To investigate the effect of epigallocatechin gallate (EGCG) on structural changes of gut microbiota in colorectal carcinogenesis.

An azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced colitis mouse model was established. Forty-two female FVB/N mice were randomly divided into the following three groups: group 1 (10 mice, negative control) was treated with vehicle, group 2 (16 mice, positive control) was treated with AOM plus vehicle, and group 3 (16 mice, EG) was treated with AOM plus EGCG. For aberrant crypt foci (ACF) evaluation, the colons were rapidly took out after sacrifice, rinsed with saline, opened longitudinally, laid flat on a polystyrene board, and fixed with 10% buffered formaldehyde solution before being stained with 0.2% methylene blue in saline. For tumor evaluation, the colon was macroscopically inspected and photographed, then the total number of tumors was enumerated and tumor size measured. For histological examination, the fixed tissues were paraffin-embedded and sectioned at 5 mm thickness. Microbial genomic DNA was extracted from fecal and intestinal content samples using a commercial kit. The V4 hypervariable regions of 16S rRNA were PCR-amplified with the barcoded fusion primers. Using the best hit classification option, the sequences from each sample were aligned to the RDP 16S rRNA training set to classify the taxonomic abundance in QIIME. Statistical analyses were then performed.

Treatment of mice with 1% EGCG caused a significant decrease in the mean number of ACF per mouse, when compared with the model mice treated with AOM/DSS (5.38 ± 4.24 vs 13.13 ± 3.02, P < 0.01). Compared with the positive control group, 1% EGCG treatment dependently decreased tumor load per mouse by 85% (33.96 ± 6.10 vs 2.96 ± 2.86, respectively, P < 0.01). All revealed that EGCG could inhibit colon carcinogenesis by decreasing the number of precancerous lesions as well as solid tumors, with reduced tumor load and delayed histological progression of CRC. During the cancerization, the diversity of gut microbiota increased, potential carcinogenic bacteria such as Bacteroides were enriched, and the abundance of butyrate-producing bacteria (Clostridiaceae, Ruminococcus, etc.) decreased continuously. In contrast, the structure of gut microbiota was relatively stable during the intervention of EGCG on colon carcinogenesis. Enrichment of probiotics (Bifidobacterium, Lactobacillu, etc.) might be a potential mechanism for EGCG’s effects on tumor suppression. Via bioinformatics analysis, principal coordinate analysis and cluster analysis of the tumor formation process, we found that the diversity of gut microbiota increased in the tumor model group while that in the EGCG interfered group (EG) remained relatively stable.

Gut microbiota imbalance might be a potential mechanism for the prevention of malignant transformation by EGCG, which is significant for diagnosis, treatment, prognosis evaluation, and prevention of colorectal cancer.

There are approximately 1.2 million people diagnosed with colorectal cancer (colon cancer) every year, and it is the third most common cancer in the human population. There is a fairly high incidence of colon cancer in developed countries[1]. The human body contains about 1000 kinds of bacteria, approximately 10¹⁴ in total, most of which are distributed in the large intestine. The total number of microbial genes is at least 150 times that of human genes[2], and both together are termed the human metagenome. Now it is considered that the gut microbiota plays an important role in the construction of the biological barrier, which helps with nutrient absorption, energy metabolism, and immune regulation[3]. Meanwhile, more and more evidence indicates that imbalance of gut microbiota plays an important role in gastrointestinal, metabolic, liver, and autoimmune diseases[4-6]. Previous studies found that similar to some tumor related microorganisms (such as Helicobacter pylori, human papilloma virus, and hepatitis B virus, etc.), many bacteria (such as toxigenic fragile bacteroides, Enterococcus faecalis, and Streptococcus spp.) may be toxic enough to cause colorectal cancer[7]. In addition, based on the study of fecal bacterial culture in patient and control populations, it was found that there were differences between the two groups[8]. With the development of molecular biology techniques, especially the next generation high throughput sequencing, detection of the complex structure of gut microbiota becomes feasible. A large number of reports have confirmed the role of bacterial flora in the pathogenesis of colorectal cancer, outlining the structure of gut microbiota in patients with colorectal cancer[9-12]. Previous studies in our group also found that there was a reduction in the abundance of colonic mucosal flora in patients with colorectal cancer, and the increase of Bactaeroides may be related to the occurrence of colorectal cancer[13]. The mechanisms by which bacteria cause colon cancer may include: inducing chronic inflammation of the intestine, producing carcinogenic metabolites, forming carcinogenic biofilms, etc[14]. Although the relationship between gut microbiota and the occurrence of colorectal cancer and its accurate mechanism remain unclear, it can be speculated that the regulation with gut microbiota as a target is a potential breakthrough point for the prevention and treatment of colorectal cancer[15]. Based on this theoretical basis, therapeutic means for the intervention of gut microbiota such as supplementation with probiotics and prebiotics, fecal bacteria transplantation, and even weight loss surgery have become research focuses[16-17].

The biological availability of epigallocatechin gallate (EGCG) is low, and its elimination half-time in blood is only 2.0-3.5 h. Most EGCG is fermented by bacteria in the large intestine, and discharged with stool. The large intestine is the main metabolic site for EGCG and is also the most active portion of the body for the interaction with bacteria. Importantly, when compared to smaller bacteria density in the proximal small intestine, the incidence of colorectal cancer is significantly higher[18]. Therefore, the interaction of EGCG with gut microbiota is very likely to affect the occurrence and development of colorectal cancer.

In this study, gut microbiota was considered a target for disease prevention and treatment, which will further broaden our understanding of the effects of drugs and diet on the body’s health by regulating gut microbiota in humans and animals.

Sporadic reports suggested that EGCG may inhibit proliferation of intestinal pathogenic bacteria (such as Clostridium perfringens, Clostridium difficile, and Bacaeroides) in vitro, but EGCG is less inhibitory to or even stimulatory to probiotic bacteria (such as Bacillus bifidus and Lactobacillus). Animal and human experiments also confirmed that EGCG may affect the growth and floral structure of specific intestinal specific bacteria, and participates in the regulation of the body’s energy metabolism[24,25]. The role of gut microbiota as an intermediate link for EGCG’s impact on colorectal cancer occurrence and development is still unclear.

EGCG acts as a means of chemical prevention of colorectal cancer, and gut microbiota is very likely to be one of its targets. Its impact on overall floral structure or specific bacteria may be related to the inhibition of colorectal cancer.

In this study, we used a mouse model of colorectal cancer (chemically induced) and intragastrically administered the mice with EGCG to inhibit colorectal cancer. At the same time, to further explore the mechanism of EGCG in the prevention of colorectal cancer, we collected the fresh feces of mice during the interference of tumor formation. Via bacterial 16s cDNA high throughput sequencing in combination with bioinformatics, we analyzed changes in the structure of gut microbiota in mice, and flora differences among the groups, to uncover the role of bacterial flora in the EGCG-mediated inhibition of carcinogenesis.

The development of colorectal cancer is generally known to take at least 3 years (progressing from normal mucosa, to adenoma, and finally to adenocarcinoma) and to include three phases, i.e., initial mutation, cancer promotion, and progression. The main purpose for the study of chemical prevention is preventive treatment of disease. ACF are a commonly acknowledged precancerous lesion of humans and rodents and is convenient for in vitro recognition after methylene blue staining. ACF often show proliferative lesions with poor differentiation, and enhanced gland cell division and proliferation. At the molecular level, mutations of the APC and ras genes are common. At the end of the eighth week in this experiment, the results showed that EGCG could effectively reduce the number of precancerous lesions of colorectal cancer. ACF in the distal intestine of the interfering group were reduced by 59%, indicating that EGCG may play a preventive role in the early stages (mutation and cancer promoting stages) of colorectal cancer. In the past, Xiao et al[26] interfered the AOM induced tumor formation process in rats with a high fat diet via green tea polyphenols (65% of the components were EGCG). That study also showed reduced ACF numbers, in particular the larger highly atypical hyperplastic ACF types. Similar results were confirmed in mice[27]. To determine the effect of EGCG on colorectal cancer formation, in another study, tumor formation in APC^min/+ was interfered by 0.08% and 0.16% of EGCG. Tumor numbers were decreased by 37% and 47%, respectively. In our study, however, at the end of the 13^th week after intervention, tumor number in the interfered group was reduced by 82.9% as compared to the control group, and tumor load was reduced by 91.3%, both indicating that the inhibitory effect of EGCG on tumor formation was more obvious than in the past. The possible reason is that the concentration of EGCG in our study was 1%, much higher than those used in previous studies. On the other hand, this also suggested that EGCG plays a preventive role in colorectal cancer occurrence in a dose-dependent manner.

We conducted further studies to uncover the effect of green tea extract EGCG on the gut microbiota of mice, using macro-genomics in combination with bioinformatics analysis. Considering the complexity of gut microbiota, this study was designed with the following research objectives in mind: (1) to preliminarily understand the compositions of the gut microbiota in mice at the 8th week ( precancerous stage) and the 11^th week (progressive stage) of colorectal cancer; (2) to preliminarily understand the change in gut microbiota during the process of colorectal cancer progression in mice as compared to healthy mice, and to clarify the effects of EGCG intervention on this change; and (3) through statistical analysis and bioinformatics, to try to find specific bacteria that can distinguish the structure of each group of bacteria, i.e., the specific bacterial target of EGCG intervention.

With the aid of the 454 sequencing platform for the detection of 16S rDNA V3 region or V3/V4 region[10], we analyzed the structure of gut microbiota in mice by amplification of the V4 region in this study, which was good for identification. The sequencing results were under strict quality control. The distribution map, dilution curve, and abundance cure of the sequences acquired all confirmed that the sequencing results could well reflect the structure of the gut microbiota in mice.

It is worthwhile to note that in 2011, Kosticet al[11], Castellarin et al[12], and Marchesi et al[28] simultaneously found that there were gut microbiota disorders at varying degrees in colorectal cancer patients, using sequencing techniques, and the Fusobacterium was enriched in cancer tissues as compared to the healthy tissues. Meanwhile, it was also found that the abundance of Fusobacterium was directly proportional to the rate of lymph node metastasis. It is noticeable from the above reports that despite the increasing concern about the relationship between colorectal cancer and bacteria and that exciting results were obtained by relevant studies, the studies focused mainly on the structure of gut microbiota or specific bacteria during colorectal cancer progression (especially during the middle and late stages), which was only a snapshot of the big picture. Little is known about the dynamic changes of the gut microbiota in the process of tumor formation, especially for the status of gut microbiota during the early stage of disease progression, which indeed is a key time point for chemical prevention.

At the same time, it will be informative for the elucidation of flora changes as a direct cause of colorectal cancer formation, or flora changes as a direct result of colorectal cancer occurrence and/or progression. In this study, we examined and compared the composition of gut microbiota in mice with precancerous lesions and in mice during tumor progression. This was helpful for further understanding the roles of different species of bacteria in carcinogenesis, cancer promotion, and tumor suppression, and therefore the potential EGCG target bacteria could be explored. In fact, during the precancerous stage, there were anomalies seen in gut microbiota structures in the mice of the MO group, mainly manifesting as enrichment of Bacaeroides and Anaerotruncus and reduced abundance of Clostridiaceae, Ruminococcus, Ochrobactrum, Veillonella alcalescens, Desulfococcus, etc. The enrichment of Bacteroides in the MO group was consistent with our previous study on mucosal flora in patients with colorectal cancer[15]. The relationship between Anaerotruncus and colon cancer occurrence is still unclear. Previous studies have suggested that Bacaeroides and its lower species could promote the occurrence and development of colorectal cancer by inducing chronic inflammation, producing tumor-promoting metabolites, or changing the expression of host mucosal glycosylation. Among them, toxigenic fragile bacteroides is representative and was considered a pathogenic bacterium of colorectal cancer, initially because it was found to be able to produce the toxins of toxigenic fragile bacteroides (bacteroides fragilis toxin, BFT). BFT can rapidly change the structure and function of intestinal mucosal epithelial cells (decomposition of tumor suppression protein and E-cadherin), enhancing the intranuclear expression of Wnt/B adhesion protein signaling pathway, and therefore inducing continuous proliferation of colon adenocarcinoma cells and the expression of oncogene MYC. Besides, BFT may induce inflammatory responses by inducing expression of cytokines in intestinal mucosal epithelia cells through the NF-κB signaling pathway. Notably, increased expression of NF-κB signaling pathway could also promote the carcinogenesis of mucosal epithelium. In animal experiments, continuous colonization of toxigenic fragile bacteroides into APC^min/+ mice could greatly promote the incidence of adenomas[29]. Sears et al[30] suggested that this type of bacterium might induce mucosal immune defense changes to promote tumor development through direct secretion of endotoxin or indirect changes of gut microbiota, and might supplant probiotics to further expand inflammatory responses, promoting occurrence of multiple pathogenic bacteria and ultimately promoting tumor progression.

Furthermore, it was found in this study that in the precancerous stage, the Clostridiaceae and bacteria of the Ruminococcus maintained their low abundances in the tumor model group, but not in the intervention group. The Clostridiaceae includes multiple important butyrate producing bacteria, such as Eubacterium rectale and Roseburia intestinalis. This type of bacterium was reported to be reduced in the intestinal tract of patients with colorectal cancer, obese patients, and patients with a high fat diet. Similarly, Ruminococcus is also butyrate producing bacteria, and they can effectively decompose and ferment the cellulose and polysaccharide in foods into short chain fatty acids, such as butyrate. Butyrate in the intestinal tract may provide the intestinal mucosa with energy, promoting sodium and potassium absorption. More importantly, it can reduce intestinal inflammation, protect the intestinal mucosal barrier, and even inhibit the growth of colon cancer cells[31,32]. A study by Jahns et al[33] on human colorectal cancer tissues showed that butyrate could inhibit COX2 gene expression in nodal tissues and the activities of related proteins; in vitro studies also found that butyrate might activate caspase 3 and caspase 9 to induce apoptosis of colorectal cancer cells, upregulate pro-apoptotic gene BAK expression, and activate mitochondrial pathways to induce cell apoptosis and the like.

The gut microbiota of mice in the same tumor model group was different. The structure of the gut microbiota of mice interfered with green tea extract EGCG basically remained relatively stable, and bioinformatics analysis showed that the structure of the flora in these mice was similar to that of healthy mice, with no obvious fluctuations in the number of bacteria. Instead, probiotics such as Lactobacilus and Bifidobacterium were increased in the EG group, and the relative abundance of the former was significantly increased after long-term EGCG intervention. Probiotics are defined by WHO/FAO as “live bacteria that can promote body health if taken in a proper amount”. In recent years, some studies have showed that probiotics may have certain antagonistic effects on tumors. For example, Lactobacillus may significantly reduce the incidence of mouse sarcoma, colorectal cancer, and bladder cancer. Its main mechanisms may include optimization of the combined gut microbiota to inhibit the growth of carcinogenic bacteria; production of anti-tumor active substances; and stimulation and enhancement of the host immune system to prevent chronic inflammation.

It could be inferred accordingly that EGCG plays a role in cancer inhibition by maintaining the stability of gut microbiota, inhibiting the proliferation of potential pathogenic bacteria, and promoting the enrichment of probiotics. However, gut microbiota is influenced by many factors, such as diet and region, for its role in health promotion. Further study is certainly required to uncover the causal relationship(s) between gut microbiota and colorectal cancer, the interaction between bacteria and the host, as well as the interaction between EGCG and bacteria.

The authors would like to thank Personal bioBiological Technology Company (Shanghai, China) for the contributions to this research as well as Dr. Ma Xueyun from the East China Normal University for technical support of animal experiments.