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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

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Tumor Biology

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.

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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

References

  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.

    Article  CAS  PubMed  Google Scholar 

  2. Feoktistova M, Leverkus M. Programmed necrosis and necroptosis signalling. FEBS J. 2015;282:19–31.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  4. Christofferson DE, Yuan J. Necroptosis as an alternative form of programmed cell death. Curr Opin Cell Biol. 2010;22:263–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Vandenabeele P, Melino G. The flick of a switch: which death program to choose? Cell Death Differ. 2012;19:1093–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Fulda S. The mechanism of necroptosis in normal and cancer cells. Cancer Biol Ther. 2013;14:999–1004.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Hu X, Han W, Li L. Targeting the weak point of cancer by induction of necroptosis. Autophagy. 2007;3:490–2.

    Article  CAS  PubMed  Google Scholar 

  11. Chu QD, King T, Hurd T. Triple-negative breast cancer. Int J Breast Cancer. 2012;2012:671–84.

    Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Liu Z, Zhang XS, Zhang S. Breast tumor subgroups reveal diverse clinical prognostic power. Sci Rep. 2014;4:4002.

    PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Hudis CA, Gianni L. Triple-negative breast cancer: an unmet medical need. Oncologist. 2011;16 Suppl 1:1–11.

    Article  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  17. Holliday DL, Speirs V. Choosing the right cell line for breast cancer research. Breast cancer research : BCR. 2011;13:215.

    Article  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Perou CM. Molecular stratification of triple-negative breast cancers. Oncologist. 2011;16 Suppl 1:61–70.

    Article  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  24. Longley DB, Johnston PG. Molecular mechanisms of drug resistance. J Pathol. 2005;205:275–92.

    Article  CAS  PubMed  Google Scholar 

  25. Reed JC. Mechanisms of apoptosis avoidance in cancer. Curr Opin Oncol. 1999;11:68–75.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  PubMed Central  Google Scholar 

  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. 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.

    Article  PubMed  PubMed Central  Google Scholar 

  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.

    CAS  PubMed  Google Scholar 

  34. Stoscheck CM. Quantitation of protein. Methods Enzymol. 1990;182:50–68.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  36. Fulda S. Therapeutic exploitation of necroptosis for cancer therapy. Semin Cell Dev Biol. 2014;35:51–6.

    Article  CAS  PubMed  Google Scholar 

  37. Micheau O, Tschopp J. Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes. Cell. 2003;114:181–90.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  PubMed  Google Scholar 

  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)

  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.

    Article  CAS  PubMed  Google Scholar 

  46. Han W, Xie J, Li L, Liu Z, Hu X. Necrostatin-1 reverts shikonin-induced necroptosis to apoptosis. Apoptosis. 2009;14:674–86.

    Article  CAS  PubMed  Google Scholar 

  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.

    CAS  PubMed  Google Scholar 

  48. Kaczmarek A, Vandenabeele P, Krysko DV. Necroptosis: the release of damage-associated molecular patterns and its physiological relevance. Immunity. 2013;38:209–23.

    Article  CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Cancer Research Laboratory, Department of Clinical Biochemistry, Faculty of Medical Science, Tarbiat Modares University, P.O. Box: 14115-331, Tehran, Iran

    Zahra Shahsavari & Fatemeh Karami-Tehrani

  2. Department of Clinical Biochemistry, Faculty of Medical Science, Shahid Beheshti University of Medical Sciences, Tehran, Iran

    Siamak Salami

  3. Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran

    Mehran Ghasemzadeh

Authors
  1. Zahra Shahsavari
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  2. Fatemeh Karami-Tehrani
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  3. Siamak Salami
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  4. Mehran Ghasemzadeh
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Correspondence to Fatemeh Karami-Tehrani.

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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.

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Shahsavari, Z., Karami-Tehrani, F., Salami, S. et al. 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. Tumor Biol. 37, 4479–4491 (2016). https://doi.org/10.1007/s13277-015-4258-5

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  • DOI: https://doi.org/10.1007/s13277-015-4258-5

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