Anti-PD-L1 Antibody and/or 17β-estradiol Treatment Induces Changes in The Gut microbiome in MC38 Colon Tumor Model

송진희,김나영,남령희,최수인,장재영,최진아,이하나 대한암학회 2023 Cancer Research and Treatment Vol.55 No.3

Purpose 17β-Estradiol (E2) supplementation suppresses MC38 tumor growth by downregulating the expression of programmed death-ligand 1 (PD-L1). This study aims to figure out the gut microbiota that respond to anti–PD-L1 and/or estrogen treatment in MC38 colon cancer model. Materials and Methods A syngeneic colon tumor model was developed by injection of MC38 cells into C57BL/6 background male and female mice. Three days before MC38 cells injection, E2 was supplemented to male mice daily for 1 week. Male and female mice with MC38 tumors (50-100 mm3) were injected with anti–PD-L1 antibody. Fresh feces were collected 26 days after injection of MC38 cells and 16S rRNA metagenomics sequencing of DNA extracted from feces was used to assess gut microbial composition. Results At the taxonomic family level, Muribaculaceae was enriched only in the MC38 male control group. In male mice, linear discriminant analysis effect size analysis at the species level revealed that the four microorganisms were commonly regulated in single and combination treatment with anti–PD-L1 and/or E2; a decrease in PAC001068_g_uc and PAC001070_s (family Muribaculaceae) and increase in PAC001716_s and PAC001785_s (family Ruminococcaceae). Interestingly, in the anti–PD-L1 plus E2 group, a decrease in opportunistic pathogens (Enterobacteriaceae group) and an increase in commensal bacteria (Lactobacillus murinus group and Parabacteroides goldsteinii) were observed. Furthermore, the abundance of Parabacteroides goldsteinii was increased in both males and females in the anti–PD-L1 group. Conclusion Our results suggest that gut microbial changes induced by the pretreatment of estrogen before anti–PD-L1 might contribute to treatment of MC38 colon cancer.

The difference of PD-L1 expression of colorectal tumor deepening on histology, sex and age

최진아,김나영,남령희,송진희,나희영,강경훈 대한내과학회 2021 대한내과학회 추계학술대회 Vol.2021 No.1

Changes in Gut Microbiome upon Orchiectomy and Testosterone Administration in AOM/DSS-Induced Colon Cancer Mouse Model

송진희,김나영,남령희,최수인,장재영,이하나 대한암학회 2023 Cancer Research and Treatment Vol.55 No.1

Purpose Sex hormones are known to affect the gut microbiota. Previously, we reported that endogenous and exogenous testosterone are associated with colorectal cancer (CRC) development and submucosal invasion. In the present study, we investigated whether the gut microbiota is affected by orchiectomy (ORX) and testosterone propionate (TP) administration using an azoxymethane/dextran sulfate sodium (AOM/DSS)-induced CRC mouse model.Materials and Methods Gut microbiota was evaluated by means of 16S rRNA gene sequencing of stool DNA extracted from feces that were obtained at 13 weeks after AOM injection (from 22-week-old animals) and stored in a gas-generating pouch.Results The increase in microbial diversity (Chao1 and Phylogenetic Diversity index) and Firmicutes/Bacteroidetes (F/B) ratio upon AOM/DSS treatment in ORX mice was significantly decreased by TP supplementation. The ratio of commensal bacteria to opportunistic pathogens was lower in the TP-administered females and ORX mice than in the AOM/DSS group. Opportunistic pathogens (Mucispirillum schaedleri or Akkermansia muciniphila) were identified only in the TP group. In addition, microbial diversity and F/B ratio were higher in male controls than in female and ORX controls. Flintibacter butyricus, Ruminococcus bromii, and Romboutsia timonensis showed similar changes in the male control group as those in the female and ORX controls.Conclusion In conclusion, testosterone determines the dysbiosis of gut microbiota, which suggests that it plays a role in the sex-related differences in colorectal carcinogenesis.

Histologic Findings and Inflammatory Reactions After Long-term Colonization of Helicobacter felis in C57BL/6 Mice

이주엽,김나영,최윤정,남령희,최윤진,권용환,Kichul Yoon,서지형,Seon Min Lee,이혜승,이동호 대한암예방학회 2014 Journal of cancer prevention Vol.19 No.3

Background: The Helicobacter felis (H. felis) mouse model has been developed for the research regarding pathogenesis of chronic gastritis and gastric cancer. The aim of this study was to investigate long-term H. felis colonization in the stomachs of C57BL/6 mice and subsequent histologic findings and inflammatory reactions including pro-inflammatory cytokines. Methods: Twenty-three female C57BL/6 mice at 4 weeks of age were gavaged with H. felis, and 13 control mice served as vehicle only. The mice were sacrificed at 4, 24, and 52 weeks after inoculation. The infection status and degree of inflammation were determined by culture and histopathology. The level of gastric mucosal myeloperoxidase (MPO), tumor necrosis factor alpha (TNF-α), and interleukin-1beta (IL-1β) were measured by ELISA. Results: The overall infection rate was 100%, as determined by the culture and histology. At 4, 24, and 52 weeks, the neutrophil and monocyte scores were significantly higher in infected mice than in control mice. At 24 weeks after inoculation, most of the infected mice showed mucosal atrophy with or without metaplasia, and a few showed focal dysplasia. Adenocarcinoma was observed in one mouse at 52 week post-infection. Gastric mucosal MPO and IL-1 levels were significantly higher in infected mice than those in control mice at 24 and 52 weeks. However, the expression of gastric mucosal TNF-α was not significantly different between the infected and control mice at any time-point. Conclusions: Long-term H. felis-infection in C57BL/6 mice provoked a severe inflammatory reaction and it progressed into atrophy, metaplasia, dysplasia and cancer. IL-1β might play an important role in the inflammatory response of mice to Helicobacter species.

No Correlation of Inflammation With Colonization of Helicobacter pylori in the Stomach of Mice Fed High-salt Diet

Ju Yup Lee,김나영,남령희,Yoon Jeong Choi,서지형,이혜승,Jane C Oh,이동호 대한암예방학회 2014 Journal of cancer prevention Vol.19 No.2

Background: Previous studies on Helicobacter pylori infection in mice have contributed to better understanding of the pathogenesis of chronic gastritis and gastric carcinoma. The aim of this study was to evaluate H. pylori colonization and subsequent inflammatory responses in the stomachs of C57BL/6 mice depending on inoculation number and the presence of high-salt diet. Methods: Eighty-four female mice with 4 weeks age were used in this study. The infected mice were gavaged with H. pylori strain Sydney-1 (SS1), and the uninfected mice were dosed with vehicle. In each of these groups, half of the mice were fed ona basal diet (0.25% salt) and the other half were fed on a high-salt diet (7.5% salt). The infected mice were inoculated 4 or 5 times, and infection status and degree of inflammation were checked by culture and histopathology, respectively, after 4 weeks. Gastric mucosal myeloperoxidase and tumor necrosis factor-alpha were measured by ELISA. Results: The overall infection rate was 95.2%; the infection rate after 5 inoculations (100%) was greater than that after 4 inoculations (91.3%). However, no differences in the degree of inflammation were found between 2 groups. The bacterial density was also significantly increased in mice that were on the high-salt diet and had been inoculated 5 times, respectively. Mean neutrophil infiltration in the infected group was 1.7±0.6 (1, minimal; 2, mild; 3, moderate; 4, marked). However, the high-salt diet was not increase the inflammatory grade in the infected group. Gastric mucosal myeloperoxidase and tumor necrosis factor-alpha levels did not increased by the high-salt diet and increased the number of inoculation. Conclusions: In spite of well colonization of H. pylori in the stomachs of C57BL/6 mice, the degree of subsequent inflammation was irrelevant to high-salt diet and frequent (5 times) inoculations.

Effect of N-Methyl-N-Nitrosourea on Helicobacter-induced Gastric Carcinogenesis in C57BL/6 Mice

이주엽,김나영,최윤정,남령희,최윤진,이선민,최다은,이혜승,김진욱,이동호 대한암예방학회 2016 Journal of cancer prevention Vol.21 No.3

Background: The aim of this study was to investigate the effect of N-methyl-N-nitrosourea (MNU) treatment followed by chronic Helicobacter pylori SS1 and H. felis colonization on the stomachs of C57BL/6 mice. The role of MNU and Helicobacter species in gastric carcinogenesis was also elucidated. Methods: A total of 69 C57BL/6 mice at 4 weeks of age were divided into 6 groups according to MNU treatment and H. pylori SS1 or H. felis infection. The mice were sacrificed at 21 and 50 weeks. The degree of inflammation was determined by histopathology. The levels of gastric mucosal myeloperoxidase, TNF-α, and interleukin-1β (IL-1β) were measured by ELISA. Results: In the H. felis groups with or without MNU, the incidence of gastric tumors was 21.1% and 35.0% at 21 and 50 weeks, respectively. No gastric tumors were observed in all control mice. At 50 weeks, 37.5% of gastric adenoma cases were observed in the H. felis alone and MNU + H. felis groups. Furthermore, 12.5% of gastric adenocarcinoma cases were observed in the MNU alone and MNU + H. felis groups. The gastric mucosal IL-1 level was significantly higher in the MNU + H. felis group at 21 weeks and H. felis group at 50 weeks, respectively, than that for control mice (P < 0.05). However, the effect of MNU on H. pylori SS1-induced gastric carcinogenesis was low compared to that on H. felis. Conclusions: Administration of MNU before H. felis infection provokes severe inflammation through IL-1β, and eventually induces gastric cancer. However, the role of MNU in H. pylori SS1-induced gastric carcinogenesis model is minor.

Anti-inflammatory and Anti-tumorigenic Effects of Açai Berry in Helicobacter felis-infected mice

이주엽,김나영,최윤정,남령희,이선민,함민희,서지형,최윤진,이혜승,이동호 대한암예방학회 2016 Journal of cancer prevention Vol.21 No.1

Background: The aim of this study was to evaluate the anti-inflammatory and anti-tumorigenic effect of açai berry after chronic Helicobacter felis colonization in the stomachs of C57BL/6 mice. Methods: A total of 57 four-week-old female C57BL/6 mice (18 control mice and 39 experimental mice) were used. The mice were administered orogastrically with vehicle only or vehicle containing H. felis, 5 times every other day. After inoculation of H. felis, mice were fed either a standard or an açai-containing diet and then sacrificed at 4, 24, and 52 weeks. The infection status and degree of inflammation were determined by culture and histopathology. The level of gastric mucosal myeloperoxidase (MPO), TNF-α, and interleukin-1β (IL-1β) were measured by ELISA. Results: At 24 weeks after inoculation, mucosal atrophy and mucous metaplasia appeared in all infected mice. At 52 weeks after inoculation, dysplastic change was noted in 10%, 25%, and 50% of mice in the H. felis-control, H. felis-açai 5%, and H. felis-açai 10% groups, respectively. The neutrophil, monocyte, atrophy, and metaplasia grades of infected mice showed no significant difference among the H. felis-infected groups. H. felis-infected mice fed with açai berry showed no significant difference compared with H. felis-infected control mice in gastric mucosal MPO, TNF-α, and IL-1β levels. Conclusions: H. felis that colonized the stomachs of C57BL/6 mice provoked inflammation, and induced mucosal atrophy, metaplasia, and dysplasia. However, açai berry did not effectively prohibit the gastric carcinogenesis which was induced by chronic H. felis infection.

Analysis of Gastric Body Microbiota by Pyrosequencing: Possible Role of Bacteria Other Than Helicobacter pylori in the Gastric Carcinogenesis

손성화,김나영,조현진,김재연,박지현,남령희,석영재,김연란,이동호 대한암예방학회 2017 Journal of cancer prevention Vol.22 No.2

Background: Gastric microbiota along with Helicobacter pylori (HP) plays a key role in gastric disease. The aim of our study is to investigate the difference of human gastric microbiota between antrum and body according to disease (control vs. gastric cancer) and HP status. Methods: Each antrum and body biopsy was collected from 12 subjects at Seoul National University Bundang Hospital. Gastric microbiota was analyzed by bar-coded 454 pyrosequencing of the 16S rRNA gene. Twelve subjects consisted of HP-negative control (n = 2), HP-negative cancer (n = 2), HP-positive control (n = 3), and HP-positive cancer (n = 5). The analysis was focused on non-HP urease-producing bacteria (UB) and non-HP nitrosating or nitroreducing bacteria (NB) between antrum and body. Results: Gastric body samples showed higher diversity compared to gastric antrum mucosa samples but there was no significant difference. The mean of operational taxonomic units was higher in HP(−) cancer than HP(+) cancer (antrum, 273.5 vs. 228.2, P = 0.439; body, 585.5 vs. 183.2, P = 0.053). The number of non-HP UB and non-HP NB was higher in HP(−) cancer groups than the others. These differences were more pronounced in the body (P = 0.051 and P = 0.081, respectively). Analysis of overlap of non-HP UB and non-HP NB revealed the higher composition of Streptococcus pseudopneumoniae, S. parasanguinis, and S. oralis in HP(−) cancer groups than the others, only in the body (P = 0.030) but not in the antrum (P = 0.123). Conclusions: Higher diversity and higher composition of S. pseudopneumoniae, S. parasanguinis, and S. oralis in HP(−) cancer group than the other groups in the body suggest that analysis of microbiota from body mucosa could be beneficial to identify a role of non-HP bacteria in the gastric carcinogenesis.

위점막 순응 및 소장 손상으로 인한 만성 비스테로이드성 소염제 유발 위염증 백서 모델 수립의 어려움

이병환 ( Byoung Hwan Lee ),김나영 ( Nayoung Kim ),남령희 ( Ryoung Hee Nam ),이주엽 ( Ju Yup Lee ),이혜승 ( Hye Seung Lee ),이창희 ( Chang Hee Lee ),박지현 ( Ji Hyun Park ),이동호 ( Dong Ho Lee ) 대한소화기학회 2014 대한소화기학회지 Vol.63 No.6

Background/Aims: The prevalence of peptic ulcer disease has not decreased mainly due to an increase in the use of NSAIDs. This study was conducted in order to determine whether a chronic NSAID-induced gastric inflammation model could be established by repeated administration of NSAID. Methods: Indomethacin (10 mg/kg) was administered once per week for six weeks in 8- and 26-week rats and animals were sacrificed every week after administration. Gross ulcer index, histologic damage index, myeloperoxidase (MPO) activity, and mucus (glucosamine) levels were measured. Small bowel damage was also evaluated. Results: Gross gastric damage index showed a peak level at three weeks and then decreased slowly in the 26-week indomethacin group. Gastric mucosal glucosamine level increased in both the 8-week (p=0.038) and 26-week groups (p=0.007). In addition, gastric mucosal MPO level decreased in the 8-week group (p=0.018) but did not show a decrease in the 26-week group. Small bowel damage began to occur at three weeks during the schedule and eight of 36 rats (22.2%) died due to perforation or peritonitis of the small bowel in the 8- and 26-week indomethacin groups, respectively. Conclusions: Due to gastric adaptation and small bowel damage, repeated administration of NSAID to experimental animals may not be an adequate method for establishment of the chronic gastric inflammation model. (Korean J Gastroenterol 2014;63:341-347)

Effect of Estradiol in an Azoxymethane/Dextran Sulfate Sodium-Treated Mouse Model of Colorectal Cancer: Implication for Sex Difference in Colorectal Cancer Development

손희진,손성화,김나영,이하나,이선민,남령희,박지현,송진희,신은,나희영,김주성,이동호,서영준 대한암학회 2019 Cancer Research and Treatment Vol.51 No.2

Purpose This study demonstrates that estradiol downregulates inflammation and inhibits colorectal cancer (CRC) development in azoxymethane/dextran sulfate sodium (AOM/DSS) mouse model. Materials and Methods AOM/DSS-treated male and female mice were sacrificed at weeks 2, 10, and 16, to assess estrogen effects on colitis and carcinogenesis. Macroscopic and histologic severity of colitis and Western blot and quantitative real-time polymerase chain reaction were evaluated, to measure inflammatory mediators and cytokines. Results Compared with AOM/DSS-treated male mice (M-AOM/DSS group), AOM/DSS-treated male mice with estradiol administration (M-AOM/DSS+estr group) displayed at week 2 significantly decreased severity of colitis. At weeks 10 and 16, AOM/DSS-treated female mice (F-AOM/DSS group) and the M-AOM/DSS+estr group showed significantly lower tumor multiplicity compared with the M-AOM/DSS group. At week 2, F-AOM/DSS group had a lower level of nuclear factor-B (NF-B) expression and higher level of nuclear factor erythroid 2-related factor 2 (Nrf2) expression, compared to the M-AOM/DSS group. At week 2, expression levels of NF-B and its related mediators decreased in the M-AOM/DSS+estr group, while levels of Nrf2 and Nrf2-related anti-oxidant enzymes increased. In addition, estradiol significantly increased Nod-like receptor protein 3 (NLRP3) inflammasome expressions in AOM/DSS-treated male mice. In contrast, at weeks 10 and 16, Nrf2 and its-related anti-oxidant enzymes and NLRP3 inflammasome were highly expressed in M-AOM/DSS group and in F-AOM/DSS group, who developed cancer. Conclusion The data suggest that estradiol inhibits the initiation of CRC by regulating Nrf2-related pathways. Moreover, these imply the dual role of Nrf2 and NLRP3 inflammasome, including promotion of tumor progression upon tumor initiation.