Gemcitabine triggers angiogenesis-promoting molecular signals in pancreatic cancer cells: Therapeutic implications

Abstract

Pancreatic tumor microenvironment (TME) is characterized by poor tumor-vasculature and extensive desmoplasia that together contribute to poor response to chemotherapy. It was recently shown that targeting of TME to inhibit desmoplasiatic reaction in a preclinical model resulted in increased microvessel-density and intratumoral drug concentration, leading to improved therapeutic response. This approach, however, failed to generate a favorable response in clinical trial. In that regard, we have previously demonstrated a role of gemcitabine-induced CXCR4 signaling as a counter-defense mechanism, which also promoted invasiveness of pancreatic cancer (PC) cells. Here, we investigated the effect of gemcitabine on endothelial cell phenotype. Gemcitabine-treatment of human-umbilical-vein-endothelial-cells (HUVECs) did not promote the growth of HUVECs; however, it was induced when treated with conditioned media from gemcitabine-treated (Gem-CM) PC cells due to increased cell-cycle progression and apoptotic-resistance. Moreover, treatment of HUVECs with Gem-CM resulted in capillary-like structure (CLS) formation and promoted their ability to migrate and invade through extracellular-matrix. Gemcitabine-treatment of PC cells induced expression of various growth factors/cytokines, including IL-8, which exhibited greatest upregulation. Further, IL-8 depletion in Gem-CM diminished its potency to promote angiogenic phenotypes. Together, these findings suggest an indirect effect of gemcitabine on angiogenesis, which, in light of our previous observations, may hold important clinical significance.

Keywords: IL-8; angiogenesis; gemcitabine; pancreatic cancer.

Figure 1. Effect of conditioned media obtained from gemcitabine- or vehicle- treated pancreatic cancer cells on endothelial cell growth, cell-cycle progression and survival

A. HUVECs (1 × 10⁴ cells/well) were seeded in 96-well plates and allowed to grow for 24 h followed by treatment with conditioned media (CM) obtained from vehicle (V-CM) or gemcitabine (Gem-CM) treated PC (Colo-357 and MiaPaCa) cells. Growth of HUVECs was measured by WST-1 assay after 24 h and 48 h of incubation in CM. Bars (mean ± SD; n = 3) represent fold change in growth. *, p < 0.05. B. Synchronized HUVECs were treated with V-CM or Gem-CM for 24 h and distribution of cells in different phases of cell cycle was analyzed by propidium iodide (PI) staining through flow cytometry. C. HUVECs (1 × 10⁶) were grown in 6-well plate for 24 h, treated with V-CM or Gem-CM for next 48 h, and subsequently stained with 7-AAD and PE Annexin V followed by flow cytometry.

Figure 2. Conditioned media from gemcitabine-treated pancreatic cancer cells facilitates capillary-like structure (CLS) formation in HUVEC

HUVECs (1 × 10⁴) were plated on Matrigel-coated 96-well plates in conditioned media (CM) obtained from vehicle (V-CM) or gemcitabine (Gem-CM) treated Colo-357 and MiaPaCa cells. After 16 h of incubation, CLS formation was examined under inverted microscope, photographed and number of CLS formation counted in 10 random fields. Bars (mean ± SD; n = 3) represent number of CLS per fields. *, p < 0.05.

Figure 3. Conditioned media from gemcitabine-treated pancreatic cancer cells promotes motility and invasion of endothelial cells

HUVECs were seeded on A. non-coated (for motility assay), or B. Matrigel-coated (for invasion assay) membranes. V-CM or Gem-CM obtained from Colo-357 and MiaPaCa were used as a chemoattractant. Migrated and invaded cells were counted and presented as average number of cells in 10 random field ± SD. Data is representative of three independent experiments.*, p < 0.05.

Figure 4. Gemcitabine induces IL-8 expression in pancreatic cancer cells

A. Colo-357 cells were treated with gemcitabine (10 μM) for 8 h. Subsequently, RNA was isolated, cDNA was prepared and cytokines/growth factors profiling was performed using qRT-PCR. B. Colo-357 and MiaPaCa cells were treated with gemcitabine (10 μM) for 8 h. Post treatment, media was replaced with fresh culture medium and incubated for next 24 h. Thereafter, total protein was isolated and subjected to immunoblot analysis to examine IL-8 expression using specific antibody. β-actin was used as a loading control. C. Level of IL-8 in conditioned media of vehicle or gemcitabine treated PC cells was measured using ELISA as described in materials and methods. Data is presented as mean ± SD; n = 3 .*p < 0.05.

Figure 5. Neutralization of IL-8 abolishes Gem-CM-induced HUVEC proliferation and CLS formation

A. HUVECs (1 × 10⁴ cells/well) were seeded in 96-well plates, treated with V-CM, Gem-CM or Gem-CM pre-treated with IL-8 neutralizing antibody or control IgG (200 ng/mL) and growth was measured by WST-1 assay after 48 h of incubation. B. HUVECs (1 × 10⁴) were plated on Matrigel-coated 96-well plates in V-CM, Gem-CM or Gem-CM pre-treated with IL-8 neutralizing antibody or control IgG. After 16 h of incubation, CLS formation was examined under inverted microscope, photographed and number of CLS formation counted in 10 random fields. Data is presented as mean ± SD; n = 3. *, p < 0.05.

Figure 6. Depletion of IL-8 in Gem-CM decreases motility and invasiveness of HUVEC

HUVECs were seeded on A. non-coated (for motility assay), or B. Matrigel-coated (for invasion assay) membranes. V-CM, Gem-CM or IL-8 depleted Gem-CM were used as chemoattractant. Bars represent mean ± SD (n = 3) of number of migrated or invaded cells per field. *, p < 0.05.