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Keywords:

  • angiogenesis;
  • aspirin;
  • cyclooxygenase-2;
  • sodium salicylate

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Summary.  To determine whether aspirin and salicylate suppress colon cancer cell-mediated angiogenesis, we evaluated the effects of aspirin and sodium salicylate on endothelial tube formation on Matrigel. Aspirin and sodium salicylate concentration-dependently inhibited human endothelial cell (EC) tube formation induced by conditioned medium collected from DLD-1, HT-29 or HCT-116 colon cancer cells. Aspirin and sodium salicylate at pharmacological concentrations were equally effective in blocking tube formation. Neutralizing antivascular endothelial growth factor (VEGF) antibodies blocked colon cancer medium-induced tube formation. VEGF receptor 2 but not receptor 1 antibodies inhibited tube formation to a similar extent as anti-VEGF antibodies. These results indicate that VEGF interaction with VEGF receptor 2 is the primary mechanism underlying colon cancer-induced angiogenesis. Aspirin or sodium salicylate inhibited VEGF-induced tube formation in a concentration-dependent manner comparable to that of inhibition of colon cancer medium-induced endothelial tube formation. It has been shown that cyclooxygenase-2 (COX-2) is pivotal in cancer angiogenesis. We found that colon cancer medium-induced COX-2 protein expression in EC and aspirin or sodium salicylate suppressed the cancer-induced COX-2 protein levels at concentrations correlated with those that suppressed endothelial tube formation. Furthermore, aspirin and sodium salicylate inhibited COX-2 expression stimulated by VEGF. These findings indicate that aspirin and other salicylate drugs at pharmacological concentrations inhibit colon cancer-induced angiogenesis which is correlated with COX-2 suppression.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

It is well recognized that angiogenesis induced by cancer plays a pivotal role in cancer expansion. The mechanism by which cancer induces angiogenesis is complex and not completely understood. Recent studies have shown that the effect of cancer on angiogenesis is largely due to the production of vascular endothelial growth factor (VEGF) [1–3]. Cancer cells attract the surrounding stromal cells including fibroblasts and endothelial cells to the cancer site, and induce VEGF expression in these cells [4]. It has also been shown that cancer cells are capable of inducing the surrounding fibroblasts to express cyclooxygenase-2 (COX-2) and the over-expressed COX-2 is pivotal for cancer expansion [5]. COX-2 over-expression also increases metastatic potential and alters cell adhesion and apoptosis [6,7]. Genetic deletion of COX-2 or pharmacological inhibition of COX-2 retards cancer growth in murine cancer models [8,9]. COX-2 contributes to cancer growth by promoting angiogenesis [10]. Using a two-chamber cancer cell and endothelial cell co-culture system, Tsujii et al. have demonstrated that cancer cell-induced endothelial tube formation was abrogated by pretreatment with a selective COX-2 inhibitor [10]. Selective COX-2 inhibition has also been shown to reduce angiogenesis in vivo[11]. COX-2 occupies a central position in prostaglandin (PG) biosynthesis [12]. It catalyzes the formation of PGH2, a common precursor for a large class of PGs and thromboxane. PGE2, prostacyclin and thromboxane A2 have been shown to induce angiogenesis [13–15], which may account for the pro-angiogenic action of COX-2. PGE2 and prostacyclin have also been shown to upregulate VEGF expression [15]. Interestingly, VEGF is capable of inducing a robust expression of COX-2 in endothelial cells [16]. Thus, VEGF-induced angiogenesis is amplified by COX-2-produced PGs via a positive feedback loop. It is possible that cancer-induced angiogenesis is amplified and sustained by a similar mechanism.

Aspirin use has been shown to reduce the incidence and mortality of human cancers, especially colon cancer [17]. Sodium salicylate, a natural product from plants and the key metabolite of aspirin in human circulating blood, also suppressed carcinogen-induced cancer in rats [18,19]. The anticancer action of aspirin was attributed to inhibition of COX-2 activity. However, there is considerable skepticism about this explanation as aspirin only weakly inhibits COX-2 activity and it has a short half-life in human circulating blood: it is deacetylated to yield salicylate. Salicylate at pharmacologically tolerable concentrations does not inhibit COX-2 or COX-1 activity [20]. The anticancer action of salicylate must be due to other mechanisms. Salicylate has been shown to suppress gene expression by blocking transactivator binding. At high concentrations (>10 mm) that are intolerable to humans, salicylate inhibited NF-κB-mediated gene expression [21,22]. At pharmacological concentrations, salicylate inhibited CCAAT/enhancer binding protein α (C/EBPα)-mediated COX-2 transcription [23] and possibly also interleukin (IL)-4 transcription [24]. We postulated that salicylate (aspirin and sodium salicylate) inhibits colon cancer cell medium-induced angiogenesis by blocking cancer- and VEGF-induced COX-2 expression. To begin to test this hypothesis, we evaluated the effects of aspirin and sodium salicylate on endothelial tube formation in a Matrigel model. Our results reveal that colon cancer cell medium induced endothelial tube formation in a VEGF/VEGF-receptor 2 (R2)-dependent manner. Sodium salicylate and aspirin blocked cancer- and VEGF-induced tube formation at comparable pharmacological concentrations which correlated with those that suppressed COX-2 protein expression.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Cell culture

Human umbilical vein endothelial cells (HUVEC) were isolated from umbilical veins and maintained in medium 199 supplemented with 20% fetal bovine serum (FBS), heparin, endothelial cell growth supplement and antibiotics (penicillin plus streptomycin) (all from Sigma, St Louis, MO, USA) as previously described [25]. Only passage 1 and 2 endothelial cells were used in our experiments. EA.hy926 endothelial cell line (EA) was maintained in Dulbecco's modified Eagle's medium (DMEM) containing 5% FBS, antibiotics and HAT media supplement (Sigma), as previously described [26].

Collection of cancer cell conditioned medium

Colon cancer cell lines, DLD-1, HT-29 and HCT-116, were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA) and grown in DMEM containing 5% FBS, penicillin and streptomycin. At 90% confluence medium was removed, cells were washed and incubated in serum-free medium for 24 h. Conditioned medium (CM) was collected, centrifuged to remove any cellular contaminants and stored at −80 °C until use.

In vitro angiogenesis assay

The assay was modified from a method previously described [27]. HUVEC or EA were grown in culture flasks until they reached about 80% confluence. HUVEC were washed and cultured in 199 medium containing 0.5% FBS and antibiotics for 24 h. EA were washed and cultured in serum-free medium with antibiotics for 24 h. The cells were treated with 0.25% trypsin–EDTA (Life Technologies, Inc., Rockville, MD, USA), and the trypsinized cells were collected, counted, and resuspended in 199 medium (HUVEC) or DMEM (EA) at a concentration of 5 × 104 cells mL−1. Aspirin, sodium salicylate (Sigma), NS-398, SC560 or SC58125 (Cayman Chemical, Ann Arbor, MI, USA) at indicated concentrations was added to the cell suspension for 30 min. Recombinant VEGF165 (R&D Systems, Minneapolis, MN, USA) at 20 ng mL−1 or an equal volume of cancer cell medium was added to the cell suspension immediately prior to seeding. The cells were seeded (5000 cells/well) in a 96-well tissue culture plate which had been evenly coated with growth factor-reduced Matrigel (BD Labware, Bedford, MA, USA). Seeded cells were incubated at 37 °C in a 5% CO2 incubator for 24 h and tube formation was examined under phase-contrast microscopy connected to an Axioplan 2 imaging system (Zeiss, Thornwood, NY, USA). HUVEC tube formation was quantified by counting under ×200 magnification the length of the tubular network in five randomly selected fields. Basal tubular length was determined and subtracted. EA tube formation was quantified by dividing the number of cells in tubes by the total number of cells in five randomly selected fields under ×200 magnification. The basal value was also subtracted.

Human VEGF-specific polyclonal goat IgG (final concentration 0.1 µg mL−1), VEGF-R1 polyclonal goat IgG (10 µg mL−1) and VEGF-R2 polyclonal goat IgG (0.3 µg mL−1) were obtained from R&D Systems. According to the manufacturer, these three antibodies at the concentrations used in our experiments are expected to block the VEGF and receptor activity, respectively. A normal non-immune goat IgG (10 µg mL−1) also obtained from R&D Systems was used as a control. Cells were pretreated with antibodies in serum-free medium for 1 h before addition of cancer CM or VEGF.

Western blot analysis

Western blot analysis was performed as described previously [28]. Cells were lyzed with lysis buffer containing 50 mm Tris–HCl pH 7.5, 150 mm NaCl, 1 mm EDTA, 1 mm PMSF, 1 µg mL−1 leupeptin, 5 µg mL−1 aprotinin, 1% Nonidet P450, 0.5% sodium deoxycholate, and 0.1% SDS. The lysate was centrifuged, the supernatant was collected and boiled for 5 min. Protein concentration was determined. Lysate proteins were separated in a 4–15% SDS–polyacrylamide minigel (BioRad, Hercules, CA, USA) and then electrophoretically transferred to a nitrocellulose membrane (BioRad). Western blots were probed with a specific rabbit polyclonal anti-COX-2 antibody (Cayman). Protein bands were detected by enhanced chemiluminescence.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Suppression of endothelial tube formation by aspirin and sodium salicylate

It has been shown that colon cancer cells co-cultured with HUVEC in a two-chamber system stimulate endothelial tube formation on collagen gel [10]. To ascertain that tube formation is induced by soluble factors released by colon cancer cells, we evaluated the effect of colon cancer cell CM on HUVEC or EA cell tube formation in a 96-well plate. CM collected from three human colon cancer cell lines grown in serum-free medium for 24 h was added to HUVEC or EA cells seeded on Matrigel. After incubation at 37 °C for 24 h in a CO2 incubator, tube formation was examined under phase contrast microscopy. CM from DLD-1, HT-29 and HCT-116 all induced HUVEC or EA cell tube formation (Fig. 1a,b). The morphology of tubes formed from these two types of endothelial cells differed in that the HUVEC tubes were thinner and longer than EA tubes (Fig. 1a,b). Pretreatment with aspirin at 1 mm completely inhibited HUVEC or EA tube formation induced by each CM (Fig. 1a,b). Sodium salicylate at 1 mm also reduced CM-induced tube formation to the basal level (Fig. 1a,b). Aspirin inhibited either type of tube formation in a concentration-dependent manner. HUVEC tube formation was significantly inhibited by aspirin at 10 µm and maximal inhibition was noted at 1 mm (Fig. 2). EA tube formation induced by CM of each colon cancer cell was similarly inhibited by aspirin with maximal inhibition at 1 mm (Fig. 2). Sodium salicylate inhibited HUVEC and EA tube formation in a concentration-dependent manner comparable to aspirin inhibition (Fig. 3).

imageimage

Figure 1. Representative microphotographs of (a) human umbilical vein endothelial cells and (b) EAhy926 cell tube formation on Matrigel gel. A, E and I are controls; B, F and J, cancer CM-induced; C, G and K, aspirin (1 mm) pretreated and cancer CM-induced; D, H and L, sodium salicylate (1 mm) pretreated and cancer CM-induced. Tube formation was determined by phase microscopy at ×200 original magnification.

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Figure 2. Concentration-dependent inhibition of colon cancer CM-stimulated human umbilical vein endothelial cells and EA cell tube formation by aspirin. Each bar denotes mean ± SD of three experiments.

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Figure 3. Concentration-dependent inhibition of colon cancer CM-stimulated human umbilical vein endothelial cells and EA cell tube formation by sodium salicylate. Each bar denotes mean ± SD of three experiments.

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Cancer medium-induced tube formation was mediated by VEGF and VEGF-R2

EA tube formation induced by DLD-1, HT-29 or HCT-116 CM was blocked by a VEGF neutralizing antibody but not a control IgG (Fig. 4). Furthermore, tube formation induced by each CM was selectively blocked by anti-VEGF-R2 (flk-1) but not anti-VEGF-R1 (flt-1) antibodies (Fig. 4). These results suggest that VEGF interaction with its physiological receptor, VEGF-R2, is responsible for colon cancer-induced tube formation. To provide additional evidence for this, we evaluated the effect of VEGF165 on endothelial tube formation. VEGF significantly increased tube formation which was blocked by VEGF-R2 or VEGF antibodies (Fig. 5).

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Figure 4. Effect of anti-vascular endothelial growth factor (VEGF), anti-flt-1 (VEGF-R2) or anti-flk1 (VEGF-R1) antibodies on colon cancer CM-mediated EA tube formation. Each bar denotes mean ± SD of three experiments.

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Figure 5. Effect of anti-vascular endothelial growth factor (VEGF), anti-VEGF-R2 or anti-VEGF-R1 antibodies on EA tube formation induced by recombinant human VEGF165. Each bar denotes mean ± SD of three experiments.

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Inhibition of VEGF-induced tube formation by aspirin and sodium salicylate

Since the effect of colon cancer CM is mediated by VEGF, we suspected that aspirin and salicylate are capable of inhibiting VEGF-induced tube formation. VEGF165 (20 ng mL−1) increased HUVEC or EA tube formation to a similar extent as cancer medium (Fig. 6 vs. Figs 2 and 3). Aspirin or sodium salicylate inhibited VEGF-induced HUVEC or EA tube formation in a concentration-dependent fashion (Fig. 6) comparable to inhibition of CM-induced tube formation.

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Figure 6. Concentration-dependent inhibition of vascular endothelial growth factor-induced human umbilical vein endothelial cells and EA cell tube formation by aspirin or sodium salicylate. Each bar denotes mean ± SD of three experiments.

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Suppression of cancer medium- and VEGF-induced COX-2 expression by aspirin and sodium salicylate

It has been suggested that COX-2 over-expression contributes to cancer-induced angiogenesis [10]. In this study, we confirmed that colon cancer medium-induced HUVEC and EA tube formation was inhibited by selective COX-2 inhibitors, NS-398 and SC58125 (data not shown). As VEGF is known to stimulate COX-2 expression and salicylate is capable of suppressing COX-2 transcription induced by proinflammatory mediators, we evaluated the effect of aspirin or sodium salicylate on VEGF- and cancer medium-induced COX-2 protein levels. Treatment of HUVEC with VEGF for 6 h resulted in a 4–5-fold increase in COX-2 protein levels (Fig. 7). Aspirin and sodium salicylate exerted a comparable, concentration-dependent inhibition of COX-2 protein levels and at 1 mm they reduced COX-2 proteins to near basal level (Fig. 7). DLD-1, HT-29 and HCT-116 CM induced COX-2 protein expression to a similar extent to that by aspirin or sodium salicylate (Fig. 8a–c). COX-2 protein expression induced by DLD-1 or HT-29 medium appeared to be more sensitive to aspirin than sodium salicylate (Fig. 8a,b), whereas COX-2 proteins induced by HCT-116 medium were equally inhibited by aspirin and sodium salicylate (Fig. 8c).

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Figure 7. Inhibition of vascular endothelial growth factor (VEGF)-induced COX-2 protein expression by aspirin or sodium salicylate. Human umbilical vein endothelial cells (HUVEC) were treated with aspirin or salicylate for 30 min followed by VEGF165 for 6 h. COX-2 proteins were analyzed by Western blots. Upper panel, representative blots; lower panel, densitometry analysis of blots from three separate experiments. Each bar is mean ± SD. C, Control.

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image

Figure 8. Inhibition of human umbilical vein endothelial cell COX-2 protein expression stimulated by (a) DLD-1, (b) HT-29 and (c) HCT-116 CM. The upper panel is representative Western blots and the lower panel, densitometric analysis. Each bar is mean ± SD of three experiments. C, Control.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

A major finding of this study is that aspirin and sodium salicylate at pharmacological concentrations inhibit colon cancer medium- and VEGF-induced endothelial tube formation. Inhibition of cancer or VEGF-induced tube formation by aspirin and salicylate correlates with suppression of COX-2 expression, suggesting that aspirin and sodium salicylate may exert their anti-angiogenesis action by suppressing COX-2 expression induced by VEGF produced and secreted by colon cancer cells. COX-2 occupies a pivotal position in amplifying the angiogenic signals provided by VEGF and other angiogenic growth factors. Since selective COX-2 inhibitors or neutralizing VEGF/VEGF-R2 antibodies abrogate endothelial tube formation induced by colon cancer cells, cancer angiogenesis is likely to be sustained by continuous expression of COX-2 and VEGF via a positive regulatory loop. By suppressing VEGF-induced COX-2 expression, aspirin and sodium salicylate may disrupt the regulatory loop, resulting in a reduction of new blood vessels in the cancer, and thereby limiting the supply of oxygen and nutrients essential for cancer growth. Our findings provide a plausible explanation for colon cancer prevention by aspirin and salicylate.

Our results show that sodium salicylate is as effective as aspirin in blocking COX-2 protein levels induced by colon cancer medium or VEGF. Sodium salicylate is equipotent to aspirin in suppressing endothelial tube formation induced by colon cancer medium or VEGF. This is surprising, since sodium salicylate is a very weak inhibitor of COX-2 or COX-1, and at the concentrations that effectively blocked tube formation (<1 mm) it is not expected to have any inhibitory action against either enzyme. Thus, the action of sodium salicylate is probably due to other mechanisms. As the anti-angiogenic action of salicylate is correlated with suppression of COX-2 protein levels, one possible mechanism is inhibition of COX-2 expression induced by VEGF released into colon cancer media. We have previously demonstrated that sodium salicylate at 10−5 m inhibits COX-2 transcription induced by phorbol 12-myristate 13-acetate or IL-1β by selectively blocking C/EBPβ binding to its cognate site on COX-2 promoter [23]. It is possible that salicylate may inhibit VEGF-induced COX-2 expression by a similar mechanism. Aspirin at pharmacological concentrations is effective in inhibiting COX-1 activity and less active in blocking COX-2 activity [29,30]. It was anticipated that aspirin might be more potent than sodium salicylate in blocking colon cancer-mediated endothelial tube formation. The reason that aspirin is not significantly more potent than salicylate may be its short half-life in circulating blood. Aspirin is rapidly deacetylated and converted to salicylate. The anticancer action of aspirin is likely to be contributed by salicylate.

Results from this study confirm the crucial role of VEGF in colon cancer cell-mediated angiogenesis. A VEGF-blocking antibody abrogated endothelial tube formation. Our results also confirm that the action of VEGF is primarily mediated by VEGF-R2 [31]. VEGF also binds to VEGF-R1 but the role of VEGF-R1 in cancer-mediated angiogenesis is not as clear. It has been suggested that VEGF-R1 may function as a non-signaling binding site to regulate VEGF availability [32]. Placental growth factor (P1GF) and VEGF-B also binds VEGF-R1. Recent reports have demonstrated that P1GF via its binding to VEGF-R1 plays an important role in pathological angiogenesis [33]. Monoclonal antibodies against VEGF-R1 are capable of blocking angiogenesis and growth of epidermoid A431 tumors [33]. Our results do not show a significant effect of a VEGF-R1 blocking antibody on colon cancer medium-mediated endothelial tube formation, suggesting that P1GF may not be involved in eliciting pathological angiogenesis in colon cancer. It will be important to determine whether different cancers use different angiogenic growth factors and receptors to promoter cancer growth.

In vitro endothelial tube formation is commonly used in evaluating the effects of pharmacological agents on angiogenesis. HUVEC grown on collagen gel or Matrigel is the most commonly used model. Our finding of a thin, long capillary-like network formed by stimulated HUVEC is consistent with reported results [10]. The EAhy926 cell line, a HUVEC fused to a human lung cancer cell line, is phenotypically related to HUVEC [34]. This cell line is capable of forming tubes on Matrigel in response to cancer CM or VEGF stimulation. The morphology of EA tubes observed in our study is similar to that reported previously [35]. It is different from that of HUVEC tubes. However, despite the different appearance, the quantitative data with respect to tube stimulation by cancer medium or VEGF and tube suppression by salicylate are comparable between these two cells. Thus, EAhy926 cells can be used as a substitute for HUVEC for analysis of in vitro endothelial tube formation and should be a useful model for studying control of pathological angiogenesis by endogenous factors and pharmacological compounds.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This work is supported by grants from National Heart Lung and Blood Institute (RO1 HL-50675) and National Neurological Diseases and Stroke (P50 NS-23327). We thank Ms Susan Mitterling for excellent editorial assistance.

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  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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