Skip to main content

Advertisement

SpringerLink
Log in

Doxorubicin sensitizes cancer cells to Smac mimetic via synergistic activation of the CYLD/RIPK1/FADD/caspase-8-dependent apoptosis

  • Published:
Apoptosis Aims and scope Submit manuscript

Abstract

Smac/Diablo is a pro-apoptotic protein via interaction with inhibitors of apoptosis proteins (IAPs) to relieve their inhibition of caspases. Smac mimetic compounds (also known as antagonists of IAPs) mimic the function of Smac/Diablo and sensitize cancer cells to TNF-induced apoptosis. However, the majority of cancer cells are resistant to Smac mimetic alone. Doxorubicin is a widely used chemotherapeutic drug and causes adverse effect of cardiotoxicity in many patients. Therefore, it is important to find strategies of combined chemotherapy to increase chemosensitivity and reduce the adverse effects. Here, we report that doxorubicin synergizes with Smac mimetic to trigger TNF-mediated apoptosis, which is mechanistically distinct from doxorubicin-induced cell death. Doxorubicin sensitizes cancer cells including human pancreatic and colorectal cancer cells to Smac mimetic treatment. The combined treatment leads to synergistic induction of TNFα to initiate apoptosis through activating NF-κB and c-Jun signaling pathways. Knockdown of caspase-8 or knockout of FADD significantly blocked apoptosis synergistically induced by Smac mimetic and doxorubicin, but had no effect on cell death caused by doxorubicin alone. Moreover, Smac mimetic and doxorubicin-induced apoptosis requires receptor-interacting protein kinase 1 (RIPK1) and its deubiquitinating enzyme cylindromatosis (CYLD), not A20. These in vitro findings demonstrate that combination of Smac mimetic and doxorubicin synergistically triggers apoptosis through the TNF/CYLD/RIPK1/FADD/caspase-8 signaling pathway. Importantly, the combined treatment induced in vivo synergistic anti-tumor effects in the xenograft tumor model. Thus, the combined therapy using Smac mimetic and doxorubicin presents a promising apoptosis-inducing strategy with great potential for the development of anti-cancer therapy.

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

Similar content being viewed by others

Data availability

Not applicable.

Code availability

Not applicable.

References

  1. Peter ME, Krammer PH (2003) The CD95(APO-1/Fas) DISC and beyond. Cell Death Differ 10(1):26–35. https://doi.org/10.1038/sj.cdd.4401186

    Article  CAS  PubMed  Google Scholar 

  2. Wajant H (2002) The Fas signaling pathway: more than a paradigm. Science 296(5573):1635–1636. https://doi.org/10.1126/science.1071553

    Article  CAS  PubMed  Google Scholar 

  3. Wang X (2001) The expanding role of mitochondria in apoptosis. Genes Dev 15(22):2922–2933

    CAS  PubMed  Google Scholar 

  4. Green DR, Reed JC (1998) Mitochondria and apoptosis. Science 281(5381):1309–1312. https://doi.org/10.1126/science.281.5381.1309

    Article  CAS  PubMed  Google Scholar 

  5. Du C, Fang M, Li Y, Li L, Wang X (2000) Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell 102(1):33–42. https://doi.org/10.1016/s0092-8674(00)00008-8

    Article  CAS  PubMed  Google Scholar 

  6. Verhagen AM, Ekert PG, Pakusch M, Silke J, Connolly LM, Reid GE, Moritz RL, Simpson RJ, Vaux DL (2000) Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell 102(1):43–53. https://doi.org/10.1016/s0092-8674(00)00009-x

    Article  CAS  PubMed  Google Scholar 

  7. Fernald K, Kurokawa M (2013) Evading apoptosis in cancer. Trends Cell Biol 23(12):620–633. https://doi.org/10.1016/j.tcb.2013.07.006

    Article  PubMed  PubMed Central  Google Scholar 

  8. Tamm I, Richter S, Scholz F, Schmelz K, Oltersdorf D, Karawajew L, Schoch C, Haferlach T, Ludwig WD, Wuchter C (2004) XIAP expression correlates with monocytic differentiation in adult de novo AML: impact on prognosis. Hematol J 5(6):489–495. https://doi.org/10.1038/sj.thj.6200549

    Article  CAS  PubMed  Google Scholar 

  9. Krajewska M, Krajewski S, Banares S, Huang X, Turner B, Bubendorf L, Kallioniemi OP, Shabaik A, Vitiello A, Peehl D, Gao GJ, Reed JC (2003) Elevated expression of inhibitor of apoptosis proteins in prostate cancer. Clin Cancer Res 9(13):4914–4925

    CAS  PubMed  Google Scholar 

  10. Yang QH, Du C (2004) Smac/DIABLO selectively reduces the levels of c-IAP1 and c-IAP2 but not that of XIAP and livin in HeLa cells. J Biol Chem 279(17):16963–16970. https://doi.org/10.1074/jbc.M401253200

    Article  CAS  PubMed  Google Scholar 

  11. Li L, Thomas RM, Suzuki H, De Brabander JK, Wang X, Harran PG (2004) A small molecule Smac mimic potentiates TRAIL- and TNFalpha-mediated cell death. Science 305(5689):1471–1474. https://doi.org/10.1126/science.1098231

    Article  CAS  PubMed  Google Scholar 

  12. Petersen SL, Wang L, Yalcin-Chin A, Li L, Peyton M, Minna J, Harran P, Wang X (2007) Autocrine TNFalpha signaling renders human cancer cells susceptible to Smac-mimetic-induced apoptosis. Cancer Cell 12(5):445–456. https://doi.org/10.1016/j.ccr.2007.08.029

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Wang L, Du F, Wang X (2008) TNF-alpha induces two distinct caspase-8 activation pathways. Cell 133(4):693–703. https://doi.org/10.1016/j.cell.2008.03.036

    Article  CAS  PubMed  Google Scholar 

  14. Vince JE, Wong WW, Khan N, Feltham R, Chau D, Ahmed AU, Benetatos CA, Chunduru SK, Condon SM, McKinlay M, Brink R, Leverkus M, Tergaonkar V, Schneider P, Callus BA, Koentgen F, Vaux DL, Silke J (2007) IAP antagonists target cIAP1 to induce TNFalpha-dependent apoptosis. Cell 131(4):682–693. https://doi.org/10.1016/j.cell.2007.10.037

    Article  CAS  PubMed  Google Scholar 

  15. Varfolomeev E, Blankenship JW, Wayson SM, Fedorova AV, Kayagaki N, Garg P, Zobel K, Dynek JN, Elliott LO, Wallweber HJ, Flygare JA, Fairbrother WJ, Deshayes K, Dixit VM, Vucic D (2007) IAP antagonists induce autoubiquitination of c-IAPs, NF-kappaB activation, and TNFalpha-dependent apoptosis. Cell 131(4):669–681. https://doi.org/10.1016/j.cell.2007.10.030

    Article  CAS  PubMed  Google Scholar 

  16. Kantarjian H, Thomas D, O’Brien S, Cortes J, Giles F, Jeha S, Bueso-Ramos CE, Pierce S, Shan J, Koller C, Beran M, Keating M, Freireich EJ (2004) Long-term follow-up results of hyperfractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone (Hyper-CVAD), a dose-intensive regimen, in adult acute lymphocytic leukemia. Cancer 101(12):2788–2801. https://doi.org/10.1002/cncr.20668

    Article  CAS  PubMed  Google Scholar 

  17. von der Maase H, Sengelov L, Roberts JT, Ricci S, Dogliotti L, Oliver T, Moore MJ, Zimmermann A, Arning M (2005) Long-term survival results of a randomized trial comparing gemcitabine plus cisplatin, with methotrexate, vinblastine, doxorubicin, plus cisplatin in patients with bladder cancer. J Clin Oncol 23(21):4602–4608. https://doi.org/10.1200/JCO.2005.07.757

    Article  CAS  PubMed  Google Scholar 

  18. Swain SM, Whaley FS, Gerber MC, Weisberg S, York M, Spicer D, Jones SE, Wadler S, Desai A, Vogel C, Speyer J, Mittelman A, Reddy S, Pendergrass K, Velez-Garcia E, Ewer MS, Bianchine JR, Gams RA (1997) Cardioprotection with dexrazoxane for doxorubicin-containing therapy in advanced breast cancer. J Clin Oncol 15(4):1318–1332. https://doi.org/10.1200/JCO.1997.15.4.1318

    Article  CAS  PubMed  Google Scholar 

  19. Matsumura Y, Gotoh M, Muro K, Yamada Y, Shirao K, Shimada Y, Okuwa M, Matsumoto S, Miyata Y, Ohkura H, Chin K, Baba S, Yamao T, Kannami A, Takamatsu Y, Ito K, Takahashi K (2004) Phase I and pharmacokinetic study of MCC-465, a doxorubicin (DXR) encapsulated in PEG immunoliposome, in patients with metastatic stomach cancer. Ann Oncol 15(3):517–525. https://doi.org/10.1093/annonc/mdh092

    Article  CAS  PubMed  Google Scholar 

  20. von Pawel J, Schiller JH, Shepherd FA, Fields SZ, Kleisbauer JP, Chrysson NG, Stewart DJ, Clark PI, Palmer MC, Depierre A, Carmichael J, Krebs JB, Ross G, Lane SR, Gralla R (1999) Topotecan versus cyclophosphamide, doxorubicin, and vincristine for the treatment of recurrent small-cell lung cancer. J Clin Oncol 17(2):658–667. https://doi.org/10.1200/JCO.1999.17.2.658

    Article  Google Scholar 

  21. A’Hern RP, Gore ME (1995) Impact of doxorubicin on survival in advanced ovarian cancer. J Clin Oncol 13(3):726–732. https://doi.org/10.1200/JCO.1995.13.3.726

    Article  PubMed  Google Scholar 

  22. Carvalho C, Santos RX, Cardoso S, Correia S, Oliveira PJ, Santos MS, Moreira PI (2009) Doxorubicin: the good, the bad and the ugly effect. Curr Med Chem 16(25):3267–3285. https://doi.org/10.2174/092986709788803312

    Article  CAS  PubMed  Google Scholar 

  23. Benjanuwattra J, Siri-Angkul N, Chattipakorn SC, Chattipakorn N (2020) Doxorubicin and its proarrhythmic effects: a comprehensive review of the evidence from experimental and clinical studies. Pharmacol Res 151:104542. https://doi.org/10.1016/j.phrs.2019.104542

    Article  CAS  PubMed  Google Scholar 

  24. Yang F, Teves SS, Kemp CJ, Henikoff S (2014) Doxorubicin, DNA torsion, and chromatin dynamics. Biochim Biophys Acta 1845(1):84–89. https://doi.org/10.1016/j.bbcan.2013.12.002

    Article  CAS  PubMed  Google Scholar 

  25. Huang J, Yang J, Maity B, Mayuzumi D, Fisher RA (2011) Regulator of G protein signaling 6 mediates doxorubicin-induced ATM and p53 activation by a reactive oxygen species-dependent mechanism. Cancer Res 71(20):6310–6319. https://doi.org/10.1158/0008-5472.CAN-10-3397

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Arola OJ, Saraste A, Pulkki K, Kallajoki M, Parvinen M, Voipio-Pulkki LM (2000) Acute doxorubicin cardiotoxicity involves cardiomyocyte apoptosis. Cancer Res 60(7):1789–1792

    CAS  PubMed  Google Scholar 

  27. Kim SY, Kim SJ, Kim BJ, Rah SY, Chung SM, Im MJ, Kim UH (2006) Doxorubicin-induced reactive oxygen species generation and intracellular Ca2+ increase are reciprocally modulated in rat cardiomyocytes. Exp Mol Med 38(5):535–545. https://doi.org/10.1038/emm.2006.63

    Article  CAS  PubMed  Google Scholar 

  28. Singal PK, Li T, Kumar D, Danelisen I, Iliskovic N (2000) Adriamycin-induced heart failure: mechanism and modulation. Mol Cell Biochem 207(1–2):77–86. https://doi.org/10.1023/a:1007094214460

    Article  CAS  PubMed  Google Scholar 

  29. He S, Wang L, Miao L, Wang T, Du F, Zhao L, Wang X (2009) Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha. Cell 137(6):1100–1111. https://doi.org/10.1016/j.cell.2009.05.021

    Article  CAS  PubMed  Google Scholar 

  30. Zhang J, Yang Y, Zhou S, He X, Cao X, Wu C, Hu H, Qin J, Wei G, Wang H, Liu S, Sun L (2019) Membrane-bound TNF mediates microtubule-targeting chemotherapeutics-induced cancer cytolysis via juxtacrine inter-cancer-cell death signaling. Cell Death Differ. https://doi.org/10.1038/s41418-019-0441-3

    Article  PubMed  PubMed Central  Google Scholar 

  31. Wertz IE, O’Rourke KM, Zhou H, Eby M, Aravind L, Seshagiri S, Wu P, Wiesmann C, Baker R, Boone DL, Ma A, Koonin EV, Dixit VM (2004) De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF-kappaB signalling. Nature 430(7000):694–699. https://doi.org/10.1038/nature02794

    Article  CAS  PubMed  Google Scholar 

  32. Hortobagyi GN (1997) Anthracyclines in the treatment of cancer: an overview. Drugs 54(Suppl 4):1–7. https://doi.org/10.2165/00003495-199700544-00003

    Article  CAS  PubMed  Google Scholar 

  33. Hahn WC, Weinberg RA (2002) Rules for making human tumor cells. N Engl J Med 347(20):1593–1603. https://doi.org/10.1056/NEJMra021902

    Article  CAS  PubMed  Google Scholar 

  34. Harper ME, Antoniou A, Villalobos-Menuey E, Russo A, Trauger R, Vendemelio M, George A, Bartholomew R, Carlo D, Shaikh A, Kupperman J, Newell EW, Bespalov IA, Wallace SS, Liu Y, Rogers JR, Gibbs GL, Leahy JL, Camley RE, Melamede R, Newell MK (2002) Characterization of a novel metabolic strategy used by drug-resistant tumor cells. FASEB J 16(12):1550–1557. https://doi.org/10.1096/fj.02-0541com

    Article  CAS  PubMed  Google Scholar 

  35. Park EJ, Kwon HK, Choi YM, Shin HJ, Choi S (2012) Doxorubicin induces cytotoxicity through upregulation of pERK-dependent ATF3. PLoS ONE 7(9):e44990. https://doi.org/10.1371/journal.pone.0044990

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Edwardson DW, Boudreau J, Mapletoft J, Lanner C, Kovala AT, Parissenti AM (2017) Inflammatory cytokine production in tumor cells upon chemotherapy drug exposure or upon selection for drug resistance. PLoS ONE 12(9):e0183662. https://doi.org/10.1371/journal.pone.0183662

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Weisberg E, Kung AL, Wright RD, Moreno D, Catley L, Ray A, Zawel L, Tran M, Cools J, Gilliland G, Mitsiades C, McMillin DW, Jiang J, Hall-Meyers E, Griffin JD (2007) Potentiation of antileukemic therapies by Smac mimetic, LBW242: effects on mutant FLT3-expressing cells. Mol Cancer Ther 6(7):1951–1961. https://doi.org/10.1158/1535-7163.MCT-06-0810

    Article  CAS  PubMed  Google Scholar 

  38. Zhang S, Li G, Zhao Y, Liu G, Wang Y, Ma X, Li D, Wu Y, Lu J (2012) Smac mimetic SM-164 potentiates APO2L/TRAIL- and doxorubicin-mediated anticancer activity in human hepatocellular carcinoma cells. PLoS ONE 7(12):e51461. https://doi.org/10.1371/journal.pone.0051461

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Kamata E, Kawamoto T, Ueha T, Hara H, Fukase N, Minoda M, Morishita M, Takemori T, Fujiwara S, Nishida K, Kuroda R, Kurosaka M, Akisue T (2017) Synergistic effects of a Smac mimetic with doxorubicin against human osteosarcoma. Anticancer Res 37(11):6097–6106. https://doi.org/10.21873/anticanres.12058

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Dr. Xiaodong Wang (National Institute of Biological Sciences (NIBS), Beijing, China) for kindly providing Smac mimetic.

Funding

This work was supported by the National Natural Science Foundation of China (31671436 and 31830051 to S.H., 31771533 to T.Y., 31900526 to X.Y. and 31600133 to W.Z.), National Basic Research Program of China (2013CB910102 to S.H.), the CAMS Innovation Fund for Medical Sciences (CIFMS; 2019-I2M-1-004, 2016-I2M-1-005 to S.H. and 2019-I2M-1-003 to X.Y.), and Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences (No. 2019PT310028), China Postdoctoral Science Foundation funded project (2019M650563 to X.Y.), a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, Natural Science Foundation of Jiangsu Province Grant (BK20160314 to S.H.) and Fok Ying Tung Education Foundation for Young Teachers (151020 to S.H.).

Author information

Author notes

    Authors and Affiliations

    1. Center of Systems Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medial College, Beijing, China

      Chengkui Yang, Qiao Ran, Yifei Zhou, Cong Zhao, Xiaoliang Yu, Fang Zhu, Yuting Ji, Qian Du, Tao Yang, Wei Zhang & Sudan He

    2. Suzhou Institute of Systems Medicine, Suzhou, 215123, Jiangsu, China

      Chengkui Yang, Qiao Ran, Yifei Zhou, Cong Zhao, Xiaoliang Yu, Fang Zhu, Yuting Ji, Qian Du, Tao Yang, Wei Zhang & Sudan He

    3. Cyrus Tang Hematology Center and Collaborative Innovation Center of Hematology, State Key Laboratory of Radiation Medicine and Protection, Soochow University, Suzhou, 215123, Jiangsu, China

      Chengkui Yang, Qiao Ran, Yifei Zhou, Shan Liu, Cong Zhao, Xiaoliang Yu, Fang Zhu, Qian Du, Tao Yang, Wei Zhang & Sudan He

    4. School of Life Science and Technology, China Pharmaceutical University, Nanjing, 210038, Jiangsu, China

      Yuting Ji

    Authors
    1. Chengkui Yang
      View author publications

      You can also search for this author in PubMed Google Scholar

    2. Qiao Ran
      View author publications

      You can also search for this author in PubMed Google Scholar

    3. Yifei Zhou
      View author publications

      You can also search for this author in PubMed Google Scholar

    4. Shan Liu
      View author publications

      You can also search for this author in PubMed Google Scholar

    5. Cong Zhao
      View author publications

      You can also search for this author in PubMed Google Scholar

    6. Xiaoliang Yu
      View author publications

      You can also search for this author in PubMed Google Scholar

    7. Fang Zhu
      View author publications

      You can also search for this author in PubMed Google Scholar

    8. Yuting Ji
      View author publications

      You can also search for this author in PubMed Google Scholar

    9. Qian Du
      View author publications

      You can also search for this author in PubMed Google Scholar

    10. Tao Yang
      View author publications

      You can also search for this author in PubMed Google Scholar

    11. Wei Zhang
      View author publications

      You can also search for this author in PubMed Google Scholar

    12. Sudan He
      View author publications

      You can also search for this author in PubMed Google Scholar

    Contributions

    All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by SH, CY, QR, YZ, SL, CZ, XY, FZ, YJ, QD, TY and WZ. The first draft of the manuscript was written by SH, CY, QR and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

    Corresponding authors

    Correspondence to Chengkui Yang or Sudan He.

    Ethics declarations

    Conflicts of interest

    The authors declare no conflict of interest.

    Ethics approval

    All animal experiments were performed in accordance with protocols by the Institutional Animal Care and Use Committee at Suzhou Institute of Systems Medicine.

    Consent to participate

    Not applicable.

    Consent for publication

    Not applicable.

    Additional information

    Publisher's Note

    Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

    Rights and permissions

    Reprints and permissions

    About this article

    Check for updates. Verify currency and authenticity via CrossMark

    Cite this article

    Yang, C., Ran, Q., Zhou, Y. et al. Doxorubicin sensitizes cancer cells to Smac mimetic via synergistic activation of the CYLD/RIPK1/FADD/caspase-8-dependent apoptosis. Apoptosis 25, 441–455 (2020). https://doi.org/10.1007/s10495-020-01604-6

    Download citation

    • Published:

    • Issue Date:

    • DOI: https://doi.org/10.1007/s10495-020-01604-6

    Keywords

    Search

    Navigation