Effect of triptolide on aromatase activity in human placental microsomes and human placental JEG-3 cells

Abstract

Triptolide (CAS 38748-32-2), a major active component of Tripterygium wilfordii Hook F, engages in multiple pharmacological activities. However, it interferes with the synthesis of sex steroid hormones. This was proven in the present study of male and female subjects with severe reproductive toxicities related to abnormal androgen or estrogen levels. The inhibitory action of triptolide on aromatase, a terminal enzyme responsible for the formation of estrogens from androgens, was determined in vitro to investigate its role in endocrine secretion. Triptolides were incubated with human placental microsomes for 20 min at concentrations of 10, 20, 30, and 40 nM. At 20 nM, triptolide significantly inhibited aromatase, as shown by [³H]₂O assay, and its concentration at 50% inhibition (IC₅₀) was determined as approximately 35 nM. Furthermore, the inhibitory effect of triptolide on aromatase was observed in human placental choriocarcinoma (JEG-3) cell lines. Treatment with 10 nM triptolide significantly inhibited the aromatase of JEG-3 cells and gave an IC₅₀ value of approximately 17 nM. Under these concentrations, decreased levels of mRNA and protein expression of aromatase in JEG-3 cells were observed using Western blot analysis and quantitative real-time reverse transcription polymerase chain reaction assay. The 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyl-tetra-zolium bromide (MTT) test revealed that treatment with triptolide for 24 h under these concentrations did not trigger evident cell death. These findings characterize the molecular mechanisms of triptolide on the regulation of sex steroid hormones.

Key words

Aromatase - Human placental JEG-3 cells - Human placental microsomes - Triptolide

• References

• 1 Corson TW, Crews CM. Molecular understanding and modern application of traditional medicines: triumphs and trials. Cell. 2007; 130 (5) 769-74
• 2 Efferth T, Li PC, Konkimalla VS, Kaina B. From traditional Chinese medicine to rational cancer therapy. Trends Mol Med. 2007; 13 (8) 353-61
• 3 Tao XL, Sun Y, Dong Y, Xiao YL, Hu DW, Shi YP et al. A prospective, controlled, double-blind, cross-over study of tripterygium wilfodii hook F in treatment of rheumatoid arthritis. Chin Med J (Engl.) 1989; 102 (5) 327-32
• 4 Tao X, Schulze-Koops H, Ma L, Cai J, Mao Y, Lipsky PE. Effects of Tripterygium wilfordii hook F extracts on induction of cyclooxygenase 2 activity and prostaglandin E2 production. Arthritis Rheum. 1998; 41 (1) 130-8
• 5 Wang Z, Jin H, Xu R, Mei Q, Fan D. Triptolide downregulates Rac1 and the JAK/STAT3 pathway and inhibits colitis-related colon cancer progression. Exp Mol Med. 2009; 41 (10) 717-27
• 6 Dy Y. One hundred and forty-four cases of rheumatoid arthritis treated with Tripterygium wilfordii. J Trad Chin Med. 1983; 3: 125-9
• 7 Qian SZ XY, Zhang JW. Recent progress in research on Tripterygium: a male antifertility plant. Contraception. 1995; 51: 121-9
• 8 Qian SZ HY, Tong JS. Studies on the effect of T. wilfordii on the reproduction of men. Chin J Androl. 1989; 3: 129-32
• 9 Lue Y, Sinha Hikim AP, Wang C, Leung A, Baravarian S, Reutrakul V et al. Triptolide: a potential male contraceptive. J Androl. 1998; 19 (4) 479-86
• 10 Zhen QS, Ye X, Wei ZJ. Recent progress in research on Tripterygium: a male antifertility plant. Contraception. 1995; 51 (2) 121-9
• 11 Harper-Wynne RossC, Sacks G, Salter N, Nasiri J. Posttesticular antifertility action of triptolide in the male rat: evidence for severe impairment of cauda epididymal sperm ultrastructure. Cancer Epidemiol Biomarkers Prev. 2002; 11 (7) 614-7
• 12 Sy Z. 5 cases report of Tripterygium wilfordii inducing premature ovarian failure. Sichuan Medicine. 1995; 16 (3) 19-29
• 13 Fu P MC, Gong Y. Study on sex hormone levels in Tripterygium wilfordii induced amenorrhea. J Clin Dermatol. 1999; 28 (6) 163-4
• 14 Shi JH WM, Liu XD. High gonadotropin and amenorrhea induced by Tripterygium wilfordii in two patients. Chin J New Drugs Clin Remed. 2003; 22 (10) 635-6
• 15 Gao YP JZ, Chen DF. The Effect of Tripterygium wilfordii Hook. f. on Uterin Leiomyoma. Acta Universitatis Medicinalis Secondae Shanghai. 2001; 21 (2) 137-40
• 16 Gao YP, Chen DF, Chang C. Effect of glycosides of Tripterygium wilfordii on uterus and blood supply of hysteromyoma patients. Shanghai Med J. 2002; 25 (4) 237-9
• 17 Fu YF LJ, Shi YP. The clinical observation on 26 cases of Endometriosis treated with Tripterygium wilfordii. Jiangsu Journal of Traditional Chinese Medicine. 1993; 14 (4) 36-8
• 18 Luo JH HZ. Tripterygium wilfordii treating the post-operation cases of endometriosis. J Clin Milit Surg. 2002; 30 (4) 41-2
• 19 Liu J, Jiang Z, Liu L, Zhang Y, Zhang S, Xiao J et al. Triptolide induces adverse effect on reproductive parameters of female Sprague-Dawley rats. Drug Chem Toxicol. 2010; 34 (l) l-7
• 20 Jing Liua Z, Xiao J, Zhang L. Effects oftriptolide from Tripterygium wilfordii on ERa and p53 expression in two human breast cancer cell lines. Phytomedicine. 2009; 29: 1006-3
• 21 Li H, Takai N, Yuge A, Furukawa Y, Tsuno A, Tsukamoto Y et al. Novel target genes responsive to the anti-growth activity of triptolide in endometrial and ovarian cancer cells. Cancer Lett. 2010; 297 (2) 198-206
• 22 Stocco C. Aromatase expression in the ovary: hormonal and molecular regulation. Steroids. 2008; 73: 473-87
• 23 Carreau S, Genissel C, Bilinska B, Levallet J. Sources of oestrogen in the testis and reproductive tract of the male. Int J Androl. 1999; 22 (4) 211-23
• 24 Saez JM. Leydig cells, endocrine, paracrine, and autocrine regulation. Endocr Rev. 1994; 15 (5) 574-626
• 25 Ishunina TA, Kruijver FP, Balesar R, Swaab DF. Differential expression of estrogen receptor alpha and beta immunoreactivity in the human supraoptic nucleus in relation to sex and aging. J Clin Endocrinol Metab. 2000; 85 (9) 3283-91
• 26 Bulun SE, Lin Z, Imir G, Amin S, Demura M, Yilmaz B et al. Regulation of aromatase expression in estrogen-responsive breast and uterine disease: from bench to treatment. Pharmacol Rev. 2005; 57 (3) 359-383
• 27 Schomberg DW, Couse JF, Mukherjee A, Lubahn DB, Sar Mayo KE, Korach KS. Targeted disruption of the estrogen receptor-a gene in female mice: characterization of ovarian responses and phenotype in the adult. Endocrinology. 1999; 140: 2733-44
• 28 Schomberg DW, Couse JF, Mukherjee A, Lubahn DB, Sar M, Mayo KE et al. Targeted disruption of the estrogen receptor-alpha gene in female mice: characterization of ovarian responses and phenotype in the adult. Endocrinology. 1999; 140 (6) 2733-44
• 29 Ni B, Zhu T, Jiang Z, Zhang R, Zhang T, Zhang L. In vitro and in silico approaches for analyzing the toxicological effect of triptolide on cx43 in Sertoli cells. Toxicol Mech Methods. 2008; 18 (9) 717-24
• 30 Simpson ER, Michael MD, Agarwal VR, Hinshelwood MM, Bulun SE, Zhao Y. Cytochromes P450 11: expression of the CYP19 (aromatase) gene: an unusual case of alternative promoter usage. FASEB J. 1997; 11 (l) 29-36
• 31 Bulun SE, Chen D, Lu M, Zhao H, Cheng Y, Demura M et al. Aromatase excess in cancers of breast, endometrium and ovary. J Steroid Biochem Mol Biol. 2007; 106 (l–5) 81-96
• 32 Kitaoka Y, Kitawaki J, Koshiba H, Inoue S, Ishihara H, Teramoto M et al. Aromatase cytochrome P450 and estrogen and progesterone receptors in uterine sarcomas: correlation with clinical parameters. J Steroid Biochem Mol Biol. 2004; 88 (2) 183-9
• 33 Hobler C, Andrade AJ, Grande SW, Gericke C, Talsness CE, Appel KE et al. Sex-dependent aromatase activity in rat offspring after pre- and postnatal exposure to triphenyltin chloride. Toxicology 2010; 276 (3) 198-205
• 34 Carreau S, Wolczynski S, Galeraud-Denis I. Aromatase, oestrogens and human male reproduction. Philos Trans R Soc Lond B Biol Sci. 2010; 365 (1546) 1571-9
• 35 Lephart ED, Simpson ER. Assay of aromatase activity. Methods Enzymol. 1991; 206: 477-83
• 36 Milczarek R, Sokolowska E, Hallmann A, Klimek J. The NADPH- and iron-dependent lipid peroxidation in human placental microsomes. Mol Cell Biochem. 2007; 295 (l–2) 105-11
• 37 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951; 193 (1) 265-75
• 38 Livak KJ, Schmittgen T.D.. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods Enzymol. 2001; 25: 402-8
• 39 Eng ET, Williams D, Mandava U, Kirma N, Tekmal RR, Chen S. Anti-aromatase chemicals in red wine. Ann N Y Acad Sci. 2002; 963: 239-46
• 40 Lipsky PE, Tao XL. A potential new treatment for rheumatoid arthritis: thunder god vine. Semin Arthritis Rheum. 1997; 26 (5) 713-23
• 41 Liu MX, Dong J, Yang YJ, Yang XL, Xu HB. [Progress in research on triptolide]. Zhongguo Zhong Yao Za Zhi. 2005; 30 (3) 170-4 Article in Chinese.
• 42 Liu J, Jiang Z, Liu L, Zhang Y, Zhang S, Xiao J et al. Triptolide induces adverse effect on reproductive parameters of female Sprague-Dawley rats. Drug Chem Toxicol. 2011; 34 (1) l-7
• 43 Lue Y, Sinha Hikim AP, Wang C. Triptolide: a potential male contraceptive. J Androl. 1998; 19: 479-86
• 44 Zhen QS, Ye X, Wei ZJ. Recent progress in research on Tripterygium: a male antifertility plant. Contraception. 1995; 51: 121-9
• 45 Fu P, Mao CS, Gong Y. Study on sex hormone levels in Tripterygium wilfordii induced amenorrhea. J Clin Dermatol. 1999; 28 (6) 163-4
• 46 Shoham Z, Schachter M. Estrogen biosynthesis–regulation, action, remote effects, and value of monitoring in ovarian stimulation cycles. Fertil Steril. 1996; 65 (4) 687-701
• 47 Yang JH, Luo SD, Wang YS, Zhao JF, Zhang HB, Li L. Triterpenes from Tripterygium wilfordii Hook. J Asian Nat Prod Res. 2006; 8 (5) 425-9
• 48 Bochkareva NV, Kondakova IV, Kolomiets LA, Muntian AB, Stukanov SL, Fomina SV. [Prognostic significance of assay of sex hormones][their receptors and enzymes involved in estrogen synthesis and metabolism in endometrial carcinoma]. Vopr Onkol. 2007; 53 (3) 315-20 Article in Russian.
• 49 Madej P, Plewka A, Plewka D, Palen P, Nowaczyk G, Bogunia E et al. The aromatase expression in myomas and myometriums of women in reproduction and perimenopausal age. Folia Histochem Cytobiol. 2009; 47 (3) 497-504
• 50 Wang L, Ye W, Liu X, Guo Y. [Combined use of steroid hormone and low dose gossypol for antifertility and its mechanism in rats]. Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2000; 22 (3) 214-9 Article in Chinese.
• 51 Zhou J, Gurates B, Yang S, Sebastian S, Bulun SE. Malignant breast epithelial cells stimulate aromatase expression via promoter II in human adipose fibroblasts. an epithelialstromal interaction in breast tumors mediated by CCAAT/ enhancer binding protein beta. Cancer Res. 2001; 61 (5) 2328-34
• 52 Brodie A, Lu Q, Nakamura J. Aromatase in the normal breast and breast cancer. J Steroid Biochem Mol Biol. 1997; 61 (3–6) 281-6
• 53 Goss P. Anti-aromatase agents in the treatment and prevention of breast cancer. Cancer Control. 2002; 9 (2 Suppl) 2-8
• 54 Buzdar A.U.. A summary of second-line randomized studies of aromatase inhibitors. J Steroid Biochem Mol Biol. 2001; 79: 109-14
• 55 Brodie AM, Njar VC. Aromatase inhibitors in advanced breast cancer: mechanism of action and clinical implications. J Steroid Biochem Mol Biol. 1998; 66 (1–2) 1-10
• 56 Santner SJ, Feil PD, Santen RJ. In situ estrogen production via the estrone sulfatase pathway in breast tumors: relative importance versus the aromatase pathway. J Clin Endocrinol Metab. 1984; 59 (1sss) 29-33
• 57 Hong Y, Chen S. Aromatase, estrone sulfatase, and 17beta-hydroxysteroid dehydrogenase: structure-function studies and inhibitor development. Mol Cell Endocrinol. 2010; 340 (2) 120-6