Indolepropionic Acid, a Metabolite of the Microbiome, Has Cytostatic Properties in Breast Cancer by Activating AHR and PXR Receptors and Inducing Oxidative Stress - PubMed (original) (raw)
. 2020 Aug 25;12(9):2411.
doi: 10.3390/cancers12092411.
Edit Mikó 1 2, Tünde Kovács 1, Laura Jankó 1, Tamás Csonka 3, Gréta Lente 1, Éva Sebő 4, Judit Tóth 5, Dezső Tóth 6, Péter Árkosy 5, Anita Boratkó 1, Gyula Ujlaki 1, Miklós Török 7, Ilona Kovács 7, Judit Szabó 8, Borbála Kiss 5, Gábor Méhes 3, James J Goedert 9, Péter Bai 1 2 10
Affiliations
- PMID: 32854297
- PMCID: PMC7565149
- DOI: 10.3390/cancers12092411
Indolepropionic Acid, a Metabolite of the Microbiome, Has Cytostatic Properties in Breast Cancer by Activating AHR and PXR Receptors and Inducing Oxidative Stress
Zsanett Sári et al. Cancers (Basel). 2020.
Abstract
Oncobiotic transformation of the gut microbiome may contribute to the risk of breast cancer. Recent studies have provided evidence that the microbiome secretes cytostatic metabolites that inhibit the proliferation, movement, and metastasis formation of cancer cells. In this study, we show that indolepropionic acid (IPA), a bacterial tryptophan metabolite, has cytostatic properties. IPA selectively targeted breast cancer cells, but it had no effects on non-transformed, primary fibroblasts. In cell-based and animal experiments, we showed that IPA supplementation reduced the proportions of cancer stem cells and the proliferation, movement, and metastasis formation of cancer cells. These were achieved through inhibiting epithelial-to-mesenchymal transition, inducing oxidative and nitrosative stress, and boosting antitumor immune response. Increased oxidative/nitrosative stress was due to the IPA-mediated downregulation of nuclear factor erythroid 2-related factor 2 (NRF2), upregulation of inducible nitric oxide synthase (iNOS), and enhanced mitochondrial reactive species production. Increased oxidative/nitrosative stress led to cytostasis and reductions in cancer cell stem-ness. IPA exerted its effects through aryl hydrocarbon receptor (AHR) and pregnane X receptor (PXR) receptors. A higher expression of PXR and AHR supported better survival in human breast cancer patients, highlighting the importance of IPA-elicited pathways in cytostasis in breast cancer. Furthermore, AHR activation and PXR expression related inversely to cancer cell proliferation level and to the stage and grade of the tumor. The fecal microbiome's capacity for IPA biosynthesis was suppressed in women newly diagnosed with breast cancer, especially with stage 0. Bacterial indole biosynthesis showed correlation with lymphocyte infiltration to tumors in humans. Taken together, we found that IPA is a cytostatic bacterial metabolite, the production of which is suppressed in human breast cancer. Bacterial metabolites, among them, IPA, have a pivotal role in regulating the progression but not the initiation of the disease.
Keywords: AHR; PXR; breast cancer; epithelial-to-mesenchymal transition; indolepropionic acid; metastasis; microbiome; nitrosative stress; oncobiome; oxidative stress.
Conflict of interest statement
The authors have no competing financial or non-financial interests to declare.
Figures
Figure 1
Supplementation of mice transplanted with 4T1 breast cancer cells with indolepropionic acid (IPA) reduces metastatic burden. Female Balb/c mice (n = 10/10, 3 months of age) were grafted with 4T1 cells and were treated with IPA (1 nmol/g q.d. p.o.) or vehicle (VEH) (n = 10/10) for 14 days; then, the mice were terminated. Upon autopsy (A), the total number and (B) total and (C) individual mass of primary tumors were determined. (D) The infiltration of the primary tumors to the surrounding tissues was scored. The (E) number of mice with metastasis were plotted, and the (F) total, as well as the (G) individual mass of the metastases were measured. In tissue sections of formalin-fixed, paraffin-embedded tissue specimens from the primary tumors (H), lymphocyte infiltration and (I) mitosis score were determined. Statistical significance was determined using Student’s _t_-test, except for panels D, E, and I, where chi-square tests were conducted. *** indicate statistically significant difference between vehicle and treated groups at p < 0.001.
Figure 2
Indolepropionic acid (IPA) reduces cell proliferation in cellular models of breast cancer. (A) 500 cells/well 4T1 cells were seeded in 6-well plates and then treated with IPA in the concentrations indicated for 7 days. Then, colonies were stained according to May–Grünwald–Giemsa and analyzed using the ImageJ software (n = 3). (B) 4T1 cells were seeded in 6-well plates (75,000 cells/well) and treated with the indicated concentrations of IPA for 24 h and stained with propidium-iodide; then, they were analyzed by flow cytometry. (C) 4T1 cells (75,000 cells/well in 6-well plates) were treated with IPA in the concentrations indicated for 24 h; the ratio of necrotic and apoptotic cells were determined with staining by propidium–iodide–FITC Annexin double staining using the V/Dead Cell Apoptosis Kit and measured by flow cytometry (n = 3). (D) SKBR-3 (5000 cells/well) were seeded in 96-well plates and then were treated with IPA in the concentrations indicated for 24 h; then, total protein content was assessed in sulforhodamine B (SRB) assays (n = 3). (E) SKBR-3 (200,000 cells/well) cells were seeded in 6-well plates and were treated with IPA in the concentrations indicated for 24 h. The ratio of necrotic and apoptotic cells were determined with staining by propidium–iodide; then, they were analyzed by flow cytometry. (F) SKBR-3 (200,000 cells/well) cells were seeded in 6-well plates and were treated with IPA in the concentrations indicated for 24 h. The ratio of necrotic and apoptotic cells were determined with staining by propidium–iodide–FITC Annexin double staining using the V/Dead Cell Apoptosis Kit and measured by flow cytometry (n = 3). (G) Human fibroblast (7500 cells/well) cells were seeded in 96-well plates and then were treated with IPA in the concentrations indicated for 24 h; then, the total protein content was assessed in SRB assays (n = 3). (H) Human fibroblast (20,000 cells/well) cells were seeded in 6-well plates and then were treated with IPA in the concentrations indicated for 24 h. Then, the ratio of necrotic and apoptotic cells were determined with staining by propidium–iodide and analyzed by flow cytometry (n = 3). Fold data were log2 transformed to achieve normal distribution. Statistical significance was determined using ANOVA test followed by Tukey’s post-hoc test, except for panels D, E, and F, where Dunnett’s post-hoc tests were conducted. * or *** indicate statistically significant difference between control and treated samples at p < 0.05 or p < 0.001, respectively.
Figure 3
Indolepropionic acid (IPA) induced oxidative stress, cellular energy stress, and decreased the proportions of cancer stem cells. 500,000 cells/well 4T1 cells were treated with IPA in the concentrations indicated for 24 h; then, (A) lipid peroxidation was measured by TBARS assay, and (B) 4HNE expression was assessed by Western blotting (representative figure, n = 3). In the same cells (C), the protein expression of NRF2 (at 68 kDa) and iNOS were determined by Western blotting (n = 3), while (D) the mRNA expression of catalase (cat) was determined by RT-qPCR (n = 3). (E) The expression of the indicated proteins (pACC, ACC, FOXO1, and PGC-1_β_) were determined by Western blotting (n = 3, except for PGC-1_β_, where n = 2). (F) 100,000 cells/well 4T1 cells were treated with the indicated concentration of IPA for 24 h; then, the proportions of aldehyde dehydrogenase-positive cells were determined in Aldefluor assays using flow cytometry (n = 3). For Western blots, a typical experiment was displayed. Fold data were log2 transformed to achieve normal distribution. Statistical significance was determined using the ANOVA test followed by Dunnett’s post-hoc test, except for panel F, where Student’s _t_-test was used. * and *** indicate statistically significant difference between control and treated samples at p < 0.05 and p < 0.001, respectively.
Figure 4
Indolepropionic acid (IPA) induced mesenchymal-to-epithelial transition (EMT). 100,000 cells/well 4T1 cells were treated with IPA in the concentrations indicated for 24 h; then, (A) cellular morphology was observed using Texas Red-X Phalloidin and DAPI staining (representative figure, n = 3). Scale bar corresponds to 25 μm. Mesenchymal cells are characterized by stress filaments that are absent in epithelial cells; for details on morphology, see [27] and Figure S3. (B) Normalized resistance was measured in electric cell–substrate impedance sensing (ECIS) impedance-based experiments (representative figure, mean ± SD, n = 1). (C,D) In IPA-treated 4T1 cells, the expression of the indicated genes were determined in (C) RT-qPCR ( n = 3) and (D) Western blotting (n = 3). _β_-actin was used as a loading control. For Western blots, a typical experiment was displayed. Fold data were log2 transformed to achieve normal distribution. A statistical significance was determined using the ANOVA test followed by Dunnett’s post-hoc test, except for panel D, where a chi-square test were conducted. *, ** and *** indicate statistically significant difference between control and treated samples at p < 0.05, p < 0.01 or p < 0.001, respectively. Abbreviations: Vimentin (Vim), fibroblast growth factor-binding protein1 (FgfBp1), snail family transcriptional repressor-1 (Snail) and β-catenin) E-cadherin, zonula occludens-1 (ZO1).
Figure 5
Indolepropionic acid (IPA)-elicited oxidative stress has central role in mediating IPA-elicited antineoplastic effects. 4T1 cells (500,000 cells/well, TBARS; 100,000 cells/well, ALDH; 1500 cells/well, SRB) were treated with IPA in the concentrations indicated for 24 h with or without the antioxidants, as indicated. Subsequently, (A) thiobarbituric acid-reactive substances, (B) the fraction of Aldefluor-positive cells and (C) total protein content was determined. Fold data were log2 transformed to achieve normal distribution. Statistical significance was determined using the ANOVA test followed by Dunnett’s post-hoc test. *, **, and *** indicate statistically significant difference between control and treated samples at p < 0.05, p < 0.01 or p < 0.01, respectively. Abbreviations: TBARS—Thiobarbituric acid-reactive substances, ALDH—Aldehyde dehydrogenase, SRB—Sulforhodamine B, GSH—reduced glutathione, NAC—N-acetyl-cysteine.
Figure 6
AHR and PXR are responsible for the IPA-elicited antineoplastic effects. 100,000 cells/well 4T1 cells were treated with IPA in the concentrations indicated for 24 h with or without the inhibitors, as indicated. Subsequently, (A) the actin cytoskeleton was stained using phallodin-Texas Red, and morphology was assessed using confocal microscopy. (B) On the same cells, the expression of the indicated proteins were determined by Western blotting. Fold data were log2 transformed to achieve normal distribution. Statistical significance was determined using an ANOVA test followed by Dunnett’s post-hoc test, except for panel A, where a chi-square test was conducted. *** indicate statistically significant difference between control and IPA treated samples at p < 0.01. ## and ### indicate significant difference between control and CH223191 or ketoconazole-treated samples at p < 0.01 or p < 0.001, respectively. Abbreviations: PXRi—PXR inhibitor, AHRi—AHR inhibitor, ACC—acyl-CoA carboxylase (an AMPK target protein).
Figure 7
Higher expression of pregnane X receptor and aryl hydrocarbon receptor prolongs survival in breast cancer. The effect of expression of (A) AHR or (B) PXR on survival in breast cancer was analyzed by kmplot.com, which is a freely accessible database. On panel (A), the effect of PXR expression on survival is depicted with the data acquired from RNAseq experiments. On panel (B), the effect of PXR expression on survival is depicted with the data acquired from microarray experiments, and patients were stratified as a function of receptor expression. On panel (C), the effect of AHR expression on survival is depicted with the data acquired from RNAseq experiments. On panel (D), the effect of AHR expression on survival is depicted with the data acquired from microarray experiments, and patients were stratified as a function of receptor expression. Total survival rates were assessed, and all samples are represented. Abbreviations: NR1I2—pregnane X receptor, Aryl hydrocarbon receptor (AHR), Pregnane X receptor (PXR) The database was assessed the 19th February 2020.
Figure 8
Higher intratumoral expression of pregnane X receptor (PXR) and nuclear aryl hydrocarbon receptor (AHR) shows correlation with low-grade and lower-mitosis breast cancers. (A) TMA was stained with the indicated antibodies. A typical staining pattern is shown. The bar is equivalent of 50 µm. Cases in TMA were scored for receptor expression using the H-score system. (B–E) H-score of the nuclear expression of the AHR receptor were related to (B) stage of the disease, (C) mitosis score, (D) tubule formation, and (E) histological subtype. (F–H) H-score of the expression of PXR receptor were related to (F) grade of the disease, (G) mitosis score, and (H) mitotic index. Statistical significance on panels B and F was determined using an ANOVA test followed by Dunnett’s post-hoc test, while on panels C, D, E, G, and H, Student’s _t_-test was used. Stage 0 (in situ carcinoma) and stage 1 patients, Mitosis score 2 and 3 patients, and tubule formation score 1 and 2 patients were handled together due to the low number of cases. Abbreviations: Aryl hydrocarbon receptor (AHR), Pregnane X receptor (PXR), no special type (NOS). *, ** and *** indicate statistically significant difference between control and treated samples at p < 0.05, p < 0.01 or p < 0.001, respectively.
Figure 9
The fecal expression of TnaA shows correlation with the pathological and clinical features of breast cancer. (A,B) The abundance of bacterial TnaA DNA was assessed in human fecal DNA samples from cohort study. The ct values lower than 45 are shown in Providencia rettgeri. Median values indicated by a line. On panel A, all patients and controls are compared, statistical significance was calculated using Student’s _t_-test. On panel B, patients were stratified based on the stage of the disease, and statistical comparison was made using an ANOVA test followed by Dunnett’s or Tukey’s post-hoc tests. Fold data were log2 transformed to achieve normal distribution. * indicate a statistically significant difference between control and treated samples at p < 0.05.
Figure 10
The fecal protein expression of E. coli TnaA is higher in patients with higher proportions of tumor-infiltrating lymphocytes. (A) E. coli total lysate was run on an SDS-PAGE gel, transferred to nitrocellulose membrane, and probed with an anti-TnaA antibody. (B,C) Fecal samples of 36 low tumor-infiltrating lymphocyte (TIL) patients (0–20% TIL) and 11 of high TIL patients (30%< TIL) were assessed by Western blotting using an anti-TnaA antibody. Protein content-normalized values were obtained. Values were tested for outliers using Grubb’s method; one value was omitted from the low TIL group. (B) Normalized TnaA expression was plotted. (C) On values from low and high TIL patients, liner regression was performed.
Figure 11
Schematic representation of IPA-elicited effects to breast cancer cells.
References
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