Molecular Biology Reports

, Volume 39, Issue 5, pp 6227–6234 | Cite as

Artesunate inhibits cell proliferation and decreases growth hormone synthesis and secretion in GH3 cells

  • Zhi-gang Mao
  • Jing Zhou
  • Hui Wang
  • Dong-sheng He
  • Wei-wei Xiao
  • Gui-zhi Liao
  • Lu-bin Qiu
  • Yong-hong Zhu
  • Hai-jun Wang
Article

Abstract

To determine the effect of artesunate (ART) on the rat pituitary adenoma GH3 cell line to evaluate its potential as a novel agent in growth hormone (GH) adenoma and to investigate its underlying mechanisms of action. The MTT assay was used to assess cell proliferation. DAPI staining was used to visualise apoptotic changes in the nucleus. We also analyzed cell apoptosis and cell cycle stage by flow cytometry, semi-quantitative RT-PCR analysis for the expression of GH mRNA and apoptosis-induced factor (AIF) mRNA, analysis of GH protein by western blot, ELISA detection of secreted GH, and the caspase inhibition assay. We found that ART inhibited the proliferation of GH3 cells in a dose- and time-dependent manner, with an IC50 of 9.53 ± 4.12 μM. The IC50s of ART against of two normal cell lines (mouse embryonic fibroblasts, and rat bone mesenchymal cells) were much higher than the IC50 recorded for the GH3 cells. ART induced apoptosis and blocked GH3 at G2/M arrest. The pan caspase inhibitor V-ZAD-FMK partly attenuated the inhibitory effect of ART. ART increased the expression of AIF mRNA and reduced GH mRNA levels, GH synthesis and the secretion of GH level in GH3 cells. ART can inhibit proliferation and induce apoptosis in GH3 cells by caspase-dependent pathways. Additionally, ART can inhibit GH synthesis and secretion. Thus, we propose ART as a probably anti-tumour candidate drug in the treatment of GH adenoma.

Keywords

Growth hormone adenomas Artesunate Apoptosis Caspase Apoptosis induce factor 

Abbreviations

ART

Artesunate

MEFs

Mouse embryonic fibroblasts

MSCs

Rat bone mesenchymal cells

GH

Growth hormone

DAPI

4′,6′-diamidino-2-phenylindole dihydrochloride

Supplementary material

11033_2011_1442_MOESM1_ESM.jpg (211 kb)
Supplementary Fig. 1S ART selectively inhibited GH3 cell growth, but its effects on MEFs and MSCs were small. Data are shown as the mean ± SD (*3.125 μM group GH3 vs. MEF P = 0.004, GH3 vs. MSC P = 0.003; 6.25 μM group GH3 vs. MEF P = 0.001, GH3 vs. MSC P = 0.003; 12.5 μM group GH3 vs. MEF P = 0.002, GH3 vs. MSC P = 0.002; 25 μM group GH3 vs. MEF P = 0.009, GH3 vs. MSC P = 0.004; and 50 μM group GH3 vs. MEF P = 0.03, GH3 vs. MSC P = 0.004) (JPEG 210 kb)
11033_2011_1442_MOESM2_ESM.jpg (246 kb)
Supplementary Fig. 2S ART induced GH3 apoptosis. Healthy GH3 cells were used as the negative control (left). The apoptotic nucleus is shown by the arrow (right). Bar = 10 μm (JPEG 246 kb)
11033_2011_1442_MOESM3_ESM.jpg (319 kb)
Supplementary Fig. 3S The FCM-detected apoptosis rate at 0, 12, 24, and 36 h after the ART treatment (*P = 0.342, 0.111, 0.023, and 0.002 at 0, 12, 24, and 36 h, respectively) (JPEG 319 kb)
11033_2011_1442_MOESM4_ESM.jpg (532 kb)
Supplementary Fig. 4S The FCM-detected GH3 cell cycle after incubation for 48 h with various concentrations of ART (A, B, C, D, and E were 0, 3.125, 6.25, 12.5, and 25 μM, respectively) (*P = 0.007, 0.001, 0.001, and 0.004 in 3.125–25 μM) (JPEG 531 kb)

References

  1. 1.
    Asa SL, Ezzat S (2002) The pathogenesis of pituitary tumours. Nat Rev Cancer 2:836–849PubMedCrossRefGoogle Scholar
  2. 2.
    Gola M, Doga M, Bonadonna S, Mazziotti G, Vescovi PP, Giustina A (2006) Neuroendocrine tumors secreting growth hormone-releasing hormone: pathophysiological and clinical aspects. Pituitary 9:221–229PubMedCrossRefGoogle Scholar
  3. 3.
    Chen B, Moreland J, Zhang J (2011) Human brain functional MRI and DTI visualization with virtual reality. Quant Imaging Med Surg 1:11–16Google Scholar
  4. 4.
    Melmed S (2009) Acromegaly pathogenesis and treatment. J Clin Invest 119:3189–3202PubMedCrossRefGoogle Scholar
  5. 5.
    Castinetti F, Morange I, Dubois N, Albarel F, Conte-Devolx B, Dufour H, Brue T (2009) Does first-line surgery still have its place in the treatment of acromegaly? Ann Endocrinol (Paris) 70:107–112CrossRefGoogle Scholar
  6. 6.
    Situ D, Wang J, Shao W, Zhu ZH (2011) Assessment and treatment of cancer pain: from Western to Eastern. Ann Palliat Med. doi:10.3978/j.issn.2224-5820,2011.10.01 Google Scholar
  7. 7.
    White NJ (2008) Qinghaosu (artemisinin): the price of success. Science 320:330–334PubMedCrossRefGoogle Scholar
  8. 8.
    Sriram D, Rao VS, Chandrasekhara KV, Yogeeswari P (2004) Progress in the research of artemisinin and its analogues as antimalarials: an update. Nat Prod Res 18(6):503–527PubMedCrossRefGoogle Scholar
  9. 9.
    Gomes MF, Faiz MA, Gyapong JO, Warsame M, Agbenyega T, Babiker A, Baiden F, Yunus EB, Binka F, Clerk C, Folb P, Hassan R, Hossain MA, Kimbute O, Kitua A, Krishna S, Makasi C, Mensah N, Mrango Z, Olliaro P, Peto R, Peto TJ, Rahman MR, Ribeiro I, Samad R, White NJ, Study 13 Research Group (2009) Pre-referral rectal artesunate to prevent death and disability in severe malaria: a placebo-controlled trial. Lancet 373:557–566PubMedCrossRefGoogle Scholar
  10. 10.
    Eastman RT, Fidock DA (2009) Artemisinin-based combination therapies: a vital tool in efforts to eliminate malaria. Nat Rev Microbiol 7:864–874PubMedGoogle Scholar
  11. 11.
    Hien TT, White NJ (1993) Qinghaosu. Lancet 341:603–608PubMedCrossRefGoogle Scholar
  12. 12.
    Youns M, Efferth T, Reichling J, Fellenberg K, Bauer A, Hoheisel JD (2009) Gene expression profiling identifies novel key players involved in the cytotoxic effect of artesunate on pancreatic cancer cells. Biochem Pharmacol 78:273–283PubMedCrossRefGoogle Scholar
  13. 13.
    Hou J, Wang D, Zhang R, Wang H (2008) Experimental therapy of hepatoma with artemisinin and its derivatives: in vitro and in vivo activity, chemosensitization, and mechanisms of action. Clin Cancer Res 14:5519–5530PubMedCrossRefGoogle Scholar
  14. 14.
    Li LN, Zhang HD, Yuan SJ, Yang DX, Wang L, Sun ZX (2008) Differential sensitivity of colorectal cancer cell lines to artesunate is associated with expression of beta-catenin and E-cadherin. Eur J Pharmacol 588:1–8PubMedCrossRefGoogle Scholar
  15. 15.
    Nam W, Tak J, Ryu JK, Jung M, Yook JI, Kim HJ, Cha IH (2007) Effects of artemisinin and its derivatives on growth inhibition and apoptosis of oral cancer cells. Head Neck 29:335–340PubMedCrossRefGoogle Scholar
  16. 16.
    Wang MH, Zheng JS, Luo YP, Mi C (2008) Growth inhibition effects of artesunate on human cervical cancer cell line HeLa. Di 3 Jun Yi Da Xue Xue Bao 30:1730–1733Google Scholar
  17. 17.
    Rinner B, Siegl V, Pürstner P, Efferth T, Brem B, Greger H, Pfragner R (2004) Activity of novel plant extracts against medullary thyroid carcinoma cells. Anticancer Res 24:495–500PubMedGoogle Scholar
  18. 18.
    Susin SA, Lorenzo HK, Zamzami N, Marzo I, Snow BE, Brothers GM, Mangion J, Jacotot E, Costantini P, Loeffler M, Larochette N, Goodlett DR, Aebersold R, Siderovski DP, Penninger JM, Kroemer G (1999) Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 397:441–446PubMedCrossRefGoogle Scholar
  19. 19.
    Noedl H, Wongsrichanalai C, Miller RS, Myint KS, Looareesuwan S, Sukthana Y, Wongchotigul V, Kollaritsch H, Wiedermann G, Wernsdorfer WH (2002) Plasmodium falciparum: effect of anti-malarial drugs on the production and secretion characteristics of histidine-rich protein II. Exp Parasitol 102:157–163PubMedCrossRefGoogle Scholar
  20. 20.
    Xu H, He Y, Yang X, Liang L, Zhan Z, Ye Y, Yang X, Lian F, Sun L (2007) Anti-malarial agent artesunate inhibits TNF-alpha-induced production of proinflammatory cytokines via inhibition of NF-kappaB and PI3 kinase/Akt signal pathway in human rheumatoid arthritis fibroblast-like synoviocytes. Rheumatology (Oxford) 46:920–926CrossRefGoogle Scholar
  21. 21.
    Lee J, Zhou HJ, Wu XH (2006) Dihydroartemisinin downregulates vascular endothelial growth factor expression and induces apoptosis in chronic myeloid leukemia K562 cells. Cancer Chemother Pharmacol 57:213–220PubMedCrossRefGoogle Scholar
  22. 22.
    Lou XE, Zhou HJ (2001) Effects of artesunate on progestrone estrogen content and decidua in rats. Yao Xue Xue Bao 36:254–257PubMedGoogle Scholar
  23. 23.
    Buommino E, Baroni A, Canozo N, Petrazzuolo M, Nicoletti R, Vozza A, Tufano MA (2009) Artemisinin reduces human melanoma cell migration by down-regulating alpha V beta 3 integrin and reducing metalloproteinase 2 production. Invest New Drugs 27:412–418PubMedCrossRefGoogle Scholar
  24. 24.
    Xiang P, Huang JT, Sa YL (2005) Culture and identification of rat marrow mesenchymal stem cells. J Bengbu Med Coll 30:1–3Google Scholar
  25. 25.
    Huang JT, Sa YL, Li HB, Liu AJ, Li HB (2003) Reformed method of feeder layer technique. Anat Res 25:78–79Google Scholar
  26. 26.
    Miyoshi T, Otsuka F, Otani H, Inagaki K, Goto J, Yamashita M, Ogura T, Iwasaki Y, Makino H (2008) Involvement of bone morphogenetic protein-4 in GH regulation by octreotide and bromocriptine in rat pituitary GH3 cells. J Endocrinol 197:159–169PubMedCrossRefGoogle Scholar
  27. 27.
    Ren SG, Melmed S (2006) Pyridoxal phosphate inhibits pituitary cell proliferation and hormone secretion. Endocrinology 147:3936–3942PubMedCrossRefGoogle Scholar
  28. 28.
    Chen T, Li M, Zhang R, Wang H (2009) Dihydroartemisinin induces apoptosis and sensitizes human ovarian cancer cells to carboplatin therapy. J Cell Mol Med 13:1358–1370PubMedCrossRefGoogle Scholar
  29. 29.
    Lu YY, Chen TS, Qu JL, Pan WL, Sun L, Wei XB (2009) Dihydroartemisinin (DHA) induces caspase-3-dependent apoptosis in human lung adenocarcinoma ASTC-a-1 cells. J Biomed Sci 16:16PubMedCrossRefGoogle Scholar
  30. 30.
    Singh NP, Panwar VK (2006) Case report of a pituitary macroadenoma treated with artemether. Integr Cancer Ther 5:391–394PubMedCrossRefGoogle Scholar
  31. 31.
    Lai H, Singh NP (2006) Oral artemisinin prevents and delays the development of 7,12-dimethylbenz[a]anthracene (DMBA)-induced breast cancer in the rat. Cancer Lett 231:43–48PubMedCrossRefGoogle Scholar
  32. 32.
    Li S, Xue F, Cheng Z, Yang X, Wang S, Geng F, Pan L (2009) Effect of artesunate on inhibiting proliferation and inducing apoptosis of SP2/0 myeloma cells through affecting NFkappaB p65. Int J Hematol 90:513–521PubMedCrossRefGoogle Scholar
  33. 33.
    Li Z, Yuan SJ, Nie LP, Tian ZY, Xu LP, Han CM (2004) Apoptosis of tumor cells induced by artesunate is associated with suppressing survivin expression. Chin J Clin Pharmacol Ther 9:607–611Google Scholar
  34. 34.
    Mukae N, Enari M, Sakahira H, Fukuda Y, Inazawa J, Toh H, Nagata S (1998) Molecular cloning and characterization of human caspase-activated DNase. Proc Natl Acad Sci USA 95:9123–9128PubMedCrossRefGoogle Scholar
  35. 35.
    Nicolier M, Decrion-Barthod AZ, Launay S, Prétet JL, Mougin C (2009) Spatiotemporal activation of caspase-dependent and -independent pathways in staurosporine-induced apoptosis of p53wt and p53mt human cervical carcinoma cells. Biol Cell 101:455–467PubMedCrossRefGoogle Scholar
  36. 36.
    Li K, Li Y, Shelton JM, Richardson JA, Spencer E, Chen ZJ, Wang X, Williams RS (2000) Cytochrome c deficiency causes embryonic lethality and attenuates stress-induced apoptosis. Cell 101:389–399PubMedCrossRefGoogle Scholar
  37. 37.
    Yoshida H, Kong YY, Yoshida R, Elia AJ, Hakem A, Hakem R, Penninger JM, Mak TW (1998) Apaf1 is required for mitochondrial pathways of apoptosis and brain development. Cell 94:739–750PubMedCrossRefGoogle Scholar
  38. 38.
    Hsu SC, Yang JS, Kuo CL, Lo C, Lin JP, Hsia TC, Lin JJ, Lai KC, Kuo HM, Huang LJ, Kuo SC, Wood WG, Chung JG (2009) Novel quinolone CHM-1 induces apoptosis and inhibits metastasis in a human osterogenic sarcoma cell line. J Orthop Res 27:1637–1644PubMedCrossRefGoogle Scholar
  39. 39.
    Tu HP, Chen YT, Chiu HC, Chin YT, Huang SM, Cheng LC, Fu E, Chiang CY (2009) Cyclosporine A enhances apoptosis in gingival keratinocytes of rats and in OECM1 cells via the mitochondrial pathway. J Periodontal Res 44:767–775PubMedCrossRefGoogle Scholar
  40. 40.
    Feng B, Zhou XB, Yang X, Ye ZL, He ZY (2006) Apoptosis of hypertrophic cardiomyocytes stimulated by hypoxia-reoxygenation is partially mediated by apoptosis-inducing factor. Sheng Li Xue Bao 58:599–605PubMedGoogle Scholar
  41. 41.
    Tokuyama Y, Reddy AP, Bethea CL (2008) Neuroprotective actions of ovarian hormones without insult in the raphe region of rhesus macaques. Neuroscience 154:720–731PubMedCrossRefGoogle Scholar
  42. 42.
    Miller M, Chen S, Woodliff J, Kansra S (2008) Curcumin (diferuloylmethane) inhibits cell proliferation, induces apoptosis, and decreases hormone levels and secretion in pituitary tumor cells. Endocrinology 149:4158–4167PubMedCrossRefGoogle Scholar
  43. 43.
    Dang VH, Nguyen TH, Lee GS, Choi KC, Jeung EB (2009) In vitro exposure to xenoestrogens induces growth hormone transcription and release via estrogen receptor-dependent pathways in rat pituitary GH3 cells. Steroids 74:707–714PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Zhi-gang Mao
    • 1
  • Jing Zhou
    • 2
  • Hui Wang
    • 2
  • Dong-sheng He
    • 1
  • Wei-wei Xiao
    • 2
  • Gui-zhi Liao
    • 2
  • Lu-bin Qiu
    • 1
  • Yong-hong Zhu
    • 2
  • Hai-jun Wang
    • 1
  1. 1.Department of Neurosurgery and Pituitary Tumor CenterThe First Affiliated Hospital, Sun Yat-sen UniversityGuangzhouChina
  2. 2.Department of Histology and EmbryologyZhongshan School of Medicine, Sun Yat-sen UniversityGuangzhouChina

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