Frontiers in Anti-Infective Drug Discovery: Volume 7 E-Book
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- Herausgeber: Bentham Science Publishers
- Kategorie: Fachliteratur
- Serie: Frontiers in Anti-Infective Drug Discovery
- Sprache: Englisch
This book series brings updated reviews to readers interested in advances in the development of anti-infective drug design and discovery. The scope of the book series covers a range of topics including rational drug design and drug discovery, medicinal ch
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Veröffentlichungsjahr: 2018
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Frontiers in Anti-Infective Drug
Discovery
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PREFACE
The 7th volume of the book series entitled, “Frontiers in Anti-infective Drug Discovery” comprises seven comprehensively written reviews on therapeutic advances against bacterial, fungal, and viral infections and cancers, and on study of the mechanism of action of various drugs, and drug candidates, including natural products.
Amedei and Russo have reviewed the role of gut microbiota in the on-set and progression of gastrointestinal (GI) cancers in chapter 1. GI cancers are among the most prevalent cancers with complex aetiologies and high mortality rate. Along with many other factors, human gut microbiota has been found to play an important role in GI cancers. GI cancers are multifactorial diseases influenced by genetic and environmental factors, as well as by Helicobacter pylori infection. Association between the gut microorganisms and GI tumours has attracted major scientific interests in recent years. Modern sequencing techniques have been used to understand various features of the complex microbial communities, as well as the mechanisms through which the gut microbiota are involved in carcinogenesis and cancer progression. The authors have provided a detailed description of how microbial dysbiosis (microbial imbalance) contributes to oncogenesis via multiple pathways, including tumour progression and response to the cancer treatments.
Dengue viral fever has emerged as one of the most important epidemics in recent times. Its re-emergence in tropical and sub-tropical regions of the world is causing considerable morbidity and mortality. Currently, no vaccine is available which can protect against four serotypes of the Dengue virus (DENV) which are known to use host’s biological systems to propagate, and cause infections. These host biological processes include cytoplasmic and nuclear structures and components. Damonte et al. have reviewed the recent literature in this important area of research in chapter 2. Their emphasis has been on the identification of host factors, and processes which are involved in viral infection.
Plant products have played a key role in the treatment of diseases since antiquity, including treatment of infections in the pre-antibiotics era. With the discovery of natural products and their synthetic analogues with good antibiotic activity, the importance of phytochemicals had diminished. However, with the emergence of antibiotic resistance, the search of new antibiotics including resistance reversal agents from the plant kingdom against novel targets, was globally initiated. Hazra et al. in chapter 3 discuss various classes of plant secondary metabolites which have the capacity to serve as resistance modifying agents (RMA), thus helping conventional antibiotics to function again. The authors have discussed various screening approaches protocols which have been successfully used for the discovery of RMAs, as well as approaches to study their possible mechanism of actions. The discovery of plant-based RMAs is thus a powerful new approach for the treatment infections caused by multi-drug resistant bacteria.
Computer-aided drug design (CADD) has emerged as an important tool in the modern drug discovery and development process. CADD approach plays a key role in drug target identification, study of mechanism of action, as well as in the prediction of drug candidates in the initial drug discovery process. However, CADD-predicted compounds are often found to be ineffective in biochemical and cellular assays. Kaczor and his colleagues have reviewed the application and limitations of CADD approach in chapter 4. They critically analyse the strengths and weaknesses of CADD based lead identification in the context of antibacterial drug discovery and emphasize the need to further improve the CADD based methods, as well the need for experimental verification of computational results at an early phase of drug discovery process.
Since the FDA approval of first aptamer-based drug for the treatment of age-related macular degeneration in 2004, interest in therapeutic uses of aptamers has increased substantially. These molecules are capable of specifically binding with proteins, and inhibiting targets such as vascular endothelial growth factor (VEGF), thrombin and other cellular disease targets. These oligonucleotide aptamers are essentially the small molecular equivalent of antibodies, and thus have the advantage of being highly specific and non-immunogenic. Evran et al. present a comprehensive review on the applications of small single-stranded RNA and DNA (oligonucleotide aptamers) as targeted therapies against virulence factors of drug resistance microorganisms in chapter 5. Many aptamers have been investigated and developed for the treatment of diseases, such as cancers, HIV, and macular degeneration. The review of Evran et al. focuses on recent research on the use of various aptamers as specific blockers of the virulence factors of drug resistant pathogens. The authors have also reviewed the literature on the use of aptamers as biosensors of bacterial toxins.
Fungal skin infections are often difficult to treat completely due to their capacity to go in recession through spore formation. Candida albicans and its multidrug resistant strains are a major cause of superficial, and deep seeded infections in humans and livestock. The review by Hameed et al. in chapter 6 addresses the approaches to overcome fungal infections by using natural products, that can act by blocking or modulating the functions of drug efflux pumps in MDR fungal pathogens. These natural products thus provide an excellent opportunity to treat MDR fungal infections, including Candida infections effectively.
Hydrogen sulfide (H2S) is an endogenous gaseous transmitter whose role in the pathophysiology of several diseases has been extensively studied. It has been linked to many important physiological functions and is known to play a significant role in various diseases involving inflammation, fibrosis, and vascular responses. Recent studies indicate the vasoactive, cytoprotective and anti-inflammatory role of hydrogen sulfide. It can regulate viral, bacterial, parasitic, and fungal infections through its function as signalling molecule. Bhatia et. al. has reviewed state-of-the-art understanding about the mechanisms underlying H2S-mediated regulation of different infectious diseases in chapter 7. This can help in developing H2S- releasing drugs as therapeutic molecules. In the absence of effective treatment for many infections, the H2S based therapeutic agents provide a new approach for infection treatment.
The above articles by prominent researchers in chosen fields have made this volume another important treatise for scientists and research scholars. We are grateful to all the authors for their excellent and scholarly contributions for the 7th volume of this internationally recognized eBook series. We hope that like the previous volumes of this internationally reputed book series, the current compilation will also receive a wide readership and appreciation.
The editorial team of Bentham Science Publishers is greatly appreciated for efficient processing and timely management of this publication. The coordination and liaison by Ms Fariya Zulfiqar (Assistant Manager Publications), and leadership of Mr. Mahmood Alam (Director Publications) are duly acknowledged.
List of Contributors
The Role of the Microbiota in the Genesis of Gastrointestinal Cancers
Edda Russo,Amedeo Amedei*Abstract
The term “Gastro-Intestinal (GI) cancer” indicates a group of tumors that affect the digestive system. Despite progress in treatment, these widespread types of malignant condition represent a serious health problem in the world. GI cancer is a multi-factorial and multi-stage involved disorder, its progression is influenced by environmental and genetic elements and the involvement of microbial population has also recently been recognized in many studies. Today, Next Generation Sequencing (NGS) approach has been used to elucidate the involvement of microorganisms in initiating and facilitating the process of GI cancer. In this chapter, we would like to clarify the role played by the gastrointestinal microflora in the genesis of GI cancers. This chapter will draw the state of the art in the study of the GI microbiota and how the dysbiosis could affect oncogenesis, tumor progression and response to cancer.
INTRODUCTION
Gastrointestinal (GI) cancers are malignant conditions of the GI tract and accessory organs of digestion, such as esophagus, biliary system stomach, small intestine, large intestine, rectum, pancreas and anus. The symptoms can include obstruction, abnormal bleeding and different associated problems. Despite several progress in treatment, GI is one of the most common form of cancer and represents an important health problem in all the world. As of 2012, esophageal cancer is the eighthmost common cancer, affecting 450,000 people worldwide [1]. Gastric cancer (GC) represents the fourth most common tumor with 1,000,000 new cases per year and 850,000 deaths [2, 3], but, GC prevalence is constantly decreasing; a possible cause could be the decrease of the H. pylori (HP) diffusion, a bacterium involved in the GC pathogenesis [4]. Neoplasms of the small intes-
tine are rare, indeed the global incidence ranges from 0.3 to 2.0 per 100,000 [5]. While the Colorectal cancer (CRC) is the third most frequent tumor worldwide and the fourth most common reason of cancer death, with about 500,000 deaths per year [3]. The multi-steps mechanisms associated with GI cancer prevention and development are still largely unknown. GI cancers are considered to be a multi-factorial disease resulting from intricate relationships between genetics, epi-genetics, immunity, environment (including geographical area and socioeconomic status), lifestyle and diet; all this factors could impact the GI microflora, altering its profiles and its functions during the tumor genesis and growth [4]. In healthy individuals, GI microflora acts as a symbiont offering protection from invading pathogens and preventing carcinogenesis [6]. When the fine balance of this commensal bacterial community is disrupted, the establishment of a dysbiosis state could cause pathological conditions in the host, including cancer [7, 8].
In 400 B.C, the words of Hippocrates (one of the most outstanding figures in the history of medicine) “Death sits in the bowels” [9], showed that the involvement of the intestinal metabolism in human health has been long acknowledged. In the past, most researches on the impact of bacteria colonization in the gut have been focused on gastrointestinal pathogens. While recent evidences still corroborate individual microorganisms influencing tumor genesis (e.g., human papilloma virus the cervical cancer, hepatitis B and C virus the hepatocellular carcinoma, Helicobacter pylori the gastric cancer) [10, 11] also microbial dysbiosis could have a large impact in malignant promotion and progression.
In this chapter, we would like to revisit the state of the art of microbiota influence in the genesis of GI cancers, discussing how disequilibria (dysbiosis) could influence the mutual relationship between the host and intestinal bacteria affecting oncogenesis, tumor progression and response to cancer treatment. We will present challenging questions to be addressed in the future of microbiota research, such as how the gut microbiota may be manipulated for therapeutic strategies.
THE HUMAN MICROBIOTA
In the past, the human body has been considered as a self-sustaining organism that can control all of its metabolic reactions. Today, scientists have shown that the human body indeed is an ecosystem containing trillions of microorganisms. The communities of microorganisms living in coexistence with their hosts has been referred as microbiota, microflora or normal flora.
The human microbiota could contain approximately 1,014 bacteria, a number that is 10 times greater than the amount of the total human cells in the body. The microflora is resident in every surface of the body exposed to the external environment such as skin and mucosa (from the GI, to respiratory and urogenital tract). The gastrointestinal tract (GIT) is the organ that contains the larger fraction of bacteria producing molecules that can be used as nutrients, making it a preferred site for colonization; indeed the colon contains over 70% of all the bacteria in the body. This human GIT ecosystem results from an evolutionary process of co-existence between the microflora and the body. The microbiota significantly influences physiological functions such as food digestion and immune system stimulation [12].
The human microbiota includes microorganisms belonging to the domains of the Archaea, Bacteria, Eukarya and their viruses. The majority of bacteria are strict anaerobes, which predominate the facultative anaerobes and aerobes. The commensal bacteria are symbiotic, but they can cause a pathological state after translocation through the mucosa or in specific conditions such as immunodeficiency. In general, the composition of the human microbiota is strictly personal, but the diversity in the structure of the bacterial population among the body sites is greater than it is between individuals. This state indicates that the human microbiota is a highly variable ecosystem that embraces different microbiological components [13, 14]. It is possible to term a bacterial community “core” of a healthy microbiota that is commonly present within different body sites.
To date, although there have been over 50 bacterial phyla described, only 2 of them dominates the human gut normal flora: the Bacteroidetes and the Firmicutes, whereas Actinobacteria, Proteobacteria, Fusobacteria, Verruco-microbia and Cyanobacteria appear in minor proportion [15]. Estimates of the amount of bacterial species present in the human intestine vary extensively between different studies, but it has been widely accepted that it contains 500 to 1,000 species. A recent study involving multiple subjects has suggested that the total human gut microbiota is composed of over 35,000 bacterial species [16]. Interestingly, a wide proportion, about 70%, of the human microbiota is com-posed of microbes that cannot be cultivated by common microbiological methods. The traditional culture-based methods capture less than 30%, of our bacterial microflora [17]. Today, genomic Next-Generation Sequencing (NGS) analysis has been crucial to analyze the bacterial microbiota profile and the metagenome, and also these techniques give more information about the impact of microflora in host metabolic reaction, cancer progression and inflammation [18, 19].
THE GASTROINTESTINAL MICROBIOTA
Composition and Activities
The human digestive system is composed of distinct regions with different functions: the oral cavity, stomach, small intestine and colon. The intestinal mucosa is the largest surface of the body that is regularly exposed to bacterial and dietary antigens. The bacterial phyla present on Earth are more than 50, but the most common human gut-associated microbiota is composed of four phyla: Firmicutes, 30.6-83% (Ruminococcus, Clostridium, Peptococcus, Eubacterium, Dorea, Lactobacillus - L, Peptostreptococcus); Bacteroidetes, 8-48% (Bacteroides); Actinobacteria, 0.7-16.7% (Bifidobacterium - BF) and Proteobacteria, 0.1-26.6% (Enterobacteriacee) [15, 20].
But the intestinal microbiota organization is not homogeneous. In the human GIT, the content of bacteria increases from mouth (less than 200 species) to the colon (bacteria reaching 1010-1012/gram of luminal content, with a predominance of anaerobe bacteria) [21]. Notably, the proportion of bacterial cells resident in the mammalian gut goes from 101 to 103 bacteria x gram (g) of contents in the stomach and duodenum, progressing to 104 to 107 bacteria x g in the jejunum and ileum and ending in 1011 to 1012 cells x g in the colon [22]. Furthermore, the bacterial structure changes between these GIT sites. Various microbial strains are enriched at different sections when comparing biopsy samples of the small intestine and colon from healthy controls. Bacilli class of the Firmicutes and Actinobacteria are increased in the specimens of the small intestine. On the contrary, Bacteroidetes and the Lachnospiraceae families of the Firmicutes were more dominant in colonic samples [16]. A thick mucus layer divides the intestinal epithelium from the lumen leading to a great latitudinal heterogeneity in the bacterial composition. The microbiota assemblage of the intestinal lumen is significantly different from the microbiota embedded in this mucus layer as well as the bacterial population resident in the immediacy of the epithelium. Several bacterial strains resident in the intestinal lumen did not access the mucus layer and epithelial crypts. Streptococcus, Bacteroides, Bifidobacterium, members of Enterobacteriacea, Enterococcus, Clostridium, Lactobacillus and Ruminococcus were all detected in feces, whereas only Clostridium, Lactobacillus and Enterococcus were observed in the mucus layer and epithelial crypts of the small intestine [23]. Different factors could contribute to the diversifications along the length of the GI tract such as bacterial factors (enzymes, metabolic activity, adhesion capacity), host elements (bile acids, mucus pH, digestive enzymes, transit time,) and non-host aspects (medication, nutrients, environmental factors) [24].
Due to the abundance of nutrients, the human oral cavity represents the ideal habitat for microorganisms. At least six billion microorganisms take place in mouth belonging to the Bacteroidetes (e.g. Bacteroides, Prevotella), Firmicutes (Gram positive; e.g., Clostridia, Bacilli,), Proteobacteria (Gram negative, e.g., Salmonella, Escherichia, Helicobacter and Yersinia), Fusobacteria (Gram negative, e.g., Fusobacterium) and Actinobacteria (Gram positive, e.g., Streptomyces, Actinomyces) [25]. The gastric microbiota is composed mostly of Actinobacteria but, due to the acidic environment, Helicobacter (e.g., H. pylori) is also present [26]. The small intestine microbiota has a qualitative composition similar to the colon microbiota, but the latter contains a higher number of microorganisms. The small intestine hosts few bacteria in its proximal part, the microbiota is composed of Gram+ Lactobacillus and Enterococcus faecalis. More microorganisms occur in the distal part, e.g., Bacteroides and coliforms. In the colon quantitatively Firmicutes and Bacteroidetes were dominant and, at the genus level, anaerobic lactic acid bacteria, e.g., Bifidobacterium bi- fidum and anaerobic Bacteroides, prevailed [25].
The GI microbiota is crucial to the physiology of the human body, as it could produce molecules able to interact with the host and performs important metabolic functions. In particular, the bacteria of the gut microbiota act as a first defense against pathogen colonization and they break down indigestible dietary components [27], promote angiogenesis, support fat metabolism, synthetize vitamins, help the development of the immune system and maintain homeostasis [28]. The bacteria population is separated from the internal gut milieu by a layer of epithelial cells, which is a physical and chemical barrier that balances the crosstalk between the immune host system and the external environment. Moreover, the epithelial surfaces have evolved mechanisms to counteract the microorganism invasion. Adaptive and innate immune responses protect the mucosa and the internal environment of the human body. Almost 80% of the immunological cells are active in the mucosal-associated immune system, most of these cells are resident in the GI tract, where the level of immunogenic components of the food and the bacterial flora is at the highest respect to other districts of the body.
Usually the bacterial flora does not cause a proinflammatory response because the immune system tolerates the commensal bacteria and preserve the homeostasis but, when these mechanisms are impaired (e.g. use of antibiotics, immuno-deficiency and unhealthy diets) or new pathogenic bacteria are introduced into this balanced system, the immune system reacts to the microbiota triggering a pathological state, facilitating inflammation and cancer progression in the intestine [29]. Different studies suggest that an imbalance of the gut microbiota and its metabolic functions are correlated with the initiating and progression of GI pathologies, including colorectal cancer, functional dyspepsia, severe diarrhea, inflammatory bowel disease (IBD), celiac disease and irritable bowel syndrome IBS [30, 31]. It is now understood that the imbalance of gut microbial population (dysbiosis) can be activated by intrinsic (e.g., stress, genetics and aging) and extrinsic factors (e.g., appendectomy, diet and antibiotic use).
Gastro-Intestinal Colonization by the Microbiota and Selection
The microbiota composition is more plastic and variable than the human genome and also more readily changeable and reactive to stimuli than most human cells. The human superorganism is composed of two constituents: 1) inheritable human gene pool, surrounded by 2) evolvable and changeable bacteria gene pool, acquired after birth, whose composition varies with time, space, health and hormonal state.
Indeed, microbial colonization of the newborns commences at moment of the birth during the passage through the birth canal and is affected by the delivery mode [32]. The bacterial settling during birth impacts the development of the gut normal flora. The intestinal microbiota of infants and the mother vaginal microbiota show some similarities such as an example they are both enriched in Prevotella, Lactobacillus or Sneathia spp [33]. On the contrary, infants delivered through cesarean section exhibited different bacteria compositions compared with vaginally delivered newborns [34].
During the first twelvemonth of life, the microbiota structure of the infant’s gut is simple and varies between different individuals and with time [35, 36] but, after 1 year of age, it looks like to young adult gut microbial assemblage [33, 35]. Experiments in mouse showed that the gut microbiota of offspring is similar to that of their mothers [36]. Other studies revealed that gut microbiota of adult monozygotic and dizygotic twins were equally similar to that of their siblings, this data suggests that the gut colonization by the microbiota from the same mother had a key role in determining the adult bacteria community composition [37]. Several other factors, as host genetics, have been found to impact the gut microbial structure. For instance, experiments in mouse revealed that the gut bacteria composition is altered in genetically obese mice vs genetically lean siblings [36]. Moreover, a mutation in the major component of the high density lipoprotein (apolipoprotein a-I) in mouse is associated to an altered gut bacteria assemblage [38]. Other studies in obese mouse showed the consumption of western diet can alter the gut microbiota profile [39]. Further limiting weight gain with dietary manipulations could reverse the effects of diet-induced obesity on the microbiota of murine gut.
The Human Microbiome Project
The microbial composition of a specific ecosystem and its function has been studied by several international consortium researchers such as the Human Microbiome Project (HMP; www.hmpdacc.org), launched in October 2007 by the National Institutes of Health. HMP is a global project that brought together a big number of scientists to different specific aims:
Characterize the microorganism communities of the major human districts (skin, mouth, nose, colon and vagina)Study the functional and metabolic pathways of microbial communitiesDetermine their functional roles in health and diseaseThis consortium published over 350 papers [40-42]. The HMP estimates that the human microbiota contains between 3,500 and 35,000 Operational Taxonomic Units (OTUs). An OTU is a cluster of organisms grouped on the basis of the sequence similarity [41]. In addition, the consortium HMP discovered novel taxa at the genus level, including the Dorea, Oscillibacter and Desulfovibrio genera, which correlated with disease conditions [41, 43, 44]. Furthermore, the HMP has supported the development of new technological and Bioinformatics tools to be used in metagenomic studies [45].
Gut Enterotypes
In 2011, Arumugam et al. [46] identified three distinct enterotypes of the human gut microbiota (Table 1). These enterotypes vary in functional composition, species and enzyme balance. Enterotype 1 produces enzymes associated with the biotin biosynthesis pathway, while Enterotype 2 and 3 produce those which are connected with the thiamine and heme biosynthesis pathways, respectively [42, 46]. Also, long-term diets correlated with enterotypes [47], indeed, food rich in protein and fat was associated with the Bacteroides enterotype, while food rich in carbohydrate and simple sugars was associated with the Prevotella enterotype. Ruminococcus enterotype did not correlate with feeding [47].
THE ROLE OF MICROBIOTA IN TUMOR DEVELOPMENT
The involvement of infectious elements in the cancer etiology has recently attracted the research attention. In 1890, the Scottish pathologist William Russell [48] reported evidence for a bacterial cause of cancer. Currently, different data have strengthened this theory suggesting a bacterial involvement in the genesis and cancer progression (often interfering with and modulating the local immune response) [49].
As previously reported, recent studies suggest that not only a single bacteria, but also global changes in the host microbiota could cause human disease [50, 51]. Different studies in germfree animals report a tumor promoting effects of the microbial community in genetically induced and spontaneous cancers as breast, lungs, skin, liver and colon tumors [52-54]. But, there are also conflicting data showing a central role of the gut microbiota in reducing proliferative responses that lead to cancer development in germfree animals [55].
In 1975, Reddy and colleagues for the first time, linked the gut microbiota to intestinal cancer development, establishing that only 20% of genetically modified germfree rodents develops chemically induced CRC. In contrast, the tumor incidence in rats with a normal microbiota was about 90% with several neoplasms [56]. Vannucci and colleagues confirmed these data showing that germfree rats, compared with similar animals with a normal microbiota, develop smaller tumors, as spontaneously as after chemically induced carcinogenesis [57]. In colitis-associated cancer and adenomatous polyposis coli (APC)-related colorectal cancer, germfree mice display decreased tumor formation and less oncogenic mutations [58]. In addition, antibiotics depletion of the gut microbiota in mice limits cancer growth in the colon and the liver [59-62] as does the eradication of specific pathogens in humans and in mice [63, 64]. All these data provide strong evidence for the microbiota role in tumor initiating and growth. Probably, the germfree rats can develop a more active anticancer immune response in the absence of the physiological inflammation induced by the gut commensal community.
Proposed Models for Microbiota-induced Carcinogenesis
Currently, researchers have proposed three mechanisms of microbiota-induced carcinogenesis:
The unbalanced proinflammatory signaling at the intestinal level induces an increased repair of the intestinal epithelium that can result in the tumor developmentSome microbial species can have direct cytotoxic effects on intestinal cells.Particular members of the microbiota can generate by-products that are toxic to the intestinal surface.To better understand the microbiota’s contribution in tumor growth, different “hypothesis models” have been proposed:
The ‘alpha bugs’ (microbiota members possessing unique virulence traits) are both directly pro-oncogenic bacteria able to remold the mucosal immune response and bacteria species that protect against cancer [65]. An example of “alpha bugs” is enterotoxigenic Bacteroides fragilis (ETBF),The ‘bacterial driver-passenger’ model describes the microbiota influence in the development of CRC. The ‘driver bacteria’ (indigenous intestinal bacteria), initiate the first phases of tumor progression, inducing DNA injury and driving genome instability. As a consequence of this process, the bacterial drivers (such as alpha bugs) are replaced by commensals bacteria with either tumor-promoting or tumor-suppressing properties (bacterial passengers). According to the “driver-passenger” model, the disease progression causes changes in the microenvironment resulting in a different selective pressure on the microbial population [66].The ‘keystone pathogen’ hypothesis. The term ‘keystone’ (firstly used in the ecological studies) refers to species whose effects on their communities are excessively large relative to their abundance and which are thought to form the ‘keystone’ of the community’s structure. According to this model, some low-abundance bacterial pathogens can induce inflammatory disease by shaping a normal microbiota into a dysbiotic one [67].Finally, inflammatory responses triggered by microbiota is able to enhance tumor progression [68]. Some microbes produce variations of mucosal permeability, inducing bacterial translocation. Different studies demonstrated the role of inflammation in creating the conditions that could change local immune responses and tissue balance. Moreover, it is well documented that the inflammatory molecules, such as TNF-α, interleukin (IL)-1), IL-8, nitric oxide, prostaglandin-2 derivatives are involved in the interplay between the immune and tissue cells undergoing transformation [69].
Antibacteria-Specific Immune Response and Cancer Promotion
As previously reported, the bacterial population is divided from the internal gut milieu by a stratum of epithelial cells, which acts as chemical and physical barrier and regulates the crosstalk between the immune host system and the external environment. This epithelial surface evolved protective mechanisms to counteract bacteria invasion. Adaptive and innate immune responses protect the mucosa and the internal environment of the human body. The normal microbiota (in eubyosis condition) does not trigger a proinflammatory reaction because commensal bacteria are usually tolerate by the immune system, but when these mechanisms are impaired, they could cause tumor development and progression [29]. So, the inflammatory and host-derived immune responses are essential actors that shape the gut microbial profile and may contribute to the dysbiosis state. Several studies demonstrated that IBD patients have an increased risk of CRC because inflammation-promoted cancerogenesis also plays an important role in CRC development [70]. Furthermore, gut microbiota has also been shown to have an impact on colitis-associated CRC progression. IL-10/ mice develop spontaneous colitis when colonized with gut microflora, but after exposure to a strong carcinogen, mice showed a very high incidence of CRC [71]. On the contrary, the contact to a carcinogen of GF IL-10/ mice did not cause a malignant neoplasia, whereas IL-10/mice mono-associated with a mildly colitogenic bacterium had a reduced incidence of CRC following exposure to a carcinogen, compared with mice colonized by the normal gut microbiota.
One of the main avenues by which the microbiota can indirectly promote tumor growth are the Th (helper) 17 cells. The bacterial flora actively shape intestinal T-cell responses to establish homeostasis. Th17 cells control microbial invasion in the gut, but specific compensatory mechanisms are required to regulate the Th17 cells. At intestinal level, the bacteria induce IL-1β production to maintain a basal level of Th17 cells in the lamina propria under physiological conditions [72], but in response to pathogenic extracellular bacterial or fungal infections, strong numbers of naive Th cells differentiate into Th17 under the influence of IL-1β, IL-6, IL-23 or TGFβ in mucosal surfaces of the intestine and respiratory tract [73]. If those mechanisms are impaired, Th17 cells become pathogenic and can induce autoimmune disease and chronic inflammation. When stimulated with IL-6 and TGF-β, the antigen-activated CD4+ T cells upregulate the transcription factor RORγt (retinoic acid receptor related orphan receptor gamma t) and secrete Th17-specific cytokines such as IL-17 and IL-22 [74]. Usually, the CD4+ T cells that express RORγt increase tight junction formation and stimulate the secretion of microbicide proteins, contributing to the barrier function of the intestinal epithelium but they can have also a protumorigenic role [74]. The functional Th17 impact in cancer is still equivocal, showing both protumorigenic and antitumorigenic activities in different cancer type [75-77].
Furthermore, Th17 cells can secrete IL-21, IL-17F, IL-22, granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon (IFN)-γ [78, 79]. Th17 responses and mainly the IL-17 action itself, were originally considered as a cancer growth promoters [80]. In a mouse model, Wu et al. demonstrate that the Th17 cells are able to promote CRC progression, induced by colon inflammation [81]. In experiments with genetically predisposed mice (APCmin/+) crossed with IL-17A-deficient mice a drastic impairment in intestinal tumorigenesis was observed [82]. Moreover, different studies revealed that APCmin/+ mice that cannot respond to IL-17 develop fewer tumors in the colon [75].
The function of Th17 cells has been investigated in patients with different tumor types, including prostate and ovarian cancer [83-86]. These studies have examined Th17 cells in peripheral blood, but it is important to notice that Th17 cells may be induced in or recruited in the cancer microenvironment [87]. A more direct proof for a microbiota role in stimulating tumor growth via Th17 cells comes from studies of enterotoxigenic Bacteroides fragilis (B. fragilis), a colonic bacterium that produces B. fragilis toxin (BFT). Several mouse models, predisposed to develop gut tumors, indicate that between colonization of B. fragilis and nontoxigenic B. fragilis, only the first causes colitis and produces colonic tumors [81]. Notably, B. fragilis induces STAT3 activation with colitis characterized by a selective Th17 response. Antibody-mediated blockade of IL-17, inhibits B. fragilis induced colitis, tumor formation and colonic hyperplasia. These data show that also a common human commensal bacterium could induce cancer by STAT3- and Th17-dependent pathway of inflammation, providing a new insight into CRC development.
Moreover, the Th17 response upon contact with specific microbes, stimulates neutrophil cells, required for the clearance of invading bacteria [88]. The Th17 response is important for protection against mucosal pathogens like Klebsiella pneumonia and Salmonella typhimurium. Deficient Th17 mice models show a pathological condition during infection with Salmonella or C. rodentium, with increased translocation of bacteria into lymphonodes [89]. Th17 are also activated by the segmented filamentous bacteria (SFB), belonging to nonculturable Clostridia-related species and flagellin-positive bacteria. These bacteria interact with the epithelial cells promoting chronic inflammation, mediated by IL-17 and IL-22 release, which favors intestinal cancer. In addition, the IL-22 has been linked to intestinal tumor in mouse models triggered by STAT3 activation and also human pancreatic cancer [90, 91]. Moreover, the conjunction of IL-22 with IFN-γ can activate inducible nitric oxide synthase (iNOS) production and procarcinogenic nitric oxygen species in human CRC cell lines [92]. Finally, the cytokine IL-23 is produced by myeloid cells in response to different bacteria molecules, such as flagellin [93]. IL-23 (able to promote Th17- type response) was increased in human colon adenocarcinoma, it promotes cancer growth through a proinflammatory response [94].
MICROBIOTA AND GI CANCERS
Gut Bacteria Dysbiosis Associated with GI Cancer
Dysbiosis (also called dysbacteriosis) is a term for a microbial imbalance or maladaption on or inside the body, such as an impaired bacterial composition. It can be caused not only by pathogenic organisms and passenger commensals, but also by aging and environmental factors such as antibiotics, xenobiotics, smoking, hormones and dietary cues [29]. Of note, these are also well-established risk factors for the development of intestinal or extraintestinal neoplasms. In addition, genetic defects that affect epithelial, myeloid or lymphoid components of the intestinal immune system could favor dysbiosis because they promote inflammatory states, such as Crohn’s disease, that increase the host risk of neoplastic conversion [95].
So, several factors that facilitate carcinogenesis also promote dysbiosis. Epidemiological studies linking intra-abdominal infections, antibiotic administration or both to an increased incidence of CRC [96] underscore the clinical importance of the association between dysbiosis and intestinal carcinogenesis. Abrogating or specifically altering the assemblage of the gut microbiota impacts the incidence and progression of CRC in both genetic and carcinogen-induced models of tumorigenesis [55, 97]. Moreover, several products of the gut microbiota directly target intestinal epithelial cells (IECs) and either mediate oncogenic effects (as reported for hydrogen sulfide and the Bacteroides fragilis toxin) or suppress tumorigenesis (as demonstrated for short-chain fatty acids, SCFA) [98].
Intestinal bugs participate in more than just colorectal carcinogenesis. Experimental alterations of the gut microbiota also influence the incidence and progression of extraintestinal cancers, including breast and hepatocellular carcinoma, presumably through inflammatory and metabolic circuitries [52, 60]. These results are compatible with the findings of epidemiological data that reveal an association between dysbiosis, its consequences or determinants (in particular the overuse of antibiotics) and an increased incidence of extracolonic neoplasms, including breast carcinoma [99, 100]. These evidences may reflect the systemic distribution of bacteria and their by-products in the course of inflammatory responses that compromise the integrity of the intestinal barrier [60]. The gut microbiota influences oncogenesis and tumor progression both locally and systemically. Although inflammatory and metabolic indications support this phenomenon, additional, uncharacterized mechanisms can contribute to the ability of dysbiosis to promote carcinogenesis (Fig. 1).
Fig. (1)) Mechanisms by which dysbiosis affects oncogenesis.Microbiota Involvement in Gastric and Esophageal Cancers, the Role of Helicobacter pylori
The esophagus is an organ through which food transits, aided by peristaltic contractions, from the pharynx to the stomach. The esophagus is divided into three main sections - the upper, middle and lower. Tumor can develop anywhere along the esophagus length. The mucus produced by glands in the wall of the esophagus help food slide down. The most widespread type of cancer seen in Western countries is esophagus adenocarcinoma generated by these glands.
During the past 3 decades, the amount of adenocarcinomas of distal esophagus and the gastroesophageal junction has been increasing. This data is attributed to smoking, gastroesophageal reflux and alcohol consumption [101]. On the contrary, H. pylori infection seems to be protective to distal esophageal cancer, leading to loss of acid secretion, hormonal deregulation or cytokine and changes in microflora composition [102, 103]. A recent Chinese research demonstrated that individuals with lower oral microbial diversity were more likely to have squamous dysplasia in the esophagus and chronic atrophic gastritis [104]. In the same study, the authors also found a correlation between esophageal squamous dysplasia with the odds ratio being significantly decreased with increasing bacterial richness. Another research performed in Northern Iran (considered part of the “esophageal cancer belt”) evaluated the gastric microflora from the gastric mucosa in patients with esophageal squamous cell carcinoma [105]. An enrichment of Erysipelotrichales and Clostridiales species, belonging to the phylum Firmicutes, was found. These species were significantly related to early squamous dysplasia and esophageal squamous cell cancer.
Most stomach cancers develop slowly in cells that line the mucosa and are called adenocarcinoma of the stomach. As the microbiota come in close contact with gastric and esophageal linings, current studies support its influences in oncogenesis. The most important example of a cancer induced by bacteria is the Helicobacter pylori-mediated gastric carcinoma [106]. This bacterium takes part of the gastric microbiota [106] and its presence induces a continuous activation immune response in the human host, resulting in inflammation of stomach mucosa that leads to cancer transformations at the gastric epithelium. Different hypothesis have been suggested by which H. pylori influences GC development.
Murine models of H. pylori infection (infected with H. felis, the homologous of H. pylori in mouse) have shown that induction of a T cell-mediated response [107] and a Th1 cytokine environment [108] are crucial to the development of the bacterium induced pathology.H. pylori could influence the production of reactive nitrogen compounds at the gastric mucosa [109], which could induce carcinogenic DNA damage [110].H. pylori could affect regulators of DNA transcription [111]. Helicobacter pylori infection increases cell proliferation which could lead to a higher frequency of mutation and less time for DNA repair [112].Mice deficient in secretory phospholipase A2 (sPLA2), showed increased apoptosis levels after infection with H. felis in mouth and the growth of aberrant gastric mucosa cell lineages [113]. Cell cycle and apoptosis are regulated by Raf-kinase inhibitor protein (RKIP) in the gastric mucosa. In infected mucosa, H. pylori phosphorylates RKIP, eliminating apoptotic control and increasing cell proliferation [114]. Another tumor suppressor gene, LOX, was shown to be methylated in mice infected by H. felis [115]. Notably, H. pylori derived from distinct stages of tumor progression in the same human subject, one during the chronic atrophic gastritis and the second following cancer transformation, showed different interaction with gastric epithelial stem cells [116]. Mongolian gerbils, whose gastric system is close to humans showed that 37% of animals infected by H. pylori developed cancer, on the contrary uninfected controls showed no tumor development [112]. A recent study suggests that long-term H. pylori infection disrupts the gut microbiota balance [117]. Lactobacillus species decrease H. pylori growth in vitro, suggesting a prevention of H. pylori infection [118].
Human studies, comparing gastric microbiota composition in cancer patients versus healthy subjects, indicate that also other microbes must be present to trigger the progression from healthy mucosa toward tumor development [119, 120]. Notably, there are many people H. pylori positive that do not develop gastric cancer [121]. GC patients showed low levels of Porphyromonas, Meisseria and Streptococcus sinesis while Lactobacillus coleohominis, Pseudomonas and Lachnospiraceae were increased [122]. L. coleohominis is a species initially reported to be beneficial in the gastric environment, a study showed the increase of Lactobacilli terminal restriction fragments (TRFs) in gastric cancer subjects, supporting the increase in the abundance of these bacteria [120].
H. pylori-associated carcinoma is one of the most preventable cancers because H. pylori eradication resolves the gastric inflammation and has been shown to decrease GC incidence [120]. In mice, the eradication of Helicobacter felis, produce a decrease in the methylation of the LOX tumor suppressor gene [115]. H. pylori infection was also linked with junctional tumors, those involving the esophagus and gastric cardia [121, 122]. More is known about the microbiota in precursor states to esophageal cancer like reflux esophagitis and Barrett metaplasia. In these conditions the dysbiosis plays a key role in the cancer development, indeed a shift from Gram+ bacteria to mostly Gram- has been shown [121]. Other microbes as Streptococcus mitis, Treponema denticola and Streptococcus anginosus induce inflammation by cytokines, possibly supporting tumor progression [119, 123].
Microbiota Involvement in Colorectal Cancer
Colorectal cancer (CRC), also referred as bowel cancer, is the third most common cancer and the fourth leading cause of cancer deaths worldwide [120]. Most bowel cancers develop from polyps in the colon or rectum but not all polyps become cancerous. Colorectal carcinogenesis is not fully understood, but it is thought to be a heterogeneous process with genetic and epigenetic alterations, influenced by diet, environment, host immunity and microbial exposures [123-125]. A large number of microbes is able to live in the human gut, forming complex communities which may play key roles in the CRC development [126, 127]. Interestingly, the high microbial density in the colon (*1012 cells/mL) compared to the small intestine (*102 cells/mL) is related with a *12-fold increase in tumor occurrence [128]. Furthermore, patients affected by IBD, who are more exposed to microbes because of the reduced intestinal barrier, have a*5-fold increased risk for CRC due to the abnormal inflammatory reaction to commensal bacteria [129]. Previous studies have suggested a potential dysbiosis of gut microbiota in CRC patients [130] and many other studies have been conducted to assess the possible pathogens involved in CRC progression [120]. Once they are identified, it would lead to a breakthrough in the prevention and CRC treatment, in particular for sporadic CRC that represents 85-90% of all CRC and that will be addressed in this topic. Currently, many evidences show that colonic microbiota is involved in tumorigenesis and its structure and characteristics are altered in CRC precancerous lesions [131]. However, it cannot be always clearly understood whether these variations are causally related to cancer progression or are a consequence of tumor-induced changes [132, 133].
In general, the gut microbiota could contribute to the CRC genesis via altered composition of its components (dysbiosis) [134], changes in the local abundance of bacteria population, harmful properties of some bacteria and change in bacterial metabolic activity [127, 132]. Many causes can influence the GI ecosystem, including physical and psychological stress, antibiotics, radiations, modified peristalsis, diet, etc. [9, 133].
The involvement of gut microbiota is firstly supported by the demonstration of the cytotoxicity/genotoxicity of the microflora in CRC by using fecal extracts from healthy controls (fecal water) [132]. Fecal water cytotoxicity determines mucosal cell proliferation [135, 136], possibly through secondary bile acids and produces DNA damage (genotoxicity) [137], proving that carcinogens exist in the colonic lumen. Genotoxicity has been significantly associated with fecal water from people following a diet high in fats and meat, but low in fibers (considered to be of high-risk for CRC), as compared to a diet low in fats and meat [136].
Other strong supporting data comes from mice studies: in germfree conditions, colitis and cancer genesis are significantly decreased or do not appear, compared with wild type mice [138]. Clostridium butyricum, Mitsuokella multiacida or BF longum have been associated with higher prevalence of colonic adenoma (68% in each case) in mice, as compared with L acidophilus (30%) [139, 140]. In addition, the tumor free germfree rats exhibit increased cytotoxic T lymphocytes, natural killer and B cells in peripheral blood, suggesting a better anticancer immune response. Azoxymethane-treated interleukin (IL)-10-/- mice develop colitis-associated CRC when infected with Bacteroides vulgatus, whereas germ-free mice do not [71]. Previous studies have indicated that different microbial species preferentially populate the cancer sites [141, 142] and the structure of gut microbiota is altered also in CRC patients [143, 144], an increase in the diversity of Clostridium spp. [144], as well as an enrichment of Bifidobacterium spp and Bacteroides has been observed. On the contrary, the gut microbiota composition in a group of patients at low risk for CRC progression was shown to be increased in Eubacterium aerofaciens and Lactobacillus spp, producing lactic acid [144]. Interestingly, recent data revealed that Bacteroides fragilis, Bacteroides uniformis and Bacteroides vulgatus were enriched in CRC patients. The genera Escherichia/Shigella, Enterococcus, Streptococcus, Klebsiella and Peptostreptococcus were increased in CRC patients, while the genus Roseburia and the family Lachnospiraceae were less abundant [145]. Streptoccocus bovis/gallolyticus antigen profiles allowed the distinction between healthy controls and CRC patients [146] and were detected also in polyposis patients, indicating that the infection occurs in the first stages of carcinogenesis [147]. The gut microflora composition of patients with polyposis (which anticipate the carcinoma progression) was also altered vs healthy controls, but similar to the population detected in the CRC.
So these data suggest that the variation of gut microflora population paves the way to the onset of carcinogenesis, but it is still not clear if the aberrant microbiota drives the malignant progression or host and diet factors promote concurrent microflora alterations in the colonic environment.
Analyses of CRC colonic mucosa-associated microbiota have shown lower counts of total BF, especially Bifidobacterium longum than those with diverticulitis [148]. In contrast with fecal results [149], the composition of the mucosa-associated microbiota in adenoma patients showed significantly increased abundance of Proteobacteria, Faecalibacterium spp, Dorea spp. and decreased levels of Bacteroidetes, Coprococcus spp and Bacteroides spp. vs controls [150]. In addition, the mucosal district of colon adenoma had a 20-fold relative reduction of mucosa-adherent microbes compared with healthy tissue [151].
Gut microbiota influence CRC course also through interaction with the inflammatory process in the colon mucosa [152]. As previously reported, the chemically induced cell proliferation due to dextran sulfate sodium (DSS) and azoxymethane (AOM) was increased in germfree mice, which lack of protective commensal bacteria. Notably, compared to pathogen free mice, tumor development in germfree mice leads to more and larger neoplasies [55]. An inflammatory milieu in the host could disrupt the eubiosis status, indeed host inflammation could modulate microflora composition through production of particular metabolites such as nitrate, that acts as a single energy source for facultative anaerobic microbes. These conditions allow them to outcompete microbial species that cannot employ nitrates [153], disrupting eubiosis and so, promoting dysbiosis.
Pro-inflammatory host responses can also prejudice immune function and barrier to permit microbial translocation through the tight junctions of the gut and intensify the inflammatory process [154]. Several hypothetical mechanisms are suggested to explain the mutual interplay between inflammation and gut microbiota in CRC initiating and progression: 1) accordingly to the ‘alpha-bug’ hypothesis (previously described), keystone pathogen bacteria, such as ETBF, shape gut microflora via Th17and IL-17 cell-mediated inflammation. This process may be obstructed by beneficial commensal bacteria [67]. 2) the bacterial driver-passenger model proposes that ‘driver’ bacteria, such as ETBF, induce or exacerbate inflammation and generate genotoxins that lead to cell mutations and proliferation. In a second step, ‘passenger’ bacteria, such as Fusobacterium spp. Colonize the adenoma supporting malignant progression [66]. Following cancer generation, the gut barrier is injured by the constant inflammation and allows microbe contacts to neoplastic tissue. These microbes and their metabolites induce further inflammatory signals, including the production of IL-17 family cytokines, promoting cancer advancement [75]. Inflammatory mediators may also stimulate macrophages to generate chromosome-breaking factors, injuring DNA and causing chromosomal instability in adjacent cells [6].
When microbial population translocate beyond an injured gut epithelium, the host immune system activates multiple pattern recognition receptors (PRRs). Cytoplasmic NOD-like receptors (NLRs) and membrane Toll-like receptors (TLRs) are PRRs crucial to the CRC progression [29]. In particular, TLR4 and TLR2 were found in murine models and associations between human genetic polymorphisms in TLR4 and TLR2 and CRC risk support a role in humans [155]. Moreover, activation of nuclear factor (NF)-κB influence CRC induction enhancing both Wnt-signaling and cytokines [7], which can transform gut epithelial nonstem cells into cancer-initiating cells. NF-κB has a complex role in CRC progression and involves several signaling pathways which have recently been widely reviewed [156]. Otherwise, in colitis-associated CRC, TLR signaling in cancer-associated fibroblasts induce an inflammatory cascade via
