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Small molecule-driven mitophagy-mediated NLRP3 inflammasome inhibition is responsible for the prevention of colitis-associated cancer - PubMed

Small molecule-driven mitophagy-mediated NLRP3 inflammasome inhibition is responsible for the prevention of colitis-associated cancer

Wenjie Guo et al. Autophagy. 2014 Jun.

Abstract

Nonresolving inflammation in the intestine predisposes individuals to the development of colitis-associated cancer (CAC). Inflammasomes are thought to mediate intestinal homeostasis, and their dysregulation contributes to inflammatory bowel diseases and CAC. However, few agents have been reported to reduce CAC by targeting inflammasomes. Here we show that the small molecule andrographolide (Andro) protects mice against azoxymethane/dextran sulfate sodium-induced colon carcinogenesis through inhibiting the NLRP3 inflammasome. Administration of Andro significantly attenuated colitis progression and tumor burden. Andro also inhibited NLRP3 inflammasome activation in macrophages both in vivo and in vitro, as indicated by reduced expression of cleaved CASP1, disruption of NLRP3-PYCARD-CASP1 complex assembly, and lower IL1B secretion. Importantly, Andro was found to trigger mitophagy in macrophages, leading to a reversed mitochondrial membrane potential collapse, which in turn inactivated the NLRP3 inflammasome. Moreover, downregulation of the PIK3CA-AKT1-MTOR-RPS6KB1 pathway accounted for Andro-induced autophagy. Finally, Andro-driven inhibition of the NLRP3 inflammasome and amelioration of murine models for colitis and CAC were significantly blocked by BECN1 knockdown, or by various autophagy inhibitors. Taken together, our findings demonstrate that mitophagy-mediated NLRP3 inflammasome inhibition by Andro is responsible for the prevention of CAC. Our data may help guide decisions regarding the use of Andro in patients with inflammatory bowel diseases, which ultimately reduces the risk of CAC.

Keywords: andrographolide; colitis; inflammasome; inflammation-associated cancer; mitophagy.

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Figures

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Figure 1. Andrographolide prevents colitis-associated tumorigenesis. Mice were injected i.p. with a single dose (7.5 mg/kg) of AOM followed by 3 cycles of 2.5% DSS given in the drinking water for 5 d. Andro (7.5 and 15 mg/kg) was given i.g. daily during the interval between DSS cycles as shown. Mice were sacrificed on d 95 after CAC induction. (A) Body weight was recorded. (B) The inside of the colon was photographed. (C) Colon tissues were fixed and stained with H&E. (D) Tumor numbers were counted. (E and F) Tumor diameter and distribution were measured. (G) The tumor load was determined by totaling the diameters of all tumors for a given animal. Values are mean ± SEM of 9 mice/group. *P < 0.05, **P < 0.01 vs. AOM+DSS group. Andro, andrographolide.

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Figure 2. Andrographolide inhibits inflammation in a colitis-associated colorectal cancer model. Mice were subjected to the AOM-DSS model. For other details, see the legend of Figure 1. (A) The expression of PCNA, p-STAT3, p-RELA/p-p65, and PTGS2/COX2 were analyzed by immunochemistry in paraffin-embedded colon sections. Data shown are representative of 3 experiments. (B) The expressions of PCNA, p-STAT3, p-RELA, and PTGS2 in colonic tissues were examined by western blotting. (C) Statistical data of the expressions of protein from 3 mice were shown. (D and E) The mRNA expressions of Hif1a, Vegfa, Tnf, Il17a, Il6, and Ptgs2 in colon sections were determined by real-time PCR. Data are presented as means ± SEM (n = 6). *P < 0.05, **P < 0.01 vs. AOM+DSS group. Andro, andrographolide.

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Figure 3. Andrographolide alleviates experimental colitis induced by DSS in mice. Mice were treated with 2.5% DSS in their drinking water for 7 d to induce acute colitis. Andro was administered daily via i.p. injection. Mice were sacrificed on d 10 after colitis induction. (A) The body weight of the mice was measured and presented as a percentage of the original body weight. (B) The disease activity index was calculated. (C) The length of the colon was measured when the mice were sacrificed. (D) The colon was photographed. (E and F) Paraffin-embedded colon sections were stained with H&E for light microscopy assessment of epithelial damage. (G) MPO activity in colonic tissues was detected. (H) RNA was extracted from colonic tissues, and mRNA expressions of Il17a, Ifng, Il1b, and Tnf were determined by real-time PCR. (I) Protein levels of various cytokines in colon homogenates were examined by ELISA. (J) Expression of p-RELA was analyzed by immunochemical staining of paraffin-embedded colon sections. (K) Expression of p-RELA among colonic proteins was examined by western blotting. Values are mean ± SEM of 6 mice/group. *P < 0.05, **P < 0.01 vs. DSS-treated group. Andro, andrographolide. Sulfa, Sulfasalazine.

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Figure 4. Andrographolide inhibits NLRP3 inflammasome activation in mice with DSS-induced colitis. Mice were treated with 2.5% DSS in their drinking water for 7 d to induce acute colitis. Andro was administered daily via i.p. injection. (A) Peritoneal macrophages from mice were isolated on d 7. After stimulation with 5 mM ATP for 30 min, proteins were collected for western blotting. *P < 0.05. (B) Sections of colonic tissues were immunostained with DAPI (blue) and anti-ITGAM/CD11b-FITC (green) and were observed by fluorescence microscopy. Scale bar: 100 μm. Data shown are representative of 3 experiments. Andro, andrographolide.

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Figure 5. Andrographolide inhibits CASP1 activation and IL1B maturation by interrupting the formation of the NLRP3 inflammasome in vitro. THP-1 (pretreated with 500 nM PMA for 3 h) or BMDM cells were cultured with 100 ng/ml LPS for 3 h, then treated with Andro (3, 10, or 30 μM) for 1 h, followed by 1 h incubation with 5 mM ATP. (A) IL1B levels in the supernatant fraction were analyzed by ELISA. Data are mean ± SEM of 3 different experiments. *P < 0.05, **P < 0.01 vs. LPS+ATP group. (B) Protein levels of pro-IL1B, IL1B p17, pro-CASP1, cleaved CASP1, PYCARD, and NRLP3 were determined by western blotting. Data shown are representative of 3 experiments. (C) CASP1 activity was measured. Data are mean ± SEM of 3 different experiments. *P < 0.05, **P < 0.01 vs. LPS+ATP group. (D) LPS-primed THP-1 cells were treated with 30 μM Andro for 1 h, followed by 2, 5, or 15 min incubation with 5 mM ATP. In the other experiment, LPS-primed THP-1 cells were treated with Andro (3, 10, or 30 μM) for 1 h, followed by 5 min incubation with 5 mM ATP. Proteins were isolated and immunoprecipitated with an antibody against PYCARD. Data shown are representative of 3 experiments. (E) LPS-primed BMDM cells were treated with 30 μM Andro for 1 h, followed by treatment with 5 mM ATP for 15 min. Cells were analyzed by immunofluorescence cytochemistry. Scale bar: 10 μm. Data shown are representative of 3 experiments. Andro, andrographolide.

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Figure 6. Andrographolide promotes mitophagy and inhibits NLRP3 inflammasome activation in macrophages. (A) THP-1 cells, pretreated with 500 nM PMA for 3 h, were cultured with 100 ng/ml LPS for 3 h (below referred to as LPS-primed THP-1 cells), then cells were treated with Andro (3, 10, or 30 μM) for 1 h, followed by 1 h incubation with 5 mM ATP. The mitochondrial membrane potential was determined by JC-1 staining. *P < 0.05, **P < 0.01 vs. ATP group. (B) LPS-primed THP-1 cells were treated with 30 μM Andro for the indicated times. Proteins were collected and the expression of p-MTOR and LC3 was detected by western blot. (C) BMDM cells were cultured with 100 ng/ml LPS for 3 h, and then cells were treated with 30 μM Andro for 1 h. LC3-II dot formation was detected by immunofluorescence. Scale bar: 10 μm. (D) LPS-primed THP-1 cells were treated with Andro (3, 10, or 30 μM) for 1 h, followed by 1 h incubation with 5 mM ATP. Then proteins were collected and analyzed by western blotting. (E) LPS-primed THP-1 cells were treated with 30 μM Andro for 1 h, followed by 1 h incubation with 5 mM ATP. Then mitochondria and cytosol were separated using a commercial kit and detected for LC3 expression. Data shown in (A) are mean ± SEM of 3 different experiments. Data shown in (B–E) are representative of 3 experiments. Andro, andrographolide.

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Figure 7. Andrographolide triggers mitophagy in macrophages. (A) BMDM cells were cultured with 30 μM Andro for 1 h (Alone and Andro group) or BMDM cells were cultured with 100 ng/ml LPS for 3 h, and then with 30 μM Andro for 1 h, followed by 15 min incubation with 5 mM ATP(LPS+ATP and LPS+ATP+Andro group). LC3 (green) and mitochondria (MitoTracker Red) colocalization were examined by immunofluorescence. Scale bar: 10 μm. (B) LPS-primed THP-1 cells were treated with 30 μM Andro for 1 h, followed by 1 h incubation with 5 mM ATP. Cells were collected for transmission electron microscopy assay. White arrow, normal mitochondria; red arrow, swollen mitochondria with disrupted cristae; blue arrow, a double membrane; green arrow, the damaged mitochondria were localized near a lysosome in the autophagolysosome. Data shown in (A and B) are representative of 3 experiments. Andro, andrographolide.

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Figure 8. Andro-induced mitophagy is responsible for inhibition of the NLRP3 inflammasome. (A and B) THP-1 cells were transfected with control shRNA or shRNA targeting BECN1. After 48 h, LPS-primed THP-1 cells were treated with Andro for 1 h, followed by 1 h incubation with 5 mM ATP. The effects of Andro on CASP1 activation (A) and IL1B maturation (B) were measured by western blotting and ELISA, respectively. *P < 0.05, **P < 0.01 vs. control group. (B–D) LPS-primed THP-1 cells were incubated with the autophagy inhibitors 3-MA (5 mM), chloroquine (CQ, 25 μM), Baf A1 (10 nM), or NH4Cl (20 mM) for 1 h, followed by Andro treatment for 1 h, and then stimulated with ATP for 1 h. The effects of Andro on IL1B maturation (B), CASP1 activation (C), and LC3 expression (D) were measured by ELISA and western blotting, respectively. Data shown in (B) are mean ± SEM of 3 different experiments. Data shown in (A, C, and D) are representative of 3 experiments. Andro, andrographolide.

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Figure 9. Andrographolide-driven mitophagy-mediated NLRP3 inflammasome inactivation is responsible for amelioration of murine models for colitis and CAC. (A–E) Mice were treated with 2.5% DSS in their drinking water for 7 d to induce colitis. Andro (5 mg/kg) was administered i.p. daily. CQ (50 mg/kg) was administered i.p. every 2 d. Mice were sacrificed on d 10 after colitis induction. Values are mean ± SEM of 6 mice/group. (A) The body weight of the mice was measured and presented as a percentage of the original body weight. (B) The colon was photographed. (C) The length of the colon was measured when the mice were sacrificed. (D) Protein levels of various cytokines in colon homogenates from DSS-induced mice at d 10 were examined by ELISA. *P < 0.05, **P < 0.01. (E) Expression of PTGS2/COX2 and p-RELA/p-p65 were examined by immunohistochemical staining of paraffin-embedded colon sections from DSS-induced mice at d 10. (F–I) Mice were injected i.p. with a single dose (7.5 mg/kg) of AOM followed by 3 cycles of 2.5% DSS given in the drinking water for 5 d. Andro (15 mg/kg) was given i.g. daily and CQ (50 mg/kg) was i.p. administered every 2 d during the interval between DSS cycles. Mice were sacrificed on d 95 after CAC induction. Values are mean ± SEM of 6 mice/group. (F) The inside of the colon was photographed. (G) Tumor numbers, size, and load were measured. *P < 0.05, **P < 0.01. (H) Macrophages were isolated from the spleen of AOM-DSS mice on d 95 using commercial magnetic beads as described in Materials and Methods. After stimulation with 5 mM ATP for 30 min, proteins were collected for western blotting. (I) Statistical data of the expressions of CASP1 and LC3 from 6 mice were shown. *P < 0.05. Andro, andrographolide.

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