Search JIM Advanced Search

Journal of Chinese Integrative Medicine ›› 2008, Vol. 6 ›› Issue (10): 1017-1023.doi: 10.3736/jcim20081006

• Original Experimental Research • Previous Articles     Next Articles

Regulation of aromatase P450 expression by puerarin in endometrial cell line RL95-2

Yan-wei Li1, Zai-long Cai2, Wei Shen1, Sheng-sheng Yang3, Chao-qin Yu1()   

  1. 1. Department of Traditional Chinese Medicine, Changhai Hospital, Second Military Medical University, Shanghai 200433, China
    2. Clinical Research Center, Changhai Hospital, Second Military Medical University, Shanghai 200433, China
    3. Department of Biochemistry and Molecular Biology, Second Military Medical University, Shanghai 200433, China
  • Received:2008-07-24 Online:2008-10-20 Published:2008-10-15

Objective: 

To study the effects of puerarin on the aromatase P450 (P450arom) mRNA expression and the effects of low-dose puerarin on transcription factors of the P450arom gene (PⅡ) 5'-flanking region.


Methods: 

The effects of puerarin on the P450arom mRNA expression were determined by real-time polymerase chain reaction (RT-PCR). The 5'-flanking region was amplified by PCR using human genomic cDNA as a template. By means of the restriction sites and sequence confirmation, the PCR product was cloned into reporter vector. Series of sequential deletion reporter constructs were transiently transfected into RL95-2 cells which were treated with or without puerarin. Luciferase activity was measured by Dual-Luciferase Reporter Assay System and Luminoskan Ascent luminometer. Furthermore, by using web-based search program, the most possible cis-acting elements and transcription factors were evaluated.


Results: 

The data demonstrated that low-dose puerarin treatment could decrease P450arom expression at mRNA level compared to dimethyl sulphoxide (DMSO) treatment (P<0.01), and puerarin (10 –7 mol/L) had a time-course effect on P450arom mRNA expression, which reached the bottom at 12 h (P<0.01). Cells transfected with the –763/+8 bp constructs showed decrease in relative luciferase activity after puerarin (10 –7 mol/L) treatment compared to DMSO treatment (P<0.05), indicating an essential regulatory site between –763 bp and –543 bp responsible for the transcription suppression by puerarin. Furthermore, the most possible transcription factors, which turned out to be AP-1(c-jun/c-fos) at –410/–401 bp were also evaluated. The activity of exogenous AP-1 was reduced after 12 hours of puerarin treatment (P<0.05). The inhibition of c-jun mRNA also showed a time-course effect, which bottomed out at 12 h in parallel with that of P450arom (P<0.01). The protein level of c-jun was also down-regulated by puerarin (10 -7mol/L) treatment at 12 h.


Conclusion: 

The suppression of P450arom expression and activity may be associated with the down-regulation of transcription factor AP-1/c-jun. This partially explains the mechanisms whereby puerarin treats endometriosis.

Key words: puerarin, aromatase P450, endometriosis

CLC Number: 

  • R711.71

Figure 1

Effects of puerarin on P450arom expression and activity A: Structure of puerarin; B: mRNA expression of P450arom in RL95-2 cells treated with different concentration of puerarin, by RT-PCR, normalized by β-actin mRNA; C: Time-course experiment on P450arom expression at 10–7 mol/L puerarin. Values were expressed as mean±SE of three separate experiments. *P<0.05, **P<0.01, vs DMSO line."

Figure 2

P450arom PⅡ responsible for the transcription suppression by puerarin A: RL95-2 cells were transfected with –1 017/+8 bp, –763/+8 bp, –543/+8 bp and –234/+8 bp constructs and were then treated with puerarin or DMSO for 12 hours. Luciferase activity was normalized by cotransfected pRL-CMV and these results represented the mean±SE of three different experiments. *P<0.05 with respect to the DMSO-treated samples; B: Sequence of the cloned 5'-flanking region of P450arom PⅡ from –417 bp to –336 bp. Putative transcription factor binding sites were identified using the TESS job and cis-acting elements were indicated in boxes. Two potential AP-1/c-jun binding sites with the highest scores were observed."

Figure 3

Suppression of 4×AP-1-luciferase activity and c-jun mRNA expression by puerarin A: To find out whether AP-1 varies along with P450arom by puerarin, the activity and mRNA levels of AP-1 were assayed. RL95-2 cells were transfected with 4×AP-1-luciferase plasmid and treated with DMSO (control) or puerarin (10–7 mol/L) for 12 hours; B: Cells were collected at indicated time point after treated with puerarin (10–7 mol/L) or DMSO and then used for RNA extraction and quantitative RT-PCR; C: Western blot analysis showed a significant decrease of c-jun protein expression by puerarin (10–7 mol/L) treatment for 12 hours, compared with the amount of β-actin. The target gene mRNA levels were normalized by the amount of β-actin mRNA levels. These results represented the mean±SE of three separate experiments. *P<0.05, **P<0.01, vs DMSO line."

[1] Bulun SE, Fang Z, Imir G , et al. Aromatase and endometriosis[J]. Semin Repod Med, 2004,22(1):45-50
doi: 10.1055/s-2004-823026
[2] Bulun SE, Imir G, Utsunomiya H , et al. Aromatase in endometriosis and uterine leiomyomata[J]. J Steroid Biochem Mol Biol, 2005,95(1-5):57-62
doi: 10.1016/j.jsbmb.2005.04.012 pmid: 16024248
[3] Bulun SE, Lin Z, Imir G , et al. Regulation of aromatase expression in estrogen-responsive breast and uterine disease: from bench to treatment[J]. Pharmacol Rev, 2005,57(3):359-383
doi: 10.1124/pr.57.3.6
[4] Mousa NA, Bedaiwy MA, Casper RF . Aromatase inhibitors in the treatment of severe endometriosis[J]. Obstet Gynecol, 2007,109(6):1421-1423
doi: 10.1016/S0002-8223(00)00427-2 pmid: 17540816
[5] Sanderson JT, Hordijk J, Denison MS , et al. Induction and inhibition of P450arom(CYP19) activity by natural and synthetic flavonoid compounds in H295R human adrenocortical carcinoma cells[J]. Toxicol Sci, 2004,82(1):70-79
doi: 10.1093/toxsci/kfh257
[6] Tsuchiya M, Miura T, Hanaoka T , et al. Effect of soy isoflavones on endometriosis: interaction with estrogen receptor 2 gene polymorphism[J]. Epidemiology, 2007,18(3):402-408
doi: 10.1097/01.ede.0000257571.01358.f9
[7] Rice S, Mason HD, Whitehead SA . Phytoestrogens and their low dose combinations inhibit mRNA expression and activity of aromatase in human granulosa-luteal cells[J]. J Steroid Biochem Mol Biol, 2006,101(4-5):216-225
doi: 10.1016/j.jsbmb.2006.06.021
[8] Yeung DK, Leung SW, Xu YC , et al. Puerarin, an isoflavonoid derived from Radix puerariae, potentiates endothelium-independent relaxation via the cyclic AMP pathway in porcine coronary artery[J]. Eur J Pharmacol, 2006,552(1-3):105-111
doi: 10.1016/j.ejphar.2006.08.078
[9] Yan LP, Chan SW, Chan AS , et al. Puerarin decreases serum total cholesterol and enhances thoracic aorta endothelial nitric oxide synthase expression in diet-induced hypercholesterolemic rats[J]. Life Sci, 2006,79(4):324-330
doi: 10.1016/j.lfs.2006.01.016
[10] Zhang S, Ji G, Liu J . Reversal of chemical-induced liver fibrosis in Wistar rats by puerarin[J]. J Nutr Biochem, 2006,17(7):485-491
doi: 10.1016/j.jnutbio.2005.09.002 pmid: 16426832
[11] Xiong FL, Sun XH, Gan L , et al. Puerarin protects rat pancreatic islets from damage by hydrogen peroxide[J]. Eur J Pharmacol, 2006,529(1-3):1-7
doi: 10.1016/j.ejphar.2005.10.024 pmid: 16321378
[12] Bo J, Ming BY, Gang LZ , et al. Protection by puerarin against MPP+-induced neurotoxicity in PC12 cells mediated by inhibiting mitochondrial dysfunction and caspase-3-like activation[J]. Neurosci Res, 2005,53(2):183-188
doi: 10.1016/j.neures.2005.06.014
[13] Xu ME, Xiao SZ, Sun YH , et al. The study of anti-metabolic syndrome effect of puerarin in vitro[J]. Life Sci, 2005,77(25):3183-3196
doi: 10.1016/j.lfs.2005.03.036 pmid: 16005472
[14] Kazmierczak W, Skalba P, Dabkowska-Huc A . et al. Expression of aromatase and 5-alpha-reductase genes in endometrial adenocarcinoma[J]. Pathol Res Pract, 2006,202(11):789-791
doi: 10.1016/j.prp.2006.09.001
[15] Berstein L, Zimarina T, Kovalevskij A , et al. CYP19 gene expression and aromatase activity in endometrial cancer tissue: importance of the type of the disease[J]. Neoplasma, 2005,52(2):115-118
[16] Huang JC, Ruan CH, Tang K , et al. Prunella stica inhibits the proliferation but not the prostaglandin production of ishikawa cells[J]. Life Sci, 2006,79(5):436-441
doi: 10.1016/j.lfs.2006.01.018
[17] Chen YP, Higgins JA, Gundersen-Rindal DE . Quantitation of a Glyptapanteles indiensis polydnavirus gene expressed in parasitized host, Lymantria dispar, by real-time quantitative RT-PCR[J]. J Virol Methods, 2003,114(2):125-133
doi: 10.1016/j.jviromet.2003.08.010
[18] Wolters M, Hahn A . Soy isoflavones--a therapy for menopausal symptoms?[J]. Wien Med Wochenschr, 2004,154(13-14):334-341
doi: 10.1007/s10354-004-0083-y pmid: 15368955
[19] Whitten PL, Patisaul HB, Young LJ . Neurobehavioral actions of coumestrol and related isoflavonoids in rodents[J]. Neurotoxicol Teratol, 2002,24(1):47-54
doi: 10.1016/S0892-0362(01)00192-1 pmid: 11836071
[20] Overstreet DH, Keung WM, Rezvani AH , et al. Herbal remedies for alcoholism: promises and possible pitfalls[J]. Alcohol Clin Exp Res, 2003,27(2):177-185
doi: 10.1097/01.ALC.0000051022.26489.CF pmid: 12605067
[21] Xu X, Zhang S, Zhang L , et al. The Neuroprotection of puerarin against cerebral ischemia is associated with the prevention of apoptosis in rats[J]. Planta Med, 2005,71(7):585-591
doi: 10.1055/s-2005-871261
[22] Fatemi HM, Al-Turki HA, Papanikolaou EG , et al. Successful treatment of an aggressive recurrent post-menopausal endometriosis with an aromatase inhibitor[J]. Reprod Biomed Online, 2005,11(4):455-457
doi: 10.1016/S1472-6483(10)61140-6
[23] Lephart ED, Setchell KD, Handa RJ , et al. Behavioral effects of endocrine-disrupting substances: phytoes-trogens[J]. ILAR J, 2004,45(4):443-454
doi: 10.1016/j.susc.2005.04.045 pmid: 15454683
[24] Myllymaki S, Haavisto T, Vainio M , et al. In vitro effects of diethylstilbestrol, genistein, 4-tert-butylphenol, and 4-tertoctylphenol on steroidogenic activity of isolated immature rat ovarian follicles[J]. Toxicol Appl Pharmacol, 2005,204(1):69-80
doi: 10.1016/j.taap.2004.08.009
[25] Ranney B . Etiology, prevention and inhibition of endometriosis[J]. Clin Obstet Gynecol, 1980,23(3):875-882
doi: 10.1097/00003081-198023030-00017 pmid: 6998632
[26] Noble LS, Simpson ER, Johns A , et al. Aromatase expression in endometriosis[J]. J Clin Endocrinol Metab, 1996,81(1):174-179
[27] Liu Y, Teng CT . Identification of the estrogen sensitive marker in human endometrial carcinoma RL95-2 cells[J]. Mol Cell Endocrinol, 1994,101(1-2):167-171
doi: 10.1016/0303-7207(94)90231-3 pmid: 9397949
[28] Mao CP, Gu ZL . Puerarin reduces increased c-fos, c-jun, and type Ⅳ collagen expression caused by high glucose in glomerular mesangial cells[J]. Acta Pharmacol Sin, 2005,26(8):982-986
doi: 10.1111/aphs.2005.26.issue-8
[1] Ling Xu, Qiao-ling Zhou, Jie Han, Dan-ying Zhang, Hui-juan Dong, Zai-long Cai, Chao-qin Yu, Dong-xia Zhai . Effects of Chinese herbal medicine Neiyi Recipe-medicated serum on angiopoiesis of endometriosis in the chick chorioallantoic membrane model. Journal of Chinese Integrative Medicine, 2012, 10(7): 800-806.
[2] Lei Chen, Xiao-ying Bi, Li-xun Zhu, Yi-qing Qiu, Su-ju Ding, Ben-qiang Deng. Flavonoids of puerarin versus tanshinone ⅡA for ischemic stroke: A randomized controlled trial. Journal of Chinese Integrative Medicine, 2011, 9(11): 1215-1220.
[3] Yang Cao, Ting-ting Zhang, Shu-wu Xie, Yan Zhu, Lan-di Shu, Zhi-li Song, Lin Cao, De-ying Dai. Effects of Caulis Sargentodoxae Granule on expressions of vascular endothelial growth factor and its receptor-2 in rats with endometriosis. Journal of Chinese Integrative Medicine, 2009, 7(4): 400-400.
[4] Chao-qin Yu, Jin Yu, Jie Han, Qiao-ling Zhou, Wei Shen. Regulatory mechanism of malignant behavior of endometriosis mediated by puerarin. Journal of Chinese Integrative Medicine, 2009, 7(1): 41-47.
[5] Ming-yu Zhang, Zhi-bin Shi, Hua-qing Yang, Shu-qiang Ma, Wei Wang, Xiao-qian Dang, Kun-zheng Wang. Effects of different doses of puerarin on vascular endothelial cell proliferation in vitro. Journal of Chinese Integrative Medicine, 2008, 6(9): 942-945.
[6] Pei-yong Zheng, Zan-song Ma, Yong-qiang Hua, Tao Liu, Lian-jun Xing, Guang Ji. Effects of puerarin on expressions of leptin receptor mRNA, phosphoryla-ted Janus kinase 2/phosphorylated signal transducers and activators of transcription 3 proteins in the liver of rats with non-alcoholic fatty liver. Journal of Chinese Integrative Medicine, 2008, 6(9): 921-927.
[7] Xin-ling Li, Fang Lian, Yan-he Liu. Effects of Quyu Jiedu Granule on expressions of tumor necrosis factor-α and interleukin-6 mRNAs in ovarian granulosa cells of endometriosis rats. Journal of Chinese Integrative Medicine, 2008, 6(9): 960-963.
[8] Fan Qu​, Jue Zhou​, Dong-Xia Yang​, Wen-guang Ma​, Bao-zhang Ma​. Effects of Yiweining Recipe on expressions of metalloproteinase-2 and cyclooxygenase-2 mRNAs in ectopic endometrium of rats with endometriosis. Journal of Chinese Integrative Medicine, 2006, 4(6): 634-638.
[9] Chao-qin Yu​, Shu-fang Shi​, Yu-huan Liu​, Rui-xia Wang​, Yan-hua Song​, Jin Yu​. Primary culture and morphologic observation of eutopic and ectopic endometrial cells from patients with endomehiosis. Journal of Chinese Integrative Medicine, 2006, 4(2): 189-193.
[10] Pi-qi Zhou, Lin Shen, Yan-ping Yang, Jing Xie. Clinical observation of puerarin in treating patients with vertebral artery insufficiency due to cervical spondylosis: A report of 123 cases. Journal of Chinese Integrative Medicine, 2006, 4(1): 80-81.
[11] Le-hong Jin, Chuan-fei Liu, Yu Zeng. Protective effects of puerarin on radiation injury of experimental rats. Journal of Chinese Integrative Medicine, 2005, 3(1): 43-45.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
[1] Wei-xiong Liang. Problems-solving strategies in clinical treatment guideline for traditional Chinese medicine and integrative medicine. Journal of Chinese Integrative Medicine, 2008, 6(1): 1-4
[2] Zhao-guo Li. Discussion on English translation of commonly used sentences in traditional Chinese medicine: part one. Journal of Chinese Integrative Medicine, 2008, 6(1): 107-110
[3] Jun Hu, Jian-ping Liu. Non-invasive physical treatments for chronic/recurrent headache. Journal of Chinese Integrative Medicine, 2008, 6(1): 31
[4] Xue-mei Liu, Qi-fu Huang, Yun-ling Zhang, Jin-li Lou, Hong-sheng Liu, Hong Zheng. Effects of Tribulus terrestris L. saponion on apoptosis of cortical neurons induced by hypoxia-reoxygenation in rats. Journal of Chinese Integrative Medicine, 2008, 6(1): 45-50
[5] . Uniform requirements for manuscripts submitted to biomedical journals: Writing and editing for biomedical publication (Chinese version, part two). Journal of Chinese Integrative Medicine, 2010, 8(11): 1001-1005
[6] Daniel Weber, Janelle M Wheat, Geoffrey M Currie. Inflammation and cancer: Tumor initiation, progression and metastasis,and Chinese botanical medicines. Journal of Chinese Integrative Medicine, 2010, 8(11): 1006-1013
[7] Hong Liu , Guo-liang Zhang, Li Shen , Zhen Zeng, Bao-luo Zhou, Cheng-hai Liu, Guang Nie . Application and evaluation of a pseudotyped virus assay for screening herbs for anti-H5Nl avian influenza virus. Journal of Chinese Integrative Medicine, 2010, 8(11): 1036-1040
[8] Zhao-guo Li . A discussion of English translation of 1995 and 1997 Chinese National Standards of Traditional Chinese Medical Terminologies for Clinical Diagnosis and Treatment. Journal of Chinese Integrative Medicine, 2010, 8(11): 1090-1096
[9] Rui Jin, Bing Zhang. A complexity analysis of Chinese herbal property theory: the multiple formations of herbal property (Part 1). Journal of Chinese Integrative Medicine, 2012, 10(11): 1198-1205
[10] Hui-min Liu, Xian-bo Wang, Yu-juan Chang, Li-li Gu. Systematic review and meta-analysis of randomized controlled trials of integrative medicine therapy for treatment of chronic severe hepatitis. Journal of Chinese Integrative Medicine, 2012, 10(11): 1211-1228