Method for screening compounds that prevent colon cancer, and method for testing colon cancer

Abstract

To provide a screening method for a substance that can prevent the development of colon cancer, and a new testing method for colon cancer.SOLUTION: We have analyzed bacteria that have distinctively increased with the development of colon cancer, identified the bacteria that affect the cell proliferation, and further clarified a mechanism by which these bacteria induce cancer development. With the proliferation of the bacteria as an index, a substance to inhibit the development of colon cancer can be screened for. Additionally, a colon cancer patient can be screened for by using the bacteria that are often detected in the intestinal bacterial flora of the colon cancer patient but seldom detected in the healthy subject.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for screening for substances that contribute to the prevention of colon cancer. Despite the large number of colon cancer patients, there is no effective preventive method. The present invention relates to a method for screening for compounds or compositions that can prevent colon cancer. Furthermore, the present invention relates to a method for detecting colon cancer by testing for specific bacteria. [Background technology]

[0002] According to statistics from the Ministry of Health, Labor and Welfare, cancer is the leading cause of death among both men and women in Japan. As the number of cancer cases and deaths in Japan is increasing due to the aging of the population, it is expected that the number of cancer cases will continue to increase in the future. By organ, there are cancers such as stomach cancer, prostate cancer, and breast cancer where the number of cases is increasing, but the number of deaths has remained flat or is only slightly increasing. On the other hand, there are cancers such as colon cancer where both the number of cases and the number of deaths are increasing.

[0003] According to the Japan Cancer Society's statistics on cancer by site, colorectal cancer is the leading cause of cancer death for women and the third leading cause for men, with approximately 50,000 deaths from colorectal cancer annually, both men and women combined (2016 statistics). Globally, colorectal cancer is increasing significantly, becoming the second most common cancer with a new incidence rate (Non-Patent Document 1). If colorectal cancer is detected early and treatment is initiated, it has a high cure rate compared to other cancers, and early detection is expected to reduce the number of deaths. Furthermore, as the number of cases is increasing, effective preventive methods are desired.

[0004] In recent years, it has become possible to analyze a large amount of nucleic acid sequences at once using next-generation sequencers. Comprehensive analysis of the 16S ribosomal RNA gene (16S rDNA) sequences of bacteria in feces has led to reports of correlations between changes in the intestinal microbiota and various diseases. Correlations between specific bacterial species and colorectal cancer have also been reported. For example, Fusobacterium nucleatum and Bacteroides fragilis have been identified as Apc min / +It has been shown that when introduced into mice, intestinal tumors develop (Non-Patent Documents 2 and 3). Furthermore, it has been reported that pks+E. coli induces colon cancer through the production of colibactin (Non-Patent Documents 4 and 5), Peptostreptococcus anaerobius induces colon cancer through the PI3K-AKT pathway (Non-Patent Document 6), Clostridium spp. induces colon cancer through the production of secondary bile acids (Non-Patent Document 7), and Bacteroides fragilis induces colon cancer through the toxins it produces (Non-Patent Document 8).

[0005] Based on these reports, it has been suggested that analyzing the intestinal microbiota and using specific bacteria are useful for screening colorectal cancer patients and preventing colorectal cancer. Regarding methods for reducing cancer risk by improving the intestinal microbiota, the inventors' group discovered the possibility that Clostridium bacteria may induce liver cancer and disclosed a method for screening food ingredients and compositions that reduce cancer risk using Clostridium bacteria as an indicator (Patent Document 1). However, many of the bacteria whose abundance correlates with the onset of colorectal cancer are obligate anaerobes, making direct analysis of bacterial activity technically difficult. Therefore, there have been few reports that identify bacteria directly involved in the onset of colorectal cancer or delve into the mechanism of action of colorectal cancer. Furthermore, because differences in dietary habits based on residential location and medical treatments significantly affect the onset of colorectal cancer, it is unclear whether bacteria that increase in the intestinal microbiota of colorectal cancer patients are directly involved in the onset of colorectal cancer. Many intestinal bacteria have been reported to increase or decrease in colorectal cancer patients, but it has been suggested that most of these bacteria are not the cause of colorectal cancer, but rather are bacteria that increase or decrease as a result of the cancer.

[0006] Due to the high incidence and mortality rate of colorectal cancer, preventive measures are highly desirable. Given the involvement of intestinal bacteria in the development of colorectal cancer, improving daily diet and maintaining a healthy intestinal microflora are desirable as preventative measures. Identifying the bacteria that cause colorectal cancer could allow screening for compounds or food components that reduce these bacteria. By ingesting the compounds or food components identified through screening, it is possible to reduce the bacteria involved in colorectal cancer development, improve the intestinal microflora, and reduce the risk of developing colorectal cancer. Furthermore, improving the intestinal microflora is also thought to be effective in preventing recurrence after treatment, such as surgery. Furthermore, if patients with colorectal cancer could be screened by detecting specific bacteria present in fecal samples, this could lead to early detection and treatment of colorectal cancer patients. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2014 / 126043 [Non-patent literature]

[0008] [Non-Patent Document 1] Ferlay, J. et al.,2019, Int.J. Cancer Vol.144, pp.1941-1953. [Non-patent document 2] Kostic, AD et al.,2013, Cell Host Microbe Vol.14,pp.207-215. [Non-patent document 3] Wu, S. et al. 2009, Nat. Med. Vol.15, pp.1016-1022. [Non-patent document 4] Nougayrede,JP et al., 2006, Science Vol.313, pp.848-851. [Non-patent document 5] Pleguezuelos-Manzano, C. et al., 2020, Nature, Vol.580, pp.269-273. [Non-patent document 6] Long,X. et al., 2019, Nat. Microbiol., Vol.4, pp.2319-2330. [Non-Patent Document 7] Fu, T. et al., 2019, Cell Vol.176, pp.1098-1112. [Non-patent document 8] Wu, S. et al., 2003, Gastroenterology Vol.124, pp.392-400. [Non-Patent Document 9] Takayama, N. et al. 2010, J. Exp.Med. Vol.207, pp.2817-2830. [Non-Patent Document 10] Takahashi, Y. et al., 2018, Stem Cell Reports, Vol.10, pp.314-328. [Non-Patent Document 11] Sato, M. et al., 2016, Front. Microbiol. Vol.7, pp.1146. [Non-Patent Document 12] Colnot, S. et al., 2004, Lab.Invest. Vol.84, pp.1619-1630. Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention aims to provide a method for identifying bacteria involved in the development of colorectal cancer, reducing the identified bacteria that cause colorectal cancer, and screening for compounds or food ingredients that prevent colorectal cancer. The present invention also relates to a testing method for detecting the identified bacteria.

[0010] Another objective of this study is to provide a method for co-culturing obligate anaerobic bacteria with cultured animal cells. Until now, obligate anaerobic bacteria, which can only grow under anaerobic conditions, could only be co-cultured for a few hours with animal cells, which require oxygen, and only limited analysis could be performed. If co-culturing for a longer period of time becomes possible, it will be possible to analyze in more detail the effects of obligate anaerobic bacteria on cells. The co-culture system for obligate anaerobic bacteria and cultured cells will be a research tool not only for analyzing intestinal bacteria, but also for analyzing the relationship between bacteria and various diseases. [Means for solving the problem]

[0011] The present invention relates to a method for screening for a substance that reduces bacteria that cause colon cancer, a method for co-culturing obligately anaerobic bacteria and cultured cells, and a method for detecting colon cancer by detecting intestinal bacteria. (1) A method for screening a substance that prevents carcinogenesis or recurrence, comprising: A screening method characterized by using inhibition of the growth of bacteria that induce cellular senescence as an indicator. (2) The screening method according to (1), wherein the bacteria are bacteria that are characteristically present in colon cancer patients. (3) The screening method according to (1) or (2), wherein the bacterium is a bacterium that produces butyric acid. (4) The screening method according to any one of (1) to (3), wherein the bacterium is Porphyromonas gingivalis or Porphyromonas asaccharolytica. (5) A screening method according to any one of (1) to (4), characterized in that a target substance or composition is added to a medium for bacterial culture, and the growth of the bacteria is used as an indicator for screening. (6) A method for co-culturing obligately anaerobic bacteria and animal cells, characterized in that the cells are cultured under 3% O2 ​​conditions. (7) The co-culture method according to (6), characterized in that obligate anaerobic bacteria are added to a culture medium for animal cells and cultured. (8) A method for screening for substances that prevent carcinogenesis or recurrence, which comprises searching for bacteria that overproduce short-chain fatty acids that are characteristically present in the intestines of colorectal cancer patients, and using the inhibition of the overproduction of short-chain fatty acids by the bacteria as an indicator. (9) The screening method according to (8), wherein the bacterium is at least one of Fusobacterium nucleatum, Porphyromonas gingivalis, and Porphyromonas asaccharolytica. (10) The screening method according to (8) or (9), wherein the short-chain fatty acid is butyric acid. (11) A screening method according to any one of (8) to (10), characterized in that a target substance or composition is added to a medium for bacterial culture, and the growth of the bacteria is used as an indicator for screening. (12) A colon cancer testing method that analyzes bacteria in a subject's fecal sample and determines that the subject is highly likely to have colon cancer if the detection frequency of one or more of the following bacteria is higher than that in healthy subjects: Parvimonas micra, Prevotella intermedia, Peptostreptococcus stomatis, Porphyromonas asaccharolytica, Porphyromonas uenonis, Solobacterium moorei, Gemella morbillorum, Peptostreptococcus anaerobius, Porphyromonas gingivalis, Alloprevotella tannerae, Dialister pneumosintes, Fusobacterium nucleatum ssp. animalis, Fusobacterium nucleatum ssp. vincentii, Fusobacterium nucleatum ssp. fusiforme, and Fusobacterium nucleatum ssp. nucleatum. [Brief explanation of the drawings]

[0012] [Figure 1] A diagram showing bacteria characteristically found in colorectal cancer patients. (A) shows the analysis results for Cohort 1. The left plot of each plot shows the relative proportion of OTUs detected in samples from healthy individuals, and the right plot shows the relative proportion of OTUs detected in samples from colorectal cancer patients. (B) shows the analysis results for Cohort 2. The left plot of each plot shows the relative proportion of OTUs detected in samples from healthy individuals, the middle plot shows the relative proportion of OTUs detected in samples from early-stage colorectal cancer patients, and the right plot shows the relative proportion of OTUs detected in samples from advanced colorectal cancer patients. (C) shows the relative proportion of OTUs before and after surgical resection in patients in Cohort 1. A diagram showing that these bacteria are hardly detected after surgical resection of the primary tumor. The left plot shows the results of analysis of samples before resection, and the right plot shows the results of analysis of samples after resection. [Figure 2] Diagram showing the analysis method using co-culture of obligate anaerobic bacteria and animal cells. (A) A schematic diagram of the co-culture method. (B) A micrograph showing cellular senescence induced by doxorubicin, a positive control. (C) A micrograph showing the results of co-culture with Escherichia coli, a gram-negative bacterium, and Lactobacillus, a gram-positive bacterium, as negative controls that do not induce cellular senescence. [Figure 3] Micrographs showing the results of co-culture of bacteria with human normal diploid fibroblast TIG-3 cells. (A) shows a case where no change in proliferation was observed, (B) shows a case where delayed cell proliferation was observed, and (C) shows a case where cell death was observed. [Figure 4] Diagram showing the method for analyzing the effects of bacterial metabolic products. (A) is a schematic diagram of the analysis method. (B) is a diagram showing the analysis schedule. (C) is a micrograph showing the results at the start of culture (day 0) when only GAM, the bacterial growth medium, and modified GAM medium (mGAM) were added as controls, and (D) is a micrograph showing the results when doxorubicin was added as a positive control, and culture medium of Escherichia coli, a gram-negative bacterium, and Lactobacillus, a gram-positive bacterium, were added as negative controls. [Figure 5] Figure showing the results of analyzing cell growth in a system in which bacterial metabolic products were added. (A) shows a case in which no change in growth was observed, (B) shows a case in which delayed cell growth was observed, and (C) shows a case in which cell senescence was observed. [Figure 6]This figure shows the results of a time-dependent analysis of cell growth in two species of bacteria in which cell aging was observed, in a system where metabolic products were added. [Figure 7] FIG. 1 shows mRNA expression profiles of cells supplemented with bacterial culture supernatant. [Figure 8] (A) is a photograph showing the results of immunostaining analysis of DNA damage markers γH2AX and pST / Q. The position of the nucleus is indicated by DAPI. (B) is a graph showing the percentage of cells in which three or more foci co-stained with γH2AX and pST / Q were observed. [Figure 9] This figure shows that cellular senescence is induced in cells treated with bacterial culture supernatant. (A) shows the mRNA expression levels of cellular senescence markers, and (B) shows Western blot analysis. (C) shows the analysis of ROS generation, and (D) shows the analysis of apoptotic cells detected by Annexin V and PI staining. (E) and (F) show the effects of bacterial culture supernatant analyzed using organoids differentiated into intestinal cells from human induced pluripotent stem cells. (E) shows representative microscopic images showing the time course of organoids cultured under each condition, and (F) shows the results of analyzing cell numbers based on ATP levels on days 9 and 12 of culture. [Figure 10] Diagrams analyzing the effects of butyrate on cells. (A) shows the effect of butyrate on cell proliferation, and (B) shows the analysis of mRNA expression levels of cellular senescence markers. [Figure 11] Analysis of the effects of short-chain fatty acids on cells. (A) shows the results of analyzing the effects of short-chain fatty acids on cells when TIG-3 cells were cultured with 1 / 30 volume of each bacterial culture supernatant. (B) shows the results of RT-qPCR analysis of the gene expression of p16INK4a, p21Cip1 / Waf1, and lamin B1, which are involved in cellular senescence, in cells cultured under the above conditions. (C) shows the butyrate concentration in the culture medium of wild-type P. gingivalis (ATCC33277) and a butyrate synthesis-deficient mutant (PGAGU118) (left), and typical micrographs (right) of TIG-3 cells cultured for 9 days with the culture medium containing each bacterial strain. [Figure 12]Figures showing the results of analyzing the presence and effects of butyrate-producing bacteria in clinical specimens. (A) shows the results of in situ hybridization using probes specific to P. gingivalis and P. asaccharolytica, and immunostaining using anti-p16 and anti-IL-6 antibodies. (B) shows the results of analyzing butyrate concentrations in cancerous and non-cancerous tissues. [Figure 13] Figure showing the results of analysis using APCΔ14 / + mice. (A) P. gingivalis or P. asaccharolytica was administered to APCΔ14 / + mice, and the number (left) and size (right) of colon tumors were analyzed. (B) Wild-type P. gingivalis (ATCC33277) or a butyrate synthesis-deficient mutant (PGAGU118) was administered to APCΔ14 / + mice, and the number (left) and size (right) of colon tumors were analyzed. The images below show representative tumor photographs in each treatment group. (C) Wild-type P. gingivalis was administered in combination with ABT-263 to APCΔ14 / + mice, and the number (left) and size (right) of colon tumors were analyzed. The images below show representative tumor photographs in each treatment group. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present inventors comprehensively analyzed the intestinal microbiota of numerous colorectal cancer patients and healthy individuals and discovered intestinal bacteria characteristic of colorectal cancer patients. Furthermore, detailed analysis of these bacteria identified bacteria that induce cellular senescence and apoptosis, known as carcinogenic stress responses. Furthermore, analysis of the metabolic products of these bacteria identified substances that induce cellular senescence responses. Based on these findings, compounds, food ingredients, and food compositions that prevent the onset of colorectal cancer can be screened using the proliferation of bacteria involved in the onset of colorectal cancer as an indicator. Furthermore, by detecting bacteria that are abundant in colorectal cancer patients in fecal samples, the risk of a subject developing colorectal cancer can be determined.

[0014] The present invention provides a method for screening compounds, food ingredients, and food compositions using, as an index, the inhibition of the growth of bacteria that have been shown to be involved in the development of colorectal cancer. The substance to be screened may be a single compound contained in a compound library, or it may be an extract from a food or a composition containing several substances.

[0015] The substances identified as a result of screening that inhibit the growth of bacteria involved in the development of colorectal cancer are thought to be able to prevent the onset of colorectal cancer by taking them as supplements or foods for specified health uses. Furthermore, taking them as supplements after colorectal cancer treatment, such as surgery, is thought to improve the intestinal flora and maintain a healthy intestinal flora, thereby contributing to the prevention of recurrence.

[0016] Furthermore, since the bacteria identified this time include bacteria that have been shown to be significantly increased in patients with early-stage colorectal cancer, this study is thought to be useful as a testing method for detecting patients with early-stage colorectal cancer.

[0017] (1) Bacteria that are characteristically increased in patients with colorectal cancer To analyze changes in the gut microbiota related to the development of colorectal cancer, we analyzed the gut microbiota profiles of colorectal cancer patients and healthy individuals. Fecal samples were collected from colorectal cancer patients and healthy individuals undergoing colonoscopy to diagnose colorectal cancer or confirm the absence of colorectal cancer lesions. To avoid unforeseen influences, fecal samples were collected from individuals who had not undergone colonoscopy, antibiotic treatment, chemotherapy, or radiation therapy. Furthermore, individuals with diseases that affect the gut microbiota, such as gastrointestinal reconstruction, inflammatory bowel disease, or severe liver damage, were excluded from the analysis.

[0018] From 1,600 individuals, 384 healthy controls and 380 colon cancer patients, including 63 early-stage cancer patients, were selected according to the above criteria and metasequencing was performed using standard methods. Sequences of the variable regions 1 and 2 (V1, V2) of the bacterial 16S rRNA gene were error-corrected using DADA2 and analyzed using QIIME2 (version 2019.10). OTUs (operational taxonomic units) with ≥97% identity were then classified using VSEARCH. Analysis using linear discriminant analysis effect size (LEfSe) revealed that 87 OTUs were increased and 24 OTUs were decreased in colon cancer patients. All 12 OTUs shown here were significantly different between colon cancer patients and healthy controls using the two-tailed Wilcoxon rank-sum test (P < 0.05 was considered significant). We also found that 12 OTUs that were detected in large numbers in colorectal cancer patients were rarely detected in healthy individuals (Fig. 1(A)). The bacterial species identified from the OTUs that were significantly detected in colorectal cancer patients are shown below (Table 1).

[0019] [Table 1]

[0020] To confirm the reproducibility of the results detected in Cohort 1, we performed a similar analysis in another independent cohort, Cohort 2. 129 healthy individuals, 136 patients with early-stage colorectal cancer, and 153 patients with advanced colorectal cancer were selected using the same criteria and analyzed (Figure 1(B)). Not only were similar results obtained in this independent cohort, but four OTUs (P. stomatis, G. morbillorum, and two F. nucleatum subsp. OTUs) were significantly increased in early-stage cancer compared to healthy individuals. Furthermore, when we analyzed the gut microbiota in Cohort 1 before and after surgical resection, these 12 OTUs were significantly reduced and barely detected after surgical resection, suggesting a strong correlation with colorectal cancer (Figure 1(C)).

[0021] (2) Identification of bacteria that cause carcinogenic stress (i) Analysis by co-culture with cells The bacteria shown in Table 1 are bacteria that are characteristically increased in patients with colorectal cancer, but it is unknown whether they are involved in the onset of colorectal cancer or whether their increase is associated with disease symptoms. Because it is known that applying carcinogenic stress to normal cells causes cellular senescence or apoptosis, we decided to co-culture cells with these bacteria and use cellular senescence or apoptosis as indicators to analyze whether they are involved in the onset of colorectal cancer.

[0022] After examining various culture conditions, we found that the intestinal bacteria analyzed here, even though they are obligate anaerobes, can be co-cultured with cells under 3% O2 ​​conditions. We co-cultured bacteria that increase in colon cancer patients with normal diploid fibroblasts derived from human fetal lung, TIG-3 (obtained from the Japan Health Science Foundation), and analyzed their effects on cell proliferation.

[0023] The bacteria used for co-culture analysis (Table 2) were obtained from the RIKEN BRC (JCM Microbial Materials Research Laboratory). They were cultured on GAM agar medium (Nissui Pharmaceutical Co., Ltd.) or mGAM agar medium (Nissui Pharmaceutical Co., Ltd.) at 37°C under anaerobic conditions for 1–3 days, then harvested and suspended in PBS. Each bacterium was infected into TIG-3 cells at different moi. The cells were co-cultured in DMEM (Nacalai Tesque, Inc.) supplemented with 10% FBS (Gibco, Thermo Fisher Scientific) (hereafter referred to as 10% FBS / DMEM) at 37°C with 3% O2, and the effect of the bacteria on cell growth was observed (Figure 2(A)).

[0024] [Table 2]

[0025] As a positive control, doxorubicin, which induces DNA damage and cell senescence, was added at a concentration of 200 ng / mL (Figure 2(B)). As a negative control, cells were co-cultured with Escherichia coli DH5α (a Gram-negative bacterium, obtained from TOYOBO) and Lactobacillus salivalius JCM1040 (a Gram-positive bacterium, obtained from RIKEN BRC), which do not induce cell senescence or apoptosis (Figure 2(C)). When cells were co-cultured with E. coli or Lactobacillus, cell death was observed due to changes in the pH and composition of the culture medium caused by bacterial overgrowth. However, typical signs of cell senescence, as seen with the addition of doxorubicin, were not observed.

[0026] Next, we similarly co-cultured 11 species of bacteria listed in Table 2, which are found to be increased in colorectal cancer patients, and analyzed their effects on cell proliferation (Fig. 3). Four species, Parvimonas micra, Porphyromonas asaccharolytica, Alloprevotella tannerae, and Prevotella intermedia, did not experience growth inhibition and continued to grow normally (Fig. 3(A)). Two species, Dialister pneumosintes and Solobacterium moorei, showed delayed cell proliferation (Fig. 3(B)). On the other hand, five species, Fusobacterium nucleatum, Gemella morbillorum, Peptostreptococcus anaerobius, Peptostreptococcus stomatis, and Porphyromonas gingivalis, were observed to undergo cell death in association with bacterial proliferation (Fig. 3(C)).

[0027] Previously, it was thought that obligately anaerobic bacteria and animal cells could be co-cultured for a few hours, but that long-term co-culture was not possible because animal cells require an oxygen supply and anaerobic bacteria cannot grow in the presence of oxygen. However, we have found that under 3% O2 ​​conditions, both animal cells and obligately anaerobic bacteria can be cultured and grown for long periods of time. Using these culture conditions, obligately anaerobic bacteria and animal cells can be co-cultured and analyzed.

[0028] (ii) Analysis using bacterial culture supernatant The five species of bacteria in which cell death was observed may be causing cancer-causing mutations. However, since cell death was also observed in co-cultures with the negative controls, E. coli and Lactobacillus, the possibility cannot be ruled out that cell death was caused by changes in the pH of the culture medium due to bacterial overgrowth or by depletion of nutrients. Furthermore, since the bacteria in which cell death was not observed may also exert carcinogenic stress on cells via metabolic products, we analyzed what changes occurred in the cells upon addition of bacterial culture supernatant (Figure 4).

[0029] Each bacterium was cultured in GAM or mGAM liquid medium under anaerobic conditions for 2–3 days, then centrifuged to collect the culture medium and filtered through a 0.22 μm membrane to obtain the bacterial culture supernatant. The bacterial culture supernatant was analyzed by adding 100 μL of bacterial culture supernatant (1 / 30 of the cell culture medium) to 3 mL of 10% FBS / DMEM medium (Figure 4(A)). TIG-3 cells were cultured in 10% FBS / DMEM supplemented with bacterial culture supernatant on days 0, 3, and 6, and then replaced with 10% FBS / DMEM (sometimes referred to as normal DMEM) without bacterial culture supernatant on day 9, and the effect on cell growth was analyzed (Figure 4(B)).

[0030] The cell status is shown for cells at the start of culture (day 0), GAM medium control, mGAM medium control (Figure 4(C)), cells with doxorubicin added as a positive control, and cells with bacterial culture supernatant obtained by culturing E. coli or Lactobacillus added as a negative control on days 9 and 12 (3 days after changing the medium to normal DMEM) (Figure 4(D)). When cells were cultured with doxorubicin added, cell growth did not recover even when the medium was changed to normal DMEM on day 9 and culture was continued. When cells were cultured with the addition of E. coli or Lactobacillus culture supernatant, cell growth was confirmed to be comparable to that of the medium-only control.

[0031] Although cell death was observed in co-cultures with Escherichia coli and Lactobacillus, cell death did not occur in systems where bacterial culture supernatant was added to confirm TIG-3 cell growth. This suggests that the cell death observed in co-cultures caused by these bacteria is due to changes in the culture medium caused by bacterial growth, and not due to bacterial metabolic products. When analyzing the effects of obligate anaerobic bacteria in co-cultures, it is necessary to adjust the moi and consider culture conditions, but it is possible to culture and analyze them with animal cells for longer periods than previously thought.

[0032] Next, we analyzed the culture supernatants of 11 bacterial species that were found to be increased in colorectal cancer patients by adding them to the culture medium. Analysis using bacterial culture supernatants showed that the culture supernatants of five bacterial species, Alloprevotella tannerae, Gemella morbillorum, Dialister pneumosintes, Prevotella intermedia, and Parvimonas micra, did not affect cell proliferation (Figure 5(A)). Furthermore, we found that the addition of culture supernatants of Peptostreptococcus anaerobius, Solobacterium moorei, and Peptostreptococcus stomatis caused cell growth retardation. As shown in Figure 5(B), when cells were supplemented with these bacterial culture supernatants, their proliferation ability was restored by switching to 10% FBS / DMEM without bacterial culture supernatant on day 9. On the other hand, the addition of bacterial culture supernatants from Fusobacterium nucleatum, Porphyromonas asaccharolytica, and Porphyromonas gingivalis did not restore cell growth.

[0033] The cell number was measured over time in an analysis system that added the culture supernatants of these two bacteria, P. asaccharolytica and P. gingivalis (Figure 6). Under the same conditions as above, cells were cultured for 9 days with or without doxorubicin (mock), with 1 / 30 addition of culture supernatants from E. coli, P. asaccharolytica, or P. gingivalis. On the 9th day, the cells were returned to normal medium and cultured. The photograph shows the typical cell appearance on the 12th day (3 days after culture in normal medium). Like doxorubicin, P. asaccharolytica and P. gingivalis culture supernatants induced irreversible cell cycle arrest in human normal diploid fibroblast TIG-3 cells.

[0034] In an experimental system using bacterial culture supernatant, we analyzed the mRNA expression profiles of cells cultured with bacterial culture supernatants from F. nucleatum, P. accharolytica, and P. gingivalis, which were observed to undergo cellular senescence, and cells cultured with GAM medium alone (shown as FNsup. vs. GAM, PAsup. vs. GAM, and PGsup. vs. GAM in Figure 7). We compared the mRNA expression profiles of doxorubicin (DXR vs. Ctrl), which causes DNA damage, and activated Ras (HrasG12 vs. Mock), whose overexpression is known to induce cellular senescence (Figure 7).

[0035] In cellular senescence induced by the addition of bacterial culture supernatant, the mRNA expression profile was found to resemble that induced by DNA damage and the DNA damage response by the addition of doxorubicin, rather than that induced by forced expression of activated Ras.

[0036] Analysis of the mRNA expression profile suggested that the addition of bacterial culture supernatant caused DNA damage and subsequent DNA damage responses. Therefore, we stained the cells with DNA damage markers to analyze whether DNA damage had occurred. Foci co-stained with γH2AX, a DNA damage marker, and pST / Q, which detects the phosphorylation of the ST / Q sequence, a substrate of ATM, were analyzed microscopically (Figure 8). Immunostaining was performed with anti-γH2AX antibody (Merck Millipore) and anti-pST / Q antibody (Cell Signaling Technology), and nuclei were stained with DAPI.

[0037] In cells treated with bacterial culture supernatants from P. asaccharolytica (Pa), P. gingivalis (Pg), and F. nucleatum (Fn), foci that co-stained with anti-γH2AX and anti-pST / Q antibodies were observed, although they were smaller than those observed in cells treated with doxorubicin (DXR) (Fig. 8(A)). When examining the percentage of cells with three or more co-stained foci, the percentage was significantly higher in cells treated with bacterial culture supernatants than in cells treated with doxorubicin, but was lower than in cells treated with medium alone (GAM) or E. coli (Fig. 8(B)). These results suggest that DNA damage is induced by the addition of bacterial culture supernatants.

[0038] Next, we analyzed the expression of cellular senescence markers in cells 9 days after the addition of bacterial culture supernatant using real-time PCR. We analyzed the expression of p16 and p21, which are involved in the activation of growth inhibitory factors, lamin B1, whose expression level is known to decrease with cellular senescence, and IL-1β and IL-6, which are secreted by senescent cells and are said to be SASP factors (Figure 9(A)). In cells treated with the culture supernatant of P. asaccharolytica (Pa), P. gingivalis (Pg), or F. nucleatum (Fn), the expression of p16, a gene that induces cellular senescence, was also analyzed. INK4a , p21 Cip1 / Waf1 Increased expression of SASP factor genes IL-1 and IL-6, and decreased expression of lamin B1 were observed.

[0039] On the 9th day of culture, cell lysates were prepared using RIPA buffer and then incubated with anti-p16 INK4a Antibody (IBL), anti-p21 Cip1 / Waf1Western blot analysis was performed using antibodies against phospho-RB (Cell Signaling Technology), anti-lamin B1 (Abcam), anti-RB (Santa Cruz), anti-phospho-RB (Cell Signaling Technology) to detect phosphorylation at Ser780, anti-p53 (Santa Cruz), anti-phospho-p53 (Cell Signaling Technology) to detect phosphorylation at Ser15, and anti-α-tubulin (Sigma-Aldrich) antibodies. In addition to the changes at the RNA level detected by PCR, we observed a decrease in phosphorylated RB and an increase in phosphorylated p53, similar to changes observed in cellular senescence.

[0040] It is also known that reactive oxygen species (ROS) production increases with cellular senescence. Therefore, we analyzed ROS production in TIG-3 cells cultured for 9 days in medium supplemented with each bacterial culture supernatant using H2DCFDA. The results showed an increase in ROS levels in cells treated with the culture supernatants of P. asaccharolytica (Pa), P. gingivalis (Pg), and F. nucleatum (Fn) (Figure 9(C)). Next, we confirmed that the effects of these bacterial culture supernatants were not due to the induction of apoptosis. Figure 9(D) shows the results of Annexin V or PI staining analysis performed on day 9 after the addition of culture supernatants of P. asaccharolytica, P. gingivalis, or E. coli. Actinomycin D was used as an apoptosis-inducing compound, and doxorubicin was used as a senescence-inducing compound. None of the cells cultured with bacterial supernatants showed positive staining with Annexin V or PI, confirming that apoptosis was not induced.

[0041] In addition, induced pluripotent stem cells (iPSCs) TkDN4-M (Non-Patent Document 9) generated from human normal cells were obtained from the University of Tokyo and used to form intestinal epithelial organoids (Non-Patent Document 10). Culture supernatants from P. asaccharolytica or P. gingivalis were added to the intestinal epithelial organoid culture medium, with unused culture medium (Mock) added at 1 / 30 volume. The culture medium was changed every three days for nine days, and then the organoid culture medium was changed for three days. The changes in organoids over time are shown in Figure 9(E), and the results of analyzing the relative cell number based on ATP content are shown in Figure 9(F). Cell proliferation was suppressed in both organoids cultured with P. asaccharolytica and P. gingivalis culture medium.

[0042] These results suggest that the culture supernatants of these three bacterial species secrete genotoxic stress factors that cause DNA damage and induce carcinogenesis. Therefore, we comprehensively analyzed the metabolites contained in the culture supernatants of these bacteria using mass spectrometry. We cultured 11 species of bacteria that are significantly increased in colorectal cancer patients and analyzed the metabolites contained in the culture supernatants. We found that short-chain fatty acids were significantly increased in the supernatants of the three bacterial species that induce cellular senescence. Among the short-chain fatty acids found in the metabolites, butyrate was found to be abundant in the metabolic products of the bacteria that induce cellular senescence and cell death. Therefore, we analyzed the effect of butyrate on cell proliferation and cellular senescence (Figure 10).

[0043] Butyrate was added to TIG-3 cells at concentrations ranging from 0 mM to 10 mM, and the effect on cell proliferation was analyzed. The medium containing butyrate was added on days 0, 3, and 6 after the start of culture, and then replaced with medium without butyrate on day 9. The figure shows the state of the cells on day 12. Cell proliferation was not inhibited up to a butyrate concentration of 1 mM, but at higher concentrations, cell senescence and cell death were observed (Figure 10(A)).

[0044] Analysis of cellular senescence markers revealed that they also changed in a butyrate concentration-dependent manner (Figure 10(B)). Since it is thought that there are other metabolic products besides butyrate that induce cellular senescence and cell death, it is possible that carcinogenic stress is caused by the combined effects of multiple metabolic products.

[0045] We investigated whether short-chain fatty acids, either individually or in mixtures, could induce cellular senescence by adding them to TIG-3 cell culture medium at concentrations equivalent to those found in bacterial metabolic products (Figure 11). The amounts of short-chain fatty acids in the culture supernatants of P. asaccharolytica, P. gingivalis, and F. nucleatum, which induce cellular senescence, and E. coli (a control), were measured, and short-chain fatty acids at the same concentrations as when added to the cell culture medium (Table 3 shows the final concentrations of each short-chain fatty acid, namely, acetic acid, propionic acid, butyric acid, isobutyric acid, and isovaleric acid, added to the medium), either individually or as a mixture (SCFA mixture), were added to TIG-3 culture medium to analyze whether they could induce cellular senescence.

[0046] [Table 3]

[0047] Figure 11(A) shows the state of TIG-3 cells on day 9 after the addition of each fatty acid. Cell senescence was observed in TIG-3 cells when butyrate was added alone at the same concentration as when the culture supernatants of P. gingivalis and F. nucleatum were suspended in the culture medium. Furthermore, when a mixture of short-chain fatty acids was added, P. asaccharolytica, P. gingivalis, and F. nucleatum showed cellular senescence similar to that observed when the culture supernatants were added. These results suggest that short-chain fatty acids, particularly butyrate, which are metabolic products of bacteria, may induce cellular senescence and contribute to the development of cancer.

[0048] Furthermore, cells cultured with short-chain fatty acids contained in the culture supernatants of P. asaccharolytica, P. gingivalis, and E. coli were analyzed by RT-qPCR to analyze the expression of genes involved in cellular senescence (Figure 11(B)). The addition of butyric acid at the same concentration as that of P. gingivalis culture supernatant at 1 / 30 increased the expression of p16 INK4a , p21 Cip1 / Waf1 The expression of lamin B1 was altered in a manner similar to that induced by cellular senescence. Furthermore, the addition of a mixture of short-chain fatty acids from the culture supernatants of P. asaccharolytica and P. gingivalis resulted in the expression of p16 INK4a , p21 Cip1 / Waf1 The expression of lamin B1 induced a stronger senescence-like response than the addition of lamin B1 alone.

[0049] Butyrate inhibits p16 INK4a , p21 Cip1 / Waf1 Since butyrate induces the expression of SASP factors and secretes them, it is thought that butyrate promotes cellular senescence. Therefore, we used butyrate-producing bacteria in which butyrate production was inhibited to examine their ability to induce cellular senescence. We analyzed the effects of wild-type P. gingivalis (ATCC33277) and a butyrate synthesis-deficient mutant (PGAGU118, Non-Patent Document 11) on cellular senescence. The butyrate concentrations in the culture medium of each bacterium (Figure 11(C), left) and typical photographs of TIG-3 cells cultured for 9 days with 1 / 30 the amount of culture supernatant of each bacterium (Figure 11(C), right) are shown. Even when culture supernatant of the butyrate synthesis-deficient mutant was added, the cells did not undergo cellular senescence and continued to grow, strongly suggesting that butyrate induces cellular senescence.

[0050] To further verify the idea that butyrate induces cellular senescence and is involved in carcinogenesis, we performed analysis using colon cancer tissue samples in which high concentrations of butyrate-producing bacteria were detected. High concentrations of butyrate (15-25 mM) are said to be present in the colonic lumen, but the intestinal epithelium is covered with mucus, which inhibits the invasion of intestinal bacteria, and it has been reported that the butyrate concentration in the intestinal epithelium is quite low. Using patient tissue samples, we analyzed the presence of butyrate-producing bacteria and the expression of proteins related to cellular senescence (Figure 12). The patient profiles of the samples used for analysis are as follows:

[0051] [Table 4]

[0052] In situ hybridization using probes specific for the butyrate-producing bacteria P. asaccharolytica and P. gingivalis revealed that these butyrate-producing bacteria were present in the patient's colon cancer tissue. Furthermore, immunohistochemistry confirmed the expression of p16 and IL-6, proteins involved in cellular senescence, in the same area, revealing increased expression of these proteins (Figure 12(A)). Furthermore, it was revealed that butyrate concentrations were higher in cancerous tissues than in non-cancerous tissues (Figure 12(B)). These results suggest that butyrate-producing bacteria induce cellular senescence in human colon tissue.

[0053] Furthermore, we investigated whether butyrate-producing bacteria promote the development of colorectal cancer and whether Apc, a cancer-prone organism, is involved in the development of colorectal cancer in the distal colon and rectum. Δ14 / + Analysis was performed using mice (Non-Patent Document 12). 8CFU of P. asaccharolytica, P. gingivalis, or F. nucleatum subsp. (as a positive control) suspended in 200 μl of PBS was administered orally every other day for 8 weeks, and the number and size of tumors were analyzed (Figure 13(A)). Both P. gingivalis and P. asaccharolytica significantly increased the number of tumors compared to the control PBS-treated group. Furthermore, the P. gingivalis-treated group developed more tumors than the P. asaccharolytica-treated group. This is thought to be strongly correlated with the amount of butyrate produced by the bacteria and their ability to induce cellular senescence.

[0054] Furthermore, using P. gingivalis and a butyrate synthesis-deficient mutant, we examined whether tumorigenesis was reduced by reducing butyrate production (Figure 13(B)). Δ14 / + Mice were force-fed with P. gingivalis and a butyrate synthesis-deficient mutant, and the number and size of tumors were analyzed (Figure 13(B)). As a result, the number and size of tumors were significantly reduced in the butyrate synthesis-deficient mutant-treated group. The photographs show typical colon tumors in model mice administered with wild-type and butyrate synthesis-deficient mutants.

[0055] Furthermore, ABT-263, a compound known to selectively remove senescent cells and inhibit cellular senescence, was administered at a dose of 100 mg / kg for a total of 4 weeks, 5 days a week with two 1-week rest periods. Δ14 / + ABT-263 was administered to mice, and the number and size of tumors were analyzed (Figure 13(C)). Although the number of tumors was reduced by ABT-263 administration, no significant difference was observed (Figure 13(C) left). However, it was observed that the tumor size was significantly reduced by ABT-263 (Figure 13(C) right).

[0056] These results indicate that P. gingivalis and P. asaccharolytica induce cellular senescence and promote carcinogenesis through the secretion of bacterial metabolic products, short-chain fatty acids, particularly butyrate. Furthermore, bacteria that were narrowed down as characteristic bacteria of colorectal cancer patients but did not affect cells alone in either the co-culture system or the system with the addition of culture supernatant may also induce cellular senescence together with other bacteria.

[0057] Furthermore, the 12 species of bacteria detected in colorectal cancer patients but rarely detected in healthy individuals may include bacteria that proliferate with carcinogenesis, but because they are thought to be highly correlated with colorectal cancer, they could be used to screen colorectal cancer patients. For example, if these bacteria are detected in a stool sample, further testing could be performed by colonoscopy, making them suitable for screening colorectal cancer patients. In particular, detecting the four species of bacteria detected in early-stage colorectal cancer patients may lead to early detection of colorectal cancer.

[0058] Based on the above experimental results, compounds, food ingredients, and food compositions can be screened using P. gingivalis, P. asaccharolytica, and F. nucleatum, which are characteristically found in colorectal cancer patients and induce cellular senescence, as indicators to identify substances that suppress carcinogenesis. Specifically, the substance to be screened can be mixed with an agar medium or liquid medium and the growth of the bacteria can be analyzed.

[0059] Furthermore, since it has been revealed that bacterial metabolic products, particularly short-chain fatty acids, cause cellular senescence, it is also possible to use the short-chain fatty acids secreted by bacteria as markers to screen for compounds, food ingredients, etc. that suppress excessive secretion of short-chain fatty acids, particularly butyric acid. Specifically, P. gingivalis, P. asaccharolytica, and F. nucleatum can be cultured in a medium supplemented with a candidate substance, and compounds and compositions can be screened using a decrease in the production of short-chain fatty acids, particularly butyric acid, as an indicator.

[0060] Considering that the detection frequency of these bacteria is significantly reduced after surgical resection of tumors as described above, it is believed that ingesting substances, compositions, or foods that can inhibit the growth of these bacteria can maintain a good intestinal environment and prevent or suppress the recurrence of colorectal cancer. Screening for compounds, food ingredients, and food compositions that inhibit the growth of these bacteria or the excessive secretion of short-chain fatty acids will lead to the development of supplements and functional foods that maintain a good intestinal flora and contribute to the prevention of colorectal cancer onset.

Claims

1. A method for screening for a substance that prevents the onset or recurrence of colorectal cancer, comprising: A screening method characterized by using as an index the inhibition of growth of Porphyromonas gingivalis or Porphyromonas asaccharolytica, which are bacteria that induce cellular senescence and produce butyric acid.

2. Adding a target substance or composition to a culture medium for bacteria; 2. The screening method according to claim 1, wherein the screening is carried out using the growth of the bacteria as an index.

Images