Artichoke Polyphenols Sensitize Human Breast Cancer Cells to Chemotherapeutic Drugs via a ROS-Mediated Downregulation of Flap Endonuclease 1.

Combined treatment of several natural polyphenols and chemotherapeutic agents is more effective comparing to the drug alone in inhibiting cancer cell growth. Polyphenolic artichoke extracts (AEs) have been shown to have anticancer properties by triggering apoptosis or reactive oxygen species- (ROS-) mediated senescence when used at high or low doses, respectively. Our aim was to explore the chemosensitizing potential of AEs in order to enhance the efficacy of conventional chemotherapy in breast cancer cells. We employed breast cancer cell lines to assess the potential synergistic effect of a combined treatment of AEs/paclitaxel (PTX) or AEs/adriamycin (ADR) and to determine the underlying mechanisms correlated to this potential therapeutic approach. Our data shows that AEs/PTX reduced cell proliferation by increasing DNA damage response (DDR) mediated by Flap endonuclease 1 (FEN1) downregulation that results into enhanced breast cancer cell sensitivity to chemotherapeutic drugs. We demonstrated that ROS/Nrf2 and p-ERK pathways are two molecular mechanisms involved in the synergistic effect of AEs plus PTX treatment. To highlight the role of ROS herein, we report that the addition of antioxidant N-acetylcysteine (NAC) significantly decreased the antiproliferative effect of the combined treatment. A combined therapy could be able to reduce the dose of chemotherapeutic drugs, minimizing toxicity and side effects. Our results suggest the use of artichoke polyphenols as ROS-mediated sensitizers of chemotherapy paving the way for innovative and promising natural compound-based therapeutic strategies in oncology.

TGFß1- miR-140-5p axis mediated up-regulation of Flap Endonuclease 1 promotes epithelial-mesenchymal transition in hepatocellular carcinoma.

Flap Endonuclease 1 (FEN1) is a known oncogene in an array of cancers, but its role in hepatocellular carcinoma (HCC) remains obscure. In this study, we report that FEN1 expression was elevated in the Cancer Genome Atlas (TCGA) database which was verified in HCC tissue and hepatoma cell lines. Pearson correlation analysis indicated that FEN1 was involved in HCC metastasis. We demonstrated that FEN1 silencing inhibits HCC cell epithelial-mesenchymal transition (EMT), invasion and migration in vitro and significantly suppressed tumor growth and metastasis in vivo. Conversely, FEN1 overexpression in HCC cells enhanced these metastatic processes. We further confirmed that FEN1 was a direct target of miR-140-5p, which was down-regulated in HCC tissues, and negatively correlated with FEN1 expression. Moreover, low miR-140-5p levels and high FEN1 expression predicted a poor clinical outcome. The effects of FEN1 overexpression could be partially abolished by miR-140-5p. miR-140-5p down-regulation and FEN1 overexpression were observed in a TGFß1 induced EMT model. TGFß1 mediated EMT could be blocked by miR-140-5p overexpression or FEN1 silencing. Taken together, our findings suggest that FEN1 is regulated by the TGFß1- miR-140-5p axis and promotes EMT in HCC.

Proteins of the retinoblastoma pathway, FEN1 and MGMT are novel potential prognostic biomarkers in pancreatic adenocarcinoma.

BACKGROUND: We studied the expression of some major proteins involved in cell-cycle regulation and DNA repair, the roles of which are not well known in pancreatic ductal adenocarcinoma (PDAC), but which have a significant impact on carcinogenesis of many other cancers. METHODS: We immunohistochemically assessed expression levels of the cell-cycle regulators Rb1, p16 and cyclin-dependent kinase 4 (CDK4), and the DNA repair enzymes O6-methylguanine-DNA-alkyltransferase (MGMT) and flap endonuclease-1 (FEN1) separately in malignant tissue and benign tissue from resection margins in 102 cases of PDAC. Nearly all (95.1%) patients had undergone pancreaticoduodenectomy. RESULTS: The studied proteins showed wide but somewhat variable expression in both benign and malignant pancreatic tissues. Strong CDK4 expression in islets of Langerhans predicted poor relapse-free survival (RFS) (HR 2.874; 95% CI 1.261-6.550; p = .012) and within T3-4 tumors CDK4 expression in adenocarcinoma cells also predicted poor disease-free survival (DFS) (RR 2.148; 95% CI 1.081-4.272; p = .029). Strong MGMT expression was associated in N1 patients with weak local relapse-free survival (RFS), DFS and overall survival; all significantly in Cox regression analysis. FEN1 was also an independent predictor of decreased DFS (in the whole study population) and worse RFS (in the patients with T3-4 tumors). CONCLUSIONS: Major cell-cycle regulator also have predictive significance, but further studies are required to evaluate this.

[Expression and activity assay of recombinant flap endonuclease 1].

Flap endonuclease 1 (FEN1) is an endonuclease that catalyzes invasive reaction. It can be used in signal-amplification reaction-based nucleic acid assay. However, the application of FEN1 is hampered due to the lack of detailed protocols to express and purify the enzyme, and to quantify the enzyme activity. In this paper, the DNA fragment coding the gene of FEN1 from Archaeoglobus fulgidus was synthesized, and inserted into the plasmid of pET24a(+) to express recombinant FEN1 with His-tag. After optimizing the expression, detailed expression protocol of FEN1 was obtained by culturing the recombinant E. coli at 37 ℃ with 200 r/min of shaking for 8 h, followed by inducing with 0.05 mmol/L IPTG at 37 ℃ for 11 h. The purified recombinant FEN1 with the molecular mass of 38 kDa was obtained by Ni-affinity chromatography. Moreover, we developed a accurate quantification method with fluorescence-labelled probes. Finally, the recombinant FEN1 was used in real-time PCR coupled with high specific invader assay for aldh2 gene genotyping to obtain the correct typing results, indicating that the recombinant FEN1 can be used in gene polymorphism detection. We provide a reliable enzyme for developing invasive reaction-based nucleic acid assay.

Nucleophosmin modulates stability, activity, and nucleolar accumulation of base excision repair proteins.

Nucleophosmin (NPM1) is a multifunctional protein that controls cell growth and genome stability via a mechanism that involves nucleolar-cytoplasmic shuttling. It is clear that NPM1 also contributes to the DNA damage response, yet its exact function is poorly understood. We recently linked NPM1 expression to the functional activation of the major abasic endonuclease in mammalian base excision repair (BER), apurinic/apyrimidinic endonuclease 1 (APE1). Here we unveil a novel role for NPM1 as a modulator of the whole BER pathway by 1) controlling BER protein levels, 2) regulating total BER capacity, and 3) modulating the nucleolar localization of several BER enzymes. We find that cell treatment with the genotoxin cisplatin leads to concurrent relocalization of NPM1 and BER components from nucleoli to the nucleoplasm, and cellular experiments targeting APE1 suggest a role for the redistribution of nucleolar BER factors in determining cisplatin toxicity. Finally, based on the use of APE1 as a representative protein of the BER pathway, our data suggest a function for BER proteins in the regulation of ribogenesis.

Concerted action of exonuclease and Gap-dependent endonuclease activities of FEN-1 contributes to the resolution of triplet repeat sequences (CTG)n- and (GAA)n-derived secondary structures formed during maturation of Okazaki fragments.

There is much evidence to indicate that FEN-1 efficiently cleaves single-stranded DNA flaps but is unable to process double-stranded flaps or flaps adopting secondary structures. However, the absence of Fen1 in yeast results in a significant increase in trinucleotide repeat (TNR) expansion. There are then two possibilities. One is that TNRs do not always form stable secondary structures or that FEN-1 has an alternative approach to resolve the secondary structures. In the present study, we test the hypothesis that concerted action of exonuclease and gap-dependent endonuclease activities of FEN-1 play a role in the resolution of secondary structures formed by (CTG)n and (GAA)n repeats. Employing a yeast FEN-1 mutant, E176A, which is deficient in exonuclease (EXO) and gap endonuclease (GEN) activities but retains almost all of its flap endonuclease (FEN) activity, we show severe defects in the cleavage of various TNR intermediate substrates. Precise knock-in of this point mutation causes an increase in both the expansion and fragility of a (CTG)n tract in vivo. Taken together, our biochemical and genetic analyses suggest that although FEN activity is important for single-stranded flap processing, EXO and GEN activities may contribute to the resolution of structured flaps. A model is presented to explain how the concerted action of EXO and GEN activities may contribute to resolving structured flaps, thereby preventing their expansion in the genome.

Fen1 is induced p53 dependently and involved in the recovery from UV-light-induced replication inhibition.

Mouse embryonic fibroblasts (MEFs) that lack p53 are hypersensitive to the cytotoxic and genotoxic effect of ultraviolet (UV-C) light. They also display a defect in the recovery from UV-C-induced DNA replication inhibition. An enzyme involved in processing stalled DNA replication forks is flap endonuclease 1 (Fen1). Gene expression profiling of UV-C-irradiated MEFs revealed fen1 to be upregulated, which was confirmed by RT-PCR and Western blot experiments. Increased Fen1 levels upon UV-C exposure are due to transcriptional activation, as revealed by inhibitor studies. Fen1 induction was dose- and time-dependent; it occurred on protein level already 3 h after irradiation. Induction of Fen1 by UV-C requires p53 since it was observed in p53 wild-type (wt) but not in p53 null (p53-/-) fibroblasts. Fen1 upregulation paralleled the increase in p53 protein level in replicating wt cells, whereas in nonreplicating cells both Fen1 and p53 were not induced by UV-C. The mouse fen1 promoter was cloned and shown to harbor a p53 consensus sequence to which p53 binds. In cotransfection experiments, p53 stimulated the expression of a fen1 promoter-reporter construct. Transgenic expression of Fen1 in p53 null cells attenuated UV-C light-induced DNA replication inhibition, supporting the hypothesis that Fen1 induction is involved in the recovery of cells from DNA damage.

Tolerated wobble mutations in siRNAs decrease specificity, but can enhance activity in vivo.

RNA interference (RNAi) has become an invaluable tool for functional genomics. A critical use of this tool depends on an understanding of the factors that determine the specificity and activity of the active agent, small interfering RNA (siRNA). Several studies have concluded that tolerance of mutations can be considerable and hence lead to off-target effects. In this study, we have investigated in vivo the toleration of wobble (G:U) mutations in high activity siRNAs against Flap Endonuclease 1 (Fen1) and Aquaporin-4 (Aqp4). Mutations in the central part of the antisense strand caused a pronounced decrease in activity, while mutations in the 5' and 3'ends were tolerated very well. Furthermore, based on analysis of nine different mutated siRNAs with widely differing intrinsic activities, we conclude that siRNA activity can be significantly enhanced by wobble mutations (relative to mRNA), in the 5' terminal of the antisense strand. These findings should facilitate design of active siRNAs where the target mRNA offers limited choice of siRNA positions.