Commentary - Journal of Cancer Immunology (2020) Volume 2, Issue 3
Prevention of Lung Cancer Growth by Water Extract from Euglena gracilis
Susumu Ishiguro, Jeffrey Comer, Masaaki Tamura*
Department of Anatomy & Physiology, Kansas State University College of Veterinary Medicine, Manhattan, KS 66506, USA
- *Corresponding Author:
- Masaaki Tamura
Received date: July 25, 2020; Accepted date: August 20, 2020
Citation: Ishiguro S, Comer J, Tamura M. Prevention of Lung Cancer Growth by Water Extract from Euglena gracilis. J Cancer
Immunol. 2020; 2(3): 128-132.
Copyright: © 2020 Ishiguro S, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
As a source of novel drugs and dietary supplements, natural products from plants and animals have been studied for more than decades. Unicellular algae, such as Chlorella, Spirulina and Euglena, are such resources. Euglena gracilis is a unicellular green alga found in fresh water. This organism possesses features characteristic of both animals and plants, having the ability to swim by means of flagella and to photosynthesize. This alga contains a rich variety of nutrients including amino acids, carbohydrates, vitamins, and minerals; therefore, it has been used as a nutritional and functional dietary supplement . Furthermore, it has been shown that a dried powder of Euglena species or its extracts in water or polar organic solvents have potential medicinal properties, such as antimicrobial , antimutagenic , anti-inflammatory [3,4], anti-fibrotic , antiviral , anti-obesity [3,7], and antitumor [8-10] activities. It has been hypothesized that carbohydrate granules made by Euglena for energy storage and consisting mainly of β-1,3-glucan, called paramylon, are primarily responsible for these biological activities. However, our recent study demonstrated that a partially purified water extract from Euglena gracilis devoid of mature paramylon granules (referred to hereafter as Euglena water extract or EWE) inhibited the growth of lung cancer cells in culture and in a mouse orthotopic lung carcinoma allograft model . In cell culture, the direct attenuating effect of EWE on lung cancer cell lines was observed in 2-dimentional (2D) and 3-dimentional (3D) spheroid culture. In 3D spheroid culture, spheroid growth of the lung cancer cells was significantly attenuated by EWE treatment compared with the PBS control group. However, EWE did not stop the spheroid growth completely suggesting that the direct cancer cell growth inhibition by EWE may be effective only in the early stage of tumor growth. In a mouse orthotopic lung carcinoma allograft model, orally administered EWE, which was started 3 weeks prior to cancer cell inoculation, prevented growth of lung carcinoma by attenuating immune regulatory cell populations including granulocytes and myeloid-derived suppressor cells (MDSCs). This preventive effect may function not only in early stage of tumorigenesis, but may last into later stages of tumor development in vivo. The notable findings in this study are (1) orally administered EWE that does not contain mature paramylon granules exhibited a preventive effect against lung tumor growth in mice, (2) EWE attenuated populations of myeloid-derived suppressor cells in cell culture and granulocyte populations in peripheral blood of mice. Both of these cell types (myeloid-derived suppressor cells and granulocytes) play an important role in immune suppression; hence, reduction in their populations may be linked to enhanced immune activity against cancer. Together, these results suggest that Euglena water extract contains a cancer growth prevention agent. However, many questions remain about the nature of this agent including: (1) Is this anti-cancer activity due to a single component of Euglena gracilis or might it be a synergistic effect of multiple components? (2) What is the chemical nature of this agent or agents? (3) Is there involvement of β-1,3-glucan (the major component of paramylon) in this unique bioactivity? (4) How does this agent attenuate granulocyte genesis? (5) Is intestinal microbiota involved in induction of this bioactivity? and (6) What is the overall effect of this agent on animal health? In this commentary, we discuss evidence suggesting that paramylon (and the β-1,3-glucan of which it consists) may not be primarily responsible for EWE bioactivity, evidence indicating the involvement of intestinal microbiota, and the overall effect of EWE on animal health. The exact chemical nature of the bioactive agent and potential mechanisms by which EWE attenuates granulocyte genesis will be addressed in future work.
The lack of involvement of β-1,3-glucan and
paramylon in lung cancer prevention by EWE
Paramylon consists of water-insoluble β-1,3-glucans having molecular masses on the order of 500 kDa. The size of paramylon granules ranges from 1-6 μm in diameter and they typically constitute approximately 80% of Euglena body weight [1,11]. Due to their large size and solid granular shapes, paramylon can be purified easily by low speed centrifugation from a suspension of whole Euglena dry powder in water . It has been suggested that paramylon acts as an immunostimulant or an immunopotentiator [12-14]. In previous studies, paramylon was shown to stimulate production of proinflammatory cytokines, such as IL-1, IL-6, tumor necrosis factor (TNF), etc. [12-14]. In our study, we demonstrated that Euglena water extract retained its cancer-prevention activity even after removal of a majority (if not all) of the paramylon granules by multistep centrifugation and filtration through a 0.22 μm pore size filter . However, it is impossible to rule out a potential contamination with the immature paramylon granules with less than 0.2 μm diameter and water soluble β-1,3-glucan oligomers in the EWE. Gissibl et al. reported that paramylon-associated bioactivity was maximized when the paramylon granules were hydrolyzed by heat, enzymes, or acid treatment into water-soluble short chain β-1,3-glucan oligomers . Indeed, they detected increased bioavailability of hydrolyzed paramylon and better protection against infection by Staphylococcus aureus or Candida albicans
via increased serum IL-12 production, resulting in longer survival of mice. However, since a heated water extract prepared from purified paramylon granules did not show any lung cancer growth prevention in a parallel mouse study (our unpublished data), the EWE-dependent prevention of lung cancer growth that we observed is not likely attributable to contaminated paramylon nor watersoluble β-1,3-glucan oligomers.
Involvement of intestinal microbiota in lung
cancer prevention by EWE
Although the lung cancer preventive agent in the EWE has yet to be identified, involvement of intestinal microbiota or their metabolites in its cancer prevention is postulated. Because EWE was administered through drinking water in our study, it is conceivable that the EWE altered both the quantity and quality of the intestinal microbiota as well as its metabolites. The intestinal microbiota of humans, typically consisting of about 1014 living organisms [16,17], has been shown to generate large amounts of various metabolic products including substances beneficial for host health (vitamins, short-chain fatty acids (SCFAs), etc.), as well as other substances in potentially harmful quantities (amines, hydrogen sulfide) . It has been shown that the intestinal
microbiota and its metabolites have an important role in maintaining host homeostasis and health [18,19]. Among
these intestinal microbiota metabolites, SCFAs can act as
ligands for G protein-coupled receptors (GPCRs) .
Because GPCRs are involved in many essential biological
reactions, GPCRs are often targets for therapeutic drugs
[21,22]. Furthermore, SCFAs are known to influence the
development and function of the immune system .
SCFAs reduce expression of T cell-activating molecules in
innate immune cells such as macrophages and dendritic
cells by inducing histone deacetylase (HDAC) inhibition.
This HDAC inhibition by SCFAs also influences peripheral
T cells, particularly regulatory T (Treg) cells. In a mouse
study, HDAC inhibition by SCFAs increased forkhead
box P3 (FOXP3) expression in Treg cells and enhanced
the suppressive function of FOXP3+ Treg cells, following amplified Treg cell-mediated attenuation of colitis .
These mechanisms are crucial for maintaining immune
homeostasis. In addition, Harusato et al. demonstrated
that exposure to beneficial microbiota in early-life was
important in establishing intestinal homeostasis that
restrains colon cancer in adulthood . This regulation against colon cancer appears to be associated with an alteration of granulocytic MDSC in colonic lamina propria. Another investigation demonstrated that an intestinal microbiota component species, Fusobacterium
nucleatum, contributed to promotion of growth of
colorectal cancer by increasing MDSC in the tumor site
. Since EWE-induced lung cancer growth prevention
required preliminary administration of the EWE three
weeks prior to allografting, no lung cancer growth
prevention was observed when the EWE was administered
after allografting. We suspect that the effect of EWE is
primarily by alteration of immune system homeostasis.
Due to accumulated experimental evidence that oral
administration of micro-algae alters the intestinal
microbiota as well as its metabolites [27,28], EWEdependent
lung cancer growth prevention is potentially
mediated by these alterations in intestinal microbiota and
Neutrophils in lung cancer prevention by EWE
Oral administration of EWE significantly attenuated the granulocyte population in mouse peripheral blood. In addition, this administration also directly attenuated granulocytic MDSCs in a primary cell culture with mouse bone marrow cells . Neutrophils, which account for a majority of the granulocytes, play a fundamental role in the innate immune response. Neutrophils immediately migrate to the inflammation site and kill invading bacterial and fungal species through phagocytosis, by release of preformed granular enzymes and proteins, and by the production of a range of reactive oxygen species . It is also known that tumor-associated neutrophils (TANs) play important roles in the tumor microenvironment, having both pro- and anti-tumor effects . Transforming growth factor-β (TGF-β), an immunosuppressive cytokine
released from tumors, transforms tumor-infiltrating
neutrophils to an N2 pro-tumorigenic phenotype, while
they are transformed back into an N1 anti-tumorigenic
phenotype by a TGF-β blockade. The N2 TANs attenuate
T cell activity, therefore, promoting tumor growth . A
high neutrophil-lymphocyte ratio in both peripheral blood
[31-34] and in the tumors [35-37] is highly correlated
with poor prognosis in chemo- and immunotherapy in
patients with malignant tumors. Therefore, the decrease
of the granulocyte population in mouse peripheral blood
observed in our study may suggest that EWE intake
generates a favorable condition for antitumor immunity
in the host. No leukocyte analysis in the tumor tissues
was carried out in this study. Future work to compare the
immune cell populations in both peripheral blood and
tumor tissues in mice with or without EWE treatment may
provide a better understanding of the antitumor effect of
EWE. On the other hand, a large decrease in the neutrophil
population of peripheral blood, called neutropenia, causes
severe health problems in both human and animals [38,39].
Neutropenia patients have an increased risk of infection
by bacterial and fungal species. In cancer, treatment with
radiation therapy, chemotherapy, or immunotherapy
sometimes cause severe infectious disease due to
neutropenia [40,41]. No noticeable health problems were
observed over five to six weeks of EWE administration in
our mouse studies. The effect of EWE administration on
the granulocyte genesis should be studied carefully in the
future using other animal species since the neutrophil
population in mouse peripheral blood constitutes only
15% of the whole leukocyte population (, average of 42
inbred strains of mice), which is significantly smaller than
the 70% fraction in human peripheral blood .
The water extract from Euglena gracilis dry powder devoid of mature paramylon granules, abbreviated EWE, prevented the growth of lung carcinoma in mice. Oral administration of the EWE may have altered intestinal microbiota and metabolites and attenuated granulocytic MDSC and granulocyte in peripheral blood, thereby preventing lung cancer growth in mice. Although its chemical nature is yet to be determined, a water soluble β-1,3-glucan or paramylon is not likely to be the cause of this bioactivity. It is urgently important to identify this bioactive substance and study both the safety and utility of EWE-dependent cancer growth prevention.
Authors acknowledge financial supports of Kansas State University Johnson Cancer Research Center [2017 JCRCURA and 2018 JCRC-URA] (MT), College of Veterinary Medicine Dean’s funds [2018 CVM-SMILE] (MT and JC), euglena research funds [2016 euglena-1] (MT and JC), and K-INBRE bridging grant [P20 GM103418] (MT and JC).
- Gissibl A, Sun A, Care A, Nevalainen H, Sunna
A. Bioproducts from Euglena gracilis: Synthesis
and applications. Frontiers in Bioengineering and
Biotechnology. 2019 May 15;7:108..
- Das BK, Pradhan J, Pattnaik P, Samantaray BR, Samal
SK. Production of antibacterials from the freshwater alga
Euglena viridis (Ehren). World Journal of Microbiology
and Biotechnology. 2005 Feb 1;21(1):45-50.
- Okouchi R, Yamamoto K, Ota T, Seki K, Imai M,
Ota R, et al. Simultaneous intake of Euglena gracilis
and vegetables exerts synergistic anti-obesity and antiinflammatory
effects by modulating the gut microbiota in
diet-induced obese mice. Nutrients. 2019 Jan;11(1):204.
- Sakanoi Y, Yamamoto K, Ota T, Seki K, Imai M, Ota
R, et al. Simultaneous intake of euglena gracilis and
vegetables synergistically exerts an anti-inflammatory
effect and attenuates visceral fat accumulation by affecting
gut microbiota in mice. Nutrients. 2018 Oct;10(10):1417.
- Nakashima A, Sugimoto R, Suzuki K, Shirakata Y,
Hashiguchi T, Yoshida C, et al. Anti-fibrotic activity of
Euglena gracilis and paramylon in a mouse model of nonalcoholic
steatohepatitis. Food Science & Nutrition. 2019
- Nakashima A, Suzuki K, Asayama Y, Konno M, Saito K,
Yamazaki N, et al. Oral administration of Euglena gracilis
Z and its carbohydrate storage substance provides survival
protection against influenza virus infection in mice.
Biochemical and Biophysical Research Communications.
2017 Dec 9;494(1-2):379-83.
- Sugimoto R, Ishibashi-Ohgo N, Atsuji K, Miwa Y,
Iwata O, Nakashima A, et al. Euglena extract suppresses
adipocyte-differentiation in human adipose-derived stem
cells. PloS one. 2018 Feb 15;13(2):e0192404.
- Panja S, Ghate NB, Mandal N. A microalga, Euglena
tuba induces apoptosis and suppresses metastasis in
human lung and breast carcinoma cells through ROSmediated
regulation of MAPKs. Cancer Cell International.
- Quesada LA, LUSTIG ES, MARECHAL LR,
BELOCOPITOW E. Antitumor activity of paramylon on
sarcoma-180 in mice. GANN Japanese Journal of Cancer
Research. 1976 Jun 30;67(3):455-9.
- Watanabe T, Shimada R, Matsuyama A, Yuasa M,
Sawamura H, Yoshida E, et al. Antitumor activity of the
ß-glucan paramylon from Euglena against preneoplastic
colonic aberrant crypt foci in mice. Food & Function.
- Ishiguro S, Upreti D, Robben N, Burghart R, Loyd
M, Ogun D, et al. Water extract from Euglena gracilis
prevents lung carcinoma growth in mice by attenuation
of the myeloid-derived cell population. Biomedicine &
Pharmacotherapy. 2020 Jul 1;127:110166.
- KONDO Y, KATO A, HOJO H, NOZOE S, TAKEUCHI
M, OCHI K. Cytokine-related immunopotentiating
activities of Paramylon, a ß-(1 3)-D-glucan from
Euglena gracilis. Journal of Pharmacobio-Dynamics.
- Phillips FC, Jensen GS, Showman L, Tonda R,
Horst G, Levine R. Particulate and solubilized ß-glucan
and non-ß-glucan fractions of Euglena gracilis induce proand
anti-inflammatory innate immune cell responses and
exhibit antioxidant properties. Journal of Inflammation
- Russo R, Barsanti L, Evangelista V, Frassanito AM,
Longo V, Pucci L, et al. Euglena gracilis paramylon activates
human lymphocytes by upregulating pro-inflammatory
factors. Food Science & Nutrition. 2017 Mar;5(2):205-14.
- Gissibl A, Care A, Parker LM, Iqbal S, Hobba G,
Nevalainen H, et al. Microwave pretreatment of paramylon
enhances the enzymatic production of soluble ß-1,
3-glucans with immunostimulatory activity. Carbohydrate
polymers. 2018 Sep 15;196:339-47.
- Kho ZY, Lal SK. The human gut microbiome–a
potential controller of wellness and disease. Frontiers in
Microbiology. 2018 Aug 14;9:1835.
- Thursby E, Juge N. Introduction to the human gut
microbiota. Biochemical Journal. 2017 Jun 1;474(11):1823-36.
- Ercolini D, Fogliano V. Food design to feed the
human gut microbiota. Journal of Agricultural and Food
Chemistry. 2018 Mar 22;66(15):3754-8.
- Visconti A, Le Roy CI, Rosa F, Rossi N, Martin
TC, Mohney RP, et al. Interplay between the human
gut microbiome and host metabolism. Nature
Communications. 2019 Oct 3;10(1):1-0.
- Husted AS, Trauelsen M, Rudenko O, Hjorth SA,
Schwartz TW. GPCR-mediated signaling of metabolites.
Cell Metabolism. 2017 Apr 4;25(4):777-96.
- Hauser AS, Chavali S, Masuho I, Jahn LJ, Martemyanov
KA, Gloriam DE, et al. Pharmacogenomics of GPCR drug
targets. Cell. 2018 Jan 11;172(1-2):41-54.
- Thompson MD, Cole DE, Capra V, Siminovitch KA,
Rovati GE, Burnham WM, et al. Pharmacogenetics of the G
protein-coupled receptors. InPharmacogenomics in Drug
Discovery and Development 2014 (pp. 189-242). Humana
Press, New York, NY.
- Rooks MG, Garrett WS. Gut microbiota, metabolites
and host immunity. Nature Reviews Immunology. 2016
- Tao R, De Zoeten EF, Özkaynak E, Chen C, Wang
L, Porrett PM, et al. Deacetylase inhibition promotes
the generation and function of regulatory T cells. Nature
Medicine. 2007 Nov;13(11):1299-307.
- Harusato A, Viennois E, Etienne-Mesmin L,
Matsuyama S, Abo H, Osuka S, et al. Early-life microbiota
exposure restricts myeloid-derived suppressor cell–driven
colonic tumorigenesis. Cancer Immunology Research.
2019 Apr 1;7(4):544-51.
- Dong X, Pan P, Zheng DW, Bao P, Zeng X, Zhang
XZ. Bioinorganic hybrid bacteriophage for modulation
of intestinal microbiota to remodel tumor-immune
microenvironment against colorectal cancer. Science
Advances. 2020 May 1;6(20):eaba1590.
- de Jesus Raposo MF, De Morais AM, De Morais RM.
Emergent sources of prebiotics: seaweeds and microalgae.
Marine Drugs. 2016 Feb;14(2):27.
- Hu J, Li Y, Pakpour S, Wang S, Pan Z, Liu J, et al. Dose
Effects of Orally Administered Spirulina Suspension on
Colonic Microbiota in Healthy Mice. Frontiers in Cellular
and Infection Microbiology. 2019 Jul 5;9:243.
- Summers C, Rankin SM, Condliffe AM, Singh N,
Peters AM, Chilvers ER. Neutrophil kinetics in health and
disease. Trends in Immunology. 2010 Aug 1;31(8):318-24.
- Fridlender ZG, Sun J, Kim S, Kapoor V, Cheng G,
Ling L, et al. Polarization of tumor-associated neutrophil
phenotype by TGF-ß:“N1” versus “N2” TAN. Cancer Cell.
2009 Sep 8;16(3):183-94.
- Faria SS, Fernandes Jr PC, Silva MJ, Lima VC, Fontes
W, Freitas-Junior R, et al. The neutrophil-to-lymphocyte
ratio: a narrative review. ecancermedicalscience. 2016;10.
- Mei Z, Shi L, Wang B, Yang J, Xiao Z, Du P, et al.
Prognostic role of pretreatment blood neutrophilto-
lymphocyte ratio in advanced cancer survivors: a
systematic review and meta-analysis of 66 cohort studies.
Cancer Treatment Reviews. 2017 Jul 1;58:1-3.
- Sacdalan DB, Lucero JA, Sacdalan DL. Prognostic
utility of baseline neutrophil-to-lymphocyte ratio in
patients receiving immune checkpoint inhibitors: a review
and meta-analysis. OncoTargets and Therapy. 2018;
- Templeton AJ, McNamara MG, Šeruga B, Vera-Badillo
FE, Aneja P, Ocaña A, et al. Prognostic role of neutrophilto-
lymphocyte ratio in solid tumors: a systematic review
and meta-analysis. JNCI: Journal of the National Cancer
Institute. 2014 Jun 1;106(6).
- Cha YJ, Park EJ, Baik SH, Lee KY, Kang J. Clinical
significance of tumor-infiltrating lymphocytes and
neutrophil-to-lymphocyte ratio in patients with stage III
colon cancer who underwent surgery followed by FOLFOX
chemotherapy. Scientific Reports. 2019 Aug 12;9(1):1-9.
- Mandelli GE, Missale F, Bresciani D, Gatta LB, Scapini
P, Caveggion E, et al. Tumor infiltrating neutrophils are
enriched in basal-type urothelial bladder cancer. Cells.
- Wang J, Jia Y, Wang N, Zhang X, Tan B, Zhang G, et al.
The clinical significance of tumor-infiltrating neutrophils
and neutrophil-to-CD8+ lymphocyte ratio in patients with
resectable esophageal squamous cell carcinoma. Journal
of Translational Medicine. 2014 Dec 1;12(1):7.
- Schnelle AN, Barger AM. Neutropenia in dogs and
cats: causes and consequences. Veterinary Clinics: Small
Animal Practice. 2012 Jan 1;42(1):111-22.
- Schwartzberg LS. Neutropenia: etiology and
pathogenesis. Clinical Cornerstone. 2006 Jan 1;8:S5-11.
- Lustberg MB. Management of neutropenia in cancer
patients. Clinical Advances in Hematology & Oncology:
H&O. 2012 Dec; 10(12):825.
- Ménétrier-Caux C, Ray-Coquard I, Blay JY, Caux
C. Lymphopenia in Cancer Patients and its Effects
on Response to Immunotherapy: an opportunity for
combination with Cytokines?. Journal for Immunotherapy
of Cancer. 2019 Dec; 7(1):1-5.
- Peters LL, Cheever EM, Ellis HR, Magnani PA,
Svenson KL, Von Smith R, et al. Large-scale, highthroughput
screening for coagulation and hematologic
phenotypes in mice. Physiological Genomics. 2002 Dec
- Dancey JT, Deubelbeiss KA, Harker LA, Finch
CA. Neutrophil kinetics in man. The Journal of Clinical
Investigation. 1976 Sep 1;58(3):705-15.