Cytosolic phospholipase A2: targeting cancer through the tumor vasculature

Abstract

Purpose: In vascular endothelial cells, low doses of ionizing radiation trigger the immediate activation of cytosolic phospholipase A2 (cPLA2). This event initiates prosurvival signaling that could be responsible for radioresistance of tumor vasculature. Thus, the development of radiosensitizers targeting these survival pathways may enhance tumor response to radiation therapy. Arachidonyltrifluoromethyl Ketone (AACOCF3), a specific cPLA2 inhibitor, was studied as a potential radiosensitizer.

Experimental design: Vascular endothelial cells (3B11 and MPMEC) and lung tumor cells (LLC and H460) were treated with 1 micromol/L AACOCF3 for 30 minutes prior to irradiation. Treatment response was evaluated by clonogenic survival, activation of extracellular signal-regulated kinase 1/2 (ERK1/2), tubule formation, and migration assays. For in vivo experiments, mice with LLC or H460 tumors in the hind limbs were treated for 5 consecutive days with 10 mg/kg AACOCF3 administered daily 30 minutes prior to irradiation. Treatment response was assessed by tumor growth delay, Power Doppler Sonography, and immunohistochemistry.

Results: In cell culture experiments, inhibition of cPLA2 with AACOCF3 prevented radiation-induced activation of ERK1/2 and decreased clonogenic survival of irradiated vascular endothelial cells but not the lung tumor cells. Treatment with AACOCF3 also attenuated tubule formation and migration in irradiated vascular endothelial cells. In both tumor mouse models, treatment with AACOCF3 prior to irradiation significantly suppressed tumor growth and decreased overall tumor blood flow and vascularity. Increased apoptosis in both tumor cells and tumor vascular endothelium was determined as a possible mechanism of the observed effect.

Conclusion: These findings identify cPLA2 as a novel molecular target for tumor sensitization to radiation therapy through the tumor vasculature.

Fig. 1. Inhibition of cPLA₂ with AACOCF₃ enhances cell death and prevents activation of pro-survival signaling in irradiated vascular endothelial cells

Clonogenic Survival. (A, B) 3B11, LLC, and H460 cells were plated and treated with 1 μM AACOCF₃ for 30 min prior to irradiation. After 1 week, colonies consisting of ≥ 50 cells were counted and normalized for plating efficiency. A) Shown are average surviving fractions and SEM from three experiments; * p < 0.05. B) Shown is a bar graph of percent survival after exposure to 2 Gy; * p < 0.05. ERK phosphorylation. C) 3B11, LLC, and H460 cells were treated with 1 μM AACOCF₃ for 30 min prior to irradiation with 3 Gy and lysed 3 min after the beginning of irradiation. Shown are the Western blot analyses with specific antibodies to phospho-ERK1/2, total ERK1/2, and actin.

Fig. 2. cPLA₂ inhibitor AACOCF₃ attenuates tubule formation and migration in irradiated vascular endothelial cells

Tubule formation in A) 3B11 cells or B) primary MPMEC. Cells were cultured onto Matrigel in the absence (control) or presence of 1 μM AACOCF₃ for 30 minutes and then irradiated with 3 Gy. A) Representative micrographs of capillary tubule formation taken 5 hours after treatment are shown. Tubule formation was quantified as the number of tubule branches per high power field (4 HPF per sample). Shown are bar graphs of the average tubule formation for 3B11 (A) and MPMEC (B) with SEM from three independent experiments; *, p < 0.05. Migration assay is shown for C) 3B11 cells or D) MPMEC cells were added to the top chamber of 24 well plates with 8 μm matrigel-coated inserts. Fresh medium was added to the bottom chamber, and both chambers were treated with vehicle (control) or 1 μM AACOCF₃ for 30 minutes prior to irradiation with 3 Gy. After 24 hours, the insert chambers were stained with DAPI and migrated cells were counted (6 HPF per sample). Shown are representative micrographs of migrated 3B11 taken 24 hours after treatment (C) and bar graphs of the average number of migrated cells per HPF for 3B11 (C) and MPMEC (D) with SEM from three independent experiments; *, p < 0.05.

Fig. 3. Inhibition of cPLA₂ with AACOCF₃ decreases tumor size in irradiated mouse models

Using heterotopic tumor models of Lewis Lung Carcinoma (LLC) (A, B) or H460 large cell carcinoma of the lung (C, D), mice were treated intraperitoneally with vehicle (control) or 10 mg/kg AACOCF₃ and tumors were irradiated 30 minutes later with 3 Gy. Treatment was repeated for 5 consecutive days. Tumor volumes were calculated using external caliper measurements. A) Shown are the mean LLC tumor volumes in each of the treatment groups (control, IR, AACOCF₃, and AACOCF₃ + IR). B) LLC; Shown is a bar graph of the average number of days taken to reach 700 mm³ in comparison to control with SEM from group of 4 to 6 mice; *, p = 0.02. C) H460; Shown is a bar graph of the average tumor volumes 13 days post-injection with SEM from group of 4 to 6 mice; *, p =0.05. D) H460; Shown is a bar graph of the average number of days taken to reach 1400 mm³ in comparison to control with SEM from group of 4 to 6 mice.

Fig. 4. cPLA₂ inhibitor AACOCF₃ attenuates vascularity in irradiated tumors

C57/BL6 mice with LLC tumors received intraperitoneal injections of vehicle or 10 mg/kg AACOCF₃ 30 minutes prior to irradiation with 3 Gy. Treatment was repeated for 5 consecutive days. Twenty-four hours after the final treatment, tumor blood flow was analyzed by three-dimensional Power Doppler sonography (A, B) or tumors were harvested, fixed in 10% formalin, sectioned into 5 μm sections and stained with anti-vWF antibody (C, D). A) Shown are representative images of tumor blood flow. B) Shown is a bar graph of the average Percent Vascular Index with SEM from group of 3 to 5 animals; *, p < 0.05. C) Shown are representative micrographs of vWF-stained vessels. D) Shown is the bar graph of the average number of stained vessels per HPF with SEM from group of 3 to 5 mice; *, p < 0.05.

Fig. 5. Treatment with AACOCF₃ results in increased apoptosis and decreased Akt phosphorylation within irradiated tumor models

C57/BL6 mice with LLC tumors were treated as described in Fig. 4. A) TUNEL staining and hematoxylin counterstaining were performed on 5 μm sections from LLC tumors. Shown are representative micrographs of tumor with blood vessels from treated mice White arrows indicate TUNEL-positive endothelial cells; red arrows indicate TUNEL-positive tumor cells. B) Phospho-Akt immunofluorescence staining (green) and DAPI counterstaining (blue) were performed on LLC tumor sections from treated mice. Shown are representative micrographs of positive staining for Akt phosphorylation in LLC tumors.

Fig. 6. Tumor vascular window model and vascular length density analysis

Lewis Lung Carcinoma (LLC) cells were implanted into the dorsal skinfold window in C57/BL6 mice. A sufficient vascular network was allowed to develop, and windows were treated with AACOCF₃ 30 minutes prior to irradiation with 3 Gy. Color photographs were taken daily to monitor blood vessel appearance. A) Shown are representative micrographs of 40X magnification of LLC tumor vascular window models at 0 hr and 72 hr after treatment. B) Changes in the quantity of blood vessels over time were compared with that observed at 0 h. Shown is a bar graph of the percent vascular length density 72 and 96 hours after treatment of implanted tumors with SEM from group of 3 to 5 mice; *, p < 0.05.