed that OGG1 326 GC was associated with poor PFS [hazard ratio (HR) 1.730, p = 0.005], while XRCC1 399 GA, or GA+AA, was associated with poor OS in short‐term period (HR 1.718, p = 0.003; HR 1.691, p = 0.003, respectively). Patients with OGG1 326/XRCC1 399 variant alleles had a higher risk to die early in short‐term period (HR 1.929, p < 0.001). Furthermore, patients with XRCC1 399 variant allele (GA+AA) had higher risk of hematologic toxicity (p = 0.009), whereas patients carrying the OGG1 326 variant (GG), or the APE1‐141 GG variant, had reduced risk of gastrointestinal toxicity (p = 0.015 and p = 0.023, respectively). The data from the current study provide evidence that OGG1 Ser326Cys, XRCC1 Arg399Gln, APE1 Asp148Glu, and APE1‐141T/G polymorphisms may be useful in predicting clinical outcomes in patients with advanced inoperable NSCLC that will undergo platinum‐based chemotherapy.

Silencing of APE1 enhances sensitivity of human hepatocellular carcinoma cells to radiotherapy in vitro and in a xenograft model.

Resistance to radiotherapy is a key limitation for the treatment of human hepatocellular carcinoma (HCC). To overcome this problem, we investigated the correlation between radioresistance and the human apurinic/apyrimidinic endonuclease (APE1), a bifunctional protein, which plays an important role in DNA repair and redox regulation activity of transcription factors. In the present study, we examined the radiosensitivity profiles of three human HCC cell lines, HepG2, Hep3B, and MHCC97L, using the adenoviral vector Ad5/F35-mediated APE1 siRNA (Ad5/F35-siAPE1). The p53 mutant cell lines MHCC97L showed radioresistance, compared with HepG2 and Hep3B cells. APE1 was strongly expressed in MHCC97L cells and was induced by irradiation in a dose-dependent manner, and Ad5/F35-siAPE1 effectively inhibited irradiation-induced APE1 and p53 expression. Moreover, silencing of APE1 significantly potentiated the growth inhibition and apoptosis induction by irradiation in all tested human HCC cell lines. In addition, Ad5/F35-siAPE1 significantly enhanced inhibition of tumor growth and potentiated cell apoptosis by irradiation both in HepG2 and MHCC97L xenografts. In conclusion, down regulation of APE1 could enhance sensitivity of human HCC cells to radiotherapy in vitro and in vivo.

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.

Induction of Apoptosis in Human Multiple Myeloma Cell Lines by Ebselen via Enhancing the Endogenous Reactive Oxygen Species Production

Ebselen a selenoorganic compound showing glutathione peroxidase like activity is an anti-inflammatory and antioxidative agent. Its cytoprotective activity has been investigated in recent years. However, experimental evidence also shows that ebselen causes cell death in several cancer cell types whose mechanism has not yet been elucidated. In this study, we examined the effect of ebselen on multiple myeloma (MM) cell lines in vitro. The results showed that ebselen significantly enhanced the production of reactive oxygen species (ROS) accompanied by cell viability decrease and apoptosis rate increase. Further studies revealed that ebselen can induce Bax redistribution from the cytosol to mitochondria leading to mitochondrial membrane potential ΔΨm changes and cytochrome C release from the mitochondria to cytosol. Furtherly, we found that exogenous addition of N-acetyl cysteine (NAC) completely diminished the cell damage induced by ebselen. This result suggests that relatively high concentration of ebselen can induce MM cells apoptosis in culture by enhancing the production of endogenous ROS and triggering mitochondria mediated apoptotic pathway.

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 induced upregulation of fibroblast growth factor 2 and its receptor 3 induces angiogenesis in human osteosarcoma cells.

Tumor angiogenesis contributes to inferior prognosis in osteosarcoma. Apurinic/apyrimidinic endonuclease 1 (APE1) and fibroblast growth factor 2 (FGF2) and its receptor 3 (FGFR3) signaling pathway plays an important role in the angiogenic process. In this study we observed that high expression of APE1, FGF2 and FGFR3, and microvessel density are positively correlated with poor prognosis of osteosarcoma patients. Furthermore, the Cox model showed that the tumor size, FGF2 and its receptor 3 (FGFR3), and microvessel density were adverse prognostic factors. Based on our clinical data, and the fact that APE1 is involved in tumor angiogenesis, we hypothesize that it is very likely that APE1 may indirectly promote angiogenesis by upregulating fibroblast FGF2 and FGFR3. Our preliminary data show small interfering RNA‐mediated silence of APE1 experiments, which further supports this hypothesis. APE1‐small interfering RNA significantly inhibited tumor angiogenesis by downregulating in vitro expression of FGF2 and FGFR3 in human umbilical vein endothelial cells in Matrigel tube formation assay, and further inhibited tumor growth in vivo in a mouse xenograft model. Thus, the proposed APE1‐FGF2 and FGFR3 pathway may provide a novel mechanism for regulation of FGF2 and FGFR3 by APE1 in tumor angiogenesis.