Research

One of the projects searching for chemopreventive agents performed in my laboratory aimed to compare the chemical composition and antitumor-related activities of polysaccharide-extracts produced by the edible mushroom Pleurotus pulmonarius from mycelium grown in liquid culture (ME) or fruiting bodies (FBE), in vitro. In addition, we determined whether orally administered of FBE and ME could attenuate or prevent the development of experimental colitis in mice and colitis- associated-colorectal cancer (CRC). The main goal of these studies was to advance the understanding of the medicinal characteristic of glucans harvested from the edible mushroom including anti- inflammatory and anti- carcinogenic properties and to learn about the mechanisms of action involved. We compared the chemical composition, anti-inflammatory and antitumor-related activities of ME and FBE glucans, in vivo and in vitro. Our findings suggest that these glucans are able to inhibit colitis, colitis-associated colon carcinogenesis through modulation of inflammation, cell proliferation and induction of apoptosis. These studies were conducted in co-operation with Prof. Yitzhak Hadar’s laboratory.

Next, we explored several Pleurotus species for their total, β and α‐glucan content. Pleurotus eryngii was found to have the highest total glucan concentrations and the highest α‐glucans proportion. We also found that the stalks (stipe) of the fruit body contained higher glucan content then the caps (pileus). Since mushrooms respond markedly to changes in environmental and growth conditions, we developed cultivation methods aiming to increase the levels of α and β‐glucans. Using olive mill solid waste (OMSW) in the cultivation substrate we were able to enrich the levels of both α‐and β-glucans. Maximal total glucan concentrations were enhanced up to twice when the growth substrate contained 80%‐100 % of OMSW compared to no OMSW.

We then performed in vitro testing of either crude or glucan extract preparations of stalks and caps originated from Pleurotus eryngii and the preparations giving the most significant anti-inflammatory effects were further tested in vivo and in vitro. We demonstrated that Pleurotus eryngii can serve as a potential rich source of glucans for nutritional and medicinal applications and that glucan content in mushroom fruiting bodies can be further enriched by applying OMSW into the cultivation substrate. Using dextran sulfate sodium (DSS)-inflammatory bowel disease (IBD) mice model, we measured the colonic inflammatory response to the different glucan preparations. We found that the histology damaging score (HDS) resulting from DSS treatment were efficiently downregulated by treatment with the fungal extracted glucans. Similar downregulatory effects were obtained for expression of various intestinal cytokines. All tested glucans were equally effective in regulating the number of CD14/CD16 monocytes. We tested the effect of glucans on lipopolysaccharide-induced production of TNF-α, which demonstrated that stalk-derived glucans were more effective than caps-derived glucans. Isolated glucans competed with anti-Dectin-1 and anti-CR3 antibodies, indicating that they contain β-glucans recognized by these receptors. In conclusion, the most effective glucans in ameliorating IBD-associated symptoms induced by DSS treatment in mice were glucan extracts prepared from the stalk of P. eryngii grown at higher concentrations of OMSW. We conclude that these stress-induced growing conditions may be helpful in selecting more effective glucans derived from edible mushrooms. 

Fig. 1 Representative histological sections of colonic mucosa stained with haematoxylin and eosin showing the anti-inflammatory effects of glucan administration. (A and B) Control, non-dextran sulfate sodium (DSS)-treated mice showing the normal histology of the colon and normal crypt structure (group 1). ↓ Normal crypt. (C and D) DSS-only mice showing extensive intestinal ulceration with severe inflammatory cell infiltrate in the lamina propria and submucosa (group 2). ←, damaged crypts. (E and F) Colonic sections of high fruiting bodies extract (FBE) mice (no DSS) and high mycelium extract (ME) mice (no DSS), respectively, showing normal colonic mucosa, submucosa and muscularis propria (groups 3 and 4). (G) High FBE + DSS (group 5) and (H) low FBE ‏+ DSS (group 6) showing normal colonic structure and accumulation of goblet cells. A marked reduction in infiltration of inflammatory cells, as well as absence of ulceration, is seen in the treated mice. (I) Colonic section of high ME ‏+ DSS (group 7): normal architecture is retained and there is a higher proportion of goblet cells compared with control mice. (J and K) Low ME ‏+ DSS showing ulceration and inflammatory reaction involving the mucosa only (group 8). Compensation with connective tissue is shown. Bars =100μm. (L) Histology damage score. Extents of inflammation and crypt damage were evaluated and summed to give a total score. The histology damage score for control, FBE and high ME mice (no DSS) is 0. Values are means of three or five mice per group, with standard errors represented by vertical bars. * Mean value was significantly different from that of group 1 control mice (P<0.05).

In a recently initiated project we demonstrated that in mice fed with diet containing Medium chain fatty acids (MCFA) we observed that transcription of activators of the complement system and other inflammatory pathways are downregulated and thereby the local and systemic inflammation are affected. The fact that MCFA are majorly transported by the portal circulation in a chylomicron independent mechanism also explains the possible role of MCFA in preventing inflammation. We observed also that IL-6 and interferon-gamma (IFN-γ) are downregulated leading to lower incidence and severity of colitis in mice fed MCFA based diet compared to other isocaloric control diets.

      Another project deals with the isolation and characterization of a mold-derived protein (ACTIBIND), bearing ribonuclease (RNase). This mold protein was firstly isolated in Prof. Oded Shoseyov’s laboratory for plant-associated activities and tested in my laboratory in in vivo and in vitro models of cancer and proved to have antiangiogenic and antitumorigenic characteristics. This protein was crystallized and analyzed by our crystallographic expert associate in Ben-Gurion University, Prof. OrnaAlmog. Sequencing this protein allowed us to identify a novel human homolog classified also as an RNase T2. A synthetic RNase T2 gene was designed, cloned, expressed and purified. This human RNase T2 exhibited similar antiangiogenic and antitumorigenic characteristics as the mold protein. Further development of this molecule for testing in humans has been transferred to a company [T2 BIOTECH Ltd (former name: Shaligal Promotion Ltd)], which aims at developing this T2RNase as a possible anticancer and antiangiogenic agent in humans, with a potentially high commercial value. We conducted many experiments to understand the molecular mechanism of action of selected peptides based on the RNase T2 molecule by joint M.Sc. and Ph.D. students.

RNAse T2 conformation                                       ACTIBIND conformation

The figure depicts that ACTIBIND inhibits MMP-2 expression and microvessel density and increases apoptosis in supermetastatic A375SM Human Melanoma xenografts. Immunohistochemical staining for the expression of CD31, MMP-2, and a TUNEL staining. In the ACTIBIND-treated tumors, a decrease in MMP-2 and microvessel density was observed, whereas the number of cells undergoing apoptosis (TUNEL) was increased in comparison with the tumors in the control group.

An additional avenue that we undertook is investigating the putative ameliorating effect of selected probiotics on inflammatory bowel disease and colon cancer. We isolated a novel high butyrate producing, non-pathogenic, putative probiotic bacterial strain from human colonic flora, identified as Enterococcus durans. Firstly, we characterized this putative probiotic and tested its role in amelioration of inflammatory bowel disease in a murine model and in an in vitro compartmentalized co-culture model. The concerted effect of the probiotic and butyrate secreted components, each working via different mechanisms, seemed to optimize and potentiate the individual properties of each component of the novel probiotic.

In the search for effective anticancer agents from foods, we have studied the effects of vitamin D, allicin from garlic and phytoestrogens. Assessing the true impact of such constituents on human health is difficult since the bioavailability of active constituents is not known. It is becoming clear that these agents exert pleiotropic effects on tumor cells, affecting various molecules involved in proliferation, invasion, and metastasis of cancer. In this regard, we demonstrated that vitamin D bioavailability through binding to vitamin D receptor can be positively regulated by estrogen, and we clearly describe a mechanism involving the plasma membrane associated molecule caveolin-1. Next, we described the mechanism of action of purified allicin on apoptotic induction through the transcriptional pathway involving Nrf2.

An additional nutrient that we are interested in are omega-3 fatty acids, a project supported by the ISF demonstrated that modulation of the transcriptional activity of HNF-4α as a function of chain length and unsaturation of its fatty acyl-CoA ligands may account for the differential effects of dietary fatty acids on colon tumorigenesis. Their efficacy in modulating colon cancer development was expected to verify the role played by HNF-4α in transducing colorectal cancer suppression by (n-3) PUFA. We aimed to assess the impact of omega-3-enriched formula on the healing of existent pressure ulcers in critically ill patients and concomitantly evaluate the expression of tissue-homing membrane proteins in circulating white blood cells. We demonstrated that granulocyte and lymphocyte expression of several membrane proteins involved in transmigration into tissues was boosted by delivery of omega-3-enriched enteral formula to these patients.  A recent supporting manuscript on this topic has been published.

An additional avenue that we undertook is investigating the putative ameliorating effect of selected probiotics on inflammatory bowel disease and colon cancer. We isolated a novel high butyrate producing, non-pathogenic, putative probiotic bacterial strain from human colonic flora, identified as Enterococcus durans. Firstly, we characterized this putative probiotic and tested its role in amelioration of inflammatory bowel disease in a murine model and in an in vitro compartmentalized co-culture model. The concerted effect of the probiotic and butyrate secreted components, each working via different mechanisms, seemed to optimize and potentiate the individual properties of each component of the novel probiotic.

In the search for effective anticancer agents from foods, we have studied the effects of vitamin D, allicin from garlic and phytoestrogens. Assessing the true impact of such constituents on human health is difficult since the bioavailability of active constituents is not known. It is becoming clear that these agents exert pleiotropic effects on tumor cells, affecting various molecules involved in proliferation, invasion, and metastasis of cancer. In this regard, we demonstrated that vitamin D bioavailability through binding to vitamin D receptor can be positively regulated by estrogen, and we clearly describe a mechanism involving the plasma membrane associated molecule caveolin-1. Next, we described the mechanism of action of purified allicin on apoptotic induction through the transcriptional pathway involving Nrf2.

An additional nutrient that we are interested in are omega-3 fatty acids, a project supported by the ISF demonstrated that modulation of the transcriptional activity of HNF-4α as a function of chain length and unsaturation of its fatty acyl-CoA ligands may account for the differential effects of dietary fatty acids on colon tumorigenesis. Their efficacy in modulating colon cancer development was expected to verify the role played by HNF-4α in transducing colorectal cancer suppression by (n-3) PUFA. We aimed to assess the impact of omega-3-enriched formula on the healing of existent pressure ulcers in critically ill patients and concomitantly evaluate the expression of tissue-homing membrane proteins in circulating white blood cells. We demonstrated that granulocyte and lymphocyte expression of several membrane proteins involved in transmigration into tissues was boosted by delivery of omega-3-enriched enteral formula to these patients.  A recent supporting manuscript on this topic has been published.

An additional issue that we are interested to investigate is the role of obesity in colorectal cancer (CRC). The incidence of obesity is increasing at an accelerating rate worldwide.  Obesity is widely recognized as an important risk factor for the development of some cancers, such as CRC. For every 2.4 unit increase in body mass index, CRC risk increases by 7%. Adipose tissue is a highly active endocrine organ that secretes a variety of molecules, collectively called “adipokines”, which are directly involved not only in the regulation of whole-body metabolism but also in inflammatory and immune responses. An important study conducted in our laboratory demonstrated that leptin affects processes related to colon cancer initiation and progression in colon cancer cells in vitro (paper 63, a highly cited paper). We conducted a series of studies in obese, insulin-resistant db/db mice, in fat-1 transgene coding for desaturation of (n-6) PUFA into (n-3) PUFA, in db/db crossed with fat-1 mice and in control mice. We conclude that suppression of intestinal HNF-4α activity by (n-3) PUFA may ameliorate diabesity-induced intestinal ontogenesis and offer an effective preventive modality for colorectal cancer. A very recent review on the role of obesity in colorectal cancer we published has impacted significantly and the immediate outcome was extremely positive remarks.

Obese adipose tissue is infiltrated by macrophages that participate in inflammatory pathways activated in the adipose tissue. Energy metabolism of tumor cells result from the interplay of the two main bioenergetic pathways, oxidative phosphorylation and glycolysis. Mitochondria are important organelles in cellular processes, including cellular respiration and energy expenditure, and programmed cell death (apoptosis). The nature of the metabolic networks that give rise to metabolic phenotypes that confer tumor cells oncogenic and metastatic properties, such as increased proliferation and the ability to avoid apoptosis, are still not well understood. Our goal in this area of research was to identify key molecular signals and interactions between adipocytes and colon cancer cells that may foster the genesis and growth of the latter. We found crosstalk mechanisms connecting inflammatory pathways to bioenergetic pathways. Linking both approaches may lead to a more complete picture relating obesity to colorectal cancer. Understanding the molecular mechanisms whereby obesity increases colon cancer risk is thus a focus for our research to be able to design more relevant strategies to prevent the increasing trend in obesity-related colon cancer.

Regarding studies concentrating on the wealth of the gastrointestinal tract and specifically the intestine we have recently focused in the disease Necrotizing Enterocolitis (NEC), which is the most common and severe intestinal inflammatory disease occurring principally in very low birth weight premature infants. We tried to elucidate the most important cellular mechanisms associated with the development of NEC. We specifically chose to focus in changes that took place at the tight junctions since based in the literature; we assumed that they have a decisive role in the pathological process associated with NEC.

We found that TIMP-1, a molecule naturally expressed in HBM inhibited Matrix Metalloproteinase-2 (MMP-2) activity, induced a significant increase in the expression of occluding. Additionally, we developed a label-free infrared surface plasmon biosensor with a double-chamber flow cell for live cell studies and successfully demonstrated the advantages of this biosensor by tracking the kinetics of intestinal cells layer response.

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We have recently started to study the role of oligosaccharides in human milk and goat milk in the inhibition of experimental necrotizing enterocolitis in neonatal mice model. Human milk is the optimal source of nutrition for infants. It contains numerous complex lipids, proteins, carbohydrates, micronutrients and bioactives that change dynamically both over a single feed and throughout the course of lactation. This reflects the changing nutritional requirements of the infant. Infants fed with human milk (HM) have a significant reduction in the incidence of NEC, reaching a ratio of up to1:10  when compared with infants fed commercial infant formula. HMOs are highly abundant group of indigestible oligosaccharides. Have been demonstrated to stimulate gut commensals' growth, prevent adhesion of enteropathogens and modulate host immunity. In humans, reduced HMO exposure during the first month of life is linked to the development of NEC. When incorporated into infant formula, HMOs promote survival and decrease the formation of NEC lesions in preclinical models. The mechanism underlying such protection
is currently unclear.

We established a model of experimental necrotizing enterocolitis in neonatal mice model.

Changes in HMO concentrations during lactation stages postpartum among 9 secretor mothers, n=8 samples for each lactation stage.
Values are means ± SEM. Mature milk ≥1 month.
*Means differ, P< 0.05. **Means differ, P< 0.005.

Goat milk oligosaccharides (GMO) as a natural source of oligosaccharides

Compared to cows and sheep, the oligosaccharides in goat's milk has a more similar structure and profile to HM. Ranging from 250 and 300 mg/L (approximately 20 times less than human milk, four times greater than bovine milk, ten times greater than sheep. Oligosaccharides isolated from caprine whey showed to have potential prebiotic activity in vitro and rodents with induced experimental colitis. It has been proved that GMOs can improve the intestinal barrier function of epithelial cell co-cultures and enhance mucin gene expression and mucin protein abundance. Five structurally similar oligosaccharides, including 2'-FL and 6'-SL, in stage 1 and stage 2 goat milk-based infant formula, similar to the detection in the fresh goat milk.  As in HM, in goat milk-based infant formula, fucosylated and sialylated oligosaccharides were found to be the dominant oligosaccharides present. Goat milk can be considered as a natural source of oligosaccharides for infants in the prevention of NEC.

Additional new projects recently initiated aims (i) to examine the putative effect of different cannabis derived molecules, in the treatment of obesity and comorbid diseases, and at the same time assess their effect on the control of intestinal microbial composition. We observed a great potential of several cannabinoids on the treatment of obesity and related metabolic diseases focusing mainly on the liver tissue. (ii) A parallel study examined the putative effect of different cannabis derived molecules, in the treatment aimed at reducing inflammation associated with inflammatory bowel disease (IBD). We observed that certain specific phytocannabinoids reduced inflammation in two different cells involved in immunological response associated with IBD. This is observed in an inverse dose dependent manner with lower concentrations exhibiting higher immunological suppression.

Additionally, we have recently demonstrated the effect of the fungal protein Ostreolysin, that can specifically increase brown adipocyte differentiation, prevent weight gain, prevent fatty liver and ameliorates glucose intolerance as a result of high fat diet (HFD), therefore may be used as a future drug for obesity. We created a recombinant version of this protein (rOly).