BACKGROUND: Altered DNA repair may be associated with aggressive tumour biology and impact upon response to chemotherapy and radiotherapy. We investigated whether expression of human AP endonuclease (APE1), a key multifunctional protein involved in DNA BER, would impact on clinicopathological outcomes in ovarian, gastro-oesophageal, and pancreatico-biliary cancer. METHODS: Formalin-fixed human ovarian, gastro-oesophageal, and pancreatico-biliary cancers were constructed into TMAs. Expression of APE1 was analysed by IHC and correlated to clinicopathological variables. RESULTS: In ovarian cancer, nuclear APE1 expression was seen in 71.9% (97 out of 135) of tumours and correlated with tumour type (P ¼ 0.006), optimal debulking (P ¼ 0.009), and overall survival (P ¼ 0.05). In gastro-oesophageal cancers previously exposed to neoadjuvant chemotherapy, 34.8% (16 out of 46) of tumours were positive in the nucleus and this correlated with shorter overall survival (P ¼ 0.005), whereas cytoplasmic localisation correlated with tumour dedifferentiation (P ¼ 0.034). In pancreatico-biliary cancer, nuclear staining was seen in 44% (32 out of 72) of tumours. Absence of cytoplasmic staining was associated with perineural invasion (P ¼ 0.007), vascular invasion (P ¼ 0.05), and poorly differentiated tumours (P ¼ 0.068). A trend was noticed with advanced stage (P ¼ 0.077). CONCLUSIONS: Positive clinicopathological correlations of APE1 expression suggest that APE1 is a potential drug target in ovarian, gastro-oesophageal, and pancreatico-biliary cancers.
Aims:Modulation of DNA base excision repair (BER) has the potential to enhance response to chemotherapy and improve outcomes in tumours such as melanoma and glioma. APE1, a critical protein in BER that processes potentially cytotoxic abasic sites (AP sites), is a promising new target in cancer. In the current study, we aimed to develop small molecule inhibitors of APE1 for cancer therapy.Methods:An industry-standard high throughput virtual screening strategy was adopted. The Sybyl8.0 (Tripos, St Louis, MO, USA) molecular modelling software suite was used to build inhibitor templates. Similarity searching strategies were then applied using ROCS 2.3 (Open Eye Scientific, Santa Fe, NM, USA) to extract pharmacophorically related subsets of compounds from a chemically diverse database of 2.6 million compounds. The compounds in these subsets were subjected to docking against the active site of the APE1 model, using the genetic algorithm-based programme GOLD2.7 (CCDC, Cambridge, UK). Predicted ligand poses were ranked on the basis of several scoring functions. The top virtual hits with promising pharmaceutical properties underwent detailed in vitro analyses using fluorescence-based APE1 cleavage assays and counter screened using endonuclease IV cleavage assays, fluorescence quenching assays and radiolabelled oligonucleotide assays. Biochemical APE1 inhibitors were then subjected to detailed cytotoxicity analyses.Results:Several specific APE1 inhibitors were isolated by this approach. The IC50 for APE1 inhibition ranged between 30 n and 50 μ. We demonstrated that APE1 inhibitors lead to accumulation of AP sites in genomic DNA and potentiated the cytotoxicity of alkylating agents in melanoma and glioma cell lines.Conclusions:Our study provides evidence that APE1 is an emerging drug target and could have therapeutic application in patients with melanoma and glioma.
Homeostasis between pro-and anti-inflammatory responses induced by bacteria is critical for the maintenance of health. In the oral cavity, proinflammatory mechanisms induced by pathogenic bacteria are well-established; however, the anti-inflammatory responses that act to restrain innate responses remain poorly characterized. Here, we demonstrate that infection with the periodontal pathogen P. gingivalis enhances the activity of JAK3 in innate immune cells, and subsequently phospho-inactivates Nedd4-2, a ubiquitin E3 ligase. In turn, Wnt3 ubiquitination is decreased, while total protein levels are enhanced, leading to a reduction in proinflammatory cytokine levels. In contrast, JAK3 inhibition or Wnt3a robustly enhances NF-κB activity and the production of proinflammatory cytokines in P. gingivalis-stimulated innate immune cells. Moreover, using gainand loss-of-function approaches, we demonstrate that downstream molecules of Wnt3a signaling, including Dvl3 and β-catenin, are responsible for the negative regulatory role of Wnt3a. In addition, using an in vivo P. gingivalis-mediated periodontal disease model, we show that JAK3 inhibition enhances infiltration of inflammatory cells, reduces expression of Wnt3a and Dvl3 in P.
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