Tumor Biology

, Volume 37, Issue 4, pp 4479–4491 | Cite as

RIP1K and RIP3K provoked by shikonin induce cell cycle arrest in the triple negative breast cancer cell line, MDA-MB-468: necroptosis as a desperate programmed suicide pathway

  • Zahra Shahsavari
  • Fatemeh Karami-Tehrani
  • Siamak Salami
  • Mehran Ghasemzadeh
Original Article

Abstract

Resistance to cell death and reprogramming of metabolism are important in neoplastic cells. Increased resistance to apoptosis and recurrence of tumors are the major roadblocks to effective treatment of triple negative breast cancer. It has been thought that execution of necroptosis involves ROS generation and mitochondrial dysfunction in malignant cells. In this study, the effect of shikonin, an active substance from the dried root of Lithospermum erythrorhizon, on the induction of necroptosis or apoptosis, via RIP1K-RIP3K expressions has been examined in the triple negative breast cancer cell line. The expression levels of RIP1K and RIP3K, caspase-3 and caspase-8 activities, the levels of ROS, and mitochondrial membrane potential have been studied in the shikonin-treated MDA-MB-468 cell line. An increase in the ROS levels and a reduction in mitochondrial membrane potential have been observed in the shikonin-treated cells. Cell death has mainly occurred through necroptosis with a significant increase in the RIP1K and RIP3K expressions, and characteristic morphological changes have been observed. In the presence of Nec-1, caspase-3 mediating apoptosis has occurred in the shikonin-treated cells. The current findings have revealed that shikonin provoked mitochondrial ROS production in the triple negative breast cancer cell line, which works as a double-edged executioner’s ax in the execution of necroptosis or apoptosis. The main route of cell death induced by shikonin is RIP1K-RIP3K-mediated necroptosis, but in the presence of Nec-1, apoptosis has prevailed. The present results shed a new light on the possible treatment of drug-resistant triple negative breast cancer.

Keywords

RIP1K RIP3K Triple negative breast cancer cell line ROS Necroptosis Apoptosis 

Abbreviations

ER

Estrogen receptor

PR

Progesterone receptor

HER-2

HER-2/neu receptor

Z-VAD-FMK

Carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone

Nec-1

Necrostatin-1

ROS

Reactive oxygen species

Δψm

Mitochondrial membrane potential

FITC

Fluorescein isothiocyanate

PI

Propidium iodide

MTT

3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide

Notes

Acknowledgments

Part of this work was supported by a Ph.D. grant from Tarbiat Modares University. The authors would like to express their gratitude to Professor Peter Vandenabeele for his valuable comments. The sincere cooperation of Mrs. Batoul Etemadi-kia, lab expert, is much obliged.

Conflicts of interest

None

References

  1. 1.
    Vanden Berghe T, Linkermann A, Jouan-Lanhouet S, Walczak H, Vandenabeele P. Regulated necrosis: the expanding network of non-apoptotic cell death pathways. Nat Rev Mol Cell Biol. 2014;15:135–47.CrossRefPubMedGoogle Scholar
  2. 2.
    Feoktistova M, Leverkus M. Programmed necrosis and necroptosis signalling. FEBS J. 2015;282:19–31.CrossRefPubMedGoogle Scholar
  3. 3.
    Radogna F, Dicato M, Diederich M. Cancer-type-specific crosstalk between autophagy, necroptosis and apoptosis as a pharmacological target. Biochem Pharmacol. 2015;94:1–11.CrossRefPubMedGoogle Scholar
  4. 4.
    Christofferson DE, Yuan J. Necroptosis as an alternative form of programmed cell death. Curr Opin Cell Biol. 2010;22:263–8.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Vandenabeele P, Melino G. The flick of a switch: which death program to choose? Cell Death Differ. 2012;19:1093–5.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Fulda S. The mechanism of necroptosis in normal and cancer cells. Cancer Biol Ther. 2013;14:999–1004.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Khan N, Lawlor KE, Murphy JM, Vince JE. More to life than death: molecular determinants of necroptotic and non-necroptotic RIP3 kinase signaling. Curr Opin Immunol. 2014;26:76–89.CrossRefPubMedGoogle Scholar
  8. 8.
    Christofferson DE, Li Y, Hitomi J, Zhou W, Upperman C, Zhu H, et al. A novel role for RIP1 kinase in mediating TNFα production. Cell Death Dis. 2012;3:e320.CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Remijsen Q, Goossens V, Grootjans S, Van den Haute C, Vanlangenakker N, Dondelinger Y, et al. Depletion of RIPK3 or MLKL blocks TNF-driven necroptosis and switches towards a delayed RIPK1 kinase-dependent apoptosis. Cell Death Dis. 2014;5:e1004.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Hu X, Han W, Li L. Targeting the weak point of cancer by induction of necroptosis. Autophagy. 2007;3:490–2.CrossRefPubMedGoogle Scholar
  11. 11.
    Chu QD, King T, Hurd T. Triple-negative breast cancer. Int J Breast Cancer. 2012;2012:671–84.Google Scholar
  12. 12.
    Prat A, Adamo B, Cheang MC, Anders CK, Carey LA, Perou CM. Molecular characterization of basal-like and non-basal-like triple-negative breast cancer. Oncologist. 2013;18:123–33.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Liu Z, Zhang XS, Zhang S. Breast tumor subgroups reveal diverse clinical prognostic power. Sci Rep. 2014;4:4002.PubMedPubMedCentralGoogle Scholar
  14. 14.
    Anders CK, Carey LA. Biology, metastatic patterns, and treatment of patients with triple-negative breast cancer. Clin Breast Cancer. 2009;9 Suppl 2:S73–81.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Hudis CA, Gianni L. Triple-negative breast cancer: an unmet medical need. Oncologist. 2011;16 Suppl 1:1–11.CrossRefPubMedGoogle Scholar
  16. 16.
    Ghosh S, Adhikary A, Chakraborty S, Bhattacharjee P, Mazumder M, Putatunda S, et al. Cross-talk between endoplasmic reticulum (ER) stress and the MEK/ERK pathway potentiates apoptosis in human triple negative breast carcinoma cells: role of a dihydropyrimidone, nifetepimine. J Biol Chem. 2015;290:3936–49.CrossRefPubMedGoogle Scholar
  17. 17.
    Holliday DL, Speirs V. Choosing the right cell line for breast cancer research. Breast cancer research : BCR. 2011;13:215.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Neve RM, Chin K, Fridlyand J, Yeh J, Baehner FL, Fevr T, et al. A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer Cell. 2006;10:515–27.CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Lehmann BD, Bauer JA, Chen X, Sanders ME, Chakravarthy AB, Shyr Y, et al. Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies. J Clin Invest. 2011;121:2750–67.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Perou CM. Molecular stratification of triple-negative breast cancers. Oncologist. 2011;16 Suppl 1:61–70.CrossRefPubMedGoogle Scholar
  21. 21.
    Moestue SA, Dam CG, Gorad SS, Kristian A, Bofin A, Maelandsmo GM, et al. Metabolic biomarkers for response to PI3K inhibition in basal-like breast cancer. Breast Cancer Res. 2013;15:R16.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Fallahian F, Karami-Tehrani F, Salami S, Aghaei M. Cyclic GMP induced apoptosis via protein kinase G in oestrogen receptor-positive and -negative breast cancer cell lines. FEBS J. 2011;278:3360–9.CrossRefPubMedGoogle Scholar
  23. 23.
    Salami S, Karami-Tehrani F. Biochemical studies of apoptosis induced by tamoxifen in estrogen receptor positive and negative breast cancer cell lines. Clin Biochem. 2003;36:247–53.CrossRefPubMedGoogle Scholar
  24. 24.
    Longley DB, Johnston PG. Molecular mechanisms of drug resistance. J Pathol. 2005;205:275–92.CrossRefPubMedGoogle Scholar
  25. 25.
    Reed JC. Mechanisms of apoptosis avoidance in cancer. Curr Opin Oncol. 1999;11:68–75.CrossRefPubMedGoogle Scholar
  26. 26.
    Singha PK, Pandeswara S, Venkatachalam MA, Saikumar P. Manumycin A inhibits triple-negative breast cancer growth through LC3-mediated cytoplasmic vacuolation death. Cell Death Dis. 2013;4:e457.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Ghavami S, Hashemi M, Ande SR, Yeganeh B, Xiao W, Eshraghi M, et al. Apoptosis and cancer: mutations within caspase genes. J Med Genet. 2009;46:497–510.CrossRefPubMedGoogle Scholar
  28. 28.
    Tavakoli-Yaraki M, Karami-Tehrani F, Salimi V, Sirati-Sabet M. Induction of apoptosis by trichostatin A in human breast cancer cell lines: involvement of 15-Lox-1. Tumour Biol. 2013;34:241–9.CrossRefPubMedGoogle Scholar
  29. 29.
    Ye YC, Wang HJ, Yu L, Tashiro S, Onodera S, Ikejima T. RIP1-mediated mitochondrial dysfunction and ROS production contributed to tumor necrosis factor alpha-induced L929 cell necroptosis and autophagy. Int Immunopharmacol. 2012;14:674–82.CrossRefPubMedGoogle Scholar
  30. 30.
    Yu X, Deng Q, Li W, Xiao L, Luo X, Liu X, et al. Neoalbaconol induces cell death through necroptosis by regulating RIPK-dependent autocrine TNFα and ROS production. Oncotarget. 2014;6:1995–2008.CrossRefPubMedCentralGoogle Scholar
  31. 31.
    Yang JT, Li ZL, Wu JY, Lu FJ, Chen CH. An oxidative stress mechanism of shikonin in human glioma cells. PLoS One. 2014;9:1–12.Google Scholar
  32. 32.
    Wiench B, Eichhorn T, Paulsen M, Efferth T. Shikonin directly targets mitochondria and causes mitochondrial dysfunction in cancer cells. Evid Based Complement Alternat Med. 2012;2012:726025.CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Jang SY, Lee JK, Jang EH, Jeong SY, Kim JH. Shikonin blocks migration and invasion of human breast cancer cells through inhibition of matrix metalloproteinase-9 activation. Oncol Rep. 2014;31:2827–33.PubMedGoogle Scholar
  34. 34.
    Stoscheck CM. Quantitation of protein. Methods Enzymol. 1990;182:50–68.CrossRefPubMedGoogle Scholar
  35. 35.
    Sadeghi RN, Karami-Tehrani F, Salami S. Targeting prostate cancer cell metabolism: impact of hexokinase and CPT-1 enzymes. Tumour Biol. 2015;36:2893–905.CrossRefPubMedGoogle Scholar
  36. 36.
    Fulda S. Therapeutic exploitation of necroptosis for cancer therapy. Semin Cell Dev Biol. 2014;35:51–6.CrossRefPubMedGoogle Scholar
  37. 37.
    Micheau O, Tschopp J. Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes. Cell. 2003;114:181–90.CrossRefPubMedGoogle Scholar
  38. 38.
    Park S, Shin H, Cho Y. Shikonin induces programmed necrosis-like cell death through the formation of receptor interacting protein 1 and 3 complex. Food Chem Toxicol. 2013;55:36–41.CrossRefPubMedGoogle Scholar
  39. 39.
    Yao Y, Zhou Q. A novel antiestrogen agent shikonin inhibits estrogen-dependent gene transcription in human breast cancer cells. Breast Cancer Res Treat. 2010;121:233–40.CrossRefPubMedGoogle Scholar
  40. 40.
    Han W, Li L, Qiu S, Lu Q, Pan Q, Gu Y, et al. Shikonin circumvents cancer drug resistance by induction of a necroptotic death. Mol Cancer Ther. 2007;6:1641–9.CrossRefPubMedGoogle Scholar
  41. 41.
    Fu Z, Deng B, Liao Y, Shan L, Yin F, Wang Z, et al. The anti-tumor effect of shikonin on osteosarcoma by inducing RIP1 and RIP3 dependent necroptosis. BMC Cancer. 2013;13:580.CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Gong K, Li W. Shikonin, a Chinese plant-derived naphthoquinone, induces apoptosis in hepatocellular carcinoma cells through reactive oxygen species: a potential new treatment for hepatocellular carcinoma. Free Radic Biol Med. 2011;51:2259–71.CrossRefPubMedGoogle Scholar
  43. 43.
    Huang WR, Zhang Y, Tang X. Shikonin inhibits the proliferation of human lens epithelial cells by inducing apoptosis through ROS and caspase-dependent pathway. Molecules. 2014;19:7785–97.CrossRefPubMedGoogle Scholar
  44. 44.
    Tian R, Li Y, Gao M. Shikonin causes cell-cycle arrest and induces apoptosis by regulating the EGFR/NF-κB signaling pathway in human epidermoid carcinoma A431 cells. Biosci Rep. 2015; 35(2)Google Scholar
  45. 45.
    Zhang DW, Shao J, Lin J, Zhang N, Lu BJ, Lin SC, et al. RIP3, an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis. Science. 2009;325:332–6.CrossRefPubMedGoogle Scholar
  46. 46.
    Han W, Xie J, Li L, Liu Z, Hu X. Necrostatin-1 reverts shikonin-induced necroptosis to apoptosis. Apoptosis. 2009;14:674–86.CrossRefPubMedGoogle Scholar
  47. 47.
    Wang GL, Chen CB, Gao JM, Ni H, Wang TS, Chen L. Investigation on the molecular mechanisms of anti-hepatocarcinoma herbs of traditional Chinese medicine by cell cycle microarray. Zhongguo Zhong yao za zhi = Zhongguo zhongyao zazhi = China Journal of Chinese Materia Medica. 2005;30:50–4.PubMedGoogle Scholar
  48. 48.
    Kaczmarek A, Vandenabeele P, Krysko DV. Necroptosis: the release of damage-associated molecular patterns and its physiological relevance. Immunity. 2013;38:209–23.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2015

Authors and Affiliations

  • Zahra Shahsavari
    • 1
  • Fatemeh Karami-Tehrani
    • 1
  • Siamak Salami
    • 2
  • Mehran Ghasemzadeh
    • 3
  1. 1.Cancer Research Laboratory, Department of Clinical Biochemistry, Faculty of Medical ScienceTarbiat Modares UniversityTehranIran
  2. 2.Department of Clinical Biochemistry, Faculty of Medical ScienceShahid Beheshti University of Medical SciencesTehranIran
  3. 3.Blood Transfusion Research CenterHigh Institute for Research and Education in Transfusion MedicineTehranIran

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