Fan PS et al / Acta Pharmacol Sin 2003 Dec; 24 (12): 1231-1234

Inhibitory effect of quercetin on proliferation of human microvascular endothelial cells in vitro¹

FAN Pan-Sheng, GU Zhen-Lun², SHENG Rui, LIANG Zhong-Qin, WANG Xiao-Xia, ZHU Yi

Department of Pharmacology, Institute of Chinese Materia Medica, Soochow University, Suzhou 215007, China

¹ Project supported by the National Natural Science Foundation of China, No 39870954.

² Correspondence to Prof GU Zhen-Lun. Phn 86-512-519-0599. Fax 86-512-519-0599. E-mail zhenlungu.2003@163.com

Received 2003-02-06 Accepted 2003-06-30

KEY WORDS quercetin; vascular endothelium; cell division; cytotoxicity; human

ABSTRACT

AIM: To investigate the role of quercetin (Que) in the proliferation of cultured human skin microvascular endothelial cells (MVEC). METHODS: Cell count and [methyl-³H]thymidine ([³H]TdR) uptake assay were used to measure the effect of Que in the proliferation of cultured MVEC. Cytotoxicity of Que on MVEC was also evaluated by ⁵¹Cr release assay. RESULTS: When MVEC were treated with Que, the proliferation was significantly inhibited in a time-course and dose-dependent manner. Que 5 µmol/L did not inhibit the proliferation of MVEC. When the concentration of Que increased to 20, 40, 80, and 160 µmol/L, the cell numbers per well were decreased and the inhibition rate was 12.2 %, 23.5 %, 35.3 %, and 54.1 % respectively with IC₅₀ of 138 µmol/L. The inhibitory rate of [³H]-TdR uptake was 18.7 %, 34.4 %, 48.9 %, and 62.5 % respectively (IC₅₀=87.5 µmol/L). ⁵¹Cr release assay showed that Que 160 µmol/L incubated with MVEC from 1 to 16 h had no clear cytotoxicity compared with control group. CONCLUSION: Que greatly inhibited the proliferation of cultured human MVEC in vitro. This effect may not be related to the cytotoxicity of Que on MVEC.

INTRODUCTION

The induction of angiogenesis is characterized by the degradation of the vascular basement membrane, endothelial cell migration, proliferation, and tube formation^[1,2]. There are growing evidences that anti-angiogenic drugs, especially those inhibiting proliferation of endothelial cells, will improve future therapies of diseases including cardiovascular diseases, chronic inflammation (rheumatoid arthritis), diabetes, psoriasis, adiposity, and other neovascularisation^[3,4].

Quercetin (Que, 3,3',5,7-pentahydroxy flavone) is by far the most abundant bioflavonoid. Very wide ranges of biological actions of Que have been reported such as anti-oxidantion^[5], anti-aggregation and anti-adhesion of platelets^[6,7], vasodilation^[8], and antitumorigenic (anti-angiogenesis) effects^[9]. Recently, Que has been demonstrated to inhibit proliferation of several types of cell lines including cancer cells^[10-12], smooth muscle cells^[13], and porcine endothelial cells^[14]. However, there is little information about the effect of Que on human endothelial cells, and particularly, the microvascular endothelial cells (MVEC) that plays a crucial role in the angiogenesis. Therefore, in the present study we have evaluated the role of Que in the proliferation and cytotoxicity of MVEC in vitro.

MATERIALS AND METHODS

Culture of MVEC MVEC were purchased from Clonetics (San Diego, USA). Cells were cultured with MCDB 131 medium on 75-cm² tissue culture flasks. The media were supplemented with 5 % fetal calf serum, endothelial cell growth supplement 30 mg/L, epidermal growth factor 10 µg/L, hydrocortisone 10 mg/L, heparin 10 kU/L, and benzylpenicillin/streptomycin as antibiotics. The medium was changed every 2-3 d until subculture. For subculture, the cells were washed twice with phosphate-buffered saline (in g/L: NaCl 8, KCl 0.2, Na₂HPO₄ 1.44, KH₂PO₄ 0.24) without calcium and magnesium, a digestion buffer (0.25 % trypsin-0.01 % edetic acid dissolved in PBS, pH 7.4) was added and incubated at 37 ºC for 3-5 min until the cells were detached from the flask. The digestion was then stopped by adding the same volume of medium containing 10 % fetal calf serum and centrifuged at 300×g for 10 min. The pellets were resuspended with medium and seeded to culture flasks or plates for experiment. Three-seven passages were used for the test.

Cell count MVEC were cultured on 24-well tissue culture plates at a number of 5×10⁴ cells per well in 1 mL media. To ensure most of cells entered the DNA synthesis state of cell cycle, the cells were treated with MCDB 131 base media with 0.5 % fetal calf serum and without any stimulators for 2 d. After wash and replacement with normal media, Que (Que was dissolved in 0.05 % NaOH at a concentration of 5 mmol/L as a stocking solution and diluted to desired concentration with media before the experiment) was added and the cells were cultured for another 20 h as desired. At the end of experiment, the cells were digested, centrifuged and re-suspended in count buffer. Cell numbers in each well were counted on a Coulter Counter.

[³H]TdR uptake assay MVEC were cultured on 96-well tissue culture plates at 1×10⁴ cells per well in 100 µL media, and treated as described above. [³H]TdR from Amersham (Minneapolis, USA) was added to MVEC at a concentration of 37 kBq per well for 16 h before harvest. At the end of experiment an automated multiwell harvester was used to aspirate, and lyse cells. DNA was transferred onto glass filter paper, while unincorporated [³H]TdR was washed out. Each row of 96-well plate was filled and aspirated 10 times to ensure complete cell transfer and removal of unincorporated thymidine. After wash, each filter strip was washed with 100 % ethanol and transferred to scintillation vials. Scintillation fluid 2 mL was added to each vial. Radioactivity of each well was determined by Beckman liquid scintillation spectroscopy. The data were shown as TBq.

⁵¹Cr release assay The cytotoxic effect of Que on MVEC was determined by a method described by Coligan et al^[15] with modification. Briefly, MVEC were cultured on 96-well plates till 80 % confluence. ⁵¹Cr diluted with media was then added at a concentration of 37 kBq per well in 100 µL growth media. The cells were then labeled in a humidified atmosphere at 37 ºC with 5 % CO₂ for 4 h. Unlabeled ⁵¹Cr was removed by washing four times with MCDB base media. Que 160 µmol/L was incubated with labeled MVEC for different periods. H₂O₂ (0.01 %) was used as a positive cytotoxic stimulator. At the end of experiment, supernatants from each well were collected and radioactivity of each well was determined by Beckman liquid scintillation spectroscopy. The data were shown as TBq.

Reagents All the reagents were from Sigma (Saint Louis, USA). [³H]Thymidine and ⁵¹Cr were purchased from Amersham (Minneapolis, USA).

Statistics Pooled data were analyzed using Microsoft Excel program. All the values were expressed as mean±SD and compared with t test.

RESULTS

Effect of Que on the proliferation of MVEC When MVEC were treated with Que, the proliferation of MVEC was significantly inhibited in a time-course manner. Que did not inhibit the proliferation until 1 h. Higher Que (160 µmol/L) had a faster inhibitory effect on the proliferation than that of lower Que (Tab 1). Cell count indicated that Que inhibited the proliferation of human MVEC cultured in vitro in a concentration-dependent manner. Que 5 µmol/L did not show any inhibitory effect. When the concentration increased from 20, 40, 80, and 160 µmol./L separately, the inhibitory rate measured by cell number increased to 12.2 %, 23.5 %, 35.3 %, and 54.1 %. The [³H]TdR uptake by MVEC showed the same results, the inhibitory rate increased to 18.7 %, 34.4 %, 48.9 %, and 62.5 % respectively (Tab 2).

Tab 1. [³H]TdR uptake by microvascular endothelial cells (MVEC) in culture treated with Que (80 and 160 µmol/L) at different time points. n=4 experiments. Mean±SD.

Tab 2. Effect of Que on the proliferation of human MVEC assayed by cell count and [³H]TdR uptake. n=4 experiments. Mean±SD. ^aP>0.05, ^bP<0.05, ^cP<0.01 vs control.

Cytotoxic effect of Que on MVEC Confluence MVEC were labeled with ⁵¹Cr at 37 ºC for 4 h. Que 160 µmol/L or 0.01 % of H₂O₂ in MCDB 131 base medium were added and incubated as designed. The cytotoxicity was mainly measured by the release of labeled ⁵¹Cr from MVEC into the medium. Que did not have significant cytotoxicity to MVEC even 16 h after incubation but 0.01 % of H₂O₂ indicated a clear cytotoxicity to MVEC in a time-dependent manner compared with control group (P<0.05, Tab 3).

Tab 3. Chromium-51 released from MVEC in culture treated with Que (160 µmol/L) at different time points. The value indicates the TBq per group. n=4 experiments. Mean±SD. ^aP>0.05, ^bP<0.05, ^cP<0.01 vs control.

DISCUSSION

The previous study showed that Que inhibited the platelet adhesion to human MVEC cultured in vitro, and this role was related to the down-regulation of PECAM expression on the endothelial cells^[6].

Several in vitro studies indicate that Que has multiple effects on cancer cells, including the inhibition of cell proliferation^[10-12], little is known about the effect of Que in non-malignant cells. Que showed a significant inhibitory effect on the proliferation of cultured MVEC in vitro monitored by cell count and [³H]TdR. MVEC proliferation or re-generation play a key role in the process of angiogenesis which is associated with lots of diseases such as cardiovascular disease, chronic inflammation, diabetes, and cancers. The anti-proliferate role of Que on MVEC will help us to understand the regulation of angiogenesis and may introduce new therapy for the treatment of diseases associated with angiogenesis. Further study need to be done in vivo to confirm the anti-angiogenesis of Que.

The mechanisms of Que in inhibiting proliferation of cultured MVEC are not clear. ⁵¹Cr release assay showed that there was no clear cytotoxicity of MVEC treated with Que. Lipopolysaccharide (LPS)-induced endothelial cytotoxicity can also be inhibited by Que in vitro^[16]. These results might indicate that the inhibitory effect of Que on the proliferation of MVEC is not due to its cytotoxicity.

Several other mechanisms, including inhibition of lactate transport, suppression of glycolysis and ATP production, inhibition of various tyrosine protein kinases, interference with ion pump systems, and various signal transduction pathways and perturbule network, have been cited for the anti-proliferative activity of Que^{[10,-11, 17-19]}. Que suppressed proliferation of Ishikawa cells through down-regulation of EGF and cyclin D1^[20]. MVEC can enter the stage of proliferation because of unbalance between pro-angiogenic and antiangiogenic factors. In our model, EGF and endothelial cell growth supplement were used as stimulators of proliferation. Further study is underway in our lab to see whether the anti-proliferative activity of Que on the MVEC is related to regulation of the expression of VEGF and other anti-angiogenic factors.

REFERENCES

• 1 Folkman J. Seminars in Medicine of the Beth Israel Hospital, Boston. Clinical applications of research on angiogenesis. N Engl J Med 1995; 333: 1757-63.
• 2 Cockerill GW, Gamble JR, Vadas MA. Angiogenesis: models and modulators. Int Rev Cytol 1995; 159: 113-60.
• 3 Hagedorn M, Andreas B. Target molecules for anti-angio genic therapy: from basic research to clinical trials. Crit Rev Onco/Hema 2000; 34: 89-110.
• 4 Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other diseases. Nat Med 1995; 1: 27-31.
• 5 Middlton EJ, Kandaswami C, Theoharides CT. The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease and cancer. Pharmacol Rev 2000; 52: 673-751.
• 6 Fan PS, Gu ZL, Liang ZQ. Effect of quercetin adhesion of platelets to microvascular endothelial cells in vitro. Acta Pharmacol Sin 2001; 22: 857-60.
• 7 Xie ML, Lu Q, Gu ZL. Effect of quercetin on platelet aggregation induced by oxyradicals. Acta Pharmacol Sin 1996; 17: 334-6.
• 8 Perez-Vizcaino F, Ibarra M, Cogolludo AL, Duarte J, Zaragoza-Arnaez F, Moreno L, et al. Endothelium-independent vasodilator effects of the flavonoid quercetin and its methylated metabolites in rat conductance and resistance arteries. J Pharmacol Exp Ther 2002; 302: 66-72.
• 9 Rodgers EH, Grant MH. The effect of the flavonoids, quercetin, myricetin and epicatechin on the growth and enzyme activites of MCF-7 human breast cancer cells. Chem Biol Interact 1998; 116: 213-28.
• 10 Takagi T, Takekoshi S, Okabe T, Nagata H. Quercetin, a flavonol, promotes disease of microtubules in prostate cancer cells: possible mechanism of its antitumor activity. Cytochemistry 1998; 31: 435-45.
• 11 Kobayashi T, Nakata T, Kuzumaki T. Effect of flavonoids on cell cycle progression in prostate cancer cells. Cancer Lett 2002; 176: 17-23.
• 12 Choi JA, Kim JY, Lee JY, Kang CM, Kwon HJ, Yoo YD, et al. Induction of cell cycle arrest and apoptosis in human breast cancer cells by quercetin. Int J Oncol 2001; 19: 837-44.
• 13 Alcocer F, Whitley D, Salazar-Gonzalez JF, Jordan WD, Sellers MT, Eckhoff DE, et al. Quercetin inhibits human vascular smooth muscle cell proliferation and migration. Surgery 2002; 131: 198-204.
• 14 Igura K, Ohta T, Kuroda, Kaji K. Resveratrol and quercetin inhibit angiogenesis in vitro. Cancer Lett 2001; 171: 11-6
• 15 Coligan JE, Margulies DH, Shevach EM, Strober W. Current protocols in immunology. 1st ed. Willy-Interscience publishers; 1992. p7, 18, 1-7, 18, 2.
• 16 Melzig MF, Loose R. Inhibition of lipopolysaccharide (LPS)-induced endothelial cytotoxicity by selected flavonoids. Planta Med 1998; 64: 397-9.
• 17 Kuo SM, Morehouse HF, Lin CP. Effect of antiproliferative flavonoids on ascorbic acid accumulation in human colon adenocarcinoma cells. Cancer Lett 1997; 116: 131-7.
• 18 Lamson DW, Brignall MS. Antioxidants and cancer, part 3: quercetin. Altern Med Rev 2000; 5: 196-208.
• 19 Hagiwara M, Inoue S, Tanaka T, Nunoki K, Ito M, Hidaka H. Differential effects of flavonoids as inhibitors of tyrosine protein kinases and serine/threonine protein kinases. Biochem Pharmacol 1988; 37: 2987-92.
• 20 Kaneuchi M, Sasaki M, Tanaka Y, Sakuragi N, Fujimoto S, Dahiya R. Quercetin regulates growth of Ishikawa cells through the suppression of EGF and cyclin D1. Int J Oncol 2003; 22: 159-64.