Abstract.
Background and Purpose: Colorectal cancer (CRC) is one of the
cancers with the highest incidence in which APC gene mutations occurs in
almost 80% of patients. This mutation leads to β-catenin aberrant
accumulation and an uncontrolled proliferation. Apoptosis evasion,
changes in the immune response and microbiota composition are also
events that arise in CRC. Tetracyclines are drugs with proven antibiotic
and immunomodulatory properties that have shown cytotoxic activity
against different tumor cell lines. Experimental Approach: The
effect of tigecycline was evaluated in vitro in HCT116 cells and in vivo
in a colitis-associated colorectal cancer (CAC) murine model.
5-fluorouracil was assayed as positive control in both studies.Key Results: Tigecycline showed an antiproliferative activity
targeting the Wnt/β-catenin pathway and downregulating STAT3. Moreover,
tigecycline induced apoptosis through extrinsic, intrinsic and
endoplasmic reticulum pathways converging on an increase of CASP7
levels. Furthermore, tigecycline modulated the immune response in CAC,
reducing the cancer-associated inflammation through a downregulation of
cytokines expression. Additionally, tigecycline favored the cytotoxic
activity of CD8+ T lymphocytes, one of the main immune defenses against
tumor cells. Lastly, the antibiotic reestablished the gut dysbiosis in
CAC mice increasing the abundance of bacterial genera and species, such
as Akkermansia and Parabacteroides distasonis , that act as
protectors against tumor development. These findings resulted in a
reduction of the numbers of tumors and an amelioration of the
tumorigenesis process in CAC. Conclusion and Implications :
tigecycline exerts a beneficial effect against CRC supporting the use of
this antibiotic for the treatment of this disease.
Keywords: colitis-associated colorectal cancer, tigecycline,
β-catenin, CD8+ T lymphocytes, microbiota.
Abbreviations: activating transcription factor 6, ATF6;
azoxymethane, AOM; Bcl-2-associated X protein, BAX; C/EBP homologous
protein, DDIT3; cancer stem cells, CSCs; colorectal associated-cancer,
CAC; Colorectal cancer, CRC; dextran sulfate sodium, DSS; disease
activity index, DAI; endoplasmic reticulum, ER; immunoglobulin heavy
chain-binding protein, HSPA5; inflammatory bowel disease, IBD;
Jun-N-terminal kinase, JNK; membrane permeabilization, MOMP; mesenteric
lymph nodes, MLN; metalloproteinases, MMP; Principal Coordinates
Analysis, PCoA; Ribosomal Database Project, RDP; Truncation of BID,
tBID; tumor protein 53, TP53; β-catenin, CTNNB1.
Bullet point summary: Tetracyclines displays immunomodulatory
properties and cytotoxic activity in tumor cell lines.
Tigecycline decreases tumorigenesis in vivo inhibiting cell
proliferation and modulating immune response and gut dysbiosis.
Tigecycline effects in experimental colorectal cancer supports its
consideration for human colorectal cancer management.
Data availability statement: The data that support the findings
of this study are available from the corresponding authors upon
reasonable request. Some data may not be made available because of
privacy or ethical restrictions.
Funding statement: This work was funded by the Junta de
Andalucía (CTS 164); the Instituto de Salud Carlos III (Spain) and Fondo
Europeo de Desarrollo Regional (FEDER), from the European Union, through
the research grants PI18/00826, P18-RT-4930, PI0206-2016, PIE16/00045
and PI19/01058; Spanish Ministry of Science and Innovation
(MCIN/AEI/10.13039/501100011033/FEDER) under grant RTI2018-101309-B-C22;
and the Chair “Doctors Galera-Requena in cancer stem cell research”
(CMC-CTS963). A.J. R-M L. H-G are predoctoral fellows funded by the
Spanish Ministry of Science and Innovation (“Programa de Doctorado:
Medicina Clínica y Salud Pública” B12.56.1). M.J. R-S and J.A. M-T are
predoctoral fellows from the Instituto de Salud Carlos III (FI17/00176).
P. D-E is a postdoctoral fellow of Junta de Andalucía (P18-RT-4930). T.
V and J. G-M are postdoctoral fellows from the University of Granada.
CIBER-EHD is funded by the Instituto de Salud Carlos III.
Author contribution statement: All authors performed the
experiments and contributed to the acquisition. F.G., A.J.R.-M. and
A.R.-N. carried out the microbiome studies. A.J.R.-M., M.E.R.-C.,
A.R.-N. and J.G. designed the experiments. All authors drafted the
manuscript, read, and approved the final manuscript.
Conflict of interest disclosure: The authors have declared no
conflicting interests.
INTRODUCTION
Colorectal cancer (CRC) is the second most diagnosed cancer in women and
the third in men. Moreover, CRC is the third most deadly among cancers,
responsible for nearly a million deaths in 2020 (GLOBOCAN, 2020). Whilst
recent developments/advances in the screening and treatment have reduced
the mortality, the survival rate remains low, especially for metastatic
CRC, with a 5-year survival rate of 12% (Xie et al. , 2020).
Furthermore, there is an elevated percentage of patients that do not
respond to the current treatments or show important adverse effects, so
the development of new adjuvant therapies against CRC is still
necessary. Different risk factors contribute to the development of CRC,
from the patient’s genetic background to aging, intestinal inflammation,
diet and other environmental factors (Carethers et al. , 2015).
The accumulation of genetic mutations leads to an altered balance
between the activation of oncogenes and the reduction of tumor
suppressor genes (Fearon et al. , 1990). For instance, the
mutations in the APC gene in the intestinal epithelium is an early event
that occurs in almost 80% of CRC patients (Korinek et al. , 1997)
leading to β-catenin aberrant activation and increasing cell
proliferation (Shang et al. , 2017). In addition, a
proinflammatory environment contributes to potentiate cell
proliferation, angiogenesis and cancer progression (Terzic et
al. , 2010). In fact, patients with inflammatory bowel disease (IBD)
have a significantly elevated risk of CRC development (Clarke et
al. , 2019).
Moreover, CRC tumorigenesis and progression has been clearly associated
with an imbalanced gut microbiome, commonly termed as dysbiosis. Thus,
changes in gut microbiome abundance have been described in patients with
CRC (Garrett, 2019) evidencing a critical role for specific bacteria in
colon tumorigenesis, like Fusobacterium nucleatum andBacteroides fragilis (Tjalsma et al. , 2012), as well as, a
depletion of some bacterial species like Faecalibacterium
prausnitzii (Lopez-Siles et al. , 2016).
Considering the involvement of the microbiome in the initiation and
progression of CRC, the modulation of the gut microbiota can be
considered as a potential strategy for prevention and treatment of CRC
(Fong et al. , 2020). In this regard, tetracyclines are a family
of broad-spectrum antibiotics that have shown beneficial effects against
intestinal inflammation (Garrido-Mesa et al. , 2018) since they
present immunomodulatory properties in addition to their antibiotic
activity as it is the case of tigecycline. Besides, these antibiotics
have shown cytostatic and cytotoxic activity against different tumor
cell lines (Kroon et al. , 1984), as well as against metastasis
due to their ability to inhibit metalloproteinases (MMP) (Saikaliet al. , 2003). Therefore, it is interesting to explore their
potential application for the treatment of CRC. Consequently, in the
present study, we evaluated the antitumor effect of tigecycline using a
combination of in vitro and in vivo studies carried out on
tumoral cells and azoxymethane (AOM) / dextran sulfate sodium (DSS)
murine model, respectively.
MATERIAL AND METHODS.
Chemicals, cell culture
and treatment.
The human colon cancer cell lines HCT116 and Caco-2 were obtained from
the Cell Culture Unit of the University of Granada (Granada, Spain).
NCM356 human colonic epithelial cells were kindly provided by Laura
Medrano González and Ezra Aksoy (William Harvey Research Institute,
Queen Mary University of London, London, UK).
Cells were cultured and treated with tigecycline (Tygacil®, Pfizer, New
York, USA), Wnt3a (Sigma-Aldrich, Madrid, Spain) and 5-fluorouracil
(5-FU) (Accord Farma, Polanco, Mexico) as described in Supplementary
Material and Methods. RNA and cytoplasmic and nuclear proteins were
isolated for Taqman qPCR and Western blot, respectively.
Cell proliferation assay.
HCT116 and NCM356 cells were treated with different doses of tigecycline
(1 µM - 75 µM) or 5-FU (5 µM - 100 µM) for 48 h. After that, cell
viability was measured using an MTS assay (CellTiter 96® AQueous One
Solution Cell Proliferation Assay, Promega, Madison, USA) according to
the manufacturer’s instructions.
Colony Formation Assay.
HCT116, Caco-2 and NCM356 cells were plated into 6-well plates at 200
cells/well for HCT116 and 400 cells/well for Caco-2 and NCM356. From the
following day, fresh media was added supplemented with different doses
of tigecycline and 5-FU, every 48 h for 1 week, when the media was
removed, the cells were fixed with absolute ethanol and the colonies
were stained with 2.3% crystal violet and counted with ImageJ software
(Free Software Foundation Inc) after taking the images.
Annexin V and propidium iodide (PI) assay.
Apoptosis and necrosis were evaluated by flow cytometry using FITC
Annexin V apoptosis detection kit with PI (Immunostep, Salamanca,
Spain). HCT116 cells were treated with different concentrations of
tigecycline (1 µM - 50 µM) for 48 h. After the treatment, attached cells
and those present in the supernatant were collected, washed with cold
PBS and stained with FITC-Annexin V and PI. Labeled cells were acquired
on a BD FACsAria Ill (BD Biosciences, Becton, Dickinson and Company,
Franklin Lakes, NY, USA) and data were analyzed using the FlowJo v10.6.2
software (FlowJo LLC, Ashland, OR, USA).
TUNEL Assay
In order to confirm the apoptotic capacity of the treatments, a TUNEL
assay was carried out in HCT116 cells treated with tigecycline or 5-FU
for 48 h (n=3) with the TUNEL assay kit-HRP DAB ab206386 (Abcam,
Cambridge, UK) following the manufacturer’s instruction
Apoptosis was also evaluated using the DeadEnd Fluorometric TUNEL system
(Promega, Madison, USA) in colonic tissue sections following the
manufacturer’s indications.
Full description of the methods can be found in Supplementary Material
and Methods.
CAC murine model, DAI evaluation and macroscopic data analysis .
All mice studies were performed following the ‘Guide for the Care and
Use of Laboratory Animals’ as promulgated by the National Institute of
Health and the protocols approved by the Ethic Committee of Laboratory
Animals of the University of Granada (Spain) (Ref. No.23/10/2019/174).
C57Bl/6J female mice (7-9 weeks old) from Charles River (Barcelona,
Spain) were housed in groups of five mice in each makrolon cage, with an
air-conditioned atmosphere, a 12 h light-dark cycle and provided with
free access to tap water and food. They were subjected to a process of
CAC induction by administering one intraperitoneal initial dose of AOM
(Sigma-Aldrich, Madrid, Spain) at 10 mg/kg followed by three cycles of
DSS (36-50 KDa, MP Biomedicals, Illkirch Cedex, France) added in the
drinking water. Each DSS cycle consisted of 2% DSS for 1 week followed
by a recovery period of 2 weeks with normal drinking water. Mice were
divided into five groups: healthy control, colorectal associated-cancer
(CAC) control, and three CAC groups treated daily with tigecycline 25
mg/kg or tigecycline 50 mg/kg by oral gavage or every three days with
5-FU 15 mg/kg intraperitoneally. The treatment started 50 days after the
beginning of the assay, on the day of initiation of the third cycle of
DSS and lasted for 7 weeks. The control groups received PBS following
the same protocol.
A detailed description of the procedures can be found in Supplementary
Material and Methods. Briefly, the disease activity index (DAI)
(Camuesco et al. , 2012) was determined daily in each DSS cycle,
and the day before euthanasia, both tumor number and size was assessed
via colonoscopy and a tumor score was given as described by Becker C et
al., (Becker et al. , 2006) by a blind observer.
When mice were sacrificed spleen,
mesenteric lymph nodes (MLN) and colon were aseptically removed. The
bowels were open longitudinally, the macroscopic tumors were counted,
and images were acquired for measuring the size of each tumor with the
ImageJ software. Colon samples were taken for RT-qPCR, Western blot,
flow cytometry and fixed for histological studies. A detailed
description of the procedures can be found in Supplementary Material and
Methods.
Microbiota.
Faeces samples from each mouse were aseptically collected on the
end-point day of the assay. Stool DNA was isolated using the QIAamp
PowerFecal Pro DNA Kit (Qiagen,
Hilden, Germany). Total DNA was amplified and a library for the V4–V5
region of 16S rRNA was constructed in accordance with the 16S
Metagenomic Sequencing Library Preparation Illumina protocol. Sequencing
was executed using the MiSeq 2 × 300 platform (Illumina Inc.,
San Diego, CA, USA) in accordance
with the manufacturer’s instructions.
The resulting sequences were completed, quality-filtered, clustered, and
taxonomically assigned on the basis of 97% similarity level against the
RDP (Ribosomal Database Project) (Dhariwal et al. , 2017) by using
the QIIME2 software package (2021.11 version) and “R” statistical
software package (version 3.6.0; https://www.r-project.org/). Alpha
diversity and beta diversity were determined by the q2-diversity plugin
in QIIME2. Differential abundance analysis was performed using the Wald
test implemented DESeq2 v1.30.1 as previously described (Love et
al. , 2014) within the “R” statistical software (version 4.0;
https://www.r-project.org/). Healthy and the three treated groups were
compared with the control CAC-group and EnhancedVolcano package
was used for constructing volcano-plots. Log2-fold-change (Log2FC)
normalized values and the adjusted P-value (Padj) were used to
construct Venn diagrams (Limma package) of differentially microbial
communities up-regulated (log2FC>1.5 andPadj<0.05) and downregulated (log2FC<1.5 andPadj<0.05) among groups.
Statistical and correlation analysis
Statistical analysis was performed using the GraphPad Prism version 7
software (GraphPad Software, Inc, San Diego, CA, USA) with statistical
significance set at P<0.05 . All data are represented as
mean (SEM) of at least 3 independent experiments/biological replicates
unless otherwise stated in the figure legends. The Mann-Whitney U test
for nonparametric data was used for the analysis of the DAI. For the
rest of the data, multiple comparisons between groups were performed
using the one-way ANOVA followed by Tukey’s test.
A heat map depicting the macroscopic parameters, molecular changes, and
patterns of microbial abundance of untreated CAC mice and those treated
with both doses of tigecycline was constructed within the “R”
statistical software using the “Hmisc” and “ggplot2” packages.
Spearman’s correlations of each parameter were previously calculated
with “R” software.
RESULTS