Antiangiogenesis effect of timosaponin AIII on HUVECs in vitro and zebrafish embryos in vivo

Article metrics

Abstract

Timosaponin AIII (Timo AIII) is a natural steroidal saponin isolated from the traditional Chinese herb Anemarrhena asphodeloides Bge with proved effectiveness in the treatment of numerous cancers. However, whether Timo AIII suppresses tumor angiogenesis remains unclear. In the present study, we investigated the antiangiogenesis effects of Timo AIII and the underlying mechanisms in human umbilical vein endothelial cells (HUVECs) in vitro and zebrafish embryos in vivo. We showed that treatment with Timo AIII (0.5–2 µM) partially disrupted the intersegmental vessels (ISVs) and subintestinal vessels (SIVs) growth in transgenic zebrafish Tg(fli-1a: EGFP)y1. Timo AIII (0.5–4 µM) dose-dependently inhibited VEGF-induced proliferation, migration, invasion, and tube formation of HUVECs, but these inhibitory effects were not due to its cytotoxicity. We further demonstrated that Timo AIII treatment significantly suppressed the expression of VEGF receptor (VEGFR) and the phosphorylation of Akt, MEK1/2, and ERK1/2 in HUVECs. Timo AIII treatment also significantly inhibited VEGF-triggered phosphorylation of VEGFR2, Akt, and ERK1/2 in HUVECs. Moreover, we conducted RNA-Seq and analyzed the transcriptome changes in both HUVECs and zebrafish embryos following Timo AIII treatment. The coexpression network analysis results showed that various biological processes and signaling pathways were enriched including angiogenesis, cell motility, cell adhesion, protein serine/threonine kinase activity, transmembrane signaling receptor activity, growth factor activity, etc., which was consistent with the antiangiogenesis effects of Timo AIII in HUVECs and zebrafish embryos. We conclude that the antiangiogenesis effect of Timo AIII is mediated through VEGF/PI3K/Akt/MAPK signaling cascade; Timo AIII potentially exerts antiangiogenesis effect in cancer treatment.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

References

  1. 1.

    Siegel RL, Miller KD, Jemal A. Cancer statistics, 2017. CA Cancer J Clin. 2017;67:7–30.

  2. 2.

    Zheng R, Zeng H, Zhang S, Chen W. Estimates of cancer incidence and mortality in China, 2013. Chin J Cancer. 2017;36:66.

  3. 3.

    Mashreghi M, Azarpara H, Bazaz MR, Jafari A, Masoudifar A, Mirzaei H, et al. Angiogenesis biomarkers and their targeting ligands as potential targets for tumor angiogenesis. J Cell Physiol. 2018;233:2949–65.

  4. 4.

    Folkman J. Tumor angiogenesis: therapeutic implications. N Engl J Med. 1971;285:1182–6.

  5. 5.

    Cook KM, Figg WD. Angiogenesis inhibitors: current strategies and future prospects. CA Cancer J Clin. 2010;60:222–43.

  6. 6.

    Sakamoto S, Ryan AJ, Kyprianou N. Targeting vasculature in urologic tumors: mechanistic and therapeutic significance. J Cell Biochem. 2008;103:691–708.

  7. 7.

    Hoff PM, Machado KK. Role of angiogenesis in the pathogenesis of cancer. Cancer Treat Rev. 2012;38:825–33.

  8. 8.

    Lee B, Jung K, Kim DH. Timosaponin AIII, a saponin isolated from Anemarrhena asphodeloides, ameliorates learning and memory deficits in mice. Pharmacol Biochem Behav. 2009;93:121–7.

  9. 9.

    Wang Y, Dan Y, Yang D, Hu Y, Zhang L, Zhang C, et al. The genus Anemarrhena Bunge: a review on ethnopharmacology, phytochemistry and pharmacology. J Ethnopharmacol. 2014;153:42–60.

  10. 10.

    Lim SM, Jeong JJ, Kang GD, Kim KA, Choi HS, Kim DH. Timosaponin AIII and its metabolite sarsasapogenin ameliorate colitis in mice by inhibiting NF-κB and MAPK activation and restoring Th17/Treg cell balance. Int Immunopharmacol. 2015;25:493–503.

  11. 11.

    Cong Y, Wang L, Peng R, Zhao Y, Bai F, Yang C, et al. Timosaponin AIII induces antiplatelet and antithrombotic activity via Gq-mediated signaling by the thromboxane A2 receptor. Sci Rep. 2016;6:38757.

  12. 12.

    Lee B, Trinh HT, Jung K, Han SJ, Kim DH. Inhibitory effects of steroidal timosaponins isolated from the rhizomes of Anemarrhena asphodeloides against passive cutaneous anaphylaxis reaction and pruritus. Immunopharmacol Immunotoxicol. 2010;32:357–63.

  13. 13.

    Wu ZT, Qi XM, Sheng JJ, Ma Ll, Ni X, Ren J, et al. Timosaponin A3 induces hepatotoxicity in rats through inducing oxidative stress and down-regulating bile acid transporters. Acta Pharmacol Sin. 2014;35:1188–98.

  14. 14.

    Han FY, Song XY, Chen JJ, Yao GD, Song SJ. Timosaponin AIII: a novel potential anti-tumor compound from Anemarrhena asphodeloides. Steroids. 2018;140:125–30.

  15. 15.

    King FW, Fong S, Griffin C, Shoemaker M, Staub R, Zhang Y-L, et al. Timosaponin AIII is preferentially cytotoxic to tumor cells through inhibition of mTOR and induction of ER stress. PLoS One. 2009;4:e7283.

  16. 16.

    Jung O, Lee J, Lee YJ, Yun JM, Son YJ, Cho JY, et al. Timosaponin AIII inhibits migration and invasion of A549 human non-small-cell lung cancer cells via attenuations of MMP-2 and MMP-9 by inhibitions of ERK1/2, Src/FAK and β-catenin signaling pathways. Bioorg Med Chem Lett. 2016;26:3963–7.

  17. 17.

    MarElia CB, Sharp AE, Shemwell TA, Clare Zhang Y, Burkhardt BR. Anemarrhena asphodeloides Bunge and its constituent timosaponin‐AIII induce cell cycle arrest and apoptosis in pancreatic cancer cells. FEBS Open Bio. 2018;8:1155–66.

  18. 18.

    Wang Y, Xu L, Lou LL, Song SJ, Yao GD, Ge MY, et al. Timosaponin AIII induces apoptosis and autophagy in human melanoma A375-S2 cells. Arch Pharm Res. 2017;40:69–78.

  19. 19.

    Kim KM, Im AR, Kim SH, Hyun JW, Chae S. Timosaponin AIII inhibits melanoma cell migration by suppressing COX‐2 and in vivo tumor metastasis. Cancer Sci. 2016;107:181–8.

  20. 20.

    Tsai CH, Yang CW, Wang JY, Tsai YF, Tseng LM, King KL, et al. Timosaponin AIII suppresses hepatocyte growth factor-induced invasive activity through sustained ERK activation in breast cancer MDA-MB-231 cells. J Evid Based Integr Med. 2013;2013:421051.

  21. 21.

    Wang N, Feng Y, Zhu M, Siu FM, Ng KM, Che CM. A novel mechanism of XIAP degradation induced by timosaponin AIII in hepatocellular carcinoma. Biochim Biophys Acta Mol Cell Res. 2013;1833:2890–9.

  22. 22.

    Kang YJ, Chung HJ, Nam JW, Park HJ, Seo EK, Kim YS, et al. Cytotoxic and antineoplastic activity of timosaponin A-III for human colon cancer cells. J Nat Prod. 2011;74:701–6.

  23. 23.

    Huang HL, Chiang WL, Hsiao PC, Chien MH, Chen HY, Weng WC, et al. Timosaponin AIII mediates caspase activation and induces apoptosis through JNK1/2 pathway in human promyelocytic leukemia cells. Tumour Biol. 2015;36:3489–97.

  24. 24.

    Chen JR, Jia XH, Wang H, Yi YJ, Wang JY, Li YJ. Timosaponin A-III reverses multi-drug resistance in human chronic myelogenous leukemia K562/ADM cells via downregulation of MDR1 and MRP1 expression by inhibiting PI3K/Akt signaling pathway. Int J Oncol. 2016;48:2063–70.

  25. 25.

    Huang B, Peng Y, Li J, Li S, Sun Y, Wang D, et al. An andrographolide derivative AGP-26b exhibiting anti-angiogenic activity in HUVECs and zebrafish via blocking the VEGFA/VEGFR2 signaling pathway. Mol Omics. 2017;13:525–36.

  26. 26.

    Zhou ZY, Huan LY, Zhao WR, Tang N, Jin Y, Tang JY. Spatholobi Caulis extracts promote angiogenesis in HUVECs in vitro and in zebrafish embryos in vivo via up-regulation of VEGFRs. J Ethnopharmacol. 2017;200:74–83.

  27. 27.

    Zheng Y, Li Y, Liu G, Qi X, Cao X. MicroRNA-24 inhibits the proliferation and migration of endothelial cells in patients with atherosclerosis by targeting importin-α3 and regulating inflammatory responses. Exp Ther Med. 2018;15:338.

  28. 28.

    Paterson EK, Courtneidge SA. Invadosomes are coming: new insights into function and disease relevance. FEBS J. 2018;285:8–27.

  29. 29.

    Singh P, Jenkins LM, Horst B, Alers V, Pradhan S, Kaur P, et al. Inhibin is a novel paracrine factor for tumor angiogenesis and metastasis. Cancer Res. 2018;78:2978–89.

  30. 30.

    Zhong ZF, Hoi PM, Wu GS, Xu ZT, Tan W, Chen XP, et al. Anti-angiogenic effect of furanodiene on HUVECs in vitro and on zebrafish in vivo. J Ethnopharmacol. 2012;141:721–7.

  31. 31.

    Chiang KC, Lai CY, Chiou HL, Lin CL, Chen YS, Kao SH, et al. Timosaponin AIII inhibits metastasis of renal carcinoma cells through suppressing cathepsin C expression by AKT/miR-129-5p axis. J Cell Physiol. 2019;234:13332–41.

  32. 32.

    Gergely JE, Dorsey AE, Dimri GP, Dimri M. Timosaponin A-III inhibits oncogenic phenotype via regulation of PcG protein BMI1 in breast cancer cells. Mol Carcinog. 2018;57:831–41.

  33. 33.

    Zhou ZY, Xu JQ, Zhao WR, Chen XL, Jin Y, Tang N, et al. Ferulic acid relaxed rat aortic, small mesenteric and coronary arteries by blocking voltage-gated calcium channel and calcium desensitization via dephosphorylation of ERK1/2 and MYPT1. Eur J Pharmacol. 2017;815:26–32.

  34. 34.

    Zhou ZY, Zhao WR, Shi WT, Xiao Y, Ma ZL, Xue JG, et al. Endothelial-dependent and independent vascular relaxation effect of tetrahydropalmatine on rat aorta. Front Pharmacol. 2019;10:336.

Download references

Acknowledgements

This study was supported by the Shanghai University of Traditional Chinese Medicine (A1-U17205010301), the National Health Commission of Shanghai (GWIV-28, ZY-(2018-2020)-FWTX-8001), and the National Natural Science Foundation of China (81603549, 81673726, and 81760860)

Author information

ZYZ conducted the experiments and wrote the manuscript. WRZ and YX conducted the experiments and analyzed the data. CH analyzed the transcriptome data. XMZ, WTS, and JZ conducted the experiments. QY and XLC designed the study. JYT designed the study and revised the manuscript.

Correspondence to Xin-lin Chen or Jing-yi Tang.

Ethics declarations

Competing interests

The authors declare no competing interests.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Keywords

  • Timosaponin AIII
  • Traditional Chinese herbal
  • Neovascularization
  • Neoplasm
  • VEGF/PI3K/Akt/MAPK
  • Transcriptome
  • Zebrafish
  • HUVECs
  • SU5416