Cytosolic phospholipase A2 and lysophospholipids in tumor angiogenesis

Abstract

Background: Lung cancer and glioblastoma multiforme are highly angiogenic and, despite advances in treatment, remain resistant to therapy. Cytosolic phospholipase A2 (cPLA(2)) activation contributes to treatment resistance through transduction of prosurvival signals. We investigated cPLA(2) as a novel molecular target for antiangiogenesis therapy.

Methods: Glioblastoma (GL261) and Lewis lung carcinoma (LLC) heterotopic tumor models were used to study the effects of cPLA(2) expression on tumor growth and vascularity in C57/BL6 mice wild type for (cPLA(2)α(+/+)) or deficient in (cPLA(2)α(-/-)) cPLA(2)α, the predominant isoform in endothelium (n = 6-7 mice per group). The effect of inhibiting cPLA(2) activity on GL261 and LLC tumor growth was studied in mice treated with the chemical cPLA(2) inhibitor 4-[2-[5-chloro-1-(diphenylmethyl)-2-methyl-1H-indol-3-yl]-ethoxy]benzoic acid (CDIBA). Endothelial cell proliferation and function were evaluated by Ki-67 immunofluorescence and migration assays in primary cultures of murine pulmonary microvascular endothelial cells (MPMEC) isolated from cPLA(2)α(+/+) and cPLA(2)α(-/-) mice. Proliferation, invasive migration, and tubule formation were assayed in mouse vascular endothelial 3B-11 cells treated with CDIBA. Effects of lysophosphatidylcholine, arachidonic acid, and lysophosphatidic acid (lipid mediators of tumorigenesis and angiogenesis) on proliferation and migration were examined in 3B-11 cells and cPLA(2)α(-/-) MPMEC. All statistical tests were two-sided.

Results: GL261 tumor progression proceeded normally in cPLA(2)α(+/+) mice, whereas no GL261 tumors formed in cPLA(2)α(-/-) mice. In the LLC tumor model, spontaneous tumor regression was observed in 50% of cPLA(2)α(-/-) mice. Immunohistochemical examination of the remaining tumors from cPLA(2)α(-/-) mice revealed attenuated vascularity (P ≤ .001) compared with tumors from cPLA(2)α(+/+) mice. Inhibition of cPLA(2) activity by CDIBA resulted in a delay in tumor growth (eg, LLC model: average number of days to reach tumor volume of 700 mm(3), CDIBA vs vehicle: 16.8 vs 11.8, difference = 5, 95% confidence interval = 3.6 to 6.4, P = .04) and a decrease in tumor size (eg, GL261 model: mean volume on day 21, CDIBA vs vehicle: 40.1 vs 247.4 mm(3), difference = 207.3 mm(3), 95% confidence interval = 20.9 to 293.7 mm(3), P = .021). cPLA(2) deficiency statistically significantly reduced MPMEC proliferation and invasive migration (P = .002 and P = .004, respectively). Compared with untreated cells, cPLA(2)α(-/-) MPMEC treated with lysophosphatidylcholine and lysophosphatidic acid displayed increased cell proliferation (P = .011) and invasive migration (P < .001).

Conclusions: In these mouse models of brain and lung cancer, cPLA(2) and lysophospholipids have key regulatory roles in tumor angiogenesis. cPLA(2) inhibition may be a novel effective antiangiogenic therapy.

Figure 1

Effect of lysophospholipids on proliferation of cPLA₂-deficient and CDIBA-treated vascular endothelial cells. cPLA₂α^+/+ (wild-type [WT]) and cPLA₂α^−/− (knockout [KO]) murine pulmonary microvascular endothelial cells (A), murine vascular endothelial 3B-11 cells (B,C), or Lewis lung carcinoma (LLC) cells (C) were grown to 60%–70% confluency. Cultures were treated with vehicle (control) or 2 μM CDIBA (3B-11 and LLC only) in the absence or presence of 10 μM lysophosphatidylcholine (LPC), 10 μM lysophosphatidic acid (LPA), or 10 μM arachidonic acid (AA) for 24 hours. Proliferation was assayed by staining the cells with an antibody against Ki-67 and immunofluorescence microscopy. The number of Ki-67–positive cells was counted in four randomly selected high-power microscopic fields from each sample in triplicate and is expressed as the mean percentage of the number of Ki-67–positive cells in the control sample in three independent experiments; error bars correspond to 95% confidence intervals. A): *P = .002 (Student t test); **P = .011 (analysis of variance [ANOVA]). B): *P < .001 (Mann–Whitney rank sum test); **P = .001 (ANOVA). C): *P = .004 (Student t test). All P values are two-sided.

Figure 2

Effects of lysophospholipids on invasion and migration of cPLA₂-deficient and CDIBA-treated vascular endothelial cells. cPLA₂α^+/+ (wild type [WT]) and cPLA₂α^−/− (knockout [KO]) murine pulmonary microvascular endothelial cells (A) or murine vascular endothelial 3B-11 cells (B) were added to the top chambers of 24-well transwell Boyden chamber plates containing 8-μm Matrigel-coated inserts. Fresh medium was added to the bottom chambers; vehicle (control) or 2 μM CDIBA (3B-11 only) was added to the medium in both chambers with or without 10 μM lysophosphatidylcholine (LPC), 10 μM lysophosphatidic acid (LPA), or 10 μM arachidonic acid (AA); and the plates were incubated for 24 hours. The inserts were removed and migrated cells on the lower surfaces of the filters were stained with DAPI (4′,6-diamidino-2-phenylindole) and counted in four randomly chosen high-power microscopic fields (HPFs) per sample. Bar graphs plot the mean number of migrated cells per HPF for triplicate samples from three independent experiments; error bars correspond to 95% confidence intervals. A): *P = .004 (Student t test); **P < .001 (analysis of variance [ANOVA]). B): *P < .001 (Student t test); **P < .001 (ANOVA). All P values are two-sided.

Figure 3

Differential effect of cPLA₂ inhibition on migration of vascular endothelial and tumor cells. A scratch assay for cell migration was performed on 3B-11 or Lewis lung carcinoma (LLC) cells grown to 70%–80% confluency. Four parallel wounds were created on each plate using a 10-μL pipette tip, and the cells were treated with dimethyl sulfoxide (control) or the cPLA₂ inhibitor CDIBA (2 μM) for 24 hours. The cells were then stained with 1% methylene blue, and cells inside and outside of the wound boundaries were counted. Migration is expressed as mean cell density within the wound. A) Representative micrographs of stained cells (at ×10 magnification). B) Bar graph of the average cell density within the wound from three independent experiments; error bars correspond to 95% confidence intervals. *P < .001 (two-sided Student t test).

Figure 4

Effect of cPLA₂ inhibition on tubule formation by vascular endothelial cells. 3B-11 cells or human umbilical vein endothelial cells (HUVEC) were cultured on Matrigel-coated 24-well plates in the absence (control) or presence of 2 μM CDIBA. Capillary tubule formation was evaluated after 6 (3B-11) or 24 (HUVEC) hours of incubation. Tubule formation was quantified as the number of tubules per high-power microscopic field (HPF) in four randomly selected HPF per sample. A) Representative micrographs of treated cells (at ×20 magnification). B) Bar graph of the mean number of tubules per HPF for 3B-11 and HUVEC from three independent experiments; error bars correspond to 95% confidence intervals. Control vs CDIBA: *P = .005 and **P = .009 (two-sided Student t test).

Figure 5

Effect of cPLA₂ inhibition on tumor growth. Lewis lung carcinoma (LLC) (A and B) or GL261 cells (C and D) were injected subcutaneously into the hind limbs of cPLA₂α^+/+ C57/BL6 mice. To inhibit cPLA₂ in vivo, approximately 2 weeks after tumor cell injection, mice received intraperitoneal injections of vehicle (control) or CDIBA at 0.5 or 1.0 mg per kg body weight once per day for five (LLC) or seven (GL261) consecutive days (n = 6–7 mice per group). cPLA₂ activity was measured by an enzymatic activity assay in plasma obtained from LLC tumor–bearing mice before treatment initiation and immediately after the final treatment. Tumor volume was measured using an external caliper. A) Mean LLC tumor volume; treatment administered on days 1–5. B) Mean number of days for LLC tumors to reach 700 mm³ in mice treated with vehicle and in mice treated with 1.0 mg CDIBA per kg body weight. *P = .04 (Student t test). C) Mean GL261 tumor volume; treatment administered on days 1–7. Control vs CIBDA: P = .03 (day 9), P = .03 (day 13), P = .01 (day 15), P = .01 (day 17), P = .02 (day 19), and P = .02 (day 21) (longitudinal analysis of least squares means). D) Mean GL261 tumor volume for each treatment group on day 21. *P = .021 (Student t test). E) cPLA₂ activity in plasma from control and CDIBA-treated mice before treatment initiation (pre) and immediately after the final treatment (post). *P = .004 (analysis of variance). Error bars correspond to 95% confidence intervals, and all P values are two-sided.

Figure 6

Tumor growth in cPLA₂α-deficient mice. Lewis lung carcinoma (LLC) or GL261 cells were injected subcutaneously into the hind limbs of cPLA₂α^+/+ or cPLA₂α^−/− C57/BL6 mice (n = 6–7 mice per group). Tumor volume was measured using power Doppler sonography at 48-hour intervals beginning 1 week after injection and ending when tumors reached a volume of 700 mm³ or a diameter of 15 mm). A) Mean LLC tumor volume for cPLA₂α^+/+ mice and for remaining tumors from cPLA₂α^−/− mice at day 16 after tumor cell injection. *P = .047 (two-sided Student t test). B) Mean GL261 tumor volumes. cPLA₂α^+/+ vs cPLA₂α^−/−. Days 11–39: P < .001 (longitudinal analysis of least squares means). Error bars correspond to 95% confidence intervals.

Figure 7

Vascularity and necrosis in tumors from cPLA₂α^−/− mice. Lewis lung carcinoma (LLC) cells were injected subcutaneously into the hind limbs of cPLA₂α^+/+ and cPLA₂α^−/− mice (n = 6–7 mice per group). Once the average tumor volume reached approximately 700 mm³, the mice were killed by cervical dislocation and their tumors were resected and fixed in 10% formalin. Fixed tumors were then sectioned, and the sections were stained with an antibody against von Willebrand factor (vWF) (an endothelial cell marker) or hematoxylin–eosin. vWF-positive vessels were counted with the use of immunofluorescence microscopy. A) Representative micrographs of positive anti-vWF staining (green) at ×40 magnification in sections of tumors from cPLA₂α^+/+ (left) and cPLA₂α^−/− (right) mice. B) Mean number of tumor blood vessels per high-power microscopic field (HPF) from cPLA₂α^+/+ and cPLA₂α^−/− mice (three mice per group; six HPFs per slide). Error bars correspond to 95% confidence intervals. *P < .001 (two-sided Student t test). C) Representative micrographs of hematoxylin–eosin-stained sections of LLC tumors from cPLA₂α^+/+ (left) and cPLA₂α^−/− (right) mice (at ×40 magnification). Black arrow indicates necrosis.

Figure 8

Pericyte coverage of blood vessels in tumors from cPLA₂α^+/+ and cPLA₂α^−/− mice. Formalin-fixed Lewis lung carcinoma (LLC) tumors from cPLA₂α^+/+ (upper rows) and cPLA₂α^−/− (lower rows) mice were sectioned and co-stained with antibodies against von Willebrand factor (left panels) and either α-smooth muscle actin (middle panels, A) or desmin (middle panels, B) and counterstained with DAPI (4′,6-diamidino-2-phenylindole). A) Representative micrographs of immunofluorescence staining for von Willebrand factor (green), α-smooth muscle actin (red), and DAPI (blue) in tumors from cPLA₂α^+/+ and cPLA₂α^−/− mice (at ×40 magnification). Right panels present merged immunofluorescence staining of von Willebrand factor and cells positive for α–smooth muscle actin (yellow). B) Representative micrographs of immunofluorescence staining for von Willebrand factor (green), desmin (red), and DAPI (blue) in tumors from cPLA₂α^+/+ and cPLA₂α^−/− mice (at ×40 magnification). Right panels present merged immunofluorescence staining of von Willebrand factor and cells positive for desmin (yellow).