Flufenamic acid promotes angiogenesis through AMPK activation

  • Authors:
    • Ruiliang Ge
    • Lei Hu
    • Yilin Tai
    • Feng Xue
    • Lei Yuan
    • Gongtian Wei
    • Yi Wang
  • View Affiliations

  • Published online on: April 10, 2013     https://doi.org/10.3892/ijo.2013.1891
  • Pages: 1945-1950
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Abstract

Angiogenesis plays critical roles in development, tumor growth and metastasis. Flufenamic acid (FFA) is an anti-inflammatory agent known to alter ion fluxes across the plasma membrane. Its role in angiogenesis has not been fully addressed to date. Here, we report that FFA treatment promotes angiogenesis both in vitro and in vivo. Applying FFA for 12 h promoted tube formation of human umbilical vein endothelial cells (HUVECs) without affecting cell proliferation. Three angiogenesis-related genes, VEGF, e-NOS and AAMP, were analyzed by RT-PCR. A significant difference was found between the FFA group and the control; the FFA group had significantly higher mRNA accumulation levels of all the three genes (p<0.05). Moreover, in the chick embryo chorioallantoic membrane (CAM) assay, FFA promoted the formation of macroscopic blood vessels. Finally, western blotting showed that the FFA-treated group had significantly higher phosphorylated AMPK levels, compared with the control (p<0.05). These results suggest that FFA promotes angiogenesis both in vitro and in vivo likely via promoting tube formation through AMPK activation.

Introduction

Angiogenesis, the formation of new blood vessels, plays important roles in the normal physiological situations, such as embryonic growth and wound healing (1,2). Angiogenesis also has profound impact on pathologic development, particularly on chronic inflammation (3). Evidence has been gathered regarding the association between angiogenesis and inflammation in pathologic conditions. These phenomena have long been coupled together in many chronic inflammation diseases including psoriasis, diabetes, Crohn’s disease, rheumatoid arthritis and cancer (410). Many of the cells that play a role during inflammation release factors that have profound effects on vascular endothelial cells (1114). On the other hand, angiogenesis sustains inflammation. Without angiogenesis, cells that present at inflammatory sites will be short of oxygen and nutrients to meet their metabolic needs (15). Thus, these two processes seem to depend on each other. Common molecular mechanisms have also been found to support this idea (16,17). According to this knowledge, direct therapeutic approaches against both chronic inflammation and angiogenesis will become our pursuit. Therefore, to further understand the cross-talk between inflammation and angiogenesis will be an important issue.

Fenamate belongs to a family of non-steroidal anti-inflammatory drugs (NSAIDs). One of the fenamates, flufenamic acid (FFA), is an inhibitor of cyclooxygenase (18) and has been shown to modulate several kinds of ion channels. FFA is commonly used as a blocker of non-selective cation current. It has been shown to inhibit the current of several members of TRP channel superfamily, to potentiate potassium current, to inhibit L-type calcium current, and to inhibit Ca2+-dependent Cl - current (1923). The regulation of FFA on C-type TRP channels (TRPC) appears complex since FFA blocks currents of TRPC3 and TRPC7 channels whereas it potentiates the current of TRPC6 channels (24,25). Interestingly, both TRPC3 and TRPC6 have been shown to mediate vascular endothelial growth factor (VEGF)-induced current and TRPC6 also mediates VEGF-induced angiogenesis (2628). Although FFA affected ion channels have been shown to be involved in angiogenesis, no report is available on the effect of this chemical on angiogenesis.

In the present study, we used HUVECs as a cell model of angiogenesis and the chicken CAM assay as an in vivo angiogenesis model. We investigated the effect of FFA on HUVECs proliferation and tube formation and its role in angiogenesis in chicken CAM.

Materials and methods

Materials

Flufenamic acid was purchased from Sigma-Aldrich (USA). BrdU monoclonal antibody was obtained from Neomarkers (USA). Texas-Red-conjugated goat anti-mouse secondary antibody was from Molecular Probes. Growth factor-free Matrigel was from BD Biosciences (USA). All cell culture media and reagents were obtained from Invitrogen (Carlsbad, CA, USA).

Cell culture

The HUVECs purchased from Sciencell (USA) were grown in ECM supplemented with 5% FBS, ECGs and PS in a humidified incubator with 5% CO2 at 37°C. The cells were trypsinized with 0.15% trypsin-EDTA. Passages 3–10 were used for experiments.

Proliferation assay

Cell proliferation was determined by both counting the cell numbers and the BrdU incorporation assay. For counting the cell numbers, HUVECs were seeded at an initial density of 2×105 per well in 6-well plates. The FFA was applied at a dose of 20, 50 and 100 μM. Cells were harvested and counted 24, 48 and 72 h after the treatment. Cell numbers were read in a Beckman Counter. For BrdU incorporation assay, 100 μM FFA was applied for 24 h. Then, cells were incubated in the medium with 10 μM BrdU for 3 h and stained with a monoclonal antibody against BrdU at 4°C for 12 h. A goat anti-mouse IgG labeled with Texas Red was used as secondary antibody. The results were expressed as the percentage of BrdU-positive cells over all the cells.

Tube formation assay

The Matrigel was applied to each well of a 24-well plate and incubated at 37°C for 60 min. The 6×104 of endothelial cells was then seeded into each well with the medium containing 0.8% FCS with or without FFA (100 μM). Images of representative 10× fields were taken and endothelial cell tubes were quantified by counting length and branches.

Western blot analysis

The cells were washed twice with PBS and total cellular protein was then extracted in lysis buffer containing 62.5 mM Tris-HCl, 2% SDS, 10% glycerol with freshly added proteinase inhibitor cocktail (Sigma, Zwijndrecht, The Netherlands). The protein concentrations were determined by BCA assay (Pierce, Waltham, MA, USA). The protein lysates (40 μg/lane) were separated by SDS-PAGE and transferred onto nitrocellulose membranes. After blocking with 3% bovine serum albumin in phosphate-buffered saline, the membranes were incubated with antiphospho-AMPKα and-AMPKβ, or antiphospho-ACC antibody. After washing, the membranes were probed with horseradish peroxidase-conjugated anti-rabbit IgG and the bands were visualized using an ECL-Plus chemiluminescence detection system (GE Healthcare, NJ, USA). To confirm equal loading of proteins, the membranes were probed for β-actin protein.

Reverse transcription polymerase chain reaction (RT-PCR)

HUVECs were lysed with TRIzol Reagent (Invitrogen) and total RNA was extracted according to the manufacturer’s instructions. First-strand cDNA was synthesized from 1.5 μg of total RNA using Moloney murine leukemia virus reverse transcriptase (M-MLV RT) according to the manufacturer’s instructions (Invitrogen). cDNA was amplified by PCR according to the manufacturer’s instructions with Taq DNA polymerase (Invitrogen) under the following conditions: 94°C for 5 min, followed by 40 cycles of 94°C for 30 sec, 56°C for 30 sec and 72°C for 90 sec, with a final elongation step of 10 min at 72°C. The primer informations are as follows: VEGF: forward, 5′-CTACCTCCACCATGCCAAGT-3′; reverse, 5′-TTT CTTGCGCTTTCGTTTTT-3′; AAMP: forward, 5′-CTTTGC ATTGCACTCAGCAT-3′; reverse, 5′-CAGTCACCATTCGGG ACTTT-3′; e-NOS: forward, 5′-GGCTCCCTCCTTCCGG CTG-3′; reverse, 5′-TAGCCGCACGACGCCCT-3′; GAPDH: forward, 5′-AGCCACTGCTGTGCTTTTAAG-3′; reverse, 5′-CCAAAACCAATGATCTCATCC-3′. The products were electrophoresed in 2% agarose gel and stained with ethidium bromide.

Angiogenesis in chick embryo CAM

Angiogenesis was assayed using the chick embryo CAM assay according to the method described previously (29). Briefly, fertilized chicken eggs were treated with ethanol (70%) and then incubated at 37°C. On day 3, 2–3-ml albumen was aspirated at the acute pole using a sterile 25-G hypodermic needle in order to allow detachment of the developing CAM from the eggshell. After the removal of albumen, we cut a square window ∼10×10 mm into the shell and sealed the window with transparent tapes. Eggs were then incubated in a horizontal position. Six days later, we opened the window and implanted a 1-mm3 sterilized gelatin sponge containing DMSO or FFA onto the CAM. On day 12, the embryos of CAM were fixed by Bouin’s fluid and the distribution and density of CAM vessels next to the site of grafting were analyzed.

Statistical analyses

All experiments were repeated three times independently. Data were presented as mean ± SEM. or as percentage of control. Statistical comparisons between groups were performed using the Student’s t-test. p<0.05 was considered statistically significant.

Results

FFA suppressed HUVEC growth

To test whether FFA plays a role in angiogenesis, we first investigated the effect of FFA on HUVEC growth. The cells were incubated with DMSO, 20, 50 and 100 μM FFA for 24, 48 and 72 h before the cell numbers were determined. As shown in Fig. 1, FFA at the concentration of 20 μM had no effect on HUVEC growth at any time-point. FFA at the concentration of 50 μM had weak effect on HUVEC growth. The cell number was slightly reduced at the concentration of 50 μM after 72 h, however, there was no significant difference compared with DMSO control. When 100 μM FFA was applied to HUVEC, the numbers of the HUVEC started to reduce after 48 h and was greatly reduced after 72 h. No apoptosis was found at this concentration (data not shown).

FFA reduced HUVEC proliferation

It has been reported that FFA inhibits cell proliferation in other cell types (3032). We next determined whether the effect of FFA on HUVEC cell number was due to its influence on cell proliferation. To address this question, we applied the BrdU incorporation assay. HUVECs were treated with 100 μM FFA for 24 h before they were incubated with 10 μM BrdU for another 3 h. BrdU is an analog of DNA precursor thymidine. When cells are proliferating, BrdU can be incorporated into DNA similarly to thymidine. In this way, the amount of the BrdU in the cells reflects the proliferation rate of the cells. As shown in Fig. 2, the percentage of BrdU-positive cell in DMSO and FFA-treated group was 12±0.86 and 8.65±0.49%, respectively. There was a significant reduction of the percentage of BrdU-positive cells in the FFA treatment group compared with the control group (p<0.05).

FFA promoted tube formation of HUVECs

In vitro assays of tube formation using endothelial cells are commonly used to study critical steps of angiogenesis (33). We then examine the effect of FFA on tube formation using HUVECs cultured on Matrigel. The tube-like structure appeared 12 h after the HUVECs were seeded. The total tube length and the number of branching points were analyzed as indexes of angiogenesis.

We applied FFA to HUVECs when they were seeded on Matrigel. As shown in Fig. 3A, after 12 h, FFA greatly increased the formation of tube structure. Both total tube length and branching points were significantly increased in FFA treatment group. The relative total tube length of FFA-treated group increased 50.4% compared with that of control group (Fig. 3B). The average branching points per field of FFA-treated group increased 135% compared with that of control group (Fig. 3C). Together, these results suggest that FFA promotes angiogenesis without promoting endothelial cell proliferation.

FFA induced AMPK activation

FFA is one of the Nary-anthranilic acid derivatives, belonging to the fenamate group of NSAIDs (34). To examine whether FFA regulates angiogenesis through AMPK activation, phospho-AMPKα and-AMPKβ and phospho-ACC were measured by western blotting. As shown in Fig. 4, incubation of HUVECs with FFA resulted in increased levels of phosphorylated AMPKα and AMPKβ, which was associated with paralleled elevation of phosphorylated ACC, one of the AMPK substrates (35). Compared with control, FFA-treated group had significantly higher phosphorylated AMPK levels (p<0.05).

Expression of angiogenesis markers

Vascular endothelial growth factor (VEGF), endothelial NO synthase (e-NOS), the angio-associated migratory cell protein (AAMP) are three angiogenesis related genes, which were strongly expressed in endothelial cells (36). The mRNA levels of VEGF, e-NOS and AAMP were measured by RT-PCR. Expression of all three angiogenesis related genes is shown in Fig. 5. There was a significant difference between FFA group and control, FFA group had a significantly higher mRNA accumulation level of all the three angiogenesis related genes (p<0.05).

The effect of FFA on angiogenesis in vivo

We further examined the possible effect of FFA on angiogenesis in vivo. In order to explore the role of FFA in vivo, we applied the chicken chorioallantoic membrane (CAM) assay. We implanted a 1-mm3 sterilized gelatin sponge which contained PBS or FFA onto the chorioallantoic membrane for ∼72 h for the blood vessel to grow into the sponge. Then the sponge was fixed and the distribution and density of CAM vessels next to the site of grafting were analyzed.

As shown in Fig. 6, the vessel density in the CAM implanted with the gelatin sponge containing PBS was 15.99±1.30. The vessel density in the CAM implanted with the gelatin sponge containing FFA was 23.74±1.82, which was a significant increase compared with the control group. These results suggest that FFA promotes angiogenesis in vivo.

Discussion

Our results indicate that FFA treatment promotes angiogenesis. In the tube formation assay of HUVEC cells, both total tube length and the number of branching points were increased in the FFA treatment group compared to the control group and cell proliferation was not significantly affected at the time when tube formation assay was performed. AMP-activated protein kinase (AMPK) is a key regulator of metabolic homeostasis (35) and has anti-inflammatory effects (37,38). In addition, it promotes angiogenesis, and protects cells from apoptosis (3941). RT-PCR was used to analyse the expression of VEGF, AAMP, e-NOS and the results showed significantly increased expression in response to FFA-stimulation. These results indicated that FFA promoted angiogenesis in vitro. Moreover, FFA can also promote angiogenesis in vivo. In the chicken CAM assay FFA significantly increased the number of vessels that grow into the gelatin sponge. In addition, we observed that FFA can significantly increase the phosphorylated levels of AMPK. Our results thus support the notion that FFA promotes angiogenesis both in vitro and in vivo through AMPK activation.

The process of angiogenesis includes the proliferation of endothelial cells and migration of these cells to form tube-like structures. At the concentration of 100 μM, 12-h FFA treatment did not affect HUVEC proliferation whereas promoted the formation of tube-like structures and it was not until 24 h that FFA treatment began to inhibit cell proliferation. The differential effect of FFA on tube formation and cell proliferation is probably due to the multiple targets of FFA. FFA has been reported to inhibit cell proliferation in several cell types (30,42). As a non-selective cation blocker, FFA blocks several channels that have been shown to be involved in the process of cell proliferation. For example, TRPM7 is required for MCF-7 cell proliferation (43). TRPC3 has been shown to be necessary for proliferation of SKOV3 cells (44). Whether the effect of FFA on HUVEC proliferation is the result of its inhibitory effect on these channels remains uncertain. The way FFA promotes HUVEC tube formation is probably through its other targets. For instance, FFA-potentiated non-selective cation channel is required for lysophosphatidylcholine-induced monocyte migration (45). FFA can potentiate the current of TRPC6 and TRPC6 has been shown to promote HUVECs tube formation (28). It is thus possible that TRPC6 participates in FFA-induced tube formation.

Common molecular mechanisms have already been shown to regulate both chronic inflammation and angiogenesis (46,47). These two processes seem to depend on each other based on literature (3,48). However, as an anti-inflammatory agent, FFA promotes angiogenesis in our system. Therefore, a careful understanding of the cross-talk between angiogenesis and chronic inflammation is very important for more effective therapies.

In conclusion, our data show that FFA treatment promotes HUVEC tube formation in vitro. In the in vivo experiment using chick CAM assay, FFA also promotes vessels to grow into the gelatin sponge. Moreover, the phosphorylated AMPK levels were significantly higher in FFA-treated group. These data suggest that FFA promotes angiogenesis both in vitro and in vivo.

References

1 

Tonnesen MG, Feng X and Clark RA: Angiogenesis in wound healing. J Investig Dermatol Symp Proc. 5:40–46. 2000. View Article : Google Scholar

2 

Breier G: Angiogenesis in embryonic development - a review. Placenta. 21(Suppl A): S11–S15. 2000. View Article : Google Scholar

3 

Costa C, Incio J and Soares R: Angiogenesis and chronic inflammation: cause or consequence? Angiogenesis. 10:149–166. 2007. View Article : Google Scholar : PubMed/NCBI

4 

Coussens LM and Werb Z: Inflammation and cancer. Nature. 420:860–867. 2002. View Article : Google Scholar : PubMed/NCBI

5 

Carmeliet P: Angiogenesis in life, disease and medicine. Nature. 438:932–936. 2005. View Article : Google Scholar : PubMed/NCBI

6 

Lusis AJ: Atherosclerosis. Nature. 407:233–241. 2000. View Article : Google Scholar : PubMed/NCBI

7 

Trayhurn P and Wood IS: Adipokines: inflammation and the pleiotropic role of white adipose tissue. Br J Nutr. 92:347–355. 2004. View Article : Google Scholar : PubMed/NCBI

8 

Wubben DP and Adams AK: Metabolic syndrome: what’s in a name? WMJ. 105:17–20. 2006.

9 

Tan TT and Coussens LM: Humoral immunity, inflammation and cancer. Curr Opin Immunol. 19:209–216. 2007. View Article : Google Scholar : PubMed/NCBI

10 

Otani A, Takagi H, Oh H, Koyama S, Matsumura M and Honda Y: Expressions of angiopoietins and Tie2 in human choroidal neovascular membranes. Invest Ophthalmol Vis Sci. 40:1912–1920. 1999.PubMed/NCBI

11 

Benelli R, Lorusso G, Albini A and Noonan DM: Cytokines and chemokines as regulators of angiogenesis in health and disease. Curr Pharm Des. 12:3101–3115. 2006. View Article : Google Scholar

12 

Nathan C: Points of control in inflammation. Nature. 420:846–852. 2002. View Article : Google Scholar : PubMed/NCBI

13 

Folkman J: Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med. 1:27–31. 1995. View Article : Google Scholar : PubMed/NCBI

14 

Mrowietz U and Boehncke WH: Leukocyte adhesion: a suitable target for anti-inflammatory drugs. Curr Pharm Des. 12:2825–2831. 2006. View Article : Google Scholar : PubMed/NCBI

15 

Lee FH, Haskell C, Charo IF and Boettiger D: Receptor-ligand binding in the cell-substrate contact zone: a quantitative analysis using CX3CR1 and CXCR1 chemokine receptors. Biochemistry. 43:7179–7186. 2004. View Article : Google Scholar : PubMed/NCBI

16 

Pacifico F and Leonardi A: NF-kappaB in solid tumors. Biochem Pharmacol. 72:1142–1152. 2006. View Article : Google Scholar : PubMed/NCBI

17 

Nam NH: Naturally occurring NF-kappaB inhibitors. Mini Rev Med Chem. 6:945–951. 2006. View Article : Google Scholar : PubMed/NCBI

18 

Flower RJ: Drugs which inhibit prostaglandin biosynthesis. Pharmacol Rev. 26:33–67. 1974.

19 

Naziroglu M, Luckhoff A and Jungling E: Antagonist effect of flufenamic acid on TRPM2 cation channels activated by hydrogen peroxide. Cell Biochem Funct. 25:383–387. 2007. View Article : Google Scholar : PubMed/NCBI

20 

Peppiatt-Wildman CM, Albert AP, Saleh SN and Large WA: Endothelin-1 activates a Ca2+-permeable cation channel with TRPC3 and TRPC7 properties in rabbit coronary artery myocytes. J Physiol. 580:755–764. 2007.PubMed/NCBI

21 

Farrugia G, Rae JL, Sarr MG and Szurszewski JH: Potassium current in circular smooth muscle of human jejunum activated by fenamates. Am J Physiol. 265:G873–G879. 1993.PubMed/NCBI

22 

Doughty JM, Miller AL and Langton PD: Non-specificity of chloride channel blockers in rat cerebral arteries: block of the L-type calcium channel. J Physiol. 507:433–439. 1998. View Article : Google Scholar : PubMed/NCBI

23 

Greenwood IA and Large WA: Comparison of the effects of fenamates on Ca-activated chloride and potassium currents in rabbit portal vein smooth muscle cells. Br J Pharmacol. 116:2939–2948. 1995. View Article : Google Scholar : PubMed/NCBI

24 

Inoue R, Okada T, Onoue H, et al: The transient receptor potential protein homologue TRP6 is the essential component of vascular alpha(1)-adrenoceptor-activated Ca(2+)-permeable cation channel. Circ Res. 88:325–332. 2001.PubMed/NCBI

25 

Jung S, Strotmann R, Schultz G and Plant TD: TRPC6 is a candidate channel involved in receptor-stimulated cation currents in A7r5 smooth muscle cells. Am J Physiol Cell Physiol. 282:C347–C359. 2002. View Article : Google Scholar : PubMed/NCBI

26 

Poteser M, Graziani A, Eder P, et al: Identification of a rare subset of adipose tissue-resident progenitor cells, which express CD133 and TRPC3 as a VEGF-regulated Ca2+ entry channel. FEBS Lett. 582:2696–2702. 2008. View Article : Google Scholar : PubMed/NCBI

27 

Hamdollah Zadeh MA, Glass CA, Magnussen A, Hancox JC and Bates DO: VEGF-mediated elevated intracellular calcium and angiogenesis in human microvascular endothelial cells in vitro are inhibited by dominant negative TRPC6. Microcirculation. 15:605–614. 2008.PubMed/NCBI

28 

Ge R, Tai Y, Sun Y, et al: Critical role of TRPC6 channels in VEGF-mediated angiogenesis. Cancer Lett. 283:43–51. 2009. View Article : Google Scholar : PubMed/NCBI

29 

Ribatti D, Nico B, Vacca A and Presta M: The gelatin sponge-chorioallantoic membrane assay. Nat Protoc. 1:85–91. 2006. View Article : Google Scholar : PubMed/NCBI

30 

Schober W, Wiskirchen J, Kehlbach R, et al: Flufenamic acid: growth modulating effects on human aortic smooth muscle cells in vitro. J Vasc Interv Radiol. 13:89–96. 2002. View Article : Google Scholar : PubMed/NCBI

31 

Tiemann U, Neels P, Pohland R, Walzel H and Lohrke B: Influence of inhibitors on increase in intracellular free calcium and proliferation induced by platelet-activating factor in bovine oviductal cells. J Reprod Fertil. 116:63–72. 1999. View Article : Google Scholar : PubMed/NCBI

32 

Weiser T and Wienrich M: Investigations on the mechanism of action of the antiproliferant and ion channel antagonist flufenamic acid. Naunyn Schmiedebergs Arch Pharmacol. 353:452–460. 1996.PubMed/NCBI

33 

Wilson BD, Ii M, Park KW, et al: Netrins promote developmental and therapeutic angiogenesis. Science. 313:640–644. 2006. View Article : Google Scholar : PubMed/NCBI

34 

Chi Y, Li K, Yan Q, et al: Nonsteroidal anti-inflammatory drug flufenamic acid is a potent activator of AMP-activated protein kinase. J Pharmacol Exp Ther. 339:257–266. 2011. View Article : Google Scholar : PubMed/NCBI

35 

Towler MC and Hardie DG: AMP-activated protein kinase in metabolic control and insulin signaling. Circ Res. 100:328–341. 2007. View Article : Google Scholar : PubMed/NCBI

36 

Del Carratore R, Carpi A, Beffy P, et al: Itraconazole inhibits HMEC-1 angiogenesis. Biomed Pharmacother. 66:312–317. 2012.PubMed/NCBI

37 

Aoki C, Hattori Y, Tomizawa A, Jojima T and Kasai K: Anti-inflammatory role of cilostazol in vascular smooth muscle cells in vitro and in vivo. J Atheroscler Thromb. 7:503–509. 2010. View Article : Google Scholar : PubMed/NCBI

38 

Shin MJ, Lee YP, Kim DW, et al: Transduced PEP-1-AMPK inhibits the LPS-induced expression of COX-2 and iNOS in Raw264. 7 cells. BMB Rep. 43:40–45. 2010. View Article : Google Scholar : PubMed/NCBI

39 

Kongsuphol P, Cassidy D, Hieke B, et al: Mechanistic insight into control of CFTR by AMPK. J Biol Chem. 284:5645–5653. 2009. View Article : Google Scholar : PubMed/NCBI

40 

Kréneisz O, Benoit JP, Bayliss DA and Mulkey DK: AMP-activated protein kinase inhibits TREK channels. J Physiol. 587:5819–5830. 2009.PubMed/NCBI

41 

Klein H, Garneau L, Trinh NTN, et al: Inhibition of the KCa3. 1 channels by AMP-activated protein kinase in human airway epithelial cells. Am J Physiol Cell Physiol. 296:C285–C295. 2009. View Article : Google Scholar : PubMed/NCBI

42 

Schlichter LC, Sakellaropoulos G, Ballyk B, Pennefather PS and Phipps DJ: Properties of K+ and Cl channels and their involvement in proliferation of rat microglial cells. Glia. 17:225–236. 1996.

43 

Guilbert A, Gautier M, Dhennin-Duthille I, Haren N, Sevestre H and Ouadid-Ahidouch H: Evidence that TRPM7 is required for breast cancer cell proliferation. Am J Physiol Cell Physiol. 297:C493–C502. 2009. View Article : Google Scholar : PubMed/NCBI

44 

Yang SL, Cao Q, Zhou KC, Feng YJ and Wang YZ: Transient receptor potential channel C3 contributes to the progression of human ovarian cancer. Oncogene. 28:1320–1328. 2009. View Article : Google Scholar : PubMed/NCBI

45 

Schilling T and Eder C: Non-selective cation channel activity is required for lysophosphatidylcholine-induced monocyte migration. J Cell Physiol. 221:325–334. 2009. View Article : Google Scholar : PubMed/NCBI

46 

Fiedler U, Reiss Y, Scharpfenecker M, et al: Angiopoietin-2 sensitizes endothelial cells to TNF-alpha and has a crucial role in the induction of inflammation. Nat Med. 12:235–239. 2006. View Article : Google Scholar : PubMed/NCBI

47 

Fiedler U and Augustin HG: Angiopoietins: a link between angiogenesis and inflammation. Trends Immunol. 27:552–558. 2006. View Article : Google Scholar : PubMed/NCBI

48 

Noonan DM, De Lerma Barbaro A, Vannini N, Mortara L and Albini A: Inflammation, inflammatory cells and angiogenesis: decisions and indecisions. Cancer Metastasis Rev. 27:31–40. 2008. View Article : Google Scholar : PubMed/NCBI

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June 2013
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Ge R, Hu L, Tai Y, Xue F, Yuan L, Wei G and Wang Y: Flufenamic acid promotes angiogenesis through AMPK activation . Int J Oncol 42: 1945-1950, 2013.
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Ge, R., Hu, L., Tai, Y., Xue, F., Yuan, L., Wei, G., & Wang, Y. (2013). Flufenamic acid promotes angiogenesis through AMPK activation . International Journal of Oncology, 42, 1945-1950. https://doi.org/10.3892/ijo.2013.1891
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Ge, R., Hu, L., Tai, Y., Xue, F., Yuan, L., Wei, G., Wang, Y."Flufenamic acid promotes angiogenesis through AMPK activation ". International Journal of Oncology 42.6 (2013): 1945-1950.
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Ge, R., Hu, L., Tai, Y., Xue, F., Yuan, L., Wei, G., Wang, Y."Flufenamic acid promotes angiogenesis through AMPK activation ". International Journal of Oncology 42, no. 6 (2013): 1945-1950. https://doi.org/10.3892/ijo.2013.1891