Resolution of eicosanoid/cytokine storm prevents carcinogen and inflammation-initiated hepatocellular cancer progression

Abstract

Toxic environmental carcinogens promote cancer via genotoxic and nongenotoxic pathways, but nongenetic mechanisms remain poorly characterized. Carcinogen-induced apoptosis may trigger escape from dormancy of microtumors by interfering with inflammation resolution and triggering an endoplasmic reticulum (ER) stress response. While eicosanoid and cytokine storms are well-characterized in infection and inflammation, they are poorly characterized in cancer. Here, we demonstrate that carcinogens, such as aflatoxin B₁ (AFB₁), induce apoptotic cell death and the resulting cell debris stimulates hepatocellular carcinoma (HCC) tumor growth via an "eicosanoid and cytokine storm." AFB₁-generated debris up-regulates cyclooxygenase-2 (COX-2), soluble epoxide hydrolase (sEH), ER stress-response genes including BiP, CHOP, and PDI in macrophages. Thus, selective cytokine or eicosanoid blockade is unlikely to prevent carcinogen-induced cancer progression. Pharmacological abrogation of both the COX-2 and sEH pathways by PTUPB prevented the debris-stimulated eicosanoid and cytokine storm, down-regulated ER stress genes, and promoted macrophage phagocytosis of debris, resulting in suppression of HCC tumor growth. Thus, inflammation resolution via dual COX-2/sEH inhibition is an approach to prevent carcinogen-induced cancer.

Keywords: bioactive lipid; carcinogenesis; cell death; inflammation resolution; soluble epoxide hydrolase.

Fig. 1.

AFB₁-generated cell death stimulates HCC via up-regulation of sEH and COX-2. Annexin V/PI analysis of (A) Hepa 1-6 murine and (B) HepG2 human HCC tumor cells treated with AFB₁ (25 µM, 48 h) compared to vehicle. Quadrants containing dead tumor cells are outlined in pink. Bar graphs represent means of percent cell death ±SEM. n = 3 per group. **P < 0.01, ****P < 0.0001 vs. vehicle. (C) Hepa 1-6 tumor growth stimulated by AFB₁-generated dead cells coinjected with a subthreshold inoculum of living cells. n = 5 to 15 mice per group. The two-tailed unpaired Student’s t test was used for final tumor measurements throughout. *P < 0.05 vs. 10⁶ living cells alone (no dead cells). (D) Percent survival of mice coinjected orthotopically into the liver with AFB₁-generated Hepa 1-6 dead cells and a subthreshold inoculum of Hepa 1-6 living cells. n = 5 mice per group. Kaplan–Meier analysis indicated significantly shortened survival of mice injected with a combination of dead and living cells as depicted by the area under the Kaplan–Meier survival curves. *P < 0.05. (Scale bar, 1 cm.) (E) Western blot analysis of caspase-3 and cleaved caspase-3 expression in killed and surviving Hepa 1-6 tumor cells treated with AFB₁ (25 µM, 48 h) or vehicle. Control = vehicle-treated Hepa 1-6 living cells. Levels of β-actin demonstrate protein loading. (F) Gene expression of sEH (EPHX2) and COX-2 (PTGS2) in human monocyte-derived macrophages coincubated with AFB₁-generated HepG2 dead cells; analyzed by qPCR and normalized by GAPDH. n = 3 per group. *P < 0.05, **P < 0.01 vs. control.

Fig. 2.

Cytokine storm triggered by carcinogen debris-stimulated macrophages is prevented by dual eicosanoid pathway inhibition. (A–C) Inflammatory and angiogenic cytokine array of conditioned media from RAW 264.7 murine macrophage treated with vehicle or PTUPB (5 µM, 2 h) and stimulated with AFB₁-generated Hepa 1-6 tumor cell debris vs. debris alone without macrophages. ELISA quantification of TNF-⍺ and CCL2/MCP-1 released by RAW 264.7 macrophages treated with vehicle or PTUPB (5 µM, 2 h) and stimulated with AFB₁-generated Hepa 1-6 tumor cell debris. Data are presented as means (pg/mL) ± SEM. n = 7 per group; 3 biological repeats. ***P < 0.001. n.d., not detectable. (D) Inflammatory cytokine array of conditioned media from human monocyte-derived macrophages treated with vehicle or PTUPB (5 µM, 2 h) and stimulated with AFB₁-generated HepG2 tumor cell debris vs. debris alone. (E) Inflammatory cytokine array of conditioned media from RAW 264.7 macrophages treated with vehicle or PTUPB (5 µM, 2 h) and stimulated with LPS-generated MS-1 endothelial cell debris vs. debris alone.

Fig. 3.

Dual COX-2/sEH inhibition stimulates macrophage phagocytosis of AFB₁-generated HCC debris. Human monocyte-derived, peritoneal or RAW 264.7 murine macrophage phagocytosis of CFDA-labeled AFB₁-generated tumor cell debris measured as relative fluorescent units and normalized to percent increase above vehicle-treated macrophages. One-way ANOVA used throughout. n = 12 per group. **P < 0.01, ***P < 0.001 vs. vehicle. (A) Celecoxib (2 h) did not stimulate RAW 264.7 (Left) nor human monocyte-derived (Right) macrophage phagocytosis of AFB₁-generated Hepa 1-6 or HepG2 debris, respectively. (B) sEH inhibitor (TPPU; 2 h) stimulated murine peritoneal (Left) and human monocyte-derived (Right) macrophage phagocytosis of AFB₁-generated Hepa 1-6 or HepG2 debris, respectively. (C) Dual COX-2/sEH inhibitor (PTUPB; 2 h) stimulated RAW 264.7 (Left) and murine peritoneal (Right) macrophage phagocytosis of AFB₁-generated Hepa 1-6 debris.

Fig. 4.

Dual COX-2/sEH inhibition suppresses AFB₁ debris-stimulated ER stress and eicosanoid storm. (A) qPCR analysis of ER stress-response gene (CHOP, BiP, PDI) expression in human monocyte-derived macrophages treated with PTUPB (5 µM, 2 h) or vehicle and stimulated with AFB₁-generated HepG2 tumor cell debris. Results were normalized by GAPDH. n = 3 per group. *P < 0.05, **P < 0.01. (B) Western blot analysis of ER stress response (BiP, PDI) and COX-2 protein expression in human monocyte-derived macrophages treated with PTUPB (5 µM, 2 h) or vehicle and stimulated with AFB₁-generated HepG2 debris. Levels of β-actin demonstrate protein loading. n = 2 per group. (C) UPLC-MS/MS–based oxylipin analysis of RAW 264.7 macrophages treated with PTUPB (5 µM, 2 h) or vehicle and stimulated with AFB₁-generated Hepa 1-6 HCC debris or AFB₁-generated Hepa 1-6 debris alone. n = 2 per group. ***P < 0.001. (D) Heatmap of eicosanoid storm via UPLC-MS/MS–based oxylipin analysis in macrophages exposed to AFB₁-generated Hepa 1-6 HCC debris. n = 2 per group.

Fig. 5.

Prevention of AFB₁ debris-stimulated HCC and cytokine storm in vivo by dual COX-2/sEH inhibitor PTUPB. (A) Tumor volume of debris-stimulated Hepa 1-6 HCC tumors systemically treated with PTUPB (30 mg/kg/d) vs. vehicle. Treatment initiated once tumors reached 100 to 200 mm³. n = 5 mice per group. The two-tailed unpaired Student’s t test was used for final tumor measurements. *P < 0.05. (B) Percent survival of mice coinjected orthotopically into the liver with AFB₁-generated Hepa 1-6 debris (4.5 × 10⁵ dead cells) and Hepa 1-6 living cells (5 × 10⁵). Systemic treatment with PTUPB (30 mg/kg/d) or vehicle initiated 10 d postinjection. n = 5 mice per group. Kaplan–Meier analysis indicated significantly prolonged survival in mice treated with PTUPB compared with control. *P < 0.05. (C) Inflammatory and (D) angiogenic cytokine array of plasma from control or PTUPB-treated mice bearing debris-stimulated orthotopic HCC. Plasma was collected on day 60 postinjection.