Research Articleβ-Carotene exerts anti-colon cancer effects by regulating M2 macrophages and activated fibroblasts
Introduction
Worldwide, colorectal cancer (CRC) is the third most commonly diagnosed adult cancer in males, and the second most commonly diagnosed cancer in females [1]. Development and progression of CRC has been associated with several risk factors, including genetics and environmental factors, including nutritional lifestyle practices [2], and pre-existing inflammatory bowel disease [3]. Recent studies indicate that CRC cells interact with their tumor microenvironment (TME), such as stromal cells, during cancer progression and metastasis [4]. Consequently, a better understanding of the biological processes of cancer cells within the TME may open new avenues for development of anti-cancer therapies.
The TME is composed of various cell types, including endothelial cells, resident cancer cells, cancer stem cells (CSCs), tumor-associated macrophages (TAMs), and tumor-associated fibroblasts (TAFs) [5,6]. In particular, TAMs and TAFs in the TME participate in and direct cancer progression and metastasis [7]. Macrophage differentiation involves polarization into two different functional phenotypes: classically activated M1 macrophages (M1) and alternatively activated M2 macrophages (M2) [8]. These polarization states are characterized by production of different cytokines, chemokines, and cell surface markers. For example, lipopolysaccharide (LPS) and interferon-γ (IFN-γ) induce macrophage polarization towards a M1 phenotype and production of pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6, IL-12, and tumor necrosis factor-α (TNF-α), to exert anti-tumor immunity [5]. In contrast, M2 macrophage polarization, inducible by exposure to IL-4 or IL-13, supports anti-inflammatory, pro-tumor immunity, and pro-metastatic activities by secreting IL-10 and transforming growth factor-β (TGF-β) [5,9].
Macrophages which infiltrate tumors are described as TAMs, and generally exhibit characteristics similar to those of M2 macrophages [5,10]. Accumulating evidence indicates that TAMs contribute to angiogenesis, tumor growth, tumor progression, and metastasis [11]. Moreover, TAMs can promote cancer cell acquisition of CSC-like properties including upregulation of CSC transcription factors and increase in number of spheres [12]. In clinical studies, high numbers of TAMs are significantly associated with poor prognosis in different types of human cancers [13]. Consequently, suppressing macrophage infiltration and M2 polarization may present potential anticancer effects.
Fibroblasts present within a tumor also exhibit different activated phenotypes, referred to as TAFs, cancer-associated fibroblasts, and activated fibroblasts (AFs). AFs, a principal TME component, express α-smooth muscle actin (α-SMA), fibroblast activation protein (FAP), fibroblast specific protein (FSP), and vimentin [14,15]. In addition, AFs contribute to tumor progression by growth factor and cytokine secretion, extracellular matrix (ECM) remodeling, and promoting in cancer cells the EMT and the acquisition of an invasive phenotype [14,16,17]. For several types of cancer, including CRC, AF density in the TME serves as an indicator of poor prognosis [18].
EMT involves transdifferentiation of normal epithelial cells into a mesenchymal cell phenotype. The latter is characterized by low E-cadherin expression and enhanced expression of N-cadherin, vimentin, and α-SMA. EMT is activated during tumor progression and metastasis, two processes which involve complex interactions between cancer cells and their TME [14,19]. In particular, AFs contribute to cancer progression and metastasis by inducing in cancer cells the acquisition of a mesenchymal phenotype with enhanced invasiveness [20].
β-Carotene (BC) is a major dietary carotenoid, antioxidant, and pro-vitamin A micronutrient [21,22]. In patients with liver [23] or cervical cancer [24], serum levels of BC are inversely associated with tumor development. Experimentally, in human myeloid leukemia and colon adenocarcinoma, BC induces cell cycle arrest and apoptosis [25,26]. In RAW264.7 macrophages, BC inhibits the production of LPS-stimulated inflammatory mediators by suppressing NF-κB pathway activation [27]. Nevertheless, despite these and other observations of the effects of BC in several tumor types, it remains to be determined whether BC can mediate an anti-cancer effect in colitis-associated CRC by regulating M2 macrophages and AFs.
Therefore, the goals of this study were to determine (a) if BC can regulate M2 macrophage polarization and fibroblast activation in CRC in vitro, and (b) if BC suppresses tumor growth and a tumor-promoting TME in vivo using a validated azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced colitis-associated CRC mouse model.
Section snippets
Cell culture and reagents
A human monocytic leukemia cell line (U937), a normal colonic fibroblast cell line (CCD-18Co), and a human colon cancer cell line (HCT116) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The three cell types were grown in RPMI 1640 medium (Welgene, Daegu, Korea), Minimum Essential Medium (MEM; Welgene), and McCoy's 5A medium (Welgene), respectively. Media were supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA) and 1%
BC inhibits M2 macrophage polarization and the IL-6/STAT3 pathway
To investigate possible anti-cancer effects of BC, we first examined whether BC can inhibit M2 macrophage polarization. Previous work has demonstrated that M2 macrophages contribute to tumor progression, growth, metastasis, and immunosuppression, particularly in relation to secretion of cytokines and chemokines [11]. We utilized U937 cells for these experiments since this cell line exhibits certain characteristics of M2 polarized macrophages including up-regulation of M2 markers following
Discussion
In the present study, we found that the dietary antioxidant BC directly inhibited HCT116 colon cancer cell viability. Moreover, BC indirectly suppressed cell proliferation and the expression of certain CSC markers in HCT116 colon cancer cells by acting on M2 macrophages. In addition, BC indirectly inhibited HCT116 colon carcinoma cell migration, invasion, and EMT by regulating the activation of CCD-18Co fibroblasts. Similarly, in vivo, BC supplementation suppressed colon cancer growth in an
Acknowledgement
Grants, sponsors, and funding sources: This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2016R1D1A1B03932018) and Brain Korea 21 Plus (Project Number: 22A20130012143).
References (73)
- et al.
Beta-elemene inhibits tumor-promoting effect of M2 macrophages in lung cancer
Biochem Biophys Res Commun
(2017) - et al.
Role of macrophages in tumour progression
Immunol Lett
(2009) - et al.
Tumor-associated macrophages promote cancer stem cell-like properties via transforming growth factor-beta1-induced epithelial-mesenchymal transition in hepatocellular carcinoma
Cancer Lett
(2014) - et al.
Vitamins E and C, beta-carotene, and other carotenoids as antioxidants
Am J Clin Nutr
(1995) - et al.
CCL2 and interleukin-6 promote survival of human CD11b+ peripheral blood mononuclear cells and induce M2-type macrophage polarization
J Biol Chem
(2009) - et al.
Constitutive activation of Stat3 signaling confers resistance to apoptosis in human U266 myeloma cells
Immunity
(1999) - et al.
Epithelial-mesenchymal transitions in development and disease
Cell
(2009) - et al.
Colorectal cancer cells activate adjacent fibroblasts resulting in FGF1/FGFR3 signaling and increased invasion
Am J Pathol
(2011) - et al.
Pulmonary fibroblasts induce epithelial mesenchymal transition and some characteristics of stem cells in non-small cell lung cancer
Ann Thorac Surg
(2013) - et al.
Tumour-associated macrophages are a distinct M2 polarised population promoting tumour progression: potential targets of anti-cancer therapy
Eur J Cancer
(2006)
Isoliquiritigenin, a flavonoid from licorice, blocks M2 macrophage polarization in colitis-associated tumorigenesis through downregulating PGE2 and IL-6
Toxicol Appl Pharmacol
IL-6 and Stat3 are required for survival of intestinal epithelial cells and development of colitis-associated cancer
Cancer Cell
Colon cancer stem cells dictate tumor growth and resist cell death by production of interleukin-4
Cell Stem Cell
Colon cancer stem cells: promise of targeted therapy
Gastroenterology
Retinoic acid inhibits pancreatic cancer cell migration and EMT through the downregulation of IL-6 in cancer associated fibroblast cells
Cancer Lett
Carcinoma-associated fibroblasts are a rate-limiting determinant for tumour progression
Semin Cell Dev Biol
Antioxidant effect of beta-carotene on hypoxia induced oxidative stress in male albino rats
J Ethnopharmacol
Beta-carotene inhibits neuroblastoma cell invasion and metastasis in vitro and in vivo by decreasing level of hypoxia-inducible factor-1alpha
J Nutr Biochem
Beta-carotene regulates NF-kappaB DNA-binding activity by a redox mechanism in human leukemia and colon adenocarcinoma cells
J Nutr
Lycopene inhibits experimental metastasis of human hepatoma SK-Hep-1 cells in athymic nude mice
J Nutr
Beta-carotene inhibits neuroblastoma tumorigenesis by regulating cell differentiation and cancer cell stemness
Biochem Biophys Res Commun
Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries
CA Cancer J Clin
Colorectal cancer epidemiology: incidence, mortality, survival, and risk factors
Clin Colon Rectal Surg
Colitis-associated cancer: the dark side of inflammatory bowel disease
Gut
Role of cancer microenvironment in metastasis: focus on colon cancer
Cancer Microenviron
Tumor-associated macrophages as major players in the tumor microenvironment
Cancers (Basel)
The tumor microenvironment: a pitch for multiple players
Front Oncol
The tumor microenvironment and its contribution to tumor evolution toward metastasis
Histochem Cell Biol
Cancer-related inflammation
Nature
Metformin prevents cancer metastasis by inhibiting M2-like polarization of tumor associated macrophages
Oncotarget
The role of tumour-associated macrophages in tumour progression: implications for new anticancer therapies
J Pathol
Fibroblasts in cancer
Nat Rev Cancer
The biology and function of fibroblasts in cancer
Nat Rev Cancer
Cancer associated fibroblasts: the dark side of the coin
Am J Cancer Res
Cancer-associated fibroblasts as another polarized cell type of the tumor microenvironment
Front Oncol
Stromal myofibroblasts predict disease recurrence for colorectal cancer
Clin Cancer Res
Cited by (37)
Role of cancer-associated fibroblasts in colorectal cancer and their potential as therapeutic targets
2023, Biochemical and Biophysical Research CommunicationsEpigenetic drugs as new emerging therapeutics: What is the scale's orientation of application and challenges?
2023, Pathology Research and Practiceβ-Carotene suppresses cancer cachexia by regulating the adipose tissue metabolism and gut microbiota dysregulation
2023, Journal of Nutritional BiochemistryDevelopment of dendrimer-like glucan-stabilized Pickering emulsions incorporated with β-carotene
2022, Food ChemistryCitation Excerpt :It presents provitamin-A activity to protect visual development (Saini, Nile, & Park, 2015). It also exhibits strong antioxidant activity and reduces the risk of cardiovascular diseases, non-alcoholic fatty liver disease, and cancer (Kimura, Mikami, Endo, Matsuzaka, Sawada, Igarashi, et al., 2020; Latief, Husain, & Ahmad, 2018; Lee, Kim, Kim, Shin, & Rubin, 2020; Kim, Miller, Coronel, & Amengual, 2020). However, β-carotene is highly sensitive to light, oxygen, and heat, due to its conjugated polyunsaturated hydrocarbon chain for oxidation, isomerization, and degradation (Boon, McClements, Weiss, & Decker, 2010).
- 1
Equal contribution to this work as first authors.