The Interaction Of APE1 And Its Related Proteins In Radiotherapy Of Human Cancer

Ionizing radiation (IR) used as a therapy (radiotherapy) belongs, besides surgery and conventional cytotoxic chemotherapy, to the three major cancer treatment modalities. More than 45% of the cancer patients are cured during their treatment and about 18% of cancers are cured with radiotherapy, but only 5% of the patients for chemotherapy. Compared with other treatment, the major limitations of Radiotherapy are not adequate for the majority of advanced or recurrent cancer patients because of the remarkable resistance of tumor cells to radiation. The consequences of IR in the human body are mainly induction of DNA damage either by direct ionization of DNA or indirectly by the generation of free radicals. The free radicals due to their unpaired electron are highly unstable and reactive. Since most of the cell is constituted from water, free radicals induced by IR are produced predominantly by the degradation of water molecules. As a consequence, reactive oxygen species (ROS) are generated. These free oxygenated radicals are strong oxidants that damage macromolecules such as DNA, but also cellular proteins and lipids. And one of the important mechanisms of the resistance of tumor cells to radiation are DNA damage and repair.The apurinic endonuclease 1/redox factor-1 (APE1) is a master regulator of cellular response to oxidative stress and plays a central role in the maintenance of genome stability acting as the major apurinic/apyrimidinic(AP)-endonuclease in the DNA base excision repair (BER) pathway that copes with DNA damage caused by both endogenous and exogenous agents. As importantly, APE1 also functions as a redox agent maintaining transcription factors involved in cancer promotion and progression in an active reduced state, and recent studies identify it as an excellent target for sensitizing tumor cells to radiotherapy and chemotherapy. B-cell translocation gene 2(BTG2) is an early growth response gene whose promoter contains p53-binding sites and is regulated by p53. It is also an antiproliferative gene and plays an important role in the suppression of carcinogenesis. Several lines of evidence suggest that the expression of BTG2 relates with the sensitivity to ionizing radiation, and BTG2 is also called Ionizing Radiation responsive gene. Nm23-H1 was initially identified as a putative metastasis suppressor on the basis of its reduced expression in certain highly metastatic cell lines and tumors. Non-metastatic protein 23 homolog 1(Nm23-H1) is also a multifunctional enzyme, although its enzymatic activity provided no evidence for a role as a metastasis suppressor in tumor progression. Extensive studies using Nm23-H1 proteins have shown that they participate in the regulation of a broad spectrum of cellular responses including development, differentiation, proliferation, apoptosis and DNA synthesis. All these studies suggest that Nm23-H1 may participate in the DNA repair of human cells. Much evidence-based literature has showed that BTG2 and Nm23-H1 might be involved in DNA repair to resistance of tumor cells to radiation and may interact with APE1. The aim of this study was to evaluate the major functions of APE1 and a detailed comprehension of the molecular targets such as BTG2 and Nm23-H1 of APE1 functions, allowed establishment of ionizing radiation of cell models to inspect and characterize in better detail the major functions of these genes.Objective1. To evaluate the expression of APE1 and BTG2 compared with p53/ cyclinD1/ cyclinE and clinicopathologic parameters in HCC.2. To further elucidate the correlation of APE1 and BTG2 expression in different human liver cancer cell lines irradiated with X-rays.3. To investigate the expression of APE1 and Nm23-H1 in non-small-cell lung carcinoma (NSCLC) compared with clinicopathologic parameters and the curative effects.4. To further elucidate the role of Nm23-H1 and APE1 in the human lung cancer A549 cell line irradiated with X-rays.Materials and Methods1. Expression feature of APE1 and BTG2 and its significance in HCC: The expression of BTG2 mRNA on tissue microarray (TMA) of HCC via in situ hybridization. Expression of APE1 and BTG2 protein was detected by S-P immunohistochemical method in normal liver tissue and HCC.2. Relation between APE1 and BTG2 in different liver cancer cells after IR: The expression of human APE1 and BTG2 protein was detected after Ad5/F35-APE1 siRNA and Ad-p53 were transfered into different liver cancer cells by western blot analysis. The interaction between APE1 and BTG2 was demonstrated by co-immunoprecipitation assays. Western blot was used to analysis the interaction between these two proteins at different time points after irradiating different human liver cancer cells with different doses of X-rays. Subcellular distribution of APE1 and BTG2 was also examined by laser confocal microscopy at 0-8h in IR treated MHCC97-L, HepG2 and Hep3B cell.3. Expression of APE1 and Nm23-H1 compared with clinicopathologic parameters and the curative effects in NSCLC : Expression of APE1 and Nm23-H1 protein was detected by immunohistochemical method in 30 patients with NSCLC. Statistical analyses were performed using a statistical software package (SPSS for Windows). The relevant variables were studied by univariate and multivariate statistical techniques. Survival difference was analyzed by the log-rank test. Multivariate analyses were performed using Cox proportional hazards model. The survival curves were drawn according to Kaplan-Meier method. To calculate statistical significance between categorical variables, we used Chi-square.4. Study of the role of Nm23-H1 and APE1 in the human lung cancer A549 cell line irradiated with X-rays: Western blot was used to detected the expression of these two proteins at different time points after irradiating human lung cancer A549 cells with different doses of X-rays. The subcellular distribution pattern of Nm23-H1 and APE1 in A549 cells after irradiation was also examined by laser confocal microscopy at different time points in IR treated A549 cell. To determine whether Nm23-H1 expression correlated with APE1 expression, we detected the expression of Nm23-H1 in A549 cells treated with Ad5/F35-APE1 siRNA by western blotting.The interaction between Nm23-H1 and APE1 was demonstrated by His-pull down and co-immunoprecipitation assays. The presence of Nm23-H1/APE1 complex in X-rays-irradiated A549 cells was also detected by DNA affinity precipitation analysis of a DNA fragment containing an AP site. The AP endonuclease activities of APE1 and Nm23-H1 were evaluated by a well-characterized oligonucleotide cleavage assay. Electrophoretic mobility shift assays (EMSAs) were performed using APE1, nuclear proteins of A549 cells transfected with different doses of pEGFP-Nm23-H1 plasmid, and oligonucleotides in DNA affinity precipitation analysis . Results1. There were the nuclear APE1, the cytoplasmic APE1 and the nuclear-cytoplasmic APE1 in HCC-tissues. Expression of nuclear APE1/ cytoplasmic APE1/ nuclear-cytoplasmic APE1, was positive in 46.6/3.88/49.51 percent in HCC-tissues, respectively. The cytoplasmic APE1 expression implied a higher tumor grade significantly.BTG2 mRNA was expressed in 71.19% HCC and BTG2 protein expression was 67.8% in HCC-tissues respectively. BTG2 expression was predominantly detected in the cytoplasm of tumour cells and the mRNA/protein expression correlated significantly with the increasing tumor grade. Expression of p53/cyclin-D1/cyclin-E was positive in 44.1/59.32/38.98 percent in HCC-tissues, respectively. There was a significant difference of BTG2 expression between normal liver tissue and HCC tissues.2. The expression of APE1 was higher in all of the liver cancer cells but the BTG2 expression was significantly higher in PLC and MHCC97-L cell than in LO2 cell. rAd-p53 enhanced the expression of BTG2 protein in all of liver cancer cells, but APE1 expression was decreased in all of liver cancer cells, especially in HepG2 and MHCC97-L cell. Ad5/F35-APE1 siRNA suppressed significantly the expression of APE1 and BTG2 in HepG2. Co-immunoprecipitation assays indicated BTG2 binds to APE1 in LO2 and HepG2 cell. The overexpression of BTG2 protein in different liver cells was induced by irradiation in a dose- dependent manner. Although the expression of APE1 protein was higher after 4 and 16 Gy X-rays irradiation at 48 hrs, the APE1 expression was significantly decreased after 16 Gy X-rays irradiation, especially in PLC cell. After irradiation with 16Gy X-rays in MHCC97-L, HepG2 and Hep3B cell, BTG2 which was initially localized in the cytoplasm of these cells was gradually increased at 1-4 hrs whereas was gradually decreased to control level at 8 hrs, especially in MHCC97-L cell. But APE1 which was mainly localized in the nuclei of cells was not changed at the same time point.3. APE1 was detected in nucleus, cytoplasm and nucleus to cytoplasm of NSCLC tissues by immunohistochemical staining technique. The positive rate of APE1 nuclear expression and cytoplasmic expression were correlated significantly with pathological type in 30 patients of NSCLC. There were not relation between the expression of APE1 and other clinicopathologic parameters. The expression of APE1 correlated to radiotherapy which affected overall survival(OS) and the cytoplasmic overexpression of APE1 was associated with poor prognosis of NSCLC. The nuclear expression of APE1 was not relationship with curative effects. Nm23-H1 was examined mainly in cytoplasm of NSCLC tissues by immunohistochemical staining technique. The expression of Nm23-H1 in 30 patients of NSCLC was correlated significantly to clinicopathologic parameters including Gender, Smokeing and pathological type. the expression of Nm23-H1 no correlated with other clinicopathologic parameters including differention grading and Age. The expression of Nm23-H1 correlated to the curative effects of radiotherapy and the overexpression of Nm23-H1 was associated with poor prognosis of NSCLC.4. The overexpression of APE1 and Nm23-H1 proteins in A549 cells was induced by irradiation in a dose- and time-dependent manner. The subcellular distribution pattern of APE1 and Nm23-H1 in A549 cells after irradiation was reversed. After irradiation with different doses of X-rays, APE1 which was initially localized in the nuclei of A549 cells was gradually increased in the cytoplasm whereas Nm23-H1 which was mainly localized in the cytoplasm was gradually increased in the nuclei of these cells. Altered Nm23-H1 expression correlates with changes in APE1 expression, detected by knockdown and overexpression.The interaction between Nm23-H1 and APE1 was demonstrated by His-pull down and co-immunoprecipitation assays. Nm23-H1 binds with APE1 to mediate AP sites and may therefore participate in the DNA BER repair pathway. Although the AP endonuclease activity of Nm23-H1 protein was too weak to be detected, the AP endonuclease activity of APE1 protein was increased with the increased expression of Nm23-H1 protein. a mobility shift clearly occurs in the presence of APE1 and nuclear proteins of A549 cells. Besides, as the doses of pEGFP-Nm23-H1 plasmid transfected into the cells were increased, formation of the APE1-DNA complex was also increased.Conclusion1. The overexpression of cytoplasmic APE1 and BTG2 protein in liver cells may play a pivotal role in the carcinogenesis and progression of HCC. Aberrant expression of BTG2 in hepatocellular carcinoma is associated with overexpression of cyclinD1/cyclinE.2. The expression of APE1 correlated to BTG2 expression in different human liver cancer cells and ionizing radiation of these cells. BTG2 protein binds to APE1 and an interaction between BTG2 and APE1 proteins may be a factor involved in a DNA repair pathway in human liver cancer cells following damage by ionizing radiation.3. The expression of nuclear/cytoplasmic APE1 and Nm23-H1 was correlated with pathological type of NSCLC. The overexpression of cytoplasmic APE1 and Nm23-H1 can be a potential marker for poor prognosis prediction of NSCLC patients after radiotherapy screening.4. Nm23-H1 protein binds to APE1 at AP sites and stimulates AP endonuclease activity following ionizing radiation of the human lung cancer A549 cells.