Cancer Chemotherapy and Pharmacology

, Volume 76, Issue 3, pp 555–565 | Cite as

Lower antioxidative capacity of multidrug-resistant cancer cells confers collateral sensitivity to protoflavone derivatives

  • Tijana Stanković
  • Balázs Dankó
  • Ana Martins
  • Miodrag Dragoj
  • Sonja Stojković
  • Aleksandra Isaković
  • Hui-Chun Wang
  • Yang-Chang Wu
  • Attila Hunyadi
  • Milica Pešić
Original Article



Multidrug resistance (MDR) may develop due to a series of adaptive responses under a new stress conditions, such as chemotherapy. Novel strategies are urgently needed to fight MDR in cancer, and chemotherapeutics that are selective for MDR cancer cells offer a promising approach. Certain protoflavones were previously found to have potential in this regard.


Cytotoxicity of six protoflavones was assessed in different P-glycoprotein overexpressing MDR cancer cell lines and in their non-MDR counterparts. The impacts of compound 5, 6-methylprotoflavone previously published and a new derivative, 6-bromoprotoflavone (compound 6), on the cell cycle distribution were evaluated, and 6 was also studied for its potential to regulate the intracellular antioxidative capacity.


Protoflavones showed a significant cytotoxicity against all cancer cell lines and selectivity toward MDR cancer cells adapted to conventional chemotherapeutics. Inverse sensitivity versus MDR selectivity pattern was observed. Treatment with H2O2 showed that MDR cancer cells are more vulnerable to oxidative stress. Compounds 5 and 6 significantly decreased the portion of MDR cells in the G1 phase. The levels of reactive oxygen and nitrogen species (ROS/RNS) between MDR and non-MDR cells significantly differed upon exposure to 6, accompanied by changes in the glutathione (GSH) levels and in the expression of manganese superoxide dismutase (MnSOD), glutathione-S-transferase π (GST π) and hypoxia-inducible factor-1α (HIF-1α).


Our results suggest that MDR cancer cells can be more vulnerable to the pro-oxidative activity of protoflavones due to an impaired antioxidative defense that might arise during the adaptation processes provoked by chemotherapy.


Multidrug resistance P-glycoprotein Protoflavones Pro-oxidative activity Reactive oxygen species Collateral sensitivity 



This research was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Grant No III41031). The authors acknowledge the support from the National Science Council (NSC), Taiwan, the Szeged Foundation for Cancer Research and the Fundação para a Ciência e a Tecnologia (FCT), Portugal (PEsT-OE/SAU/UI0074/2011 and PEsT-OE/SAU/UI0074/2014). This work was performed within the framework of COST Actions CM1106 (Chemical Approaches to Targeting Drug Resistance in Cancer Stem Cells; providing an STSM grant to A. Martins) and CM1407 (Challenging organic syntheses inspired by nature—from natural products chemistry to drug discovery). A bilateral mobility grant provided by the Hungarian Academy of Sciences and the NSC, Taiwan (MOST 104-2911-I-037-501), is also acknowledged.

Compliance with ethical standards

Conflict of interest



  1. 1.
    Hall MD, Marshall TS, Kwit AD, Miller Jenkins LM, Dulcey AE, Madigan JP, Pluchino KM, Goldsborough AS, Brimacombe KR, Griffiths GL, Gottesman MM (2014) Inhibition of glutathione peroxidase mediates the collateral sensitivity of multidrug-resistant cells to tiopronin. J Biol Chem 289(31):21473–21489. doi: 10.1074/jbc.M114.581702 PubMedCentralCrossRefPubMedGoogle Scholar
  2. 2.
    Sharom FJ (2008) ABC multidrug transporters: structure, function and role in chemoresistance. Pharmacogenomics 9(1):105–127. doi: 10.2217/14622416.9.1.105 CrossRefPubMedGoogle Scholar
  3. 3.
    Gottesman MM (2002) Mechanisms of cancer drug resistance. Annu Rev Med 53:615–627. doi: 10.1146/ CrossRefPubMedGoogle Scholar
  4. 4.
    Hall MD, Handley MD, Gottesman MM (2009) Is resistance useless? Multidrug resistance and collateral sensitivity. Trends Pharmacol Sci 30(10):546–556. doi: 10.1016/ PubMedCentralCrossRefPubMedGoogle Scholar
  5. 5.
    Pluchino KM, Hall MD, Goldsborough AS, Callaghan R, Gottesman MM (2012) Collateral sensitivity as a strategy against cancer multidrug resistance. Drug Resist Updat 15(1–2):98–105. doi: 10.1016/j.drup.2012.03.002 PubMedCentralCrossRefPubMedGoogle Scholar
  6. 6.
    Szakacs G, Hall MD, Gottesman MM, Boumendjel A, Kachadourian R, Day BJ, Baubichon-Cortay H, Di Pietro A (2014) Targeting the Achilles heel of multidrug-resistant cancer by exploiting the fitness cost of resistance. Chem Rev 114(11):5753–5774. doi: 10.1021/cr4006236 PubMedCentralCrossRefPubMedGoogle Scholar
  7. 7.
    Nakagawa-Goto K, Chang PC, Lai CY, Hung HY, Chen TH, Wu PC, Zhu H, Sedykh A, Bastow KF, Lee KH (2010) Antitumor agents. 280. Multidrug resistance-selective desmosdumotin B analogues. J Med Chem 53(18):6699–6705. doi: 10.1021/jm100846r PubMedCentralCrossRefPubMedGoogle Scholar
  8. 8.
    Hunyadi A, Martins A, Danko B, Chang FR, Wu YC (2014) Protoflavones: a class of unusual flavonoids as promising novel anticancer agents. Phytochem Rev 13(1):69–77. doi: 10.1007/s11101-013-9288-2 CrossRefGoogle Scholar
  9. 9.
    Chen WY, Hsieh YA, Tsai CI, Kang YF, Chang FR, Wu YC, Wu CC (2011) Protoapigenone, a natural derivative of apigenin, induces mitogen-activated protein kinase-dependent apoptosis in human breast cancer cells associated with induction of oxidative stress and inhibition of glutathione S-transferase pi. Invest New Drugs 29(6):1347–1359. doi: 10.1007/s10637-010-9497-0 CrossRefPubMedGoogle Scholar
  10. 10.
    Wang HC, Lee AY, Chou WC, Wu CC, Tseng CN, Liu KY, Lin WL, Chang FR, Chuang DW, Hunyadi A, Wu YC (2012) Inhibition of ATR-dependent signaling by protoapigenone and its derivative sensitizes cancer cells to interstrand cross-link-generating agents in vitro and in vivo. Mol Cancer Ther 11(7):1443–1453. doi: 10.1158/1535-7163.MCT-11-0921 CrossRefPubMedGoogle Scholar
  11. 11.
    Danko B, Martins A, Chuang DW, Wang HC, Amaral L, Molnar J, Chang FR, Wu YC, Hunyadi A (2012) In vitro cytotoxic activity of novel protoflavone analogs—selectivity towards a multidrug resistant cancer cell line. Anticancer Res 32(7):2863–2869PubMedGoogle Scholar
  12. 12.
    Pesic M, Markovic JZ, Jankovic D, Kanazir S, Markovic ID, Rakic L, Ruzdijic S (2006) Induced resistance in the human non small cell lung carcinoma (NCI-H460) cell line in vitro by anticancer drugs. J Chemother 18(1):66–73. doi: 10.1179/joc.2006.18.1.66 CrossRefPubMedGoogle Scholar
  13. 13.
    Podolski-Renic A, Andelkovic T, Bankovic J, Tanic N, Ruzdijic S, Pesic M (2011) The role of paclitaxel in the development and treatment of multidrug resistant cancer cell lines. Biomed Pharmacother 65(5):345–353. doi: 10.1016/j.biopha.2011.04.015 CrossRefPubMedGoogle Scholar
  14. 14.
    Stojkovic S, Podolski-Renic A, Dinic J, Stankovic T, Bankovic J, Hadzic S, Paunovic V, Isakovic A, Tanic N, Pesic M (2015) Development of resistance to antiglioma agents in rat C6 cells caused collateral sensitivity to doxorubicin. Exp Cell Res. doi: 10.1016/j.yexcr.2015.05.018 PubMedGoogle Scholar
  15. 15.
    Hunyadi A, Chuang DW, Danko B, Chiang MY, Lee CL, Wang HC, Wu CC, Chang FR, Wu YC (2011) Direct semi-synthesis of the anticancer lead-drug protoapigenone from apigenin, and synthesis of further new cytotoxic protoflavone derivatives. PLoS ONE 6(8):e23922. doi: 10.1371/journal.pone.0023922 PubMedCentralCrossRefPubMedGoogle Scholar
  16. 16.
    O’Driscoll L, Daly C, Saleh M, Clynes M (1993) The use of reverse transcriptase-polymerase chain reaction (RT-PCR) to investigate specific gene expression in multidrug-resistant cells. Cytotechnology 12(1–3):289–314CrossRefPubMedGoogle Scholar
  17. 17.
    Larrea E, Beloqui O, Munoz-Navas MA, Civeira MP, Prieto J (1998) Superoxide dismutase in patients with chronic hepatitis C virus infection. Free Radic Biol Med 24(7–8):1235–1241CrossRefPubMedGoogle Scholar
  18. 18.
    Nardinocchi L, Puca R, Sacchi A, D’Orazi G (2009) Inhibition of HIF-1alpha activity by homeodomain-interacting protein kinase-2 correlates with sensitization of chemoresistant cells to undergo apoptosis. Mol Cancer 8:1. doi: 10.1186/1476-4598-8-1 PubMedCentralCrossRefPubMedGoogle Scholar
  19. 19.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25(4):402–408. doi: 10.1006/meth.2001.1262 CrossRefPubMedGoogle Scholar
  20. 20.
    NicAmhlaoibh R, Heenan M, Cleary I, Touhey S, O’Loughlin C, Daly C, Nunez G, Scanlon KJ, Clynes M (1999) Altered expression of mRNAs for apoptosis-modulating proteins in a low level multidrug resistant variant of a human lung carcinoma cell line that also expresses mdr1 mRNA. Int J Cancer 82(3):368–376CrossRefPubMedGoogle Scholar
  21. 21.
    Prochazkova J, Kubala L, Kotasova H, Gudernova I, Sramkova Z, Pekarova M, Sarkadi B, Pachernik J (2011) ABC transporters affect the detection of intracellular oxidants by fluorescent probes. Free Radic Res 45(7):779–787. doi: 10.3109/10715762.2011.579120 CrossRefPubMedGoogle Scholar
  22. 22.
    Krzyzanowski D, Bartosz G, Grzelak A (2014) Collateral sensitivity: ABCG2-overexpressing cells are more vulnerable to oxidative stress. Free Radic Biol Med 76:47–52. doi: 10.1016/j.freeradbiomed.2014.07.020 CrossRefPubMedGoogle Scholar
  23. 23.
    Kaewpila S, Venkataraman S, Buettner GR, Oberley LW (2008) Manganese superoxide dismutase modulates hypoxia-inducible factor-1 alpha induction via superoxide. Cancer Res 68(8):2781–2788. doi: 10.1158/0008-5472.CAN-07-2635 PubMedCentralCrossRefPubMedGoogle Scholar
  24. 24.
    Wang M, Kirk JS, Venkataraman S, Domann FE, Zhang HJ, Schafer FQ, Flanagan SW, Weydert CJ, Spitz DR, Buettner GR, Oberley LW (2005) Manganese superoxide dismutase suppresses hypoxic induction of hypoxia-inducible factor-1alpha and vascular endothelial growth factor. Oncogene 24(55):8154–8166. doi: 10.1038/sj.onc.1208986 PubMedGoogle Scholar
  25. 25.
    Podolski-Renic A, Jadranin M, Stankovic T, Bankovic J, Stojkovic S, Chiourea M, Aljancic I, Vajs V, Tesevic V, Ruzdijic S, Gagos S, Tanic N, Pesic M (2013) Molecular and cytogenetic changes in multi-drug resistant cancer cells and their influence on new compounds testing. Cancer Chemother Pharmacol 72(3):683–697. doi: 10.1007/s00280-013-2247-1 CrossRefPubMedGoogle Scholar
  26. 26.
    Chen HM, Chang FR, Hsieh YC, Cheng YJ, Hsieh KC, Tsai LM, Lin AS, Wu YC, Yuan SS (2011) A novel synthetic protoapigenone analogue, WYC02-9, induces DNA damage and apoptosis in DU145 prostate cancer cells through generation of reactive oxygen species. Free Radic Biol Med 50(9):1151–1162. doi: 10.1016/j.freeradbiomed.2011.01.015 CrossRefPubMedGoogle Scholar
  27. 27.
    Chiu CC, Chang HW, Chuang DW, Chang FR, Chang YC, Cheng YS, Tsai MT, Chen WY, Lee SS, Wang CK, Chen JY, Wang HM, Chen CC, Liu YC, Wu YC (2009) Fern plant-derived protoapigenone leads to DNA damage, apoptosis, and G(2)/m arrest in lung cancer cell line H1299. DNA Cell Biol 28(10):501–506. doi: 10.1089/dna.2009.0852 CrossRefPubMedGoogle Scholar
  28. 28.
    Chen YJ, Chen HP, Cheng YJ, Lin YH, Liu KW, Chen YJ, Hou MF, Wu YC, Lee YC, Yuan SS (2013) The synthetic flavonoid WYC02-9 inhibits colorectal cancer cell growth through ROS-mediated activation of MAPK14 pathway. Life Sci 92(22):1081–1092. doi: 10.1016/j.lfs.2013.04.007 CrossRefPubMedGoogle Scholar
  29. 29.
    Liu Z, Yuan Q, Zhang X, Xiong C, Xue P, Ruan J (2012) RY10-4, a novel anti-tumor compound, exhibited its anti-angiogenesis activity by down-regulation of the HIF-1alpha and inhibition phosphorylation of AKT and mTOR. Cancer Chemother Pharmacol 69(6):1633–1640. doi: 10.1007/s00280-012-1873-3 CrossRefPubMedGoogle Scholar
  30. 30.
    Sarsour EH, Kalen AL, Goswami PC (2014) Manganese superoxide dismutase regulates a redox cycle within the cell cycle. Antioxid Redox Signal 20(10):1618–1627. doi: 10.1089/ars.2013.5303 PubMedCentralCrossRefPubMedGoogle Scholar
  31. 31.
    Menon SG, Sarsour EH, Spitz DR, Higashikubo R, Sturm M, Zhang H, Goswami PC (2003) Redox regulation of the G1 to S phase transition in the mouse embryo fibroblast cell cycle. Cancer Res 63(9):2109–2117PubMedGoogle Scholar
  32. 32.
    Lorendeau D, Dury L, Genoux-Bastide E, Lecerf-Schmidt F, Simoes-Pires C, Carrupt PA, Terreux R, Magnard S, Di Pietro A, Boumendjel A, Baubichon-Cortay H (2014) Collateral sensitivity of resistant MRP1-overexpressing cells to flavonoids and derivatives through GSH efflux. Biochem Pharmacol 90(3):235–245. doi: 10.1016/j.bcp.2014.05.017 CrossRefPubMedGoogle Scholar
  33. 33.
    Chang HL, Su JH, Yeh YT, Lee YC, Chen HM, Wu YC, Yuan SS (2008) Protoapigenone, a novel flavonoid, inhibits ovarian cancer cell growth in vitro and in vivo. Cancer Lett 267(1):85–95. doi: 10.1016/j.canlet.2008.03.007 CrossRefPubMedGoogle Scholar
  34. 34.
    Chang HL, Wu YC, Su JH, Yeh YT, Yuan SS (2008) Protoapigenone, a novel flavonoid, induces apoptosis in human prostate cancer cells through activation of p38 mitogen-activated protein kinase and c-Jun NH2-terminal kinase 1/2. J Pharmacol Exp Ther 325(3):841–849. doi: 10.1124/jpet.107.135442 CrossRefPubMedGoogle Scholar
  35. 35.
    Chen YJ, Kay N, Yang JM, Lin CT, Chang HL, Wu YC, Fu CF, Chang Y, Lo S, Hou MF, Lee YC, Hsieh YC, Yuan SS (2013) Total synthetic protoapigenone WYC02 inhibits cervical cancer cell proliferation and tumour growth through PIK3 signalling pathway. Basic Clin Pharmacol Toxicol 113(1):8–18. doi: 10.1111/bcpt.12057 CrossRefPubMedGoogle Scholar
  36. 36.
    Chen YJ, Cheng YJ, Hung AC, Wu YC, Hou MF, Tyan YC, Yuan SS (2013) The synthetic flavonoid WYC02-9 inhibits cervical cancer cell migration/invasion and angiogenesis via MAPK14 signaling. Gynecol Oncol 131(3):734–743. doi: 10.1016/j.ygyno.2013.10.012 CrossRefPubMedGoogle Scholar
  37. 37.
    Chen J (2014) Reactive oxygen species and drug resistance in cancer chemotherapy. Austin J Clin Pathol 1(4):1017Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Tijana Stanković
    • 1
  • Balázs Dankó
    • 2
  • Ana Martins
    • 3
    • 4
  • Miodrag Dragoj
    • 1
  • Sonja Stojković
    • 1
  • Aleksandra Isaković
    • 5
  • Hui-Chun Wang
    • 6
  • Yang-Chang Wu
    • 6
    • 7
    • 8
    • 9
  • Attila Hunyadi
    • 2
  • Milica Pešić
    • 1
  1. 1.Institute for Biological Research, Department of NeurobiologyUniversity of BelgradeBelgradeSerbia
  2. 2.Institute of PharmacognosyUniversity of SzegedSzegedHungary
  3. 3.Department of Medical Microbiology and ImmunobiologyUniversity of SzegedSzegedHungary
  4. 4.Unidade de Parasitologia e Microbiologia Médica, Institute of Hygiene and Tropical MedicineUniversidade Nova de LisboaLisbonPortugal
  5. 5.Faculty of MedicineUniversity of BelgradeBelgradeSerbia
  6. 6.Graduate Institute of Natural ProductsKaohsiung Medical UniversityKaohsiungTaiwan
  7. 7.School of Pharmacy, College of PharmacyChina Medical UniversityTaichungTaiwan
  8. 8.Chinese Medicine Research and Development CenterChina Medical University HospitalTaichungTaiwan
  9. 9.Center for Molecular MedicineChina Medical University HospitalTaichungTaiwan

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