Perspective Cite This: J. Agric. Food Chem. 2018, 66, 1737−1742
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Use of Caenorhabditis elegans To Study the Potential Bioactivity of Natural Compounds Vivian Hsiu-Chuan Liao* Department of Bioenvironmental Systems Engineering, National Taiwan University, No. 1, Section 4, Roosevelt Road, Taipei 106, Taiwan ABSTRACT: There is growing need and interest in finding specific compounds in natural products that have health benefits. Despite ongoing efforts to discover such compounds, the scientific evidence lags behind the vision, and it is important to find an effective paradigm for discovering such compounds. The model organism Caenorhabditis elegans offers a promising solution for studying the potential bioactivity and molecular mechanisms of natural compounds in vivo. This perspective discusses its use to study potential human health benefits, with focus on antioxidative, anti-aging, antimetabolic disorders (diabetes and obesity), and antineurodegenerative activities (Alzheimer’s disease and Parkinson’s disease), with practical examples. Finally, future directions in using a C. elegans-based model for discovering bioactive compounds for health promotion are discussed. KEYWORDS: Caenorhabditis elegans, natural compounds, bioactivity, health benefits, in vivo
1. INTRODUCTION Natural chemical substances produced by living organisms or found in nature (plants, animals, microflora, and minerals) encompass an extremely wide and diverse range of chemical compounds. As a result of the huge diversity in chemical structures, natural products have been rich sources and inspiration for a substantial fraction of human therapeutics and have played a significant role in drug discovery. For example, some widely used drugs are derived from natural products, such as metformin, vincristine, acetyldigoxin, and atropine. Hence, the search for bioactive compounds from natural sources to improve health and prevent diseases continues to play an important role in new medicinal therapies. Whereas pharmaceutical drugs are designed to cure or treat a specific disease, natural bioactive compounds that are used to promote health are found in agricultural products and food.1 There is increasing evidence that such bioactive natural compounds may help to promote health or reduce the risk of chronic lifestyle diseases.1 For example, several bioactive plantderived compounds have been intensively investigated for their potential human health benefits, such as tea phenolics, ascorbic acid, epigallocatechin gallate (EGCG), and curcumin. Their potential for antioxidative stress, anticancer, and anti-inflammatory activities have been explored. The desire to improve health and prevent diseases continues to drive the search for efficacious bioactive agricultural and food compounds. Efforts to discover such compounds have been deeply engaged in investigating the detailed chemical and biological properties, yet the scientific evidence lags behind the vision to exploit the potential health benefits.1 Challenges lie in the detailed chemical characterization of the molecular structures of the compounds, unraveling the bioavailability and bioefficacy of bioactive molecules and understanding how they promote health.1 Therefore, it is important to find an effective and reliable in vivo paradigm for discovering such compounds. The nematode Caenorhabditis elegans offers a promising solution for studying the potential bioactivity and molecular © 2018 American Chemical Society
mechanisms of natural compounds in vivo. This perspective discusses the use of C. elegans as a model organism in this capacity. The focus is on using C. elegans to study potential human health benefits related to antioxidative activity, antiaging activity, antimetabolic disorders (diabetes and obesity), and antineurodegenerative disorders (Alzheimer’s disease and Parkinson’s disease), with practical examples.
2. NEMATODE C. ELEGANS AS A MODEL ORGANISM C. elegans is a small, transparent nematode that lives in soil. It is a genetically tractable multicellular organism that has been a popular model for biological and basic medical research for several decades. It has been successfully used as a model system to address fundamental questions in many aspects of biology, such as development, cell fate specification, neurobiology, tumorigenesis, RNA-mediated interference (RNAi) of gene expression, and aging. C. elegans can be either self-fertilizing hermaphrodites or males, but males account for only about 0.1% of the population. An adult hermaphrodite consists of 959 somatic cells with a complete cell lineage map, all of which are visible with a microscope throughout the life of the organism. C. elegans has a short life cycle of ∼3 days to develop into fertile adults (Figure 1), a lifespan of ∼3 weeks, and an ability to produce ∼300 genetically identical progeny. It has a nervous system containing 302 neurons with a complete connectome. In addition, C. elegans has many different organs and tissues, including muscle, a hypoderm, an intestine, a reproductive system, a secretory−excretory system, and glands. In the laboratory, C. elegans is usually grown on small Petri agar plates or in liquid media with auxotrophic Escherichia coli OP50 as a food source. This makes it very easy and costReceived: Revised: Accepted: Published: 1737
December 5, 2017 January 31, 2018 February 2, 2018 February 2, 2018 DOI: 10.1021/acs.jafc.7b05700 J. Agric. Food Chem. 2018, 66, 1737−1742
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Figure 1. C. elegans hermaphrodite life cycle at 20 °C. The reproductive life cycle of hermaphrodite includes four larval stages (L1−L4), each ending in a molt. The dauer larva is a diapause stage representing an alternative L3 stage, which is entered when unfavorable conditions, such as crowding or low food availability, occur.
Table 1. Comparison of Commonly Used Model Organisms in Biomedical Research organism
C. elegans
Drosophila
mouse
life cycle adult size brood size genome size fully annotated genome no ethical constraints genetic screens growth conditions transgenic organism generation mutants can be frozen and revived easily gene homology for human diseases number of neurons in adult distinct tissues and cell diversity amenable to drug testing high-throughput drug screening
3−4 days 1−1.3 mm ∼140 eggs/day 97 Mb ○ ○ routine plates and liquid weeks ○ 65% 302 ○ ○ ○
11−12 days 3−4 mm ∼120 eggs/day 180 Mb ○ ○ routine vials weeks
50−60 days 6−10 cm 6−12 pups/month 3000 Mb
77% > 100000 ○ ○
> 90% > 70000000 ○ ○
effective to grow. Other advantages of C. elegans include the ability of mutants to be frozen indefinitely and revived easily, easy delivery of RNAi, the ability to readily create transgenic strains, free online resources, such as WormBook (http://www. wormbook.org/), and databases, such as WormBase (http:// www.wormbase.org/). An important feature for the usefulness of C. elegans as a model organism in vivo is its relevance to human disease. It is estimated that over 83% of the C. elegans proteome has human homologues as well as counterparts for an estimated ∼65% of human disease genes.2 Therefore, C. elegans has been used extensively as a key model for investigating molecular and cellular aspects of a growing number of complex human diseases, such as Alzheimer’s disease, Parkinson’s disease, diabetes, and cancer.3 To translate the experimental results to humans, research on mammals has some advantages, but there are limitations in mammalian animals, such as ethical constraints, methodological difficulties, long life cycle, small brood size, large genome size, large number of neurons in adult, and difficulties in genetic screens. Therefore, in both biological and biomedical studies, C.
difficult cages months
elegans provides several advantages over vertebrate models, such as mice (Table 1). Table 1 compares model organisms that are commonly used in biomedical research.
3. USE OF C. ELEGANS TO STUDY ANTIOXIDATIVE ACTIVITY Oxidative stress is characterized as an imbalance between the production of intracellular reactive oxygen/nitrogen species (ROS/RNS) and antioxidant defense activity in an organism as well as a disturbance in the cell redox balance. ROS/RNS include superoxide anion radicals, singlet oxygen, hydrogen peroxide, hydroxyl, alkoxyl, and lipid peroxyl radicals, nitric oxide, and peroxynitrite. Excessive free radicals are associated with damage to many biomolecules, including lipids, proteins, and nucleic acids. Free-radical-induced damage in oxidative stress has been linked to a number of chronic health problems, such as cancer, diabetes, neurodegenerative diseases, cardiovascular diseases, and inflammatory diseases.4 Increasing evidence suggests that the consumption of antioxidant-rich foods or medicinal plants can retard or help to avoid the incidence of some diseases.5 Therefore, there is growing effort and great 1738
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promising candidate chemical constituents for aging research. Many natural compounds (either pure forms or extracts) have been reported to have anti-aging activity, such as slowing cellular senescence or aging and extending lifespan.10 Some natural compounds, such as curcumin, resveratrol, and α-lipoic acid, have received great interest for their various anti-aging activities in different models, including C. elegans.10 The study of C. elegans has provided a wealth of information for understanding the role of genetics in modulating aging. In addition to the advantages mentioned, there are several other unique features that make C. elegans an ideal model organism for aging research. For example, the organism has a relatively short lifespan (∼3 weeks), which is largely invariant. This allows for identifying mutants with shorter or longer average lifespans. Second, the somatic cells are postmitotic in adult animals, making them useful for studying chronological aging. Furthermore, several important signaling pathways involved in aging and longevity have been studied extensively, such as insulin/IGF-1 and dietary restriction (DR), which allows for the analysis of molecular mechanisms involved in aging. Various assays have been developed to study aging in C. elegans. These include lifespan analyses in solid and liquid media and assays for measuring age-related changes.11 C. elegans shows certain phenotypes that are correlated with aging, such as muscle decline, which is usually analyzed with locomotory behaviors and pharyngeal pumping assays, various types of stress, which are analyzed using oxidative stress, ultraviolet (UV) stress, and heat stress assays, proteostasis, which can be analyzed with a paralysis assay, and lipofuscin accumulation, which is measured with lipofuscin autofluorescence in the intestine.11 Therefore, to evaluate the potential anti-aging activity of natural compounds in C. elegans, it is important to measure both the lifespan and age-related changes, which might suggest potential mechanisms for the influence on longevity. However, it is noted that compounds with antioxidant activity are not necessary to extend the lifespan of C. elegans, for which organic selenium Glu-SeMet has been previously reported.12 Interestingly, Glu-SeMet shows an ability to improve aging indicators that are mediated by the selenoprotein TRXR-1,12 suggesting the potential of natural compounds to improve “healthy aging”.
interest in the search for effective, non-toxic, natural compounds with antioxidative activity with associated health benefits. The antioxidant properties of natural compounds are investigated through either chemical- or cell-based in vitro or in vivo methods.6 There are various in vitro antioxidant activity assays, and each one has a specific target within the matrix with advantages and disadvantages.6 Although in vitro chemical methods are fairly straightforward, they lack information about the bioavailability of the test compounds. For most in vivo models, the tested samples are usually administered to test animals, such as mice or rats, which is usually followed by the sacrifice of the animals and the use of blood or tissues for the antioxidative activity assay.6 In C. elegans, the signal transduction pathways for oxidative stress are highly conserved, including the insulin signaling pathway, target of rapamycin (TOR) signaling pathway, and autophagy pathway, as well as the mechanisms that involve the detoxification of ROS, such as superoxide dismutase and catalase.7 C. elegans is thus an attractive in vivo model, where the whole organism can be used to evaluate the antioxidative activity of natural compounds. In recent years, an increasing number of studies have used C. elegans to explore the antioxidative activity of natural compounds, many of which have previously shown antioxidative activity in other in vitro or in vivo models. Examples include curcumin, monascin, selenium, EGCG, α-lipoic acid, quercetin, etc. This demonstrates the usefulness of C. elegans for studying the antioxidative activity of natural compounds. The antioxidative activity of natural compounds in C. elegans can be evaluated by assays, such as performing the oxidative stress resistance assay, measuring the intracellular ROS level, and analyzing the responses of transgenic strains expressing antioxidant genes, such as superoxide dismutase (SOD-3) and glutathione S-transferase (GST-4). Recently, Possik and Pause8 developed a protocol to measure oxidative stress resistance of C. elegans in liquid in a 96-well microtiter plate, which might facilitate the investigation of the potential antioxdative activity of natural compounds, while a large number of samples for screening is needed.
4. USE OF C. ELEGANS TO STUDY ANTI-AGING ACTIVITY Aging is an inevitable process characterized by accumulating functional declines of physiological integrity that lead to impaired function and ultimately result in death. Aging has been linked to several chronic human diseases, including various cancers, type 2 diabetes mellitus (T2DM), and cardiovascular and neurodegenerative diseases. Therefore, there is great interest and urgency in studying how to delay the process of aging and eliminate or prevent age-related diseases. Many mutations have been identified to prolong the lifespan in model organisms ranging from yeast to mammals.9 The rate of aging is regulated at least in part by genetic pathways and biochemical processes that are evolutionarily conserved.9 For example, the signaling pathways of aging, including insulin/ insulin-like growth factor (IGF) signaling (IIS) pathway, germline signaling pathway, and TOR pathway, are evolutionarily conserved in metazoan model organisms, such as C. elegans, Drosophila, and mice. Thus, compounds with anti-aging activity may be useful in treating or delaying age-related human diseases. Natural compounds have a special advantage as a resource with highly diverse structural scaffolds that might offer
5. USE OF C. ELEGANS TO STUDY ANTIMETABOLIC DISORDER ACTIVITY: DIABETES AND OBESITY In recent decades, there has been increasing prevalence of metabolic disorders, such as obesity and T2DM, which affect millions of people worldwide. In fact, there is increasing evidence to support the relationships between T2DM, obesity, Alzheimer’s disease, and cancer. Diabetes mellitus is characterized by poor control of glucose homeostasis, including insufficient or inefficient insulin secretary response and hyperglycemia. Diabetes is commonly divided into type 1 diabetes mellitus (T1DM), which is caused by insufficient insulin secretion, and T2DM, which is a consequence of insulin resistance and hyperglycemia. Clinically, diabetic patients with T2DM are more common (90−95%).13 The pathogenic mechanisms of diabetes are complicated and involve several distinct signaling pathways, including the insulin signaling pathway, carbohydrate metabolism pathway, endoplasmic reticulum (ER) stress pathways, and inflammation-related pathways. Recently, an increasing number of active components from natural products have been reported to exhibit antidiabetic activity and regulate pathophysiological signaling 1739
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6. USE OF C. ELEGANS TO STUDY ANTINEURODEGENERATIVE DISORDER ACTIVITY: ALZHEIMER’S DISEASE AND PARKINSON’S DISEASE Neurodegenerative disorders, such as Alzheimer’s disease and Parkinson’s disease, seriously affect millions of people worldwide. These age-associated disorders lead to a progressive loss of neurons and neuronal dysfunction. The pathophysiology involves a combination of genetic and environmental factors. Thus far, the medications to completely cure these diseases are unavailable or ineffective.21 Effective compounds and a practical experimental model are needed to decipher the molecular determinants of these disorders. There is molecular conservation in neuronal signaling pathways, such as dopamine (DA) signaling between invertebrates and vertebrates,21 as well as a diverse range of chemical entities of natural compounds. Thus, the use of C. elegans to study the beneficial effects of natural compounds on neurodegenerative disorders might provide a promising paradigm. Alzheimer’s disease is the most common neurodegenerative disorder and is characterized by the loss of memory and cognitive impairments. The histopathological hallmarks of Alzheimer’s patients include deposition of β-amyloid (Aβ) plaques and neurofibrillary tangles of τ microtubule protein.22 Aβ peptides derive from the sequential proteolytic cleavage of amyloid precursor protein (APP).22 The oligomers Aβ1−42 are toxic species and are thus a biomarker for Alzheimer’s disease progression.22 In addition, many factors are associated with Alzheimer’s disease, such as oxidative stress, inflammation, metabolic disturbances, and reduction of cholinergic neuron activity.23 Several natural compounds have been reported to have protective effects against Aβ toxicity in various experimental models. Examples are quercetin, EGCG, curcumin, and resveratrol, and some of them are in clinical trials.23 Although it is unlikely that C. elegans can completely capture the pathology of Alzheimer’s disease, it has several models that can be used to assess Aβ- and τ-induced toxicity, which have two crucial hallmarks.21,22 Transgenic C. elegans strains expressing human Aβ or human τ are used to assess the toxicity.21,22 These models have led to the discovery of a number of candidate compounds for modulating the disease. Natural compounds or extracts, such as curcumin and resveratrol, have been reported to reduce Aβ or τ toxicity. These compounds have been shown to have protective effects against Aβ toxicity in mammalian models, and in particular, compounds, such curcumin and resveratrol, have gone through clinical trials.23 Thus, C. elegans is useful for studying the potential bioactivity of compounds against Alzheimer’s disease. Parkinson’s disease is the second most common neurodegenerative disorder after Alzheimer’s disease, and it is mainly characterized by motor impairment, the progressive loss of dopaminergic neurons, and the accumulation of Lewy bodies in the brain.21 The cause and pathogenic mechanisms of Parkinson’s disease are not well understood, and thus far, there is no effective treatment. Several factors have been linked to its pathogenesis, such as oxidative stress, neuroinflammation, impaired function in the ubiquitin−proteasome system, and mitochondrial impairment.24 The presence of Lewy bodies in neurons is an important neurohistological characteristic of the disease and is considered as a preclinical or presymptomatic marker.25 Self-assembling α-synuclein (α-syn) is the most
pathways involved in diabetes. Examples include monascin, quercetin, and resveratrol. The activity has been reported in various model organisms, and some of these products have gone through clinical trials.14 The IIS pathway and the effect of lower levels of its activity in increasing lifespan are conserved across diverse metazoa.15 In C. elegans, the pathway regulates fat storage, reproduction, and lifespan. DAF-2 is the single orthologue of the human insulin and IGF-1 receptor.15 Growing evidence suggests that impaired insulin signaling plays a crucial role in the pathogenesis of obesity and T2DM.16 C. elegans thus provides a promising model to examine the molecular mechanisms of glucose toxicity that lead to diabetic complications. Enhanced blood glucose levels are generally observed in diabetes and are recognized as the major cause of diabetic complications. Several natural compounds or extracts are reported to prevent high glucose-induced toxicity in C. elegans, such as quercetin.17 Quercetin is also reported to have a protective effect on hyperglycemia in diabetic mice.18 This suggests the usefulness of C. elegans for investigating the potential antidiabetic activity of natural compounds. Another prevalent metabolic disorder is obesity, which is a significant risk for various chronic diseases, such as T2DM, heart disease, hyperlipidemia, and certain cancers.19 The causes of obesity are complicated and include genetic susceptibility, excessive caloric intake, and sedentary lifestyle. Currently, there are only a few U.S. Food and Drug Administration (FDA)approved medications for obesity, and most have undesired side effects.13 Natural compounds might be good candidates for anti-obesity treatments as a result of their fewer side effects compared to synthetic drugs.13 Several natural compounds or extracts have been reported to have anti-obesity activity, and some of them have gone through clinical trials. Examples include yerba mate, Euiiyin-tang, red wine polyphenol supplement, quercetin, and resveratrol.14 Factors controlling energy metabolism and fat regulatory pathways are evolutionarily conserved between mammals and C. elegans, which has thus emerged in the past decade as a genetically and metabolically tractable model to decipher the homeostatic mechanisms of lipid regulation that lead to obesity. Several methods have been employed to examine lipid storage in C. elegans. Fixed staining methods use colorimetric dyes or fluorescent dyes followed by quantification of the amount of bound dye to reflect fat content. Biochemical methods use lipid extracts in C. elegans and thin-layer chromatography (TLC) or gas chromatography/mass spectrometry (GC/MS).19 Recently, several natural compounds or extracts have been reported to reduce fat accumulation in C. elegans, such as proanthocyanidin trimer gallate.20 This suggests that C. elegans is useful for studying the potential anti-obesity activity of natural compounds. Besides age and genetic predisposition, obesity has been suggested as a significant risk factor for developing insulin resistance, which is a key feature of T2DM. Therefore, compounds that simultaneously address obesity and diabetes are highly desirable and anticipated. Such candidates include red wine polyphenol supplements, quercetin, resveratrol, and cinnamon, and some of them have gone through clinical trials.13,14 1740
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effective paradigm for identifying genes and bioactive compounds before studies in mammalian models or clinical trials that might facilitate the development for successful health promotion.
abundant protein in Lewy bodies and is closely associated with Parkinson’s disease.21 A growing number of studies have indicated that several natural compounds protect against the neurotoxins 6-hydroxydopamine (6-OHDA) or 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP) in animal models. Examples are green tea polyphenols, EGCG, curcumin, and resveratrol.26 Several unique features make C. elegans a valuable model for investigating Parkinson’s disease. With only 302 neurons, including 8 dopaminergic neurons, C. elegans is quite simple compared to billions of neurons in the brains of mammals or even fruit flies (Drosophila), which have ∼10 000 neurons (Table 1). The pathways involved in dopamine neurons are evolutionally conserved. As a result of the transparency of C. elegans, neuronal cell death can be readily observed within living organisms. Several transgenic strains have been generated to examine α-syn aggregation and dopaminergic neuron degeneration, which are two pathological hallmarks of the disease.27 These transgenic strains include a strain expressing human αsyn and a strain expressing green fluorescent protein (GFP) specifically in the dopaminergic neurons.27 Recently, a few studies have used C. elegans models of Parkinson’s disease to examine the potential activity of natural compounds against Parkinson’s disease, such as β-amyrin.28
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AUTHOR INFORMATION
Corresponding Author
*Telephone: +886-2-33665239. Fax: +886-2-33663462. E-mail:
[email protected]. ORCID
Vivian Hsiu-Chuan Liao: 0000-0002-4676-9953 Notes
The author declares no competing financial interest.
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REFERENCES
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7. CONCLUDING REMARKS AND FUTURE DIRECTIONS This perspective has highlighted the advantages of using C. elegans to study the potential bioactivity of natural compounds. The perspective has also described how researchers have used this versatile model organism to investigate several aspects of human health benefits as well as how these natural compounds have contributed to our understanding in promoting health. Mammalian models remain invaluable experimental tools for the discovery of new compounds, especially considering the wide range of clinical features and many analogues to the organs and circulatory system in humans. However, mammalian models are usually time-consuming, expensive, and complex, thereby hindering the efficiency of discovering compounds, especially for screening a large number of candidates. A cellbased in vitro assay is another common research tool that is used to observe bioactivity in cell-based in vitro assays, but the results might not translate to in vivo health effects.1 To address the limitations of mammalian models and cell cultures, C. elegans seems to be a practical, promising, versatile, and relevant model for providing multifaceted aspects to study the potential bioactivity of natural compounds as well as the underlying molecular determinants of the associated health effects. In the future, in addition to the human health benefits aforementioned in the perspective, C. elegans can be further explored to study other potential bioactivities of natural compounds, such as circadian rhythms, anticancer, antimicrobial, and polyglutamine-expansion disorders, e.g., Huntington’s disease. Moreover, C. elegans can be explored as a model for high throughput in the discovery of natural compounds to promote health benefits. Therefore, future large-scale screening of bioactive compounds for candidates leading to potential bioactivity is possible. Natural compounds that can simultaneously promote multiple health benefits are highly desirable and anticipated. Therefore, future studies using a C. elegansbased model to simultaneously investigate multiple bioactivities of a specific natural compound are desirable. In the future, a C. elegans-based model can serve as the first pass screen and an 1741
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