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.