Daniela Grácio

Oxidative Stress and DNA Damage: Implications in Inflammatory Bowel Disease.

Abstract:This review will focus on published human studies on oxidative stress and DNA damage in inflammatory bowel disease (IBD), both ulcerative colitis and Crohns disease, assessing their role in the pathophysiology of these diseases. Search was performed over PubMed and ScienceDirect databases to identify relevant bibliography, using keywords including “oxidative stress,” “DNA damage,” “IBD,” and “oxidative DNA damage.” Whether as cause or effect, mechanisms underlying oxidative stress have the potential to condition the course of various pathologies, particularly those driven by inflammatory scenarios. IBDs are chronic inflammatory relapsing conditions. Oxidative stress has been associated with some of the characteristic clinical features exhibited in IBD, namely tissue injury and fibrosis, and also to the ulcerative colitis–associated colorectal cancer. The possible influence of oxidative stress over therapeutic behavior and response, as well as their contribution to the oxidative burden and consequences, is also addressed. Due to the high prevalence and incidence of IBD worldwide, and also to its associated morbidity, complications, and disease and treatment costs, it is of paramount importance to better understand the pathophysiology of these diseases.

Infliximab therapy increases the frequency of circulating CD16+ monocytes and modifies macrophage cytokine response to bacterial infection

Crohns disease (CD) has been correlated with altered macrophage response to microorganisms. Considering the efficacy of infliximab treatment on CD remission, we investigated infliximab effects on circulating monocyte subsets and on macrophage cytokine response to bacteria. Human peripheral blood monocyte‐derived macrophages were obtained from CD patients, treated or not with infliximab. Macrophages were infected with Escherichia coli, Enterococcus faecalis, Mycobacterium avium subsp. paratuberculosis (MAP) or M. avium subsp avium, and cytokine levels [tumour necrosis factor (TNF) and interleukin (IL)‐10] were evaluated at different time‐points. To evaluate infliximab‐dependent effects on monocyte subsets, we studied CD14 and CD16 expression by peripheral blood monocytes before and after different infliximab administrations. We also investigated TNF secretion by macrophages obtained from CD16+ and CD16− monocytes and the frequency of TNF+ cells among CD16+ and CD16− monocyte‐derived macrophages from CD patients. Infliximab treatment resulted in elevated TNF and IL‐10 macrophage response to bacteria. An infliximab‐dependent increase in the frequency of circulating CD16+ monocytes (particularly the CD14++CD16+ subset) was also observed (before infliximab: 4·65 ± 0·58%; after three administrations: 10·68 ± 2·23%). In response to MAP infection, macrophages obtained from CD16+ monocytes were higher TNF producers and CD16+ macrophages from infliximab‐treated CD patients showed increased frequency of TNF+ cells. In conclusion, infliximab treatment increased the TNF production of CD macrophages in response to bacteria, which seemed to depend upon enrichment of CD16+ circulating monocytes, particularly of the CD14++CD16+ subset. Infliximab treatment of CD patients also resulted in increased macrophage IL‐10 production in response to bacteria, suggesting an infliximab‐induced shift to M2 macrophages.

Increased viability but decreased culturability of Mycobacterium avium subsp. paratuberculosis in macrophages from inflammatory bowel disease patients under Infliximab treatment.

Mycobacterium avium subsp. paratuberculosis (MAP) has long been implicated as a triggering agent in Crohn’s disease (CD). In this study, we investigated the growth/persistence of both M. avium subsp. hominissuis (MAH) and MAP, in macrophages from healthy controls (HC), CD and ulcerative colitis patients. For viability assessment, both CFU counts and a pre16SrRNA RNA/DNA ratio assay (for MAP) were used. Phagolysosome fusion was evaluated by immunofluorescence, through analysis of LAMP-1 colocalization with MAP. IBD macrophages were more permissive to MAP survival than HC macrophages (a finding not evident with MAH), but did not support MAP active growth. The lower MAP CFU counts in macrophage cultures associated with Infliximab treatment were not due to increased killing, but possibly to elevation in the proportion of intracellular dormant non-culturable MAP forms, as MAP showed higher viability in those macrophages. Increased MAP viability was not related to lack of phagolysosome maturation. The predominant induction of MAP dormant forms by Infliximab treatment may explain the lack of MAP reactivation during anti-TNF therapy of CD but does not exclude the possibility of MAP recrudescence after termination of therapy.

Association between Polymorphisms in Antioxidant Genes and Inflammatory Bowel Disease

Inflammation is the driving force in inflammatory bowel disease (IBD) and its link to oxidative stress and carcinogenesis has long been accepted. The antioxidant system of the intestinal mucosa in IBD is compromised resulting in increased oxidative injury. This defective antioxidant system may be the result of genetic variants in antioxidant genes, which can represent susceptibility factors for IBD, namely Crohn’s disease (CD) and ulcerative colitis (UC). Single nucleotide polymorphisms (SNPs) in the antioxidant genes SOD2 (rs4880) and GPX1 (rs1050450) were genotyped in a Portuguese population comprising 436 Crohn’s disease and 367 ulcerative colitis patients, and 434 healthy controls. We found that the AA genotype in GPX1 is associated with ulcerative colitis (OR = 1.93, adjusted P-value = 0.037). Moreover, we found nominal significant associations between SOD2 and Crohn’s disease susceptibility and disease subphenotypes but these did not withstand the correction for multiple testing. These findings indicate a possible link between disease phenotypes and antioxidant genes. These results suggest a potential role for antioxidant genes in IBD pathogenesis and should be considered in future association studies.

Short- and long-term regulation of intestinal Na+/H+ exchange by Toll-like receptors TLR4 and TLR5

Inappropriate activation of pattern recognition receptors has been described as a potential trigger in the development of inflammatory bowel disease (IBD). In this study, we evaluated the activity and expression of Na(+)/H(+) exchanger (NHE) subtypes in T84 intestinal epithelial cells during Toll-like receptor 4 (TLR4) activation by monophosphoryl lipid A and TLR5 by flagellin. NHE activity and intracellular pH were evaluated by spectrofluorescence. Additionally, kinase activities were evaluated by ELISA, and siRNA was used to specifically inhibit adenylyl cyclase (AC). Monophosphoryl lipid A (MPLA) (0.01-50.00 μg/ml) and flagellin (10-500 ng/ml) inhibited NHE1 activity in a concentration-dependent manner (MPLA short term -25.2 ± 5.0%, long term -31.9 ± 4.0%; flagellin short term -14.9 ± 2.0%, long term -19.1 ± 2.0%). Both ligands triggered AC3, PKA, PLC, and PKC signal molecules. Long-term exposure to flagellin and MPLA induced opposite changes on NHE3 activity; flagellin increased NHE3 activity (∼10%) with overexpression of membrane protein, whereas MPLA decreased NHE3 activity (-17.3 ± 3.0%). MPLA and flagellin simultaneously had synergistic effects on NHE activity. MPLA and flagellin impaired pHi recovery after intracellular acidification. The simultaneous exposure to MPLA and flagellin induced a substantial pHi reduction (-0.55 ± 0.03 pH units). Activation of TLR4 and TLR5 exerts marked inhibition of NHE1 activity in intestinal epithelial cells. Transduction mechanisms set into motion during TLR4-mediated and long-term TLR5-mediated inhibition of NHE1 activity involve AC3, PKA, PLC, and PKC. However, short- and long-term TLR4 activation and TLR5 activation might use different signaling pathways. The physiological alterations on intestinal epithelial cells described here may be useful in the development of better IBD therapeutics.

An overview on the role of autophagy in cancer therapy

Autophagy is a highly regulated catabolic process through which cells recycle their own constituents by delivering them into lysosomes. Several studies have demonstrated that autophagy plays a wide variety of physiological and pathophysiological roles in cells. In cancer, autophagy has been described to have paradoxical roles, acting both as tumor suppressor and as tumor promoter. In particular, it may exert different functions in response to cancer therapy, causing cancer resistance or increasing sensitivity to chemotherapeutic drugs and radiation. Therefore, autophagy could provide new means for the enhancement of antitumor drugs and radiation effectiveness. Correspondence to: Valdemar Máximo, Cancer Signaling and Metabolism research group, Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP), 4200-135 Porto, Portugal, Tel: 225 570 700; Fax: 225 570 799; E-mail: vmaximo@ipatimup.pt Received: January 24, 2017; Accepted: February 16, 2017; Published: February 18, 2017 Introduction Autophagy (self-eating) is a highly conserved catabolic process with critical functions in the maintenance of cellular homeostasis under normal growth conditions and in the preservation of cell viability under stress [1]. Autophagy is an intracellular process in which cellular components, such as proteins and organelles, are delivered to the lysosome leading to the degradation and recycling of cytosolic compounds, thus providing cells with essential amino acids, nucleotides, and fatty acids, that enable production of elements required for energy and macromolecule biosynthesis [2,3]. There are three main types of autophagy, differing mainly in the mechanism by which the cytosolic material is presented to the lysosome [1]: i) macroautophagy, ii) microautophagy and iii) chaperone-mediated autophagy (CMA). In macroautophagy, double-membrane vesicles, called autophagosomes, sequester cytosolic material. Those vesicles merge with lysosomes (forming the autophagolysosome), their cargo is degraded, by lysosomal hydrolases, and the recycled macromolecular precursors are transported back into the cytoplasm, where they can be used as metabolic intermediates. In microautophagy, no intermediary vesicles are present, and the cytoplasmic material is directly engulfed by the lysosome [2]. In CMA, specific proteins, associated to heat shock protein (HSP) hsc70 and its co-chaperones, are translocated to the lysosome. Those proteins contain a specific amino acid motif (KFERQ, or biochemically related), which is recognized by the HSP, and once unfolded, they are translocated directly into the lysosome, via the lysosome-associated membrane protein 2A (LAMP2A) [4,5] Several studies have already demonstrated that autophagy plays more roles than the initially expected, including: cellular adaptation to starvation, intracellular protein and organelle clearance, development, anti-aging, elimination of microorganisms, cell death and antigen presentation [6]. Deregulation of autophagy has been associated to several diseases, including neurodegenerative diseases, diabetes and cancer [7]. In this short review, we will mainly address the role of autophagy (and its different functional forms) in cancer, and its implication in cancer therapy. The majority of the studies published on autophagy, particularly those related to cancer therapy refer to “macroautophagy”. In fact, the broad term ‘autophagy’ usually means “macroautophagy”, unless otherwise specified, and therefore, in this review, we will also use this terminology [1]. Nevertheless, it is important to mention that recent studies have shown that CMA may be also important for tumor growth, progression and therapy and that pharmacological approaches that inhibit macroautophagy may also affect CMA [8,9]. Autophagy in cancer Cancer was one of the first diseases to be associated to autophagy [10-14]. Nevertheless, the exact molecular mechanisms and the role of autophagy in cancer cells is not yet clearly defined, being even paradoxical. While at early stages, autophagy usually acts as a tumor suppressor allowing cells to discard damaged cellular contents, decreasing ROS and DNA damage, in more advanced stages of tumor development, it may help cancer cells to survive under lowoxygen and low-nutrient conditions, acting as a tumor promoter [3,15]. Actually, the dependence of tumor cells on autophagy is highly variable. While some tumor models (like pancreatic cancer) display increased autophagy levels in basal situations (including in plenty nutrient conditions), with autophagy having a role in the maintenance of tumor growth [16], results from other studies, comparing the levels of autophagy in tumor cells with their corresponding non-tumor cells, show disparate data between different tumor models (for a thorough Grácio D (2017) An overview on the role of autophagy in cancer therapy Volume 2(1): 2-4 Hematol Med Oncol, 2017 doi: 10.15761/HMO.1000117 review please see [17]). Importantly, autophagy plays also a role in cancer response to therapy since cancer therapies mostly inflict stress and damage to cells to induce cell death [18]. The outcomes of therapy-induced autophagy in cancer cells may represent also a “double-edge sword” and depends on the particular type of cancer, on the stage of disease progression or even on the type and duration of autophagy [18-21]. Indeed, several studies showed that increased autophagy leads to resistance to both chemoand radiotherapy, while several others show that many anticancer drugs induce autophagy-related cell death in cancer cells [22,23]. The fact that many of the currently used clinically approved anticancer strategies have been described as inducing autophagy, makes the understanding of the functional role of autophagy within a specific cancer context much more relevant, as it could provide new means for the enhancement of antitumor drugs and radiation effectiveness. Functional forms of autophagy and their implications for cancer therapy Although, traditionally, autophagy has been seen as a pro-survival (cytoprotective) mechanism, different studies have shown that it may result in other outcomes. Currently, at least four distinct functional forms of autophagy have been described [24,25]: i) Cytoprotective, when cells die or arrest if autophagy is inhibited; ii) Cytotoxic, when autophagy induction results in cell death and its blockage results in cell survival; iii) Cytostatic, when autophagy induction results in cell growth arrest and iv) Nonprotective, if autophagy does not affect cell growth once blocked. These forms are distinguished on only based on their functional characteristics, having similar morphologic, biochemical or molecular profiles [24]. Autophagy modulation as a therapeutic strategy to improve anticancer strategies As already referred, the different functional forms of autophagy affect the cellular response to anticancer therapies. The knowledge whether autophagy is cytoprotetive or is cytotoxic/cytostatic, will help defining strategies for its modulation (through its decrease or increase, respectively) to interfere with the cellular sensitivity to therapy. Targeting cytoprotective autophagy has been at the basis for multiple clinical trials. Indeed, if increased autophagy confers tumor resistance to death-inducing agents, its inhibition will allow an enhanced response to treatment [26]. There are several autophagy inhibitors already identified and that have been classified as: earlystage inhibitors, if blocking autophagosome formation [such as 3-Methyladenine (3-MA), wortmannin, and LY294002] orlate-stage inhibitors, acting at the level of the autophagosome-lysosome fusion and degradation steps [such as chloroquine (CQ), hydroxychloroquine (HCQ), bafilomycin A1, and monensin]. Studies using, not only these pharmacological autophagy inhibitors, but also genetic silencing or knockdown of autophagy-associated genes, resulted in increased tumor cell sensitivity to the autophagy-inducing stimulus, usually via the promotion of apoptosis [24,26]. Several clinical trials have been evaluating the use of autophagy inhibitors (particularly HCQ) in combination to chemoand radiotherapy to improve its efficacy [27,28]. A study carried out in melanoma patients using HCQ in combination with the mTOR inhibitor (temsirolimus) showed an improvement of the median progressionfree survival to 3.5 months and increased the rate of stable disease in patients [27,29]. Also, its combination with a proteasome inhibitor (bortezomib) in relapsed/refractory myeloma patients resulted in a higher rate of partial response and stable disease [30]). More recently, the use of HCQ in combination with gemcitabine in pancreatic ductal adenocarcinoma patients caused significant decreases in the disease biomarker, CA 19–9, with the mean overall survival being extended to nearly 3 years [28,31]. Although clinical trials with these compounds indicate that autophagy inhibition in patients is possible, there is still room for improvement, since CQ/HCQ have also shown significant variability of autophagy inhibition levels among patients. Moreover, these type of compounds, although being already FDA approved, have to be administered in higher concentrations to inhibit autophagy and are retained for long periods of time in patients (some studies showing patients retaining HCQ) in their system up to 5 years [28,32]. On the other hand, autophagy induction may help improve the effect of anticancer therapies when autophagy is cytotoxic, by inducing cell death by itself or by the activation of other cell death mechanism, namely apoptosis [33,34]. Several drugs/natural extracts, some of which already used in the clinic, have been described to induce autophagymediated cell death in different cancer cells [23]. For example, the combination of Vitamin D with radiation promoted cytotoxic autophagy in breast tumor cells [35,36]. Resveratrol and curcumin caused cell death in several h