Skip to main content

Advertisement

SpringerLink
Log in

Diterpenoid natural compound C4 (Crassin) exerts cytostatic effects on triple-negative breast cancer cells via a pathway involving reactive oxygen species

  • Original Paper
  • Published:
Cellular Oncology Aims and scope Submit manuscript

Abstract

Purpose

Triple-negative breast cancers (TNBC) lack expression of three common cell surface receptors, i.e., estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER2). Accordingly, TNBCs are associated with fewer treatment options and a relatively poor prognosis. Having screened a National Cancer Institute natural compound library, the purpose of this study was to investigate the bioactivity of compound C4 (Crassin) in TNBC cells.

Methods

Cell viability assays were performed in two TNBC cell lines, MDA-MB-231 and 4T1, following C4 treatment in the presence or absence of the antioxidant N-acetyl-L-cysteine (NAC). Phosphorylation of Akt and ERK was assessed by Western blotting. Apoptosis, necrosis, autophagy, necroptosis, ferroptosis and cytostasis assays were performed to explain viability deficits resulting from C4 exposure.

Results

We found that the viability of the TNBC cells tested decreased in a concentration- and time-dependent fashion following C4 treatment. This decrease coincided with an unexpected increase in the expression of the cell survival effectors pAkt and pERK. In addition, we found that both the decreased cell viability and the increased pAkt/pERK levels could be rescued by the antioxidant NAC, suggesting a central role for reactive oxygen species (ROS) in the mechanism of action of C4. Necrosis, apoptosis, necroptosis and ferroptosis could be ruled out as cell death mechanisms. Instead, we found that C4 induced cytostasis downstream of ROS activation. Finally, we observed a synergistic effect between C4 and the chemotherapeutic drug doxorubicin in TNBC cells.

Conclusions

From our in vitro data we conclude that C4 exerts cytostatic effects on triple-negative breast cancer cells via a pathway involving reactive oxygen species. Its potential value in combination with cytotoxic therapies merits deeper investigation in pre-clinical models.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

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

Similar content being viewed by others

References

  1. R.D. Chacon, M.V. Costanzo, Triple-negative breast cancer. Breast Cancer Res. 12(Suppl 2), S3 (2010)

    Article  PubMed  PubMed Central  Google Scholar 

  2. W.D. Foulkes, I.E. Smith, J.S. Reis-Filho, Triple-negative breast cancer. N. Engl. J. Med. 363, 1938–1948 (2010)

    Article  CAS  PubMed  Google Scholar 

  3. H. Greenlee, Natural products for cancer prevention. Semin. Oncol. Nurs. 28, 29–44 (2012)

    Article  PubMed  PubMed Central  Google Scholar 

  4. A.R. Amin, O. Kucuk, F.R. Khuri, D.M. Shin, Perspectives for cancer prevention with natural compounds. J. Clin. Oncol. 27, 2712–2725 (2009)

    Article  PubMed  PubMed Central  Google Scholar 

  5. M.C. Wani, H.L. Taylor, M.E. Wall, P. Coggon, A.T. McPhail, Plant antitumor agents. VI. The isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifolia. J. Am. Chem. Soc. 93, 2325–2327 (1971)

    Article  CAS  PubMed  Google Scholar 

  6. H. Parekh, H. Simpkins, The transport and binding of taxol. Gen. Pharmacol. 29, 167–172 (1997)

    Article  CAS  PubMed  Google Scholar 

  7. C. Khanna, M. Rosenberg, D.M. Vail, A review of paclitaxel and novel formulations including those suitable for use in dogs. J. Vet. Intern. Med. 29, 1006–1012 (2015)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Q.Q. Yuan, S. Tang, W.B. Song, W.Q. Wang, M. Huang, L.J. Xuan, A.-H. Crassins, Diterpenoids from the roots of croton crassifolius. J. Nat. Prod. 80, 254–260 (2017)

    Article  CAS  PubMed  Google Scholar 

  9. S. Donatello, L. Hudson, D.C. Cottell, A. Blanco, I. Aurrekoetxea, M.J. Shelly, P.A. Dervan, M.R. Kell, M. Stokes, A.D. Hill, A.M. Hopkins, An imbalance in progenitor cell populations reflects tumour progression in breast cancer primary culture models. J. Exp. Clin. Cancer Res. 30, 45 (2011)

    Article  PubMed  PubMed Central  Google Scholar 

  10. S. Nobili, D. Lippi, E. Witort, M. Donnini, L. Bausi, E. Mini, S. Capaccioli, Natural compounds for cancer treatment and prevention. Pharmacol. Res. 59, 365–378 (2009)

    Article  CAS  PubMed  Google Scholar 

  11. N. Hasima, L.I. Aun, M.N. Azmi, A.N. Aziz, E. Thirthagiri, H. Ibrahim, K. Awang, 1′S-1′-acetoxyeugenol acetate: a new chemotherapeutic natural compound against MCF-7 human breast cancer cells. Phytomedicine 17, 935–939 (2010)

    Article  CAS  PubMed  Google Scholar 

  12. A.J. Robles, L. Du, R.H. Cichewicz, S.L. Mooberry, D.N.A. Maximiscin induces damage, activates DNA damage response pathways, and has selective cytotoxic activity against a subtype of triple-negative breast cancer. J. Nat. Prod. 79, 1822–1827 (2016)

  13. E.J. Kim, S.Y. Park, J.Y. Lee, J.H. Park, Fucoidan present in brown algae induces apoptosis of human colon cancer cells. BMC Gastroenterol. 10, 96 (2010)

    Article  PubMed  PubMed Central  Google Scholar 

  14. H. Yamaguchi, H.G. Wang, The protein kinase PKB/Akt regulates cell survival and apoptosis by inhibiting Bax conformational change. Oncogene 20, 7779–7786 (2001)

    Article  CAS  PubMed  Google Scholar 

  15. G. Song, G. Ouyang, S. Bao, The activation of Akt/PKB signaling pathway and cell survival. J. Cell. Mol. Med. 9, 59–71 (2005)

    Article  CAS  PubMed  Google Scholar 

  16. S.R. Datta, A. Brunet, M.E. Greenberg, Cellular survival: a play in three Akts. Genes Dev. 13, 2905–2927 (1999)

    Article  CAS  PubMed  Google Scholar 

  17. A. Brunet, A. Bonni, M.J. Zigmond, M.Z. Lin, P. Juo, L.S. Hu, M.J. Anderson, K.C. Arden, J. Blenis, M.E. Greenberg, Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96, 857–868 (1999)

    Article  CAS  PubMed  Google Scholar 

  18. I. Dolado, A.R. Nebreda, AKT and oxidative stress team up to kill cancer cells. Cancer Cell 14, 427–429 (2008)

    Article  CAS  PubMed  Google Scholar 

  19. Y.Q. Hou, Y. Yao, Y.L. Bao, Z.B. Song, C. Yang, X.L. Gao, W.J. Zhang, L.G. Sun, C.L. Yu, Y.X. Huang, G.N. Wang, Y.X. Li, Juglanthraquinone C induces intracellular ROS increase and apoptosis by activating the Akt/foxo signal pathway in HCC cells. Oxidative Med. Cell. Longev. 2016, 4941623 (2016)

    Google Scholar 

  20. Q. Liu, J. Qiu, M. Liang, J. Golinski, K. van Leyen, J.E. Jung, Z. You, E.H. Lo, A. Degterev, M.J. Whalen, Akt and mTOR mediate programmed necrosis in neurons. Cell Death Dis. 5, e1084 (2014)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. C. Peyssonnaux, A. Eychene, The Raf/MEK/ERK pathway: new concepts of activation. Biol. Cell. 93, 53–62 (2001)

    Article  CAS  PubMed  Google Scholar 

  22. D.G. Kirsch, A. Doseff, B.N. Chau, D.S. Lim, N.C. de Souza-Pinto, R. Hansford, M.B. Kastan, Y.A. Lazebnik, J.M. Hardwick, Caspase-3-dependent cleavage of Bcl-2 promotes release of cytochrome c. J. Biol. Chem. 274, 21155–21161 (1999)

    Article  CAS  PubMed  Google Scholar 

  23. A.G. Porter, R.U. Janicke, Emerging roles of caspase-3 in apoptosis. Cell Death Differ. 6, 99–104 (1999)

    Article  CAS  PubMed  Google Scholar 

  24. S.J. Dixon, B.R. Stockwell, The role of iron and reactive oxygen species in cell death. Nat. Chem. Biol. 10, 9–17 (2014)

    Article  CAS  PubMed  Google Scholar 

  25. Y. Xie, W. Hou, X. Song, Y. Yu, J. Huang, X. Sun, R. Kang, D. Tang, Ferroptosis: process and function. Cell Death Differ. 23, 369–379 (2016)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. D.E. Christofferson, J. Yuan, Necroptosis as an alternative form of programmed cell death. Curr. Opin. Cell Biol. 22, 263–268 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. S. Fulda, Regulation of necroptosis signaling and cell death by reactive oxygen species. Biol. Chem. 397, 657–660 (2016)

    CAS  PubMed  Google Scholar 

  28. H.A. Wahba, H.A. El-Hadaad, Current approaches in treatment of triple-negative breast cancer. Cancer Biol. Med. 12, 106–116 (2015)

  29. G.C. Guo, J.X. Wang, M.L. Han, L.P. Zhang, L. Li, microRNA-761 induces aggressive phenotypes in triple-negative breast cancer cells by repressing TRIM29 expression. Cell. Oncol. 40, 157–166 (2017)

  30. E. Robles-Escajeda, U. Das, N.M. Ortega, K. Parra, G. Francia, J.R. Dimmock, A. Varela-Ramirez, R.J. Aguilera, A novel curcumin-like dienone induces apoptosis in triple-negative breast cancer cells. Cell. Oncol. 39, 265–277 (2016)

  31. I. Fkih, M.P. M’hamed, F. Ponelle, F. Penault-Llorca, A. Kenani, Y.-J. Bignon, Identification of miR-10b, miR-26a, miR-146a and miR-153 as potential triple-negative breast cancer biomarkers. Cell. Oncol. 38, 433–442 (2015)

  32. T.F. Franke, C.P. Hornik, L. Segev, G.A. Shostak, C. Sugimoto, PI3K/Akt and apoptosis: size matters. Oncogene 22, 8983–8998 (2003)

    Article  CAS  PubMed  Google Scholar 

  33. H. Zhou, X.M. Li, J. Meinkoth, R.N. Pittman, Akt regulates cell survival and apoptosis at a postmitochondrial level. J. Cell Biol. 151, 483–494 (2000)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. P.D. Ray, B.W. Huang, Y. Tsuji, Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling. Cell. Signal. 24, 981–990 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Y. Son, Y.K. Cheong, N.H. Kim, H.T. Chung, D.G. Kang, H.O. Pae, Mitogen-activated protein kinases and reactive oxygen species: how can ROS activate MAPK pathways? J. Signal. Transduct. 2011, 792639 (2011)

  36. S. Cagnol, J.C. Chambard, ERK and cell death: mechanisms of ERK-induced cell death--apoptosis, autophagy and senescence. FEBS J. 277, 2–21 (2010)

    Article  CAS  PubMed  Google Scholar 

  37. R.S. Keshari, A. Verma, M.K. Barthwal, M. Dikshit, Reactive oxygen species-induced activation of ERK and p38 MAPK mediates PMA-induced NETs release from human neutrophils. J. Cell. Biochem. 114, 532–540 (2013)

    Article  CAS  PubMed  Google Scholar 

  38. V. Lobo, A. Patil, A. Phatak, N. Chandra, Free radicals, antioxidants and functional foods: impact on human health. Pharmacogn. Rev. 4, 118–126 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. J.F. Turrens, Mitochondrial formation of reactive oxygen species. J. Physiol. 552, 335–344 (2003)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. A. Valencia, J. Moran, Reactive oxygen species induce different cell death mechanisms in cultured neurons. Free Radic. Biol. Med. 36, 1112–1125 (2004)

    Article  CAS  PubMed  Google Scholar 

  41. F. Ciardiello, R. Caputo, R. Bianco, V. Damiano, G. Pomatico, S. De Placido, A.R. Bianco, G. Tortora, Antitumor effect and potentiation of cytotoxic drugs activity in human cancer cells by ZD-1839 (Iressa), an epidermal growth factor receptor-selective tyrosine kinase inhibitor. Clin. Cancer Res. 6, 2053–2063 (2000)

    CAS  PubMed  Google Scholar 

  42. M. Villasana, G. Ochoa, S. Aguilar, Modeling and optimization of combined cytostatic and cytotoxic cancer chemotherapy. Artif. Intell. Med. 50, 163–173 (2010)

    Article  PubMed  Google Scholar 

  43. S.S. Bacus, A.V. Gudkov, M. Lowe, L. Lyass, Y. Yung, A.P. Komarov, K. Keyomarsi, Y. Yarden, R. Seger, Taxol-induced apoptosis depends on MAP kinase pathways (ERK and p38) and is independent of p53. Oncogene 20, 147–155 (2001)

    Article  CAS  PubMed  Google Scholar 

  44. Y.H. Choi, Y.H. Yoo, Taxol-induced growth arrest and apoptosis is associated with the upregulation of the Cdk inhibitor, p21WAF1/CIP1, in human breast cancer cells. Oncol. Rep. 28, 2163–2169 (2012)

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This research was financially supported by Science Foundation Ireland (SFI), grant number 13/IA/1994 (to AMH). Y. Smith was funded by the Health Research Board of Ireland (HRA-POR-2014-545, to AMH). We thank the NCI/DTP Open Chemical Repository (https://dtp.cancer.gov) for the opportunity to work with the natural compound NSC210236, and Dr. Adrienne Gorman for advice on cell death assays.

Author information

Authors and Affiliations

  1. Department of Surgery, Royal College of Surgeons in Ireland, Dublin, Ireland

    Cathy E. Richards, Sri H. Vellanki, Yvonne E. Smith & Ann M. Hopkins

  2. Royal College of Surgeons in Ireland, RCSI Smurfit Building, Beaumont Hospital, Dublin 9, Ireland

    Ann M. Hopkins

Authors
  1. Cathy E. Richards
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sri H. Vellanki
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yvonne E. Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ann M. Hopkins
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ann M. Hopkins.

Ethics declarations

Conflict of interest

The authors declare no conflict of interest.

Electronic supplementary material

ESM 1

Supplementary Figure 1: C4 alters cellular morphology in MDA-MB-231 cells. MDA-MB-231 cells were plated at either 300,000 or 100,000 per well and treated 48h later with C4 (5μM) or vehicle control (DMSO) for either 24h (A) or 48h (B) for 48h. Cells were then imaged at 40x magnification on an Olympus CKX41 microscope using Cell B imaging software. Supplementary Figure 2: C4 alters nuclear morphology in MDA-MB-231 cells and 4T1 cells. MDA-MB-231 cells were plated at 75,000 per well and treated 48h later and 4T1 cells plated at 50,000 cells per well and treated 24h later with C4 (5μM) or vehicle control (DMSO) for either 24h (A) or 48h (B). Nuclei were then stained with DAPI and imaged at 40x magnification on an Olympus CKX41 microscope using Cell B imaging software. (PPTX 4792 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Richards, C.E., Vellanki, S.H., Smith, Y.E. et al. Diterpenoid natural compound C4 (Crassin) exerts cytostatic effects on triple-negative breast cancer cells via a pathway involving reactive oxygen species. Cell Oncol. 41, 35–46 (2018). https://doi.org/10.1007/s13402-017-0357-1

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13402-017-0357-1

Keywords

Search

Navigation