• Molecular & Cellular Toxicology
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• v.5 no.3
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• pp.222-229
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• 2009

Our previous finding that pre-initiation treatment of indole-3-carbinol (I3C) represents a chemopreventive effect in dimethylbenz[a]anthracene (DMBA)-induced mammary carcinogenesis has prompted us to test the global expression of genes at an early stage. Rats were continuously fed 300 ppm I3C in their diet at 6 weeks of age and were injected with DMBA at 7 weeks of age, and were sacrificed at 8 weeks of age. Global gene expression analysis using oligonucleotide microarrays was conducted to detect altered genes in DMBA- or DMBA plus I3C-treated mammary glands. Altered genes were identified by fold changes of 1.2 and by t-test (P<0.05) from the log ratios of the hybridization intensity of samples between control (Group 1) and DMBA (Group 2), and from those of samples between DMBA (Group 2) and DMBA plus I3C (Group 3). From these genes, we chose altered genes that were up- or down-regulated by DMBA treatment and recovered to the control level by I3C treatment. For early stage of carcinogenesis, I3C treatment induced the recovery to normal levels of several genes including cell cycle pathway (cyclin B2, cell division cycle 2 homolog A), MAP signaling pathway (fibroblast growth factor receptor 1, platelet derived growth factor receptor, beta polypeptide), and insulin signaling (protein phosphatase 1, regulatory (inhibitor) subunit 3B and flotillin 2), which were up-regulated by DMBA treatment. In addition, I3C treatment induced the recovery to normal levels of several genes including those of MAPK signaling (transforming growth factor, beta receptor 1 and protein phosphatase 3, catalytic subunit, beta isoform), which were down-regulated by DMBA treatment. These results suggest that the targeting of these genes presents a possible approach for chemoprevention in DMBA-induced mammary carcinogenesis.

• Molecules and Cells
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• v.41 no.4
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• pp.290-300
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• 2018

Using an in vitro model of intestinal organoids derived from intestinal crypts, we examined effects of indole-3-carbinol (I3C), a phytochemical that has anticancer and aryl hydrocarbon receptor (AhR)-activating abilities and thus is sold as a dietary supplement, on the development of intestinal organoids and investigated the underlying mechanisms. I3C inhibited the in vitro development of mouse intestinal organoids. Addition of ${\alpha}$-naphthoflavone, an AhR antagonist or AhR siRNA transfection, suppressed I3C function, suggesting that I3C-mediated interference with organoid development is AhR-dependent. I3C increased the expression of Muc2 and lysozyme, lineage-specific genes for goblet cells and Paneth cells, respectively, but inhibits the expression of IAP, a marker gene for enterocytes. In the intestines of mice treated with I3C, the number of goblet cells was reduced, but the number of Paneth cells and the depth and length of crypts and villi were not changed. I3C increased the level of active nonphosphorylated ${\beta}$-catenin, but suppressed the Notch signal. As a result, expression of Hes1, a Notch target gene and a transcriptional repressor that plays a key role in enterocyte differentiation, was reduced, whereas expression of Math1, involved in the differentiation of secretory lineages, was increased. These results provide direct evidence for the role of AhR in the regulation of the development of intestinal stem cells and indicate that such regulation is likely mediated by regulation of Wnt and Notch signals.

• Journal of Nutrition and Health
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• v.27 no.4
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• pp.347-355
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• 1994

Indolo[3,2-b]carbazole(ICZ) is a potent Ah receptor agonist with biological activities similar in several respects to those of the potent environmental toxin, TCDD. ICZ is produced during the oilgomerization of indole-3-carbinol(I3C), a breakdown product of the glucobrassicin present in food plants of the Brassica genus. In the present study we examined ICZ levels in tissues and excreta of rats treated with I3C or dietary cabbage of established glucobrasicin content, and in feces of conventional and germfree rats fed on a basal diet, and of humans. We also examined the levels of cytochrome P4501A1 induction, as determined by the ethoxyresorufin ο-deethylase assay, in tissues of animals that received cabbage-supplemented diets, or which were treated with purified I3C or ICZ. Our findings indicated that incorporation of either homogenized or whole freeze-dried cabbage in the feed led to large increases(16-60 fold) in the levels of ICZ in the feces and lower gastrointestinal tract of rats. We observed that whereas ICZ is readily detectable at about the same levels(2.00$\pm$0.50 ppb) in the feces of conventional rats fed on a purified diet and in human feces, levels of ICZ in the feces of germfree animals fed on the basal diet were at the limits of detection(0.40$\pm$0.20 ppb), indication that gut bacteria are important for the production of ICZ from essential dietary constituents in the basal diet. We showed that in contrast to the near 7000-fold difference in CYP1A1 inducing potencies of ICZ and TCDD in cells in culture, their inducing potencies differ by only about an order of magnitude in rats. Nonetheless, the levels of ICZ remaining in livers twenty hours after I3C treatment appear too low to account for the induced activity. This result indicates that ICZ may be rapidly cleared from the liver or that substances other than, or in addition to, ICZ be responsible for the enzyme-inducing activity of orally administered I3C or its precursors.

• Journal of Life Science
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• v.23 no.2
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• pp.207-212
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• 2013

In this paper, we investigated whether resveratrol could induce the expression of cysteine-rich angiogenic inducer 61 (CYR61), which is a member of the CCN families. We showed that resveratrol up-regulated CYR61 protein expression in three different human colorectal cancer cell lines. In addition, resveratrol induced CYR61 protein expression in a dose- and time-dependent manner in a HCT116 cell line. To investigate the relationship between various biological activities of resveratrol and CYR61 expression, HCT116 cells were incubated with several NSAIDs, antioxidants, or resveratrol. Interestingly, resveratrol only induced CYR61 protein expression. The expression of CYR61 was not related to the presence of p53. A promoter assay revealed that the 786-bp promoter region (-732/+54) contains a regulatory region and that indole-3-carbinol and 6-gingerol could not induce CYR61 expression. In conclusion, our results indicate up-regulation of CYR61 is extremely resveratrol specific. The results can help to shed light on the unique biological function of resveratrol.

• Journal of Nutrition and Health
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• v.41 no.3
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• pp.224-231
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• 2008

We examined the effect of indole-3-carbinol (I3C, $C_9H_9NO$), an autolysis product of a glucosinolate and a glucobrassicin in vegetables, on MMP-2, -9 activities and TIMP-l and -2 inductions via microtubule-associated protein kinase (MAPK) signaling pathway in prostate cancer cell line, PC3 cells. Our results indicated that I3C inhibited cell growth of PC3 cells in dose (0,50, 100 ,${\mu}M$) and time (0,24,48 and 72 h) dependent manners. Using gelatin zymography for MMP activity, we demonstrated that I3C significantly decrease MMP-2 and -9 activities in PC3 cells. We also observed that I3C decreased the proteins and mRNA levels of MMP-2 and -9 in PC3 cells as well. Inversely, expressions of TIMP-l and -2 protein and mRNA in PC3 cells were increased by I3C in a dose dependent manner. In another experiment, we showed that I3C inhibited PC3 cells invasiveness by using marigel invasion assay and we also found that I3C suppressed MMP transcriptional activity by MAPK signaling pathways. Taken together, our results suggest that I3C may contribute to the potential beneficial food component to prevent the cancer metastasis in prostate cancer cells. (KoreanJNutr2008; 41(3): 224~23I)

• Proceedings of the Korean Society of Toxicology Conference
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• 2001.10b
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• pp.15-15
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• 2001

In the present study, we examined the chemopreventive effects of indole-3-carbinol (I3C), a constituent of cruciferous vegetables (the Family of Cruciferae) such as cabbages, cauliflowers and broccoli on multiple intestinal neoplasia (Min) genetic mouse model and on mouse colon carcinogenesis induced by azoxymethane (AOM) as well as on rat mammary carcinogenesis induced by 7, 12-dimethybenz［$\alpha$］anthracene (DMBA).(omitted)

• Proceedings of the Korean Society of Toxicology Conference
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• 2001.10a
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• pp.58-58
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• 2001

In the present study, we examined the chemopreventive effects of indole-3-carbinol (I3C), a constituent of cruciferous vegetables (the Family of Cruciferae) such as cabbages, cauliflowers and broccoli on multiple intestinal neoplasia (Min) genetic mouse model and on mouse colon carcinogenesis induced by azoxymethane (AOM) as well as on rat mammary carcinogenesis induced by 7, 12-dimethybenz［$\alpha$］anthracene (DMBA).(omitted)

• Journal of the Korean Society of Food Science and Nutrition
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• v.41 no.12
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• pp.1701-1707
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• 2012

3,3'-Diindolylmethane (DIM) is a major in vivo derivative of the putative anticancer agent indole-3-carbinol, which is present in cruciferous vegetables and has been reported to have anti-carcinogenic properties. An abnorrmally elevated level of cyclooxygenase-2 (COX-2) has been implicated in the pathogenesis of carcinogenesis. To investigate the mechanism by which DIM exhibits anti-carcinogenic effects, we investigated the effects of DIM on COX-2 expression in MCF-10A human mammary epithelial cells treated with the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA). DIM inhibited TPA-induced COX-2 expression and suppressed the synthesis of prostaglandin $E_2$, one of the major products of COX-2. Nuclear factor-kappa B ($NF-{\kappa}B$) is a transcription factor known to play a role in regulation of COX-2 expression. Treatment of MCF-10A cells with TPA increased nuclear translocation of phospho-p65, with the maximal levels being reached at 1 hour, while DIM inhibited the TPA-induced nuclear translocation of phospho-p65. Overall, we demonstrated that DIM suppresses phorbol ester-induced $PGE_2$ production and COX-2 expression in MCF-10A cells. The reduction in COX-2 levels by DIM maybe mediated through inhibition of $NF-{\kappa}B$ signaling.

• Proceedings of the Korea Environmental Mutagen Society Conference
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• 2001.10b
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• pp.85-86
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• 2001

• Proceedings of the Korean Society of Toxicology Conference
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• 2002.05b
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• pp.106.2-132
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• 2002

형질전환 (유전자 결핍; Knockout) Min 마우스를 이용하여 대장암 발생에서 배추, 양배추 주성분인 indole-3-carbinol (I3C)의 대장암 예방효과를 규명하고자 하였다. 실험동물로는 C57BL/6J-Apc$^{min/+}$(Min 마우스)계의 5내지 6주령의 수컷 이형접합체 형질전환 마우스 70마리와 C57BL/6J계의 동일 산자, 동일 주령의 수컷 wildtype 비형질전환 마우스 10kfl를 The Jackson Laboratory 사 (Bar harber, ME, USA)로부터 직접 구입하였다. C57BL/6J-Apc$^{min/+}$계 수컷 이형접합체 형질전환 (Min)마우스 70마리를 각 군 20내지 25마리씩 세군으로 나누었다. Group 1에는 20마리, Group 2에는 25마리, Group 3에는 25마리를 배치하고, I3C 투여 실험군 (Group 1과 2)에는 실험시작시에 AIN-76A 분말사료에 I3C가 각각 100 및 300ppm이 함유되도록 조제하여 공급하였다. 그리고 실험군(Group 3)에는 실험시작부터 종료시까지 AIN-76A 정제고형사료(Teklad사, WI, USA)를 자유로이 급이하였다. 각군간의 체중, 사료 및 음수소비량을 매 2주마다 측정하였고, 10주간 (16주령)의 실험종료시에는 최종체중과 간장, 신장, 비장 등의 장기무게를 측정하여 상대장기 무게비를 산출하였다. 대조군으로서 C57BL/6J계의 동일 산자, 동일 주령의 수컷 wildtype 비형질전환 마우스 10마리는 같은 조건의 사육실에서 AIN-76A 정제고형사료를 33주간 자유로이 급이하였다. 실험동물은 부검전에 하룻밤 동안 절식하고 이산화탄소 흡입 마취하에서 흉대동맥을 절단하여 방혈하고 각 장기(심장, 폐, 위)를 적출하여 생리심염수에 넣어 장기무게를 측정하고 포르말린에 고정하였다. 소장과 대장의 검사를 위하여 위의 식도부위와 직장을 실로 결찰하여 적출하고 생리심염수를 주입하여 팽창시켜, 십이지장, 공장, 및 회장, 그리고 대장으로 나누어 여과지에 펼친 후 포르말린에 고정하였다. 소장과 대장은 육안 및 자동 영상분석길ㄹ 이용한 분석이 끝난 후에 각 부위별로 4-6개의 절편을 작제하여 포르말린에 재고정하고, 통상적인 조직처리과정, 파리핀 포매 및 3-4$\mu$m 두께의 조직절편을 제작하여 H&E 염색을 실시하여 현미경으로 검경하였다. 약 1주일간의 포르말린 고정이 끝난 소장 및 대장을 부위별, 별 종양개수 및 분포를 자동영상분석기(Kontron Co. Ltd., Germany)로 분석하였다. 체의 변화, 장기무게, 사료소비량 및 마리당 종양의 개수에 대한 통계학적 유의성 검증을 위하여 Duncan's t-test로 통계처리 하였고, 종양 발생빈도에 대하여는 Likelihood ration Chi-square test로 유의성을 검증하였다. C57BL/6J-Apc$^{min/+}$계 수컷 이형접합체 형질전환 마우스에 AIN-76A 정제사료만을 투여한 대조군의 대장선종의 발생률은 84%(Group 3; 21/25례)로써 I3C 100ppm 및 300ppm을 투여한 경우에 있어서는 각군 모두 60%(Group 1; 12/20 례, Group 2; 15/25 례)로 감소하는 경향을 나타내었다. 대장선종의 마리당 발생개수에 있어서는 C57BL/6J-Apc$^{min/+}$계 수컷 이형접합체 형질전환 마우스에 AIN-76A 정제사료만을 투여한 대조군은 1.40$\pm$0.24(100%)에 비하여 I3C 저농도 투여 실험군(Group 1; 0.85$\pm$0.23; 61%, P<0.01), 그리고 I3C 고농도 투여 실험군(Group 2 ; 1.32$\pm$0.29 ; 94%)의 순으로 감소하였다. 선종의 크기별 종양의 발생개수의 분포에 있어서 I3C 저농도 투여 실험군에 있어서는 선종의 크기가 3mm이하의 수가 현저하게 감소하였다. C57BL/6J-Apc$^{min/+}$계 수컷 이형접합체 형질전환 마우스에 AIN-76A 정제사료만을 투여한 대조군의 부위별 소장선종의 발생수는 십이지장부위를 제외하고 각 군에서 유의한 변화는 관찰되지 않았다. 십이지장 종양의 발생개수에서만 I3C 저농도 투여 실험군(Group 1 ; 3.11$\pm$0.85)이 대조군 (Group 3: 1.48$\pm$0.35) 및 I3C 고농도 투여 실험군(Group 2: 1.56$\pm$0.47)에 비하여 유의성 있게 증가하였다. (P<0.05). 따라서 I3C은 소장에서는 암예방 효과가 뚜렷하지 않으나, 대장에 대한 암에방 효과가 있을 것으로 생각된다. 소장 및 대장을 제외한 간장, 신장, 비장, 심장, 폐 그리고 위 등의 기타 장기에서의 조직병리학적 변화는 관찰되지 않았다. 소장 및 대장의 종양은 선종(polyps)으로 관찰되었다. 지난 10여년간 형질전환 및 유전자 결핍 실험동물의 종류가 기하 급수적으로 증가하여 이용되고 있다. 가족성 대장 선종성 용종증(FAP)의 대표적인 모델로 이용되고 있는 C57BL/6J-Apc$^{min/+}$계 수컷 이형접합체 형질전환 마우스를 사용하여 배추나 양배추의 주요성분인 Indole-3-carbinol(I3C)의 대장암 예방효과가 있는지를 검색하여 본 결과 AIN-76A정제사료만을 투여한 대조군의 대장선종의 발생률 84%에 비하여 I3C 100 및 300ppm을 투여한 실험군에 있어서 각군 모두 60%로서 감소하는 경향을 나타내었으며, 대장선종의 마리당 발생개수에 있어서는 대조군의 1.40$\pm$1.041를 100%로 환산하였을 경우 I3C 저농도 및 고농도 투여 실험군에서는 각각 약 61%와 94%를 나타내여 감소하였다. 특히 대장선종의 크기별 분포에 있어서 선종의 크기가 3mm이하의 수가 현저하게 감소하였다. 따라서 저농도 I3C의 투여는 실험적 유전성 가족성 대장 선종성 용종증 모델에 있어서 어느정도 암 예방효과가 있는 것으로 생각된다. 그러나 소장 선종의 발생에는 별 영향이 없는 것으로 생각된다. 그러나 본 실험에 사용된 C57BL/6J-Apc$^{min/+}$계 수컷 이형접합체 형질전환 마우스는 실험개시 시점이 7내지 8주령이 경과하여 이미 태생기부터 소장 및 대장의 선종 발생이 진행되어 온 것을 감안하고 특히 비스테로이드계 항염증 소염제(NSAIDS)와 같은 강력한 COX-2억제제가 아님을 고려하면, 상당한 선종의 발생을 억제할 수 있는 가능성이 매우 높다고 생각한다. 또한 이제까지 배추나 양배추 성분의 복합성분들에 대한 실험적 대장암 모델에서의 촉진효과 등에 대한 보고들이 있어 온 점을 고려할 때 위암(Kim 등, 1994) 간암(Kim 등, 1994), 유방암(Grubbs, 등, 1995; Bradlow 등, 1995)에 대한 예방효과가 있을 것으로 생각된다. 앞으로 이러한 종양조직내에서의 COX-2 및 iNOS mRNA와 단백질의 발현정도를 분자병리학적으로 연구중에 있으며, 향후 십자화과식물 성분인 indole-3-carbinol이 마우스뿐 만 아니라 랫드의 화학발암물질에 의한 대장종양에 대한 억제효과 있는지 연구 필요가 있다. Min 마우스와 같은 형질 전환(유전자결핍;knockout) 실험동물을 이용한 새로운 중기 발암성 시험범의 확립을 통한 각종 환경 유해물질의 발암성 유무 및 COX-2 억제작용이 있는 식품인자의 암예방 후보물질을 체계적으로 검색하는데 유용하게 활용될 수 있을 것으로 생각한다.