Analytical and Bioanalytical Chemistry

, Volume 410, Issue 14, pp 3325–3335 | Cite as

Effects of glaucocalyxin A on human liver cancer cells as revealed by GC/MS- and LC/MS-based metabolic profiling

  • Yue Liu
  • Shan Lu
  • Liang Zhao
  • Xin Dong
  • Zhenyu Zhu
  • Yongsheng Jin
  • Haisheng Chen
  • Feng Lu
  • Zhanying Hong
  • Yifeng Chai
Research Paper


Studies have documented the potential antitumor activities of glaucocalyxin A (GLA), an ent-kaurene diterpenoid isolated from Rabdosia japonica. However, the metabolic mechanism underlying the antitumor activity of GLA remains largely unknown. The effects of GLA on the metabolome of human liver cancer cells using GC/MS- and LC/MS-based metabolic profiling have been investigated. An untargeted metabolomics approach in conjunction with orthogonal projection to latent structures–discriminant analysis (OPLS-DA) has been developed to characterize the metabolic modifications induced by GLA treatment in human hepatoma cell line SMMC7721. Results demonstrated that cells cultured in the presence or absence of GLA displayed different metabolic profiles: the treatment induced an increased purine metabolism, pyrimidine metabolism, and sphingolipid metabolism and a decreased amino acid metabolism. At the same time, GLA treatment induced cell apoptosis and cell cycle arrested at G2/M phase in a dose-dependent manner. In addition, two representative apoptosis-inducing cytotoxic agents were selected as positive control drugs to validate the reasonableness and accuracy of our metabolomic investigation on GLA. The study displayed a systemic metabolic alteration induced by GLA treatment, showing the impaired physiological activity of SMMC7721 cells, which also indicated anti-proliferative and apoptotic effects of GLA. In the meantime, GC/MS- and LC/MS-based metabolomics applied to cell culture enhanced our current understanding of the metabolic response to GLA treatment and its mechanism; such an approach could be transferred to study the mechanism of other anticancer drugs.

Graphical abstract

A systemic metabolic alteration induced by glaucocalyxin A (GLA) treatment of SMMC-7721 cells


Glaucocalyxin A Human liver cancer cell Gas chromatography–mass spectrometry Liquid chromatography–mass spectrometry Metabolic profiling Metabolomics 



The authors appreciate the financial support from Shanghai Sailing Program (No.17YF1424700), National Natural Science Foundation of China (No. 8157131073, No. 81703674), and Shanghai Science and technology innovation action plan (No. 15401900700).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2018_996_MOESM1_ESM.pdf (199 kb)
ESM 1 (PDF 198 kb)


  1. 1.
    Sun HD, Huang SX, Han QB. Diterpenoids from Isodon species and their biological activities. Nat Prod Rep. 2006;23(5):673–98.CrossRefPubMedGoogle Scholar
  2. 2.
    Hong SS, Lee SA, Han XH, Hwang JS, Lee C, Lee D, et al. ent-Kaurane diterpenoids from Isodon japonicus. J Nat Prod. 2008;71(6):1055–8.CrossRefPubMedGoogle Scholar
  3. 3.
    Xiang Z, Wu X, Liu X, Jin Y. Glaucocalyxin A: a review. Nat Prod Res. 2014;
  4. 4.
    Li W, Tang X, Yi W, Li Q, Ren L, Liu X, et al. Glaucocalyxin a inhibits platelet activation and thrombus formation preferentially via GPVI signaling pathway. PLoS One. 2013;8(12):e85120.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Gao LW, Zhang J, Yang WH, Wang B, Wang JW. Glaucocalyxin a induces apoptosis in human leukemia HL-60 cells through mitochondria-mediated death pathway. Toxicol in Vitro. 2011;25(1):51–63.CrossRefPubMedGoogle Scholar
  6. 6.
    Kim B-W, Koppula S, Hong S-S, Jeon S-B, Kwon J-H, Hwang B-Y, et al. Regulation of microglia activity by Glaucocalyxin-A: attenuation of lipopolysaccharide-stimulated neuroinflammation through NF-κB and p38 MAPK signaling pathways. PLoS One. 2013;8(2):e55792.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Xiao X, Cao W, Jiang X, Zhang W, Zhang Y, Liu B, et al. Glaucocalyxin A, a negative Akt regulator, specifically induces apoptosis in human brain glioblastoma U87MG cells. Acta Bioch Bioph Sin. 2013;45(11):946–52.CrossRefGoogle Scholar
  8. 8.
    Tang, Jin, Hu, Liu, Yu. Glaucocalyxin A inhibits the growth of liver cancer focus and SMMC7721 cells. Oncol Lett. 2016;11(2):1173–8.CrossRefPubMedGoogle Scholar
  9. 9.
    Li M, Jiang XG, Gu ZL, Zhang ZB. Glaucocalyxin A activates FasL and induces apoptosis through activation of the JNK pathway in human breast cancer cells. Asian Pac J Cancer Prev. 2013;14(10):5805–10.CrossRefPubMedGoogle Scholar
  10. 10.
    Nicholson JK, Wilson ID. Understanding global 'systems biology: metabonomics and the continuum of metabolism. Nat Rev Drug Discov. 2003;2(8):668–76.CrossRefPubMedGoogle Scholar
  11. 11.
    Fernie AR, Trethewey RN, Krotzky AJ, Willmitzer L. Metabolite profiling: from diagnostics to systems biology. Nat Rev Mol Cell Biol. 2004;5(9):763–9.CrossRefPubMedGoogle Scholar
  12. 12.
    Zhao Y, Butler EB, Tan M. Targeting cellular metabolism to improve cancer therapeutics. Cell Death Dis. 2013;4:e532.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Miccheli AT, Miccheli A, Di Clemente R, Valerio M, Coluccia P, Bizzarri M, et al. NMR-based metabolic profiling of human hepatoma cells in relation to cell growth by culture media analysis. BBA-Gen Subjects. 2006;1760(11):1723–31.CrossRefGoogle Scholar
  14. 14.
    Massimi M, Tomassini A, Sciubba F, Sobolev AP, Devirgiliis LC, Miccheli A. Effects of resveratrol on HepG2 cells as revealed by 1H-NMR based metabolic profiling. BBA-Gen Subjects. 2012;1820(1):1–8.CrossRefGoogle Scholar
  15. 15.
    Bailey NJC, Oven M, Holmes E, Nicholson JK, Zenk MH. Metabolomic analysis of the consequences of cadmium exposure in Silene cucubalus cell cultures via 1H NMR spectroscopy and chemometrics. Phytochemistry. 2003;62(6):851–8.CrossRefPubMedGoogle Scholar
  16. 16.
    D’Alessandro A, Zolla L. Metabolomics and cancer drug discovery: let the cells do the talking. Drug Discov Today. 2012;17(1–2):3–9.CrossRefPubMedGoogle Scholar
  17. 17.
    Lei S, Huang F, Zhao A, Chen T, Chen W, Xie G, et al. The ratio of dihomo-γ-linolenic acid to deoxycholic acid species is a potential biomarker for the metabolic abnormalities in obesity. FASEB J. 2017;
  18. 18.
    Chen T, Xie G, Wang X, Fan J, Qiu Y, Zheng X et al. Serum and urine metabolite profiling reveals potential biomarkers of human hepatocellular carcinoma. Mol Cell Proteomics. 2011;10(7):M110.004945.Google Scholar
  19. 19.
    Tan Y, Yin P, Tang L, Xing W, Huang Q, Cao D et al. Metabolomics study of stepwise hepatocarcinogenesis from the model rats to patients: potential biomarkers effective for small hepatocellular carcinoma diagnosis. Mol Cell Proteomics. 2012;11(2):M111.010694.Google Scholar
  20. 20.
    Li Y, Ruan Q, Li Y, Ye G, Lu X, Lin X, et al. A novel approach to transforming a non-targeted metabolic profiling method to a pseudo-targeted method using the retention time locking gas chromatography/mass spectrometry-selected ions monitoring. J Chromatogr A. 2012;1255:228–36.CrossRefPubMedGoogle Scholar
  21. 21.
    Constantinou C, Chrysanthopoulos PK, Margarity M, Klapa MI. GC−MS metabolomic analysis reveals significant alterations in cerebellar metabolic physiology in a mouse model of adult onset hypothyroidism. J Proteome Res. 2011;10(2):869–79.CrossRefPubMedGoogle Scholar
  22. 22.
    Xiao JF, Varghese RS, Zhou B, Nezami Ranjbar MR, Zhao Y, Tsai T-H, et al. LC/MS based serum metabolomics for identification of hepatocellular carcinoma biomarkers in Egyptian cohort. J Proteome Res. 2012;11:5914–23.CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Xiao JF, Zhou B, Ressom HW. Metabolite identification and quantitation in LC-MS/MS-based metabolomics. TrAC Trend Anal Chem. 2012;32:1–14.CrossRefGoogle Scholar
  24. 24.
    Liu Y, Hong Z, Tan G, Dong X, Yang G, Zhao L, et al. NMR and LC/MS-based global metabolomics to identify serum biomarkers differentiating hepatocellular carcinoma from liver cirrhosis. Int J Cancer. 2014;135(3):658–68.CrossRefPubMedGoogle Scholar
  25. 25.
    Zhang L, Zhang S. Modulating Bcl-2 family proteins and caspase-3 in induction of apoptosis by Paeoniflorin in human cervical Cancer cells. Phytother Res. 2011;25(10):1551–7.CrossRefPubMedGoogle Scholar
  26. 26.
    Liu Q, Wang X, Zhang Y, Li C-J, Hu L-H, Shen X. Leukamenin F suppresses liver fibrogenesis by inhibiting both hepatic stellate cell proliferation and extracellular matrix production. Acta Pharmacol Sin. 2010;31:839.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Lorenz MA, Burant CF, Kennedy RT. Reducing time and increasing sensitivity in sample preparation for adherent mammalian cell metabolomics. Anal Chem. 2011;83(9):3406–14.CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Zhu Z, Wang H, Shang Q, Jiang Y, Cao Y, Chai Y. Time course analysis of Candida albicans metabolites during biofilm development. J Proteome Res. 2013;12(6):2375–85.CrossRefPubMedGoogle Scholar
  29. 29.
    Liao W, Tan G, Zhu Z, Chen Q, Lou Z, Dong X, et al. Combined metabonomic and quantitative real-time PCR analyses reveal systems metabolic changes in Jurkat T-cells treated with HIV-1 Tat protein. J Proteome Res. 2012;11(11):5109–23.CrossRefPubMedGoogle Scholar
  30. 30.
    Liao W, Tan G, Zhu Z, Chen Q, Lou Z, Dong X, et al. HIV-1 Tat induces biochemical changes in the serum of mice. Virology. 2012;422(2):288–96.CrossRefPubMedGoogle Scholar
  31. 31.
    Bijlsma S, Bobeldijk I, Verheij ER, Ramaker R, Kochhar S, Macdonald IA, et al. Large-scale human metabolomics studies: a strategy for data (pre-) processing and validation. Anal Chem. 2005;78(2):567–74.CrossRefGoogle Scholar
  32. 32.
    Wiklund S, Johansson E, Sjöström L, Mellerowicz EJ, Edlund U, Shockcor JP, et al. Visualization of GC/TOF-MS-based metabolomics data for identification of biochemically interesting compounds using OPLS class models. Anal Chem. 2008;80(1):115–22.CrossRefPubMedGoogle Scholar
  33. 33.
    Ong ES, Zou L, Li S, Cheah PY, Eu KW, Ong CN. Metabolic profiling in colorectal cancer reveals signature metabolic shifts during tumorigenesis. Mol Cell Proteomics. 2010;
  34. 34.
    Riccardi C, Nicoletti I. Analysis of apoptosis by propidium iodide staining and flow cytometry. Nat Protocols. 2006;1(3):1458–61.CrossRefPubMedGoogle Scholar
  35. 35.
    Pantalacci S, Tapon N, Leopold P. The Salvador partner hippo promotes apoptosis and cell-cycle exit in drosophila. Nat Cell Biol. 2003;5(10):921–7.CrossRefPubMedGoogle Scholar
  36. 36.
    Fu Y-R, Yi Z-J, Yan Y-R, Qiu Z-Y. Hydroxycamptothecin-induced apoptosis in hepatoma SMMC-7721 cells and the role of mitochondrial pathway. Mitochondrion. 2006;6(4):211–7.CrossRefPubMedGoogle Scholar
  37. 37.
    Song J, Qu Z, Guo X, Zhao Q, Zhao X, Gao L, et al. Hypoxia-induced autophagy contributes to the chemoresistance of hepatocellular carcinoma cells. Autophagy. 2009;5(8):1131–44.CrossRefPubMedGoogle Scholar
  38. 38.
    Sellick C, Knight D, Croxford A, Maqsood A, Stephens G, Goodacre R, et al. Evaluation of extraction processes for intracellular metabolite profiling of mammalian cells: matching extraction approaches to cell type and metabolite targets. Metabolomics. 2010;6(3):427–38.CrossRefGoogle Scholar
  39. 39.
    Kohara H, Tabata M, Kiura K, Ueoka H, Kawata K, Chikamori M, et al. Synergistic effects of topoisomerase I inhibitor, 7-ethyl-10-hydroxycamptothecin, and irradiation in a cisplatin-resistant human small cell lung cancer cell line. Clin Cancer Res. 2002;8(1):287–92.PubMedGoogle Scholar
  40. 40.
    Lam W, Bussom S, Cheng Y-C. Effect of hypoxia on the expression of phosphoglycerate kinase and antitumor activity of troxacitabine and gemcitabine in non-small cell lung carcinoma. Mol Cancer Ther. 2009;8(2):415–23.CrossRefPubMedGoogle Scholar
  41. 41.
    Kirovski G, Stevens AP, Czech B, Dettmer K, Weiss TS, Wild P, et al. Down-regulation of Methylthioadenosine phosphorylase (MTAP) induces progression of hepatocellular carcinoma via accumulation of 5′-deoxy-5′-methylthioadenosine (MTA). Am J Pathol. 2011;178(3):1145–52.CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Insel PA, Zhang L, Murray F, Yokouchi H, Zambon AC. Cyclic AMP is both a pro-apoptotic and anti-apoptotic second messenger. Acta Physiol. 2012;204(2):277–87.CrossRefGoogle Scholar
  43. 43.
    Nicchia GP, Frigeri A, Nico B, Ribatti D, Svelto M. Tissue distribution and membrane localization of Aquaporin-9 water channel evidence for sex-linked differences in liver. J Histochem Cytochem. 2001;49(12):1547–56.CrossRefPubMedGoogle Scholar
  44. 44.
    Nihei K, Koyama Y, Tani T, Yaoita E, Ohshiro K, Adhikary LP, et al. Immunolocalization of Aquaporin-9 in rat hepatocytes and Leydig cells. Arch Histol Cytol. 2001;64(1):81–8.CrossRefPubMedGoogle Scholar
  45. 45.
    Pyne NJ, Pyne S. Sphingosine 1-phosphate and cancer. Nat Rev Cancer. 2010;10(7):489–503.CrossRefPubMedGoogle Scholar
  46. 46.
    Ogretmen B, Hannun YA. Biologically active sphingolipids in cancer pathogenesis and treatment. Nat Rev Cancer. 2004;4(8):604–16.CrossRefPubMedGoogle Scholar
  47. 47.
    Peluso G, Nicolai R, Reda E, Benatti P, Barbarisi A, Calvani M. Cancer and anticancer therapy-induced modifications on metabolism mediated by carnitine system. J Cell Physiol. 2000;182:339–50.CrossRefPubMedGoogle Scholar
  48. 48.
    Revoltella RP, Dal Canto B, Caracciolo L, D'Urso CM. L-carnitine and some of its analogs delay the onset of apoptotic cell death initiated in murine C2.8 hepatocytic cells after hepatocyte growth factor deprivation. Biochim Biophys Acta. 1994;1224(3):333–41.CrossRefPubMedGoogle Scholar
  49. 49.
    Lo T-F, Tsai W-C, Chen S-T. MicroRNA-21-3p, a Berberine-induced miRNA, directly down-regulates human methionine adenosyltransferases 2A and 2B and inhibits hepatoma cell growth. PLoS One. 2013;8(9):e75628.CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Frau M, Feo F, Pascale RM. Pleiotropic effects of methionine adenosyltransferases deregulation as determinants of liver cancer progression and prognosis. J Hepatol. 2013;59(4):830–41.CrossRefPubMedGoogle Scholar
  51. 51.
    Smith MW, Yue ZN, Geiss GK, Sadovnikova NY, Carter VS, Boix L, et al. Identification of novel tumor markers in hepatitis C virus-associated hepatocellular carcinoma. Cancer Res. 2003;63(4):859–64.PubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Yue Liu
    • 1
    • 2
  • Shan Lu
    • 3
  • Liang Zhao
    • 4
  • Xin Dong
    • 1
  • Zhenyu Zhu
    • 1
    • 2
  • Yongsheng Jin
    • 1
  • Haisheng Chen
    • 1
  • Feng Lu
    • 1
  • Zhanying Hong
    • 1
    • 2
  • Yifeng Chai
    • 1
    • 2
  1. 1.School of PharmacySecond Military Medical UniversityShanghaiChina
  2. 2.Shanghai Key Laboratory for Pharmaceutical Metabolite ResearchShanghaiChina
  3. 3.Department of Biochemistry and Molecular BiologySecond Military Medical UniversityShanghaiChina
  4. 4.Department of Pharmacy, Eastern Hepatobiliary Surgery HospitalSecond Military Medical UniversityShanghaiChina

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