Investigating the tumour cell growth inhibitory potential of Portuguese wild mushrooms: Boletus sp., Suillus luteus and Ganoderma lucidum

FCUP 3 Investigating the tumour cell growth inhibitory potential of Portuguese wild mushrooms: Boletus spp., Suillus luteus and Ganoderma lucidum A AC CK KN NO OW WL LE ED DG GE EM ME EN NT TS S First of all, I would like to emphasize my deep gratefulness to Prof. Helena Vasconcelos, my supervisor. She always was very welcoming from the very beginning and her ideas, advice, patience and experience proved to be essential for the completion of this work. Doctor Tiago Santos had an equally important role in this project as well. Some of the experiments were performed by him and the results of which kindly given to me, not to mention his support and suggestions. Prof. Isabel Ferreira and her team were also just as crucial; if not for them and their dedication, this project would not have been possible, nor would it be as carefully revised. I would also like to thank Doctor Raquel Lima and Diana Sousa for all the support and guidance they both offered when it was needed, particularly with the analysis in the flow cytometer, and Prof. Anabela Cordeiro, Prof. Lucília Saraiva, Prof. Paula Ferreira and Prof. Luísa Peixe for kindly offering me access to equipment necessary to this work. I am very grateful to Prof. Pedro Fernandes and Prof. Paula Gameiro as well for all the support and advice they gave me when I most needed it. I am also thankful to Professor Maria S. José Nascimento, as it was because of her lectures in Virology that led me to meet Prof. Helena. And last yet not least, I must also thank Prof. Luís Vieira since it was due to his kind and well-thought suggestion that I chose to carry out this project, which has been a completely new adventure for me. FCUP 4 Investigating the tumour cell growth inhibitory potential of Portuguese wild mushrooms: Boletus spp., Suillus luteus and Ganoderma lucidum A AB BS ST TR RA AC CT T KEYWORDS: cancer, mushrooms, cytotoxicity, anti-tumour compounds, tumour cell growth inhibitory potential, cell proliferation Many of the conventional therapies for cancer treatment derive from natural products, a truly limitless source of bioactive compounds. However, due to the many unfavourable sideeffects of (and drug resistance to) the currently used chemotherapy, the scientific community is still investigating other natural products as potential sources of anti-tumour compounds. Fungi are an attractive source of physiological drug precursors displaying numerous pharmacological activities. Mushrooms, for example, contain a vast diversity of biomolecules with nutritional and/or medicinal characteristics, including compounds with anti-tumour properties. Suillus luteus has been documented for its cytotoxic compounds in a murine cancer cell line, but no data has been published in human cell lines until very recently. Also, clinical trials support that using Ganoderma lucidum, a well-known species documented as a medicinal mushroom, was helpful to patients who underwent radioand chemotherapy. In this project, extracts from six Boletus species were screened in two human cancer cell lines. Results showed that they have no tumour cell growth inhibitory potential up to the highest concentration tested (400µg/mL). The cellular effects of an extract of S. luteus were also studied in a non-small cell lung cancer cell line. Results showed that this extract inhibited cell proliferation and did not seem to cause an increase in programmed cell death. Analysis of cell cycle profile showed a G1 phase arrest and the expression of proteins involved in DNA damage repair increased in cells treated with this extract when compared with control cells. Also, preliminary results indicated that treatment with this extract did not seem to increase the sensitivity of cells to the effects of a molecular targeted drug, erlotinib. Finally, the cellular effects of a G. lucidum extract were investigated in a gastric cancer cell line. Results show that the extract inhibited cellular proliferation, but did not seem to interfere with programmed cell death. Cell cycle profile analysis also showed a G1 phase FCUP 5 Investigating the tumour cell growth inhibitory potential of Portuguese wild mushrooms: Boletus spp., Suillus luteus and Ganoderma lucidum arrest. Additionally, the levels of proteins involved in DNA damage repair were slightly increased in extract-treated cells when compared to controls. Moreover, LC3-II expression (an essential protein in the autophagic process) is notably higher in cells treated with the extract. Preliminary results also indicated that treatment with this extract did not seem to increase cell sensitivity to the effects of a conventional chemotherapeutical drug, 5fluorouracil. FCUP 6 Investigating the tumour cell growth inhibitory potential of Portuguese wild mushrooms: Boletus spp., Suillus luteus and Ganoderma lucidum R RE ES SU UM MO O PALAVRAS-CHAVE : cancro, cogumelos, citotoxicidade, substâncias anti-tumorais, potencial de inibição de crescimento celular tumoral, proliferação celular Muitas das terapias convencionais de tratamento de cancro provêm de produtos naturais, uma fonte praticamente inesgotável de substâncias bioativas. Porém, uma vez que a quimioterapia convencional causa efeitos secundários bastante agressivos para o doente (e surgem muitos casos de resistência a fármacos), a comunidade científica continua a investigar outros produtos naturais como potenciais fontes de substâncias anti-tumorais. Os fungos são uma fonte atraente de precursores fisiológicos de fármacos que apresentam propriedades farmacológicas variadas. Os cogumelos, por exemplo, contêm uma grande diversidade de biomoléculas com características nutricionais e/ou medicinais, incluindo substâncias com propriedades anti-tumorais. O Suillus luteus foi descrito como tendo compostos citotóxicos numa linha de células tumorais de ratinho, mas até muito recentemente não existiam dados publicados relativamente a linhas celulares tumorais humanas. Ensaios clínicos defendem que o uso de Ganoderma lucidum, uma espécie bem conhecida e descrita como sendo medicinal, foi benéfico em pacientes submetidos a radioe quimioterapia. Neste projeto, extratos de seis espécies de Boletus foram sujeitos a um processo de screening em duas linhas tumorais humanas. Os resultados demonstraram que nenhum possui potencial de inibição de crescimento celular tumoral na gama de concentrações testada (até um máximo de 400µg/mL). Os efeitos do extrato de S. luteus a nível celular foram também estudados numa linha de cancro do pulmão de células não pequenas. Este extrato inibiu a proliferação celular, mas não pareceu aumentar os níveis de morte celular programada. A análise do perfil de ciclo celular revelou uma paragem em fase G1 e a expressão de proteínas envolvidas na reparação de danos do DNA aumentou nas células tratadas com este extrato quando comparadas com as células dos controlos. De acordo com resultados preliminares, o tratamento com este extrato não pareceu aumentar a sensibilidade das células a efeitos FCUP 7 Investigating the tumour cell growth inhibitory potential of Portuguese wild mushrooms: Boletus spp., Suillus luteus and Ganoderma lucidum causados por uma molécula considerada como uma “terapêutica dirigida” para cancro do pulmão, o erlotinib. Por fim, os efeitos de um extrato de G. lucidum foram investigados numa linha de cancro gástrico. O extrato inibiu a proliferação destas células, ainda que não pareça interferir com morte celular programada. A análise do perfil de ciclo celular mostra uma paragem em fase G1. Adicionalmente, os níveis de proteínas envolvidas na reparação de danos do DNA aumentaram ligeiramente nas células tratadas com o extrato em comparação com os controlos. Para além disso, a expressão de LC3-II (uma proteína essencial no processo autofágico) encontra-se notavelmente elevada nas células tratadas com o extrato. Resultados preliminares são indicativos de que este extrato não pareceu aumentar a sensibilidade das células aos efeitos causados por um fármaco de “quimioterapia convencional”, o 5-fluorouracilo. FCUP 8 Investigating the tumour cell growth inhibitory potential of Portuguese wild mushrooms: Boletus spp., Suillus luteus and Ganoderma lucidum I IN ND DE EX X Acknowledgements .............................................................................................................. 3 Abstract ................................................................................................................................ 4 Resumo ................................................................................................................................ 6 Index .................................................................................................................................... 8 Index of Figures .................................................................................................................... 9 Index of Tables ................................................................................................................... 10 List of Abbreviations ........................................................................................................... 11 Introduction......................................................................................................................... 12 Materials and Methods ....................................................................................................... 34 Results and Discussion ...................................................................................................... 40 Conclusions ........................................................................................................................ 54 References ......................................................................................................................... 55 FCUP 9 Investigating the tumour cell growth inhibitory potential of Portuguese wild mushrooms: Boletus spp., Suillus luteus and Ganoderma lucidum L LI IS ST T O OF F F FI IG GU UR RE ES S Figure 1 .............................................................................................................................. 13 Figure 2 .............................................................................................................................. 26 Figure 3 .............................................................................................................................. 27 Figure 4 .............................................................................................................................. 28 Figure 5 .............................................................................................................................. 42 Figure 6 .............................................................................................................................. 43 Figure 7 .............................................................................................................................. 45 Figure 8 .............................................................................................................................. 46 Figure 9 .............................................................................................................................. 48 Figure 10 ............................................................................................................................ 49 Figure 11 ............................................................................................................................ 50 Figure 12 ............................................................................................................................ 51 Figure 13 ............................................................................................................................ 53 FCUP 10 Investigating the tumour cell growth inhibitory potential of Portuguese wild mushrooms: Boletus spp., Suillus luteus and Ganoderma lucidum L LI IS ST T O OF F T TA AB BL LE ES S Table 1 ...................................................................................................................................... 40 Table 2 ...................................................................................................................................... 44 Table 3 ...................................................................................................................................... 51 FCUP 11 Investigating the tumour cell growth inhibitory potential of Portuguese wild mushrooms: Boletus spp., Suillus luteus and Ganoderma lucidum L LI IS ST T O OF F A AB BB BR RE EV VI IA AT TI IO ON NS S 2GI50: two times growth inhibition at 50% 5-FU: 5-fluorouracil Bad: Bcl-2-associated death promoter Bak: Bcl-2 homologous antagonist killer Bax: Bcl-2-associated X protein BCG: Bacillus Calmette-Guérin Bcl-2: B-cell lymphoma-2 Bcl-xL: B-cell lymphoma-extra large Bid: BH3 interacting domain death agonist protein BrdU: 5-bromo-2’-deoxyuridine BSA: bovine serum albumine Cdk: cyclin-dependent kinases DAPI: 4’,6-diamidino-2-phenylindole DMSO: dimethyl-sulfoxide ECL: enhanced chemiluminescence EDTA: ethylenediaminetetraacetic acid FBS: fetal bovine serum FSC: forward scatter GI50: growth inhibition at 50% HCl: hydrochloric acid HRP: horseradish peroxidase IC50: inhibitory concentration at 50% IgG: immunoglobulin G LC3: Light Chain 3 miR-378: microRNA-378 NCI: National Cancer Institute PBS: phosphate buffered saline PFA: paraformaldehyde PI: propidium iodide Rb: retinoblastoma RT: room temperature SE: standard deviation error SDS: sodium dodecyl sulfate SRB: sulforhodamine B SSC: side scatter TCA: trichloroacetic acid TCC: transitional cell carcinoma TUNEL: terminal deoxynucleotidyl transferase 2’-deoxyuridine-5’- triphosphate nick end labelling FCUP 18 Investigating the tumour cell growth inhibitory potential of Portuguese wild mushrooms: Boletus spp., Suillus luteus and Ganoderma lucidum Cancer cells developed several strategies to circumvent or at least limit apoptosis. Indeed, elucidation of the signalling circuitry behind apoptosis has revealed that it can be triggered in response to various physiologic stresses experienced during the course of tumourigenesis (hypoxia, nutrient deprivation, DNA damage, defective DNA repairing, oncogene deregulation...) or as a result of anti-cancer therapy [2, 3]. Loss of p53 function is one of the most common alterations found in cancers, thus eliminating a critical “DNA damage sensor” and “transducer” of diverse signals into tumoursuppressive apoptotic or growth-arresting responses [2, 3]. In fact, the gene coding for this protein is more often reported to be mutated in human cancers than any other gene [48, 49]. Alternatively, tumour cells can also increase the expression of anti-apoptotic regulators or down-regulate pro-apoptotic protein levels (which may increase survival signals) or even short-circuit the extrinsic pathway [3]. Yet another way of escaping apoptosis is via the stabilization of mitochondrial integrity and function, which helps suppressing the release of cytochrome c and the following activation of the cascades of caspases [50]. There is also a curious interconnection between apoptosis and autophagy. The Beclin-1 protein, which is essential for the induction of the latter phenomenon [51-53], is able to bind to Bcl-2 and Bcl-xL and stress-sensor-coupled proteins can displace this association, thus triggering autophagy. Therefore, stress-transducing proteins (such as Bid, Bcl-2-associated death promoter (Bad) and many others) are able to induce apoptosis and/or autophagy according to the cellular physiologic state [3]. Much like apoptosis, autophagy usually occurs at low levels, but it can be induced by certain cellular states of stress as a vital physiologic response. The most obvious stress is nutrient deficiency [51, 52] and by triggering autophagy, cells are able to disassemble organelles, degrade possibly toxic cytoplasmic constituents and recycle the resulting molecules for biosynthesis and homeostasis of energy metabolism, supporting the survival of cancer cells in their stressed and usually nutrient-limited environment [3, 54]. Nevertheless and as already mentioned above, autophagy can also lead into an autophagic cell death, but several authors believe that it is simply a situation where cells die with autophagosomes and autolysosomes, but not directly because of autophagy [31, 55, 56]. Indeed, autophagy may be involved in lethal signalling, but rather than a process leading FCUP 19 Investigating the tumour cell growth inhibitory potential of Portuguese wild mushrooms: Boletus spp., Suillus luteus and Ganoderma lucidum into cell death, it usually is a “vain attempt” of dying cells to adapt to stressful conditions [57]. However, cellular stresses such as lack of nutrients, chemoand radiotherapy and some cytotoxic drugs can also strongly induce an apparently cytoprotective autophagy [3]. Instead of accentuating the killing actions of these stress-inducing situations, they hinder them [58-61]. Moreover, severely stressed cancer cells shrank to a state of reversible dormancy due to an autophagic response [61, 62]. Thus, autophagy seems to have paradoxical, conflicting effects on cancer cells [60, 61]. 2. THERAPEUTIC APPROACHES IN CANCER Although conventional chemotherapeutic drugs, commonly known as cytotoxics, are most frequently used for cancer therapy, they exhibit many undesirable adverse side-effects such as body weight loss, neutropenia and immunosuppression, and may consequently increase the risk of metastasis and bacterial infection despite their pharmacological benefits [63]. As cancer is in fact a multi-step process involving various epigenetic and genetic alterations as well as countless pathways, the multi-target or combination therapies may provide higher efficacy and a better treatment outcome than single target or single drug therapies [64]. Additionally, rational, mechanism-based therapies, such as the ones targeting cell cycle regulation and checkpoint abnormalities normally found in cancer, may possibly lead to potential treatment strategies with fewer side-effects, thus less nonspecific toxicity [3, 65]. Indeed, many “modern” cancer drugs are now selectively directed towards specific molecular targets that at some point enable a particular cancer characteristic and/or hallmark. These are known as molecular therapies. Unfortunately, the resulting clinical responses have sometimes been transitory and followed by almost-inevitable relapses [3]. A targeted therapeutic agent inhibiting one key signalling pathway in a particular tumour may not incapacitate a hallmark completely as each of these acquired capabilities are usually regulated by countless others, many of them with a redundant function. This allows the survival of some cancer cells until they are able to adapt (by mutation, epigenetic alterations, subversion of the surrounding tumour microenvironment...) to this selective pressure imposed by the new therapy, thus leading to the observed relapsing [3]. FCUP 20 Investigating the tumour cell growth inhibitory potential of Portuguese wild mushrooms: Boletus spp., Suillus luteus and Ganoderma lucidum On the other hand, cancer cells may also change their reliance on a particular hallmark in response to this type of treatment [3]. Indeed, for example it is known that some inhibitors of angiogenesis drove tumours into adapting and heightening their invasion and metastasis abilities in some preclinical models [66-71], which may turn out to be a major limitation for these therapies. Another example is the use of apoptosis-inducing agents, which might lead cancer cells into hyperactive mitogenic signalling, thus allowing them to compensate for the initial positive response [3]. In summary, a tumour is highly heterogeneous (due to the expansion of multiple clones harbouring different mutations) and highly dynamic (due to genetic instability and accumulation of mutations). These characteristics make it very hard to destroy all tumour cells with the currently available treatments and even though some tumours initially respond well to therapies, many of them end up becoming drug resistant due to the adaptive evolutions of the tumour mass [72]. 3. NATURAL PRODUCTS AS A SOURCE OF COMPOUNDS WITH ANTI-TUMOUR POTENTIAL Penicillin, aspirin, artemisinin, huperzine A and taxol are all typical (and successful) examples of naturally derived products (or secondary metabolites), a very important molecular structural resource with an abundance of chemical novelty and diversity for drug discovery [73, 74]. Most natural products are chiral and occur as single enantiomers. Many drugs have this advantage, allowing for only one of the enantiomers to have any relevant biological activity, possibly reducing adverse side-effects, increasing target specificity (countless enzymes and cell receptors are exclusively stereospecific) and even bioavailability. In addition, the immense structural variety of the countless and different bioactive natural compounds suggests the possibility of developing chemical modifications in order to optimize a pharmaceutical response against a specific molecular target [75]. Moreover, in some compounds identified in natural products, multiple and complex pharmacological actions on different molecular targets seem to be the main reason for the vast and very diverse therapeutic effects that have been reported, influencing many receptors and FCUP 21 Investigating the tumour cell growth inhibitory potential of Portuguese wild mushrooms: Boletus spp., Suillus luteus and Ganoderma lucidum triggering numerous downstream signalling events leading to high pharmacological efficiency and specificity [76]. Another advantage of natural products is that the isolation and purification of natural or even hemisynthetic bioactive components requires common and simple analytical procedures [75, 77-84]. The advantage of discovering new drugs or lead compounds through the study of natural products also comes from statistical data: out of the 520 new drugs approved in the United States between 1983 and 1994, 157 were from natural products and more than 60% of antibiotics and anti-cancer drugs originated from natural products. About 40% of the chemical scaffolds of published natural products are quite unique and have not been previously made by synthetic chemistry [85]. In 1990, about 80% of drugs used to treat human diseases were either natural products or analogues inspired by them. However and due to the side-effects of chemotherapy, there has been an increasing number of studies on natural products or herbal medicines that are considered safe and some have been proven to be able to block, reverse or prevent the development of invasive cancers [86]. New compounds may substitute less active and/or more toxic compounds. They may also add adjuvant treatment to conventional therapies, synergizing and/or potentiating standard treatments such as chemotherapy. This helps decreasing doses, therefore toxicity, of the main therapy and also reduces the probability of developing drug resistance, but essentially, natural products may simply bring comfort and palliative care to patients under heavy treatment [87]. Conventionally, pharmaceutical drugs have been used to cure diseases or to alleviate the symptoms of disease while nutrition aims to prevent diseases by providing the body with the optimal balance of macroand micronutrients needed for good health [88]. The entrance in the market of functional foods – which improve health or well-being, or reduce disease risk by targeting some physiological functions constructively, maintaining and promoting health, longevity and quality of life both isolated or in combination with prescription drugs [89, 90] – has further blurred this distinction [91]. But even though functional foods are currently popular, the majority of people are still unsure of their real benefits [88]. FCUP 22 Investigating the tumour cell growth inhibitory potential of Portuguese wild mushrooms: Boletus spp., Suillus luteus and Ganoderma lucidum It might be possible to understand the mechanisms by which components of foods and plants affect cancer by tracing the multi-step molecular process of development, which may be influenced by nutritional factors [92]. These could also influence cancer prevention, the quality of life of patients and even the risk of cancer recurrence. There are many sources of natural products. Fungi, for example, are a vast and yet largely untapped source of physiologically functional foods and drug precursors displaying a wide range of pharmacological activities [93]. Antibiotics (tetracycline, erythromycin), antiparasitics (avermectin), antimalarials (quinine), lipid control agents (lovastatin and analogues), immunosuppressants for organ transplants (cyclosporin, rapamycin), analgesics and antipyretics (aspirin) and anti-cancer drugs (taxol, doxorubicin) revolutionized medicine [94] and many of these were in fact discovered in fungi. Within the vast population of fungi, benefits of mushroom compounds on different clinical conditions have attracted the interest of the scientific community in order to understand the molecular mechanisms responsible for their actions [95]. Nevertheless, there is a surprising lack of epidemiologic and experimental studies addressing the biologic activities of mushrooms following oral administration to animals and/or humans [76]. 3.1. MUSHROOMS Mushrooms (such as Ganoderma lucidum, Lentinula edodes, Fomes fomentarius, Fomitopsis officinalis and many others) have been used all over the world for millennia as traditional medicines [96]. They are widely appreciated all over the world for their aroma, flavour, nutritional properties [97, 98] as well as their pharmacological value as sources of important bioactive compounds [99-102]. However, credible evaluation of their true nutritional value has so far been limited by the incomplete knowledge of their composition [97], which may be affected by mushroom strain/type, composition of growth media, time of harvest, management techniques, handling conditions and preparation of the substrates [103]. Many biological activities of mushrooms, namely from various classes of their primary and secondary metabolites, have been found. Indeed, anti-bacterial, anti-cancer, anti-fungal, anti-viral, anti-tumour, cytostatic, immunomodulating, anti-allergic, anti-atherogenic, hypoglycemic, anti-inflammatory, antioxidant, radical scavenging, anti-parasitic, FCUP 23 Investigating the tumour cell growth inhibitory potential of Portuguese wild mushrooms: Boletus spp., Suillus luteus and Ganoderma lucidum detoxification, anti-diabetic and hepatoprotective properties have been reported [87, 93, 102, 104-115]. This is why Occidental research for disease prophylaxis and/or treatment or adjunctive therapy has now begun to reassess this almost unexploited source. High-quality, long-term double-blinded, placebo-controlled studies with large trial populations and strong statistical power are needed to demonstrate safety and efficacy and which mushroom extracts and/or compounds are most effective for specific ailments [95]. These types of compounds are usually polysaccharides, glycopeptide/protein complexes, proteoglycans, proteins, polyphenols, sesquiterpenes and triterpenoids found in fruiting bodies, mycelia, spores and culture broth of macrofungi [76, 116]. Another very important detail to consider is that these substances can be obtained from many origins, such as wild and cultivated fruiting bodies and mycelial biomass, and supernatant of submerged cultures using bioreactors [117-121]. Also, it is not well understood whether these vast bioactive effects are caused only by a single component of a particular mushroom or if they are the result of an additive, or even synergistic outcome due to several compounds [95]. It is still unclear if dried bodies and powders are more or less effective and/or safer than extracts or if the extracts are better than isolated compounds. Occasionally, such comparisons have been performed, but only by using the same amount of material regardless of its state of purification and with no apparent attempts to administer equivalent amounts of a specific constituent [76]. Anti-tumoural activity is the most significant therapeutic one found to be associated with some mushrooms. Indeed, several dozens of species have been found to be highly efficient on various tumours and cancer types [87, 122-126] even though the underlying molecular mechanisms of the bioactive metabolites are far from being fully understood [127]. In Asia, several mushroom polysaccharide compounds have proceeded through clinical trial phases I, II and III and are now being successfully used to treat not only cancer, but also other diseases [85, 95, 128-130]. For example, lentinan, a polysaccharide isolated from L. edodes, has been approved since 1985 as an adjuvant for gastric cancer in Japan [128], having been successful in prolonging patient survival [131-138]. Nonetheless, despite the numerous clinical trials for lentinan, not many have been placebo-controlled and double-blinded [85]. FCUP 24 Investigating the tumour cell growth inhibitory potential of Portuguese wild mushrooms: Boletus spp., Suillus luteus and Ganoderma lucidum Compounds derived from Grifola frondosa, such as the maitake D-fraction, have antitumoural activity in gastrointestinal, lung, liver and breast cancers [139-142] and a number of clinical trials have been carried out in the United States and Japan in breast, prostate, lung, liver and gastric cancer patients regarding this extract [143]. 3.1.1. BIODIVERSITY IN THE NORTHEAST OF PORTUGAL The Northeast of Portugal, more specifically in the region of Trás-os-Montes, is well-known for its diversity of climate conditions and variety of soils. This assumes a vital role in mushroom production, thus explaining why this region is recognized as one of the richest in wild as well as edible species. These mushrooms are usually consumed fresh, dried during the off-season or canned, offering a diversity of organoleptic characteristics and palatable sensations [144]. Two main groups of fungi dominate these habitats: the saprotrophic and ectomycorrhizal [145]. The first plays an important part in the ecosystem as a major decomposer of plant residues, releasing nutrients that sustain and stimulate plant growth [146]. On the other hand, ectomycorrhizal fungi are able to enhance the absorption of mineral nutrients and water and increase plant resistance to different environmental stresses, which culminates in plant growth increase [147]. Some authors also pointed out a beneficial effect on biological control of larval root herbivores from the latter fungi population [148]. 3.1.2. MUSHROOMS WITH POTENTIAL ANTI-TUMOUR PROPERTIES There are very few available epidemiologic studies regarding the risk of cancer associated with the intake of some mushrooms [149-151]. The first one published was a hospital-based case control and found that there was a significant decrease in the risk of gastric cancer with a higher mushroom intake [150]. Unfortunately, this study lacks many details on statistical methods, making it difficult to confirm such a claim [76]. Another hospital-based case study also reported similar associations with this type of cancer, but only in the groups with the highest intake frequency (at least once a week) [152]. Results from a study on breast cancer suggest the possibility of preventing this type of cancer by consuming a diet high in mushroom intake since the authors found a decreased risk of developing this cancer in such conditions [151]. Others evaluated the association FCUP 25 Investigating the tumour cell growth inhibitory potential of Portuguese wild mushrooms: Boletus spp., Suillus luteus and Ganoderma lucidum between the daily intake of mushrooms, the average consumption frequency of dietary mushrooms and breast cancer risk in women histologically confirmed to have breast cancer. The authors found a strong inverse dose-response relationship in postmenopausal women, but not in premenopausal women, possibly due to the fact that some mushrooms have aromatase inhibitors (beneficial to menopausal women since aromatases favour estrogen production from androgens) [153]. However, whereas the latter study adjusted the results according to health-related behaviours (education, family history of breast cancer, body mass index, number of children, regular exercise, smoking, drinking, multivitamin supplement use and dietary factors), other residual effects may still have influenced them, such as the impact of commercial food products that contain mushroom components. This is also true for the other reports mentioned above. Therefore, there is a strong need for more studies on dietary mushroom intake among diverse and large populations. 3.1.2.1. Boletus GENUS Of all the forest species gathered in the wild, mushrooms from the Boletus genus are the most frequently harvested for human consumption in European countries, including Portugal [154]. Their popularity is mostly due to some of their sensory characteristics, particularly their aroma, taste and texture [155]. Among the many species of the Boletaceae family, B. edulis is undoubtedly regarded as having the finest flavour. Species related to this one involve a dozen or so varieties (Figure 2), such as B. aereus and B. reticulatus (formerly known as B. aestivalis) for example [155]. B. luridiformis, previously known as B. erythropus, is found in Northern Europe and North America and although edible when cooked, it may cause gastric upset when raw. B. impolitus, however, is edible, but rare while B. fragrans is merely rumoured to be edible. B. regius, commonly known as the royal bolete or red-capped butter bolete, is found in China, Europe and North America and is also highly appreciated. Unfortunately, very little information is documented on anti-tumoural activity of any of these species. FCUP 26 Investigating the tumour cell growth inhibitory potential of Portuguese wild mushrooms: Boletus spp., Suillus luteus and Ganoderma lucidum Figure 2 – Photographs of various species of the Boletus genus. (A) B. aereus1. (B) B. reticulatus2. (C) B. luridiformis3. (D) B. fragrans4. (E) B. impolitus5. (F) B. regius6. Nutritionally rich and a source of important metabolites, these edible fungi could be directly used in the human diet as healthy foods, taking advantage of the synergistic and/or additive effects of the natural antioxidants present in their composition. Non-edible Boletus species might also be interesting sources of extractable bioactive molecules to be used as additives in the food industry or even as components in pharmaceutical and cosmetic formulations. Bolesatine, a toxic glycoprotein purified from B. satanas [156], is a remarkable example. This lectin exerted potent mitogenic activity on human peripheral blood lymphocytes and induced the release of interleukin-1a, interleukin-2 and tumour necrosis factor-α from mononuclear cell cultures [157]. Some authors believe that studies searching for new active low molecular weight compounds should be more focused on species of the Boletales order [125]. However, some caution is required in such studies since subtle differences in some molecules (such 1 Image taken from http://en.wikipedia.org/wiki/File:Bronze_Roehrling.jpg. 2 Image taken from http://en.wikipedia.org/wiki/File:Boletus.JPG. 3 Image taken from http://en.wikipedia.org/wiki/File:Boletus_erythropus_2010_G3.jpg. 4 Image taken from http://boletales.com/genera/boletus/b-fragrans/. 5 Image taken from http://en.wikipedia.org/wiki/File:Boletus_impolitus_2009_G1.jpg. 6 Image taken from http://en.wikipedia.org/wiki/File:Boletus_regius_37212.jpg. A B C D E F FCUP 27 Investigating the tumour cell growth inhibitory potential of Portuguese wild mushrooms: Boletus spp., Suillus luteus and Ganoderma lucidum as organic acids) were found between two close geographical origins (Vinhais and Bragança, in Northeast Portugal), which indicates that geographical location may also interfere with the composition of this species [158]. 3.1.2.2. Suillus luteus Figure 3 – S. luteus7. Suillus luteus is a common fungus, fruiting abundantly in autumn and frequently harvested for food even if it may cause allergic reactions such as other Suillus species (Figure 3) [159]. Many studies have been carried out in order to elucidate the chemical composition of the bioactive molecules of this species (such as phenolic compounds, vitamins, sugars and fatty acids) as well as its antioxidant potential [98, 158, 160, 161]. Nevertheless, S. luteus has never been studied for its cytotoxic potential until a few years ago when a study by Tomasi and collaborators showed that methanolic extracts of this mushroom presented cytotoxic activity against the L1210 cell line (murine lymphocytic leukemia) [125]. Another study also reported growth inhibition of the SK-MEL-1 cancer cell line (human melanoma) following treatment with a phytosphingosine-type ceramide, suillumide, isolated from S. luteus [162]. A recently published study showed that a methanolic extract of S. luteus studied for tumour cell growth inhibitory potential caused inhibition of cell proliferation on the HCT-15 cell line, even appearing to cause DNA damage as an increase in cellular levels of p-H2AX was detected [163]. 7 Image taken from http://en.wikipedia.org/wiki/File:Butterroehrling.jpg. FCUP 34 Investigating the tumour cell growth inhibitory potential of Portuguese wild mushrooms: Boletus spp., Suillus luteus and Ganoderma lucidum M MA AT TE ER RI IA AL LS S A AN ND D M ME ET TH HO OD DS S 1. MUSHROOM SPECIES AND EXTRACT PREPARATION The methanolic extracts of Boletus aereus, Boletus reticulatus, Boletus luridiformis, Boletus fragrans, Boletus impolitus, Boletus regius, Ganoderma lucidum and Suillus luteus were obtained from the fruiting bodies of representative specimens collected in Bragança, Northeast of Portugal. The collection of the mushrooms and the preparation of the extracts were performed by collaborators from Instituto Politécnico de Bragança. Regarding the Boletus genus, two types of methanolic extracts were obtained, one using a cold extraction method (-20ºC) and the other performed at room temperature (RT). 2. CELL LINES Three human tumour cell lines were used in this work: AGS (gastric cancer), MCF-7 (breast cancer) and NCI-H460 (non-small cell lung cancer). Adherent cells were cultured at 37°C in 5% CO2 in RPMI-1640 medium (Lonza) supplemented with 5% of heat-inactivated fetal bovine serum (FBS, Gibco). Exceptionally, throughout the work with G. lucidum, 10% of FBS was used. All assays were initiated with cells in exponential growth and with viabilities over 90%. 3. EXTRACT SCREENING AND GROWTH INHIBITION The effect of the extracts on the growth of human tumour cell lines was evaluated according to the procedure adopted in the in vitro anti-cancer drug screening of the National Cancer Institute (NCI) using the sulforhodamine B (SRB, Sigma-Aldrich Chemical Co.) assay to assess growth inhibition [241], with a few adaptations. This colorimetric assay estimates cell number by staining cellular proteins with the protein-binding dye SRB. Each cell line was plated in 96-well plates and allowed to attach for 24h at an appropriate cell density (5.0x103 cells/well (for both NCI-H460 and MCF-7) and 7.5x103 cells/well for AGS). Cells were then treated with either complete medium (RPMI-1640 supplemented with 5% FBS), dimethyl-sulfoxide (DMSO) or various extract concentrations for the next 48h. FCUP 35 Investigating the tumour cell growth inhibitory potential of Portuguese wild mushrooms: Boletus spp., Suillus luteus and Ganoderma lucidum After the incubation period, adherent cells were fixed with trichloroacetic acid (TCA, Panreac) and rinsed with distilled water. After being stained with SRB, the fixated cells were then rinsed with 1% acetic acid (Merck). The bound stain was solubilised with 10mM Trisbase (Sigma-Aldrich Chemical Co.) and the absorbance measured at 510nm by a microplate reader (BioTek© Syneray HT) followed by analysis with the Gen5© software. The concentration inhibiting cell growth at 50% (GI50) was calculated as previously described [242]. Growth inhibitory activity of the extracts was inferred by comparing the absorbance of the wells containing cells treated with the extract with the absorbance of wells with untreated cells 48h following the treatment and subsequently comparing these results with the readings collected from cells fixed at time zero (the time of the extract application) [243]. At least three independent and reproducible assays were carried out (n=3) for each cell line. The GI50 concentrations were calculated as the mean from those assays. Doxorubicin (Sigma-Aldrich Chemical Co.) was used as a positive control. Cells treated with DMSO and non-treated cells were used as controls. For each potentially active extract, further assays were performed after the initial drug screening. 4. PROLIFERATION, CELL CYCLE PROGRESSION AND PROGRAMMED CELL DEATH Each cell line was plated in 6-well plates and allowed to attach for 24h at an appropriate density (1.5x105 cells/well) in medium supplemented with 10% FBS for AGS or 5% FBS for NCI-H460. Cells were then treated with either complete medium, DMSO or various extract concentrations during 24h and/or 48h. After this period of treatment, cells were analysed for proliferation, cell cycle profile and programmed cell death. 4.1. BrdU INCORPORATION At either the 23rd or 47th hour, adherent cells were exposed to 10µM 5-bromo-2’- deoxyuridine (BrdU, Sigma-Aldrich Chemical Co.) at 37°C and 5% CO2 for 1h, carefully washed with phosphate buffer saline (PBS, Amresco), before being trypsinized (Triple Express, Gibco) and the pellet re-suspended in 4% paraformaldehyde (PFA, Merck) for 30 FCUP 36 Investigating the tumour cell growth inhibitory potential of Portuguese wild mushrooms: Boletus spp., Suillus luteus and Ganoderma lucidum minutes, followed by another centrifugation and re-suspension with PBS. Samples were kept at 4ºC before the BrdU assay was performed as follows. Cytospins were prepared in a microscopic slide using a Shandon© cytospin centrifuge, rinsed thrice with PBS, each during 5 minutes at RT, before being exposed to 2M hydrochloride acid (HCl, Merck) for DNA denaturation. After two washing steps, first with PBS and then PBS supplemented with 0.5% Tween20 (Promega) and 0.05% bovine serum albumin (BSA, Sigma-Aldrich Chemical Co.), slides were incubated with mouse anti-BrdU (Palex Medical SA, 1:10) for 1h at RT and further incubated with fluorescein-labeled rabbit anti-mouse antibody (1:100, Palex Medical SA) for 30 minutes. Analysis of the slides was performed using a Nikon Eclipse TS 100 fluorescence microscope after application of Vectashield Mounting Media with 4’,6-diamidino-2-phenylindole (DAPI, Vector Laboratories). A minimum of 500 cells per slide were counted and analysed using the Image J© programme [244, 245] for a semi-quantitative evaluation of the levels of proliferation. 4.2. CELL CYCLE PROFILE ANALYSIS The cells were harvested and then centrifuged following the 24h or 48h incubation period and the pellet re-suspended in 70% iced ethanol. The fixed samples remained at 4°C at least overnight until further analysis by a FACSCalibur© flow cytometer (Becton, Dickinson and Company) after the addition of propidium iodide (PI 1:200, Sigma-Aldrich Chemical Co.) and RNAse (1:20, Invitrogen) in PBS. Flow cytometry analysis was carried out and the results were analysed by collaborators, with the help of the author, as described next. A minimum of 10000 events were analysed using BD CellQuest Pro© in which single cells were gated from debris and aggregates by a forward scatter/side-scatter (FSC/SSC) dot plot and the fluorescence (PI:563/617) was obtained. Post-acquisition analysis was carried out using the FlowJo© 7.2 software (Tree Star, Ashland, USA) where the isolation of the single cell population was considered in a similar way as described above. The integration of the peaks was done automatically by the programme and then manually adjusted so that the fluorescence mean value at G2 corresponded to a two-fold increase of the mean value at G1. FCUP 37 Investigating the tumour cell growth inhibitory potential of Portuguese wild mushrooms: Boletus spp., Suillus luteus and Ganoderma lucidum 4.3. TUNEL ASSAY After the incubation period, cells were carefully washed with PBS before being harvested and the pellet re-suspended in PBS. This was followed by another centrifugation and the sample was divided into two: i) part of the sample was re-suspended in 4% PFA for 30 minutes, followed by yet another centrifugation and re-suspension with PBS for the terminal deoxynucleotidyl transferase 2’-deoxyuridine-5’-triphosphate nick end labelling (TUNEL) assay; ii) the remaining cells were immediately frozen for protein expression assessment following the first washing, as described below. Samples for the TUNEL assay were kept at 4ºC before performing the assay, as follows: samples were cytospinned, rinsed thrice with PBS and exposed to a permeabilization solution consisting of 0.1% Triton X-100 (Promega) and 0.1% sodium citrate (Merck) in PBS at 4°C for 2 minutes, washed and exposed to the TUNEL reaction mix consisting of TUNEL dilution buffer, TUNEL labelled solution and the terminal deoxynucleotidyl transferase enzyme (“in situ cell death detection”, Roche), for 1h at 37°C. Slides were mounted with Vectashield Mounting Media with DAPI in order to visualise the nuclei with a blue fluorescence. Analysis of the slides was performed as described above for the BrdU incorporation assay using the same fluorescence microscope. A minimum of 500 cells per slide were counted and analysed using the Image J programme for a semiquantitative evaluation of the levels of programmed cell death. 4.4. ANNEXIN-FITC/PI STAINING After the 48h incubation period, cells were harvested and the pellet re-suspended in 400µL of binding buffer 4x. Samples were kept at 4°C and then analysed by an annexin-V FITC/PI double staining method previously described [246]. Briefly, cells were incubated for 10 minutes at RT in the dark with annexin-V (1:40, Bender MedSystems, Vienna, Austria). Prior to flow cytometric analysis, PI was added (1:21, Sigma-Aldrich Chemical Co.) for 2 minutes and always kept at 4ºC. Flow cytometry analysis was carried out and results were analysed by collaborators, with the help of the author, as described next. A minimum of 10000 events where then analysed using BD CellQuest Pro in which single cells were gated from debris and aggregates by a FSC/SSC dot plot and the fluorescence FCUP 38 Investigating the tumour cell growth inhibitory potential of Portuguese wild mushrooms: Boletus spp., Suillus luteus and Ganoderma lucidum (FITC:488/519) versus (PI:488/617) was obtained. Results were analyzed using the FlowJo 7.2 software [247]. 4.5. CONCOMITANT EFFECT OF CHEMOTHERAPEUTIC DRUGS AND MUSHROOM EXTRACTS The GI50 concentrations of the chemotherapeutic agents 5-fluorouracil (5-FU, Sigma-Aldrich Chemical Co.) and erlotinib (Selleck Chemicals Co., Ltd) were determined by the SRB assay in the NCI-H460 and AGS cell lines and found to be 4.75µM and 4.27µM respectively. These concentrations were used in the study of the concomitant effect of both the extracts and the drugs on these same cell lines. The extract concentrations used in these studies were the GI50 for S. luteus (30.33µg/mL) and C1 for G. lucidum (53.3µg/mL). The percentage of cell viability in each well was determined according to the trypan blue (Sigma-Aldrich Chemical Co.) dye exclusion test using a Neubauer chamber. Results are the mean ± standard deviation error (SE) of at least three independent experiments and are represented as a percentage of cells treated with medium only (Blank cells). 5. PROTEIN EXPRESSION ASSESSMENT As stated above, samples were obtained from part of the cells grown for the TUNEL assay and frozen at -20°C for further treatment. Cells were lysed in Winman's buffer [1% NP-40 (Sigma-Aldrich Chemical Co.), 0.1M TrisHCl (Merck) pH 8.0, 0.15M sodium chloride (Merck) and 5M ethylenediaminetetraacetic acid (EDTA, Sigma-Aldrich Chemical Co.)] with EDTA-free protease inhibitor cocktail (Roche) for 9 cycles of 5 minutes each, with 1 minute vortex in between them. Proteins were quantified with the DC Protein Assay kit (BioRad), separated in 12% (resolving) and 5% (stacking) tris-glycine sodium dodecyl sulfate (SDS, Merck)-polyacrylamide (BioRad) gels and transferred to a nitro-cellulose membrane (GE Healthcare). Membranes were incubated with goat anti-actin antibody (1:2000, Santa Cruz Biotechnology), mouse p53 Ab-2 (1:200, Neo-markers), rabbit p-H2AX (1:200, Santa Cruz Biotechnology) and rabbit anti-Light Chain 3 B (LC3 1:1000, Cell Signalling) and then incubated with the respective secondary antibodies: goat anti-mouse immunoglobulin Ghorseradish peroxidase (IgG-HRP) (1:2000, Santa Cruz Biotechnology), goat anti-rabbit FCUP 39 Investigating the tumour cell growth inhibitory potential of Portuguese wild mushrooms: Boletus spp., Suillus luteus and Ganoderma lucidum IgG-HRP (1:2000, Santa Cruz Biotechnology), donkey anti-goat IgG-HRP (1:2000, Santa Cruz Biotechnology), always in 5% non-fat dried molic milk in Tris-Buffered Saline (Merck) and Tween20. Actin served as the loading control. The signal was detected with the Amersham enhanced chemiluminescence kit (ECL, GE Healthcare), the Hyperfilm ECL (GE Healthcare) and the Kodak GBX developer and fixer (Sigma-Aldrich Chemical Co.) [248]. 6. STATISTICAL ANALYSIS For each assay, three to six independent experiments were performed. Results were expressed as mean values ± SE determined by Microsoft Office Excel©. Differences in p values below 0.05 were considered statistically significant. FCUP 40 Investigating the tumour cell growth inhibitory potential of Portuguese wild mushrooms: Boletus spp., Suillus luteus and Ganoderma lucidum R RE ES SU UL LT TS S A AN ND D D DI IS SC CU US SS SI IO ON N 1. STUDY OF HUMAN TUMOUR CELL GROWTH INHIBITORY EFFECT OF MUSHROOMS FROM THE Boletus GENUS 1.1. EXTRACT SCREENING FOR CELL GROWTH INHIBITORY ACTIVITY As stated before, the methanolic extracts obtained at two different temperatures from six Boletus species were subjected to a growth inhibitory screening using the SRB assay on two human tumour cell lines: lung (NCI-H460) and breast (MCF-7) cancer cells. The results for this screening are presented on Table 1. Table 1 – Results from the screening of various Boletus mushrooms for cell growth inhibitory activity in two human tumour cell lines. GI50 (μg/mL) Species Temperature of extraction (°C) NCI-H460 MCF-7 B. aereus -20 >400 >400 25 >400 >400 B. reticulatus -20 >400 >400 25 >400 >400 B. luridiformis -20 >400 >400 25 >400 >400 B. fragrans -20 >400 >400 25 >400 >400 B. impolitus -20 >400 >400 25 >400 >400 B. regius -20 >400 >400 25 >400 >400 Results are from two to three independent experiments with the six Boletus mushrooms that were studied. Both types of methanolic extracts (prepared at -20°C or 25°C) were tested at various concentrations up to a maximum concentration of 400μg/mL in order to determine the concentration that caused 50% of cell growth inhibition (GI50). Together with the mushroom extracts to be screened for their cell growth inhibitory activity, doxorubicin was used as a positive control in all assays. Also, two other controls were added to the experiments: cells treated only with culture medium (Blank treatment) and cells treated with DMSO, the solvent used to dissolve the extracts. The final concentration of DMSO (0.4% of the total well volume) presented no toxicity to the cells. FCUP 41 Investigating the tumour cell growth inhibitory potential of Portuguese wild mushrooms: Boletus spp., Suillus luteus and Ganoderma lucidum The cells were directly observed under an inverted optic microscope before proceeding with the assay following 48h of continuous treatment with the extracts (or controls). Those treated with doxorubicin showed evident morphological differences from cells treated with either medium or DMSO (together with diminished cell number): these cells were much bigger than control cells although no other phenotypic change was noted. In contrast, cells treated with the extracts presented a similar size to the cells used as controls under this microscope. In addition, the wells with extract-treated MCF-7 cells appeared to have an equal (or even higher) amount of cells as the corresponding control wells. Regarding the results obtained in the NCI-H460 cell line, a GI50 value of 190µg/mL was found for the B. aereus mushroom extracted at -20°C, but only in one out of three independent experiments. In the other two, the GI50 was greater than the maximum concentration tested (400µg/mL) and therefore considered to be >400 µg/mL. Also in the NCI-H460 cell line, the cold methanolic extract of B. impolitus was the only other extract screened three times as the highest concentration tested, 400µg/mL, almost reached 50% of cell growth inhibition in the first SRB assay. Nevertheless, in the other two experiments, the determined GI50 was consistently greater than that concentration. The screening on the MCF-7 cells gave similar results and therefore the GI50 for these extracts was considered to be >400 µg/mL in both cell lines. Taking this into consideration, none of the studied extracts showed tumour cell growth inhibitory potential. As such, no further assays were performed using these extracts. 2. STUDY OF THE EFFECT OF A METHANOLIC EXTRACT OF S. luteus IN A NON-SMALL CELL LUNG CANCER CELL LINE As mentioned in the literature review, the S. luteus methanolic extract has previously been studied for tumour cell growth inhibitory potential [163]. That study has shown that this extract was more potent in the NCI-H460 cell line (GI50=30.33µg/mL) than in three other cell lines. Therefore, in this chapter, this concentration of GI50 was used in order to perform other assays intending to investigate the effect of this extract on NCI-H460 cellular proliferation, cell cycle profile and levels of programmed cell death. Doxorubicin was used as a positive control in some assays. In addition, cells treated with DMSO and non-treated FCUP 42 Investigating the tumour cell growth inhibitory potential of Portuguese wild mushrooms: Boletus spp., Suillus luteus and Ganoderma lucidum cells (Blank) were used as controls as DMSO did not cause a statistically significant effect when compared to untreated cells. The cells were closely observed under an inverted microscope at least once a day for the duration of the experiment in order to assure they maintained their morphologic features, to avoid possible bacterial contaminations and also to make sure they did not reach full confluency. 2.1. CELLULAR PROLIFERATION Figure 5 – Graphic representation of the proliferation of NCI-H460 cells treated with the methanolic extract of S. luteus. Cells were treated during 48h with the extract concentration that inhibited cell growth by 50% (GI50, 30.33µg/mL), twice that concentration (2GI50, 60.66µg/mL) and the DMSO concentration corresponding to the 2GI50 (2DMSO, control). Untreated cells (Blank) and the DMSO-treated cells served as controls. Results are the means ± SE of three independent experiments and statistical significance was tested with a two-tailed paired Student’s t-test comparing extract-treated cells with DMSO-treated cells. DMSO treatments were compared with the blank treatment. * indicates p ≤ 0.05. The proliferation levels of NCI-H460 cells that were treated during 48h with the methanolic extract of S. luteus were decreased (Figure 5). Indeed, proliferation levels diminished from 20.87% in the untreated cells (Blank) to 12.36% in cells treated with the GI50 concentration. There was an even more accentuated and statistically significant inhibition of proliferation 0 10 20 30 40 50 60 70 80 90 100 % of cell proliferation Treatment * FCUP 43 Investigating the tumour cell growth inhibitory potential of Portuguese wild mushrooms: Boletus spp., Suillus luteus and Ganoderma lucidum (to 8.37%) when cells were treated with the 2GI50 concentration of this extract, indicating that the effect of this extract on cellular proliferation is dose-dependent. Such data shows that this mushroom extract decreases cellular proliferation of the NCIH460 cells but the pathways regulating this effect need elucidation. 2.2. CELL CYCLE PROFILE ANALYSIS So as to further understand the effect of the S. luteus extract on the NCI-H460 line, their cell cycle progression was also studied. Figure 6 – Graphic representation of the cell cycle profile analysis of NCI-H460 cells treated with the methanolic extract of S. luteus. Cells were treated during 48h with the extract concentration that inhibited cell growth by 50% (GI50, 30.33µg/mL), twice that concentration (2GI50, 60.66µg/mL) and the corresponding concentrations of DMSO (DMSO and 2DMSO, respectively). Untreated cells (Blank) and DMSO-treated cells served as controls. Results are the means ± SE of three independent experiments and statistical significance was tested with a two-tailed paired Student’s t-test comparing extract-treated cells with corresponding DMSO treated cells. DMSO treatments were compared with the blank treatment. * indicates p ≤ 0.05. Cells treated with the extract presented a cell cycle arrest in G1. Indeed, the percentage of cells in this phase of the cell cycle was higher in cells treated with the GI50 concentration (64.42%, Figure 6) and, more significantly, with the 2GI50 concentration (75.18%) than in the 50 50 FCUP 50 Investigating the tumour cell growth inhibitory potential of Portuguese wild mushrooms: Boletus spp., Suillus luteus and Ganoderma lucidum 51.24%). In agreement with these results, the cell population in the G2/M phase was significantly lower (3.53%) in cells treated with the C2 concentration of the extract than in untreated cells (Blank, 9.93%). These results suggest that a pathway involved in maintaining the cells in the G1 phase might be affected by the extract. Unfortunately, the concentration of DMSO that corresponded to the C2 concentration of the extract caused a statistically significant decrease in G1 in relation to the Blank. 3.3. APOPTOSIS Blank DMSO 2DMSO C1 C2 Doxorubicin Figure 11 – Flow cytometry analysis of apoptotic cell death following annexin-FITC/PI staining. Cells were treated during 48h with two different concentrations of this extract: C1 (53.3µg/mL) and twice that concentration (C2, 106.6µg/mL). The corresponding concentrations of DMSO were also tested (DMSO and 2DMSO, respectively). Untreated cells (Blank) and the DMSO-treated cells served as controls. Doxorubicin was used as a positive control in the experiments. FL1-H stands for annexin V-FITC fluorescence and FL2-H is the PI fluorescence. The images are representative of three independent experiments. There was no increase in programmed cell death 48h following treatment of cells with the G. lucidum extract in relation to controls (Table 3). Figure 11 also depicts this illation: the population of PI-/annexin V-FITC+ cells undergoing apoptosis is similar in both treated and untreated cells. These results indicate that the extract does not induce programmed cell FCUP 51 Investigating the tumour cell growth inhibitory potential of Portuguese wild mushrooms: Boletus spp., Suillus luteus and Ganoderma lucidum death above the normal low basal levels, as the cell cycle profile analysis results had indicated. Table 3 – Levels of apoptotic cell death obtained by quantification of the results shown in Figure 11. Treatment Apoptotic cells (%) Blank 7.9 ± 1.1 DMSO 7.4 ± 1.8 2DMSO 5.9 ± 1.1* C1 6.6 ± 2.3 C2 9.5 ± 3.2 Doxorubicin 34.6 ± 4.2* Cells were treated during 48h with two different concentrations of this extract: C1 (53.3µg/mL) and twice that concentration (C2, 106.6µg/mL). The corresponding concentrations of DMSO were also tested (DMSO and 2DMSO, respectively). Untreated cells (Blank) and the DMSOtreated cells served as controls and doxorubicin was used as a positive control. Results are the mean ± SE of three independent experiments and statistical significance was tested with a two-tailed paired Student’s t-test comparing treatments and the corresponding DMSO controls or DMSO controls and Blank. * indicates p ≤ 0.05. 3.4. PROTEIN EXPRESSION ASSESSMENT As with S. luteus, effects on the expression of some proteins caused by the methanolic extract of G. lucidum was also determined by Western blotting (FIGURE 12). Figure 12 – Protein expression of AGS cells treated with the methanolic extract of G. lucidum. Levels of p-H2AX, LC3 and actin following a 48h treatment with two different concentrations of this extract: C1 (53.3µg/mL) and twice that concentration (C2, 106.6µg/mL). Untreated cells (B, blank) and cells treated with the corresponding DMSO concentrations (D1 and D2) were used as controls. Doxorubicin (Dox) was used as a positive control in the experiments. Actin served as the loading control. Blots are representative of three independent experiments. B Dox D1 D2 C1 C2 p-H2AX LC3-I LC3-II Actin FCUP 52 Investigating the tumour cell growth inhibitory potential of Portuguese wild mushrooms: Boletus spp., Suillus luteus and Ganoderma lucidum No obvious differences in the p53 expression levels were observed in relation to the controls following 48h of treatment with either C1 or C2 extract concentrations (data not shown). Consequently, no additional analysis of known targets of this protein (p21 or E2F3, for example) was made. p-H2AX expression levels slightly increased in the extract-treated cells when compared with the untreated AGS cells. The increase was more evident with the C2 concentration (FIGURE 12). This effect may be indicative of DNA damage, which should be detected by p53. But similar to what was found for the S. luteus (in the previous chapter), the levels of p53 in the treated cells were unaffected, suggesting that the molecular mechanisms of the bioactive compounds of this mushroom probably are p53-independent as well. Further experiments would be necessary to confirm this hypothesis, for example silencing p53 gene expression by RNA interference and confirming if the effect of the extract was maintained under such conditions. Interestingly, the expression of LC3-II (which has an essential role in autophagy) noticeably increased in the extract-treated cells when compared with the untreated cells (FIGURE 12). This increase in LC3-II implies that these cells may be undergoing an autophagic process. Despite these results, the cellular effects and the pathways that are affected are possibly very complex and more detailed studies are necessary to study the effect of the extract in autophagy. FCUP 53 Investigating the tumour cell growth inhibitory potential of Portuguese wild mushrooms: Boletus spp., Suillus luteus and Ganoderma lucidum 3.5. CONCOMITANT EFFECT OF 5-FLUOROURACIL AND G. lucidum Figure 13 – Graphic representation of the concomitant effect of 5-FU and the methanolic extract from G. lucidum on the percentage of viable AGS cells. Cells were treated during 48h with the C1 concentration of G. lucidum (53.3µg/mL), with the GI50 concentration of 5-FU (4.75µM), with both the extract and the drug at the same time (Both), with the highest concentration of DMSO used in the individual treatments (DMSO) or with the concentration of DMSO corresponding to the concomitant treatment (2DMSO). Untreated cells (Blank) and the DMSO-treated cells served as controls. Results are the means ± SE of three independent experiments and statistical significance was tested by a two-tailed paired Student’s t-test comparing treated cells with corresponding DMSO-treated cells. DMSO treatments were compared with the blank treatment. * indicates p ≤ 0.05, ** indicates p ≤ 0.01 and *** indicates p ≤ 0.001. As this mushroom extract presented promising tumour cell growth inhibitory activity, an experiment was carried out in order to investigate if it could sensitize cells to the effect of a known chemotherapeutical agent commonly used in the treatment of gastric cancer, 5-FU. Results clearly showed that there was no obvious decrease in viable cell number when cells were treated concomitantly with the G. lucidum extract and 5-FU for 48h (Figure 13). Indeed, even though the percentage of viable cells was reduced following treatment with either the extract or the drug, when cells were treated with both the extract and 5-FU there was no further decrease in the number of viable cells. FCUP 54 Investigating the tumour cell growth inhibitory potential of Portuguese wild mushrooms: Boletus spp., Suillus luteus and Ganoderma lucidum C CO ON NC CL LU US SI IO ON NS S While some authors believe that the discovery of new bioactive compounds should be focused on mushrooms of the Boletales order [125], the screening of extracts from six Boletus species revealed that they have no significant tumour cell growth inhibitory potential in both the NCI-H460 and MCF-7 cancer cell lines. The methanolic extract from S. luteus caused inhibition of proliferation on NCI-H460 cells and cell cycle profile analysis showed a G1 phase arrest, but no changes in the normal levels of programmed cell death were found. The levels of p-H2AX, a phosphorylated histone deeply involved in DNA damage repair, also increased following treatment with this extract in comparison to controls, namely in cells treated with the highest concentration tested. This suggests that this effect may be dependent on concentration. In addition, treatment with this extract did not seem to increase the sensitivity of cells to the effects of erlotinib in the concentrations tested. Regarding the effect of the G. lucidum methanolic extract, it inhibited proliferation in AGS cells, but showed no effect upon programmed cell death. Analysis of cell cycle profile demonstrated that treatment with this extract caused a G1 phase arrest. Expression of pH2AX had only a slight increase in cells treated with this extract, but the autophagy-related LC3-II protein increased markedly with treatment. Also, preliminary results indicated that this extract does not seem to increase cell sensitivity to the effects of 5-FU, at least in the tested concentrations. 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