DHA-induced stress response in human colon cancer cells – Focus on oxidative stress and autophagy

ABSTRACTPolyunsaturated fatty acids (PUFAs) are important constituents of the diet and health benefits of omega-3/n−3 PUFAs, especially eicosapentaenoic acid (EPA, 20:5 n−3) and docosahexaenoic acid (DHA, 22:6 n−3) have been well documented in relation to several diseases. Increasing evidence suggests that n−3 PUFAs may have anticancer activity and improve the effect of conventional cancer therapy. The mechanisms behind these effects are still unclear and need to be elucidated.We have examined the DHA-induced stress response in two human colon cancer cell lines, SW620 and Caco-2. SW620 cells are growth-inhibited at early time points by DHA, while the growth of Caco-2 cells almost remains unaffected by the same treatment. Gene expression analysis of SW620 cells treated with DHA revealed changes at early time points; transcripts involved in oxidative stress and autophagy were among the first to be differentially expressed. We find that oxidative stress is induced in both cell lines, although at different time points and to different extent. DHA induced nuclear translocation of the oxidative stress sensor NFE2L2 in both cell lines, indicating an induction of an anti-oxidative response. However, vitamin E did not counteract ROS-production or the translocation of NFE2L2 to the nucleus. Neither vitamin E nor the antioxidants butylated hydoxyanisole (BHA) and butylated hydoxytoluene (BHT) did affect the growth inhibition in SW620 cells after DHA-treatment. Also, siRNA-mediated down-regulation of NFE2L2 did not sensitize SW620 and Caco-2 cells to DHA. These results indicate that oxidative stress response is not the cause of DHA-induced cytotoxicity in SW620 cells.Using biochemical and imaging based functional assays, we found a low basal level of autophagy and no increase in autophagic flux after adding DHA to the SW620 cells. However, Caco-2 cells displayed a higher level of autophagy, both in the absence and presence of DHA. Inhibition of autophagy by siRNA mediated knock down of ATG5 and ATG7 sensitized both SW620 and Caco-2 cells to DHA. Stimulation of autophagy by rapamycin in SW620 and Caco-2 cells resulted in decreased DHA-sensitivity and inhibition of autophagy in Caco-2 cells by chloroquine resulted in increased DHA-sensitivity.These results suggest that autophagy is important for the DHA sensitivity of colon cancer cells and imply possible therapeutic effects of this fatty acid against cancer cells with low autophagy

Divergent β-hairpins determine double-strand versus single-strand substrate recognition of human AlkB-homologues 2 and 3

Human AlkB homologues ABH2 and ABH3 repair 1-methyladenine and 3-methylcytosine in DNA/RNA by oxidative demethylation. The enzymes have similar overall folds and active sites, but are functionally divergent. ABH2 efficiently demethylates both single- and double-stranded (ds) DNA, whereas ABH3 has a strong preference for single-stranded DNA and RNA. We find that divergent F1 β-hairpins in proximity of the active sites of ABH2 and ABH3 are central for substrate specificities. Swapping F1 hairpins between the enzymes resulted in hybrid proteins resembling the donor proteins. Surprisingly, mutation of the intercalating residue F102 had little effect on activity, while the double mutant V101A/F102A was catalytically impaired. These residues form part of an important hydrophobic network only present in ABH2. In this functionally important network, F124 stacks with the flipped out base while L157 apparently functions as a buffer stop to position the lesion in the catalytic pocket for repair. F1 in ABH3 contains charged and polar residues preventing use of dsDNA substrate. Thus, E123 in ABH3 corresponds to F102 in ABH2 and the E123F-variant gained capacity to repair dsDNA with no loss in single strand repair capacity. In conclusion, divergent sequences outside of the active site determine substrate specificities of ABH2 and ABH3

Tissue distribution and transcriptional regulation of CCN5 in the heart after myocardial infarction

Abstract CCN5 is a divergent member of the cellular communication network factor (CCN) family in that it lacks the carboxyl terminal cystine knot domain common to the other CCN family members. CCN5 has been reported to antagonize the profibrotic actions of CCN2 and to inhibit myocardial collagen deposition and fibrosis in chronic pressure overload of the heart. However, what mechanisms that regulate CCN5 activity in the heart remain unknown. Recombinant, replication defective adenovirus encoding firefly luciferase under control of the human CCN5 promoter was prepared and used to investigate what mechanisms regulate CCN5 transcription in relevant cells. Tissue distribution of CCN5 in hearts from healthy mice and from mice subjected to myocardial infarction was investigated. Contrary to the profibrotic immediate early gene CCN2, we find that CCN5 is induced in the late proliferation and maturation phases of scar healing. CCN5 was identified principally in endothelial cells, fibroblasts, smooth muscle cells, and macrophages. Our data show that CCN5 gene transcription and protein levels are induced by catecholamines via β 2 -adrenergic receptors. Myocardial induction of CCN5 was further confirmed in isoproterenol-infused mice. We also find that CCN5 transcription is repressed by TNF-α, an inflammatory mediator highly elevated in early phases of wound healing following myocardial infarction. In conclusion, CCN5 predominates in endothelial cells, fibroblasts, and macrophages of the differentiating scar tissue and its transcription is conversely regulated by β 2 -adrenergic agonists and TNF-α

Connective tissue growth factor (CCN2) is a matricellular preproprotein controlled by proteolytic activation.

Connective tissue growth factor (CTGF; now often referred to as CCN2) is a secreted protein predominantly expressed during development, in various pathological conditions that involve enhanced fibrogenesis and tissue fibrosis, and in several cancers and is currently an emerging target in several early-phase clinical trials. Tissues containing high CCN2 activities often display smaller degradation products of full-length CCN2 (FL-CCN2). Interpretation of these observations is complicated by the fact that a uniform protein structure that defines biologically active CCN2 has not yet been resolved. Here, using DG44 CHO cells engineered to produce and secrete FL-CCN2 and cell signaling and cell physiological activity assays, we demonstrate that FL-CCN2 is itself an inactive precursor and that a proteolytic fragment comprising domains III (thrombospondin type 1 repeat) and IV (cystine knot) appears to convey all biologically relevant activities of CCN2. In congruence with these findings, purified FL-CCN2 could be cleaved and activated following incubation with matrix metalloproteinase activities. Furthermore, the C-terminal fragment of CCN2 (domains III and IV) also formed homodimers that were ∼20-fold more potent than the monomeric form in activating intracellular phosphokinase cascades. The homodimer elicited activation of fibroblast migration, stimulated assembly of focal adhesion complexes, enhanced RANKL-induced osteoclast differentiation of RAW264.7 cells, and promoted mammosphere formation of MCF-7 mammary cancer cells. In conclusion, CCN2 is synthesized and secreted as a preproprotein that is autoinhibited by its two N-terminal domains and requires proteolytic processing and homodimerization to become fully biologically active

The marine n-3 PUFA DHA evokes cytoprotection against oxidative stress and protein misfolding by inducing autophagy and NFE2L2 in human retinal pigment epithelial cells

<p>Accumulation and aggregation of misfolded proteins is a hallmark of several diseases collectively known as proteinopathies. Autophagy has a cytoprotective role in diseases associated with protein aggregates. Age-related macular degeneration (AMD) is the most common neurodegenerative eye disease that evokes blindness in elderly. AMD is characterized by degeneration of retinal pigment epithelial (RPE) cells and leads to loss of photoreceptor cells and central vision. The initial phase associates with accumulation of intracellular lipofuscin and extracellular deposits called drusen. Epidemiological studies have suggested an inverse correlation between dietary intake of marine n-3 polyunsaturated fatty acids (PUFAs) and the risk of developing neurodegenerative diseases, including AMD. However, the disease-preventive mechanism(s) mobilized by n-3 PUFAs is not completely understood. In human retinal pigment epithelial cells we find that physiologically relevant doses of the n-3 PUFA docosahexaenoic acid (DHA) induce a transient increase in cellular reactive oxygen species (ROS) levels that activates the oxidative stress response regulator NFE2L2/NRF2 (nuclear factor, erythroid derived 2, like 2). Simultaneously, there is a transient increase in intracellular protein aggregates containing SQSTM1/p62 (sequestosome 1) and an increase in autophagy. Pretreatment with DHA rescues the cells from cell cycle arrest induced by misfolded proteins or oxidative stress. Cells with a downregulated oxidative stress response, or autophagy, respond with reduced cell growth and survival after DHA supplementation. These results suggest that DHA both induces endogenous antioxidants and mobilizes selective autophagy of misfolded proteins. Both mechanisms could be relevant to reduce the risk of developing aggregate-associate diseases such as AMD.</p

Human AlkB Homolog 1 Is a Mitochondrial Protein That Demethylates 3-Methylcytosine in DNA and RNA*

The Escherichia coli AlkB protein and human homologs hABH2 and hABH3 are 2-oxoglutarate (2OG)/Fe(II)-dependent DNA/RNA demethylases that repair 1-methyladenine and 3-methylcytosine residues. Surprisingly, hABH1, which displays the strongest homology to AlkB, failed to show repair activity in two independent studies. Here, we show that hABH1 is a mitochondrial protein, as demonstrated using fluorescent fusion protein expression, immunocytochemistry, and Western blot analysis. A fraction is apparently nuclear and this fraction increases strongly if the fluorescent tag is placed at the N-terminal end of the protein, thus interfering with mitochondrial targeting. Molecular modeling of hABH1 based upon the sequence and known structures of AlkB and hABH3 suggested an active site almost identical to these enzymes. hABH1 decarboxylates 2OG in the absence of a prime substrate, and the activity is stimulated by methylated nucleotides. Employing three different methods we demonstrate that hABH1 demethylates 3-methylcytosine in single-stranded DNA and RNA in vitro. Site-specific mutagenesis confirmed that the putative Fe(II) and 2OG binding residues are essential for activity. In conclusion, hABH1 is a functional mitochondrial AlkB homolog that repairs 3-methylcytosine in single-stranded DNA and RNA

Human ABH3 structure and key residues for oxidative demethylation to reverse DNA/RNA damage

Methylating agents are ubiquitous in the environment, and central in cancer therapy. The 1-methyladenine and 3-methylcytosine lesions in DNA/RNA contribute to the cytotoxicity of such agents. These lesions are directly reversed by ABH3 (hABH3) in humans and AlkB in Escherichia coli. Here, we report the structure of the hABH3 catalytic core in complex with iron and 2-oxoglutarate (2OG) at 1.5 Å resolution and analyse key site-directed mutants. The hABH3 structure reveals the β-strand jelly-roll fold that coordinates a catalytically active iron centre by a conserved His(1)-X-Asp/Glu-X(n)-His(2) motif. This experimentally establishes hABH3 as a structural member of the Fe(II)/2OG-dependent dioxygenase superfamily, which couples substrate oxidation to conversion of 2OG into succinate and CO(2). A positively charged DNA/RNA binding groove indicates a distinct nucleic acid binding conformation different from that predicted in the AlkB structure with three nucleotides. These results uncover previously unassigned key catalytic residues, identify a flexible hairpin involved in nucleotide flipping and ss/ds-DNA discrimination, and reveal self-hydroxylation of an active site leucine that may protect against uncoupled generation of dangerous oxygen radicals