Apurinic/apyrimidinic Endonuclease 1 Levels Research Articles

Extreme Expression of DNA Repair Protein Apurinic/Apyrimidinic Endonuclease 1 (APE1) in Human Breast Cancer As Measured by Liquid Chromatography and Isotope Dilution Tandem Mass Spectrometry.

Apurinic/apyrimidinic endonuclease 1 (APE1) is a DNA repair protein and plays other important roles. Increased levels of APE1 in cancer have been reported. However, available methods for measuring APE1 levels are indirect and not quantitative. We previously developed an approach using liquid chromatography and tandem mass spectrometry with isotope dilution to accurately measure APE1 levels. Here, we applied this methodology to measure APE1 levels in normal and cancerous human breast tissues. Extreme expression of APE1 in malignant tumors was observed, suggesting that breast cancer cells may require APE1 for survival. Accurate measurement of APE1 may be essential for the development of novel treatment strategies and APE1 inhibitors as anticancer drugs.

Inhibitors of the apurinic/apyrimidinic endonuclease 1 (APE1)/nucleophosmin (NPM1) interaction that display anti-tumor properties.

The apurinic/apyrimidinic endonuclease 1 (APE1) is a protein central to the base excision DNA repair pathway and operates in the modulation of gene expression through redox-dependent and independent mechanisms. Aberrant expression and localization of APE1 in tumors are recurrent hallmarks of aggressiveness and resistance to therapy. We identified and characterized the molecular association between APE1 and nucleophosmin (NPM1), a multifunctional protein involved in the preservation of genome stability and rRNA maturation. This protein-protein interaction modulates subcellular localization and endonuclease activity of APE1. Moreover, we reported a correlation between APE1 and NPM1 expression levels in ovarian cancer, with NPM1 overexpression being a marker of poor prognosis. These observations suggest that tumors that display an augmented APE1/NPM1 association may exhibit increased aggressiveness and resistance. Therefore, targeting the APE1/NPM1 interaction might represent an innovative strategy for the development of anticancer drugs, as tumor cells relying on higher levels of APE1 and NPM1 for proliferation and survival may be more sensitive than untransformed cells. We set up a chemiluminescence-based high-throughput screening assay in order to find small molecules able to interfere with the APE1/NPM1 interaction. This screening led to the identification of a set of bioactive compounds that impair the APE1/NPM1 association in living cells. Interestingly, some of these molecules display anti-proliferative activity and sensitize cells to therapeutically relevant genotoxins. Given the prognostic significance of APE1 and NPM1, these compounds might prove effective in the treatment of tumors that show abundant levels of both proteins, such as ovarian or hepatic carcinomas.

Functional analysis of the involvement of apurinic/apyrimidinic endonuclease 1 in the resistance to melphalan in multiple myeloma

BackgroundMelphalan resistance has been considered one of the major obstacles to improve outcomes in multiple myeloma (MM) therapy; unfortunately, the mechanistic details of this resistance remain unclear. Melphalan is a highly effective alkylating agent which causes many types of DNA lesions, including DNA base alkylation damage that is repaired by base excision repair (BER). We postulated that human apurinic/apyrimidinic endonuclease 1 (APE1), an essential BER enzyme, plays a vital role in acquired melphalan resistance. However, because APE1 is a multifunctional protein with redox activity and acetylation modification in addition to its major repair activity, the particular APE1 function that may play a more important role in melphalan resistance is unknown.MethodsTwo MM cell lines, RPMI-8226 and U266 were used to measure the difference in APE1 levels in melphalan-resistant and sensitive derivatives. APE1 functional mutants for DNA repair, redox and acetylation were employed to investigate the roles of individual APE1 activities in acquired melphalan resistance.ResultsOur results indicate that APE1 is overexpressed in both MM melphalan-resistant cells. Knocking down APE1 sensitizes the melphalan resistant MM cells to melphalan treatment. The exogenous expression of DNA repair mutant H309N and acetylation mutant K6R/K7R of APE1 failed to restore the melphalan resistance of the APE1 knockdown RPMI-8226 cells. The AP endonuclease activity and multidrug resistance protein 1 (MDR1) regulatory activity may play roles in the melphalan resistance of MM cells.ConclusionsThe present study has identified that the DNA repair functions and the acetylation modification of APE1 are involved in melphalan resistance of MM cells and has also shed light on future therapeutic strategies targeting specific APE1 functions by small molecule inhibitors.

Apurinic/apyrimidinic endonuclease 1, the sensitive marker for DNA deterioration in dextran sulfate sodium-induced acute colitis

Mutations in mismatch repair (MMR) genes are commonly associated with the development of colorectal cancer. Additionally, base excision repair, which involves apurinic/apyrimidinic endonuclease 1 (APE1), recognizes and eliminates oxidative DNA damage. Here, we investigated the possible roles of APE1 in dextran sulfate sodium (DSS)-induced acute colitis using the young rat model. Four-week-old Sprague–Dawley rats were administered 2% DSS in drinking water for 1 week. MMR and APE1 expression levels were assessed by western blotting and immunohistochemistry. Following DSS treatment, growth of young rats failed and the animals had loose stools. Together with the histological changes associated with acute colitis, APE1 and MSH2 levels increased significantly at 3 and 5 days after DSS treatment, respectively. The difference between APE1 and MSH2 expression was significant. DSS-induced DNA damage and subsequent repair activity were evaluated by staining for 8-hydroxy-deoxyguanosine (8-OHdG) and APE1, respectively; 8-OHdG immunoreactivity increased throughout the colonic mucosa, while APE1 levels in the surface epithelium increased at an earlier timepoint. Taken together, our data suggest that changes in APE1 expression after DSS treatment occurred earlier and were more widespread than changes in MMR expression, suggesting that APE1 is more sensitive for prediction of DNA deterioration in DSS-induced colitis.

Differential expression of hepatic apurinic/apyrimidinic endonuclease 1, a DNA repair enzyme, in chronic hepatitis

To determine hepatic expression of apurinic/apyrimidinic endonuclease 1 (APE-1) and 8-hydroxydeoxyguanosine (8-OHdG) in patients with chronic hepatitis B and C. Liver biopsies were obtained from 27 patients with chronic hepatitis B virus (HBV), 30 with chronic hepatitis C virus (HCV), 6 with autoimmune hepatitis (AIH), and 6 with primary biliary cirrhosis (PBC). Normal liver tissue was obtained from surgical resection specimens of four patients. Hepatic APE-1 protein and mRNA expression were assayed by Western blot and by real-time polymerase chain reaction, respectively. Hepatocellular APE-1 and 8-OHdG expression were determined by immunohistochemistry. The staining intensity of hepatocellular nuclear APE-1 was lower in the HBV group than in the other groups (P < 0.05). Hepatic APE-1 protein levels were reduced in the HBV group relative to the other groups. Hepatic APE-1 mRNA levels were also lower in the HBV group. The proportion of hepatocytes with 8-OHdG-positive nuclei was increased in the HCV, AIH and PBC groups (P < 0.05), but not in the HBV group. Hepatocellular nuclear APE-1 levels were positively correlated with hepatocellular 8-OHdG levels in both the HBV and HCV groups (HBV, r = 0.34, P < 0.05; HCV, r = 0.54, P < 0.01). An imbalance between oxidative DNA damage and APE-1 expression may contribute to hepatocarcinogenesis in chronic viral hepatitis.

Apurinic/apyrimidinic endonuclease-1 (APE-1) is overexpressed via the activation of NF-κB-p65 in MCP-1-positive esophageal squamous cell carcinoma tissue

Apurinic/apyrimidinic endonuclease-1 (APE-1), a key enzyme responsible for DNA base excision repair (BER), has been linked to cancer chemoradiosensitivity. The phosphorylation of p65 plays a role in the activation of this pathway. In this study, we investigated APE-1 expression and its interaction with p65 in esophageal squamous cell carcinoma (ESCC) tissue. The expression of APE-1, p65, p65 nuclear localization sequence (p65-NLS), and monocyte chemoattractant protein-1 (MCP-1) was assessed by immunohistochemical analysis in 67 human ESCC tissue samples. Real-time PCR and western blotting were also performed. p65 siRNA was evaluated to determine the role of p65 in the regulation of APE-1 expression. We found nuclear localization of APE-1 in 89.6% (60/67) of ESCC tissue samples. We also observed the colocalization of p65-NLS and APE-1 in esophageal cancer tissue. In KYSE220 cells, pretreatment of MG-132 significantly abrogated upregulation of p65 and APE-1 levels induced by MCP-1, and treatment with 10 and 20 nM p65 siRNA significantly inhibited APE-1 mRNA expression. siRNA for p65 treatment significantly increased the apoptotic index in 5-FU-treated KYSE220 cells. We conclude that APE-1 is overexpressed and mainly localized in the nuclear compartment of cancer cells, and partly regulated by p65 in the NF-κB pathway in ESCC tissue.

Celecoxib Inhibits Apurinic/Apyrimidinic Endonuclease-1 Expression and Prevents Gastric Cancer in Helicobacter pylori-Infected Mongolian Gerbils

Background and Aims: The aim of this study was to see whether administration of celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, could prevent the development of gastric cancer via inhibition of apurinic/apyrimidinic endonuclease-1 (APE-1) expression induced by Helicobacter pylori infection. Methods: 70 Mongolian gerbils were divided into 6 groups. Group 1 gerbils served as controls (n = 6). Ten gerbils were given N-methyl-N-nitrosourea (MNU), 30 ppm, 5 times biweekly (group 2). Short-term H. pylori infection was induced in 6 gerbils which were sacrificed 8 weeks after H. pylori infection (group 3). Long-term H. pylori infection was induced in 6 other gerbils which were sacrificed 44 weeks after H. pylori infection (group 4). Twenty gerbils were given MNU pretreatment 5 times biweekly and long-term H. pylori infection (group 5). In addition, after H. pylori inoculation, group 6 gerbils also received celecoxib with their diet for 26 weeks. APE-1 expression alone or with COX-2 in gastric tissues was evaluated by Western blot and immunohistological analysis. Myeloperoxidase (MPO) activity and thiobarbituric-acid-reactive substance (TBARS) levels were also evaluated. Results: APE-1 was localized in gastric epithelial cells and mesenchymal cells including macrophages in H. pylori-infected gerbils. The numbers of APE-1-positive cells in group 4 and 5 were significantly increased compared to those of group 3. Celecoxib treatment significantly reduced MPO activity, TBARS levels and the incidence of gastric cancer. APE-1 and IκBα phosphorylation levels were significantly increased in MNU-pretreated H. pylori-infected gerbils compared to those in MNU-only gerbils. Celecoxib significantly reduced APE-1 and IκBα phosphorylation levels in MNU-pretreated H. pylori-infected gerbils. COX-2 and APE-1 were coexpressed in the macrophages of H. pylori-infected gerbils. Conclusion: Celecoxib prevented gastric cancer in MNU-pretreated H. pylori-infected gerbils with a reduction in APE-1 expression thereby suggesting the implication of APE-1 in gastric carcinogenesis in this model.

Expression of Apurinic/Apyrimidinic Endonuclease‐1 (APE‐1) in H. pylori‐Associated Gastritis, Gastric Adenoma, and Gastric Cancer

Apurinic/apyrimidinic endonuclease-1 (APE-1) is a key enzyme in DNA base excision repair (BER), linked to cancer chemosensitivity. However, little is known about the localization of APE-1 in Helicobacter pylori-infected gastric mucosa or its role in the development of gastric cancer. To investigate the role of APE-1 in the development of gastric cancer, we examined APE-1 expression and localization in cultured cells and gastric biopsies from patients with H. pylori-infected gastritis or gastric adenoma, and from surgically resected gastric cancer. APE-1 mRNA and protein expression were determined in H. pylori (CagA+) water-extract protein (HPWEP)-stimulated MKN-28 cells, gastric adenocarcinoma cell-line (AGS) cells, and human peripheral macrophages by real-time polymerase chain reaction and Western blot analysis. APE-1 expression and 8-OHdG as a measure of oxidative DNA damage were evaluated by immunostaining. Localization of APE-1 and IkappaBalpha phosphorylation in gastric adenoma and gastric cancer tissues were evaluated by single- and double-label immunohistochemistry. In studies in vitro, HPWEP-stimulation significantly increased APE-1 mRNA expression levels in both MKN-28 cells and human peripheral macrophages. Hypo/reoxygenation treatment significantly increased APE-1 protein expression in HPWEP-stimulated MKN-28 cells. HPWEP stimulation significantly increased both APE-1 expression and IkappaBalpha phosphorylation levels in MKN-28 and AGS cells. In human tissues, APE-1 expression in H. pylori-infected gastritis without goblet cell metaplasia was significantly increased as compared to that in tissues from uninfected subjects. Eradication therapy significantly reduced both APE-1 and 8-OHdG expression levels in the gastric mucosa. APE-1 expression was mainly localized in epithelial cells within gastric adenoma and in mesenchymal cells of gastric cancer tissues. APE-1 expression in gastric cancer tissues was significantly reduced compared to that in H. pylori-infected gastric adenoma, while 8-OHdG index and IkappaBalpha phosphorylation levels did not differ between these two neoplastic tissue types. Co-localization of APE-1 and IkappaBalpha phosphorylation was observed not in gastric cancer cells but in gastric adenoma cells. H. pylori infection is associated with increased APE-1 expression in human cell lines and in gastric tissues from subjects with gastritis and gastric adenomas. The observed distinct expression patterns of APE-1 and 8-OHdG in gastric adenoma and gastric cancer tissues may provide insight into the progression of these conditions and warrants further investigation.

Experimental Study Enhancing the Chemosensitivity of Multiple Myeloma to Melphalan by Using a Tissue-Specific APE1-Silencing RNA Expression Vector

Purpose:Because of a developing resistance to chemotherapy agents, multiple myeloma (MM) has been an incurable disease until now. As a means to overcome MM tumor cell resistance and/or sensitize tumor cells to chemotherapeutic treatments currently used, we examined the role of human apurinic/apyrimidinic endonuclease 1 (APE1) in resistance and prognosis in patients with MM. Patients and Methods:Multiple myeloma cells were analyzed by using bone marrow specimens from 32 patients with MM and 10 normal volunteers. Results:The positive rate of APE1 protein expression was 65.6% in the bone marrow specimens of patients with MM with known clinical outcome. Positive rate of APE1 expression beyond grade 2 in the relapsed/refractory group was significantly higher than that in the untreated group. No positive results of grade > 2 were detected in bone marrow specimens from patients with noncancerous disease. It was also confirmed that the amount of APE1 protein in KM3 cells was positively correlated with the dose and action time of melphalan. Because APE1 was overexpressed in refractory/relapsed MM cells, siRNA-targeted technology was used to decrease APE1 levels in KM3 cells, with protein levels deceasing to 80%-90% within 24 hours and continuing to decease for 72 hours. The best dose and time of inhibiting expression of APE1 protein were 3 μg and 2 days long. A decrease in APE1 levels in siRNA-treated KM3 cells led to enhanced cell sensitization to melphalan. Conclusion:The findings herein present prognostic and therapeutic implications for treating relapsed/refractory MM. The APE1-silencing RNA results demonstrate the feasibility of the therapeutic modulation of APE1 using a variety of molecules and approaches.