Basic Study
Copyright ©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Nov 21, 2020; 26(43): 6782-6794
Published online Nov 21, 2020. doi: 10.3748/wjg.v26.i43.6782
Lactobacillus bulgaricus inhibits colitis-associated cancer via a negative regulation of intestinal inflammation in azoxymethane/dextran sodium sulfate model
Denise Sayuri Calheiros Silveira, Luciana Chain Veronez, Luís Carlos Lopes-Júnior, Elen Anatriello, Mariângela Ottoboni Brunaldi, Gabriela Pereira-da-Silva
Denise Sayuri Calheiros Silveira, Luciana Chain Veronez, Department of Biochemistry and Immunology, University of São Paulo at Ribeirão Preto Medical School, Ribeirão Preto 14049-900, SP, Brazil
Luís Carlos Lopes-Júnior, Health Sciences Center, Federal University of Espírito Santo - UFES, Vitória 29043-900, ES, Brazil
Elen Anatriello, Institute of Science and Technology, Federal University of São Paulo, UNIFESP, São José dos Campos 12231-280, SP, Brazil
Mariângela Ottoboni Brunaldi, Department of Pathology and Forensic Medicine, University of São Paulo, Ribeirão Preto 14040-902, SP, Brazil
Gabriela Pereira-da-Silva, Department of Maternal-Infant Nursing and Public Health, University of São Paulo at Ribeirão Preto College of Nursing, Ribeirão Preto 14040-902, SP, Brazil
Author contributions: Silveira DSC and Pereira-da-Silva G contributed substantially to the design of the study; Veronez LC and Silveira DSC were in charge of the experimental protocol, analysis and interpretation of the results; Lopes-Júnior LC and Anatriello E contributed to interpretation of the results, statistical analysis and discussion of the manuscript; Brunaldi MO performed the histopathological analyzed as well as interpreted the data; all authors interpreted the data and have contributed to writing, discussion and revised the manuscript critically; and all authors have given final approval of the version of the article to be published; Silveira DSC and Pereira-da-Silva G had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.
Supported by Brazilian National Council for Scientific and Technological Development (CNPq), No. 140152/2013-0.
Institutional review board statement: All authors declare that the Institutional Review Board approval was not applicable for this manuscript, once this study does not involve human beings.
Institutional animal care and use committee statement: All animal experiments conformed to the National Council for Animal Experiment Control accepted principles for the care and use of laboratory animals [ethics committee on the use of animals (CEUA), protocol No. 14.1.418.53.1].
Conflict-of-interest statement: The authors declare that they have no competing interests.
Data sharing statement: No additional data are available.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
Corresponding author: Gabriela Pereira-da-Silva, BSc, MSc, PhD, Professor, Research Scientist, Department of Maternal-Infant Nursing and Public Health, University of São Paulo at Ribeirão Preto College of Nursing, Av. Bandeirantes, 3900, Ribeirão Preto 14049-900, SP, Brazil.
Received: May 7, 2020
Peer-review started: May 7, 2020
First decision: May 15, 2020
Revised: May 28, 2020
Accepted: October 1, 2020
Article in press: October 1, 2020
Published online: November 21, 2020
Research background

Intestinal inflammatory disorders are associated with the infiltration of immune cells and the proinflammatory release of cytokines that play a critical role in the onset and progression of colitis-associated cancer (CAC). Recent studies suggested that the intestinal microbiota has an essential role in carcinogenesis. Probiotic supplementation is an alternative means of favorably modulating the intestinal microbiota. Currently, it has become increasingly evident that intestinal microbiota plays a crucial role in the pathogenesis of inflammatory bowel diseases (IBD) and colorectal cancer. Moreover, increasing evidence suggests that probiotics prevent inflammation and carcinogenesis and several bacteria strains have been used for the prevention and treatment of the infectious colitis, IBD. Thus, probiotic modulation of intestinal microbiota has emerged as a potential chemo-preventive agent.

Research motivation

Although supplementation with probiotics have been reported to prevent CAC, little is known about the administration of strains of Lactobacillus bulgaricus (L. bulgaricus), as well as their impact on neoplastic changes in the intestinal mucosa. Our study may contribute to address the gaps in the literature of how this probiotic, dose and supplementation time used for this experimental model impact on colitis, serum cytokines and neoplastic development.

Research objectives

The purpose of this study is to investigate the effect of the probiotic L. bulgaricus during the development of an experimental model of CAC. Overall, this study intents to strengthen data from preclinical studies, encouraging clinical trials to investigate their role in preventing colitis and CAC in humans.

Research methods

We used an experimental model of CAC. For mice treatment, 1 × 109 CFU were diluted in 200 μL of PBS and orally given to each mouse, 3 times a week during all experimental period. Prior to tumor induction, C57BL/6 mice were randomly distributed in 2 groups (n = 10) and treated with PBS (control group) or L. bulgaricus (Lb group) by gavage (0.2 mL/mouse) for one week. For CAC induction, mice were intraperitoneally (i.p.) injected with a single dose (10 mg/kg in 300 μL solution) of azoxymethene (Sigma-Aldrich), followed by 3 cycles of one week of 2.5% dextran sulfate sodium (DSS) diluted in drinking water intercalated for 2 wk of normal water. Mice were euthanized 12th week after CAC induction. Intestinal inflammation in vivo, or disease score, was determined by scoring clinical signs. The severity of intestinal inflammation was assessed by measuring the length of the entire large intestine. Also, the dimensions of the colorectal tumors were measured with pachymeter and the volumes were calculated by the formula: (width)2 × length/2. For histological analysis, distal colon parts were fixed in 4% p-formaldehyde in phosphate-buffered formalin and unblocked in paraffin. Tissue sections (4.0 μm) were prepared from the paraffin-embedded tissue blocks, stained with hematoxylin and eosin and evaluated in a blinded fashion by an experienced pathologist. Cytokines levels were determined from colon and/or tumor samples by ELISA. Statistical analyses were performed using GraphPad Prism version 6.0. A 2-tailed P value < 0.05 was considered to be statistically significant.

Research results

We have shown that L. bulgaricus treatment inhibited the total tumor volume and mean size of tumors. Although we did not observe differences in body weight loss between control and L. bulgaricus-treated, we found differences in clinical signals in L. bulgaricus-treated mice, which showed a lower clinical score on the 13th and 15th days after tumor initiation. In addition to the attenuation of intestinal inflammation score, we observed that the treatment with L. bulgaricus reduced the DSS-induced shortening of the colon. In segments of the large intestine that did not present tumors (inflamed colon) we also observed a reduction of at least 2-fold in the levels of the cytokines TNF-α, IL-1β, IL-23 and IL-17 in L. bulgaricus-treated mice in comparison to controls. In contrast, increased concentrations of IFN-γ were also observed in Lb group. Regarding the cytokines measured in tumor tissues, we observed a pattern similar to that found in the inflamed colon with a negative regulation of proinflammatory cytokines in mice treated with the probiotic and an increase in IFN-γ levels in this group. Overall, these findings highlight the protective effect of L. bulgaricus in the regulation of gut inflammation and preventing CAC development. Thus, further clinical trials are needed to confirm these preclinical insights.

Research conclusions

We found an anti-inflammatory role and consequent antitumor effect of L. bulgaricus on CAC that may be used as a promising tool for the prevention and treatment of CAC. In summary, L. bulgaricus treatment during colitis-associated colorectal carcinogenesis model may be responsible for anti-inflammatory and antitumor role by lowering proinflammatory cytokine expression.

Research perspectives

The present study has shown that L. bulgaricus inhibited CAC via a negative regulation of intestinal inflammation. Hence, has demonstrates promising evidence on L. bulgaricus probiotic has a preventive potential in CAC development. Therefore, clinical trials are needed to confirm this hypothesis and increase the therapeutic arsenal against CAC.