Cellular Oncology

, Volume 42, Issue 2, pp 157–171 | Cite as

Nanoquinacrine sensitizes 5-FU-resistant cervical cancer stem-like cells by down-regulating Nectin-4 via ADAM-17 mediated NOTCH deregulation

  • Anmada Nayak
  • Sarita Das
  • Deepika Nayak
  • Chinmayee Sethy
  • Satya Narayan
  • Chanakya Nath KunduEmail author
Original Paper



Cervical cancer is a major cause of cancer-related death in women world-wide. Although the anti-metabolite 5-FU is widely used for its treatment, its clinical utility is limited due to the frequent occurrence of drug resistance during metastasis. Cancer stem-like cells (CSCs), present in the heterogeneous population of CC cells, are thought to contribute to this resistance. Nectin-4, a CSC marker, is known to play an important role in the cellular aggressiveness associated with metastatic CC. This study was designed to assess the role of Nectin-4 in the acquisition of 5-FU resistance by metastatic CC cells, including its relation to the NOTCH signalling pathway.


5FU-resistant CC cell lines were deduced from ME-180 and SiHA cells by continuous exposure to a single concentration of 5-FU. Thymidylate synthase (TS) positive cells were isolated from the 5-FU resistant cells, after which a metastatic model was developed. The role of Nectin-4 in the sensitization of 5-FU resistant metastatic CC cells upon incubation with Nano-formulated Quinacrine (NQC) was investigated using multiple bioassays including MTT, FACS, ELISA, immunoflurescence, Western blotting, comet and in vivo plasmid-based short patch and long patch base excision repair assays.


We found that the expression level of Nectin-4, as well as that of other CSC markers (Oct-4, β-catenin, SOX2) and representative NOTCH signalling components (NOTCH-1, Jagged-1, γ-secretase, ADAM-17) were elevated in the 5-FU resistant metastatic cells compared to those in control cells. Increased nuclear translocation of Nectin-4 and increased proliferation and invasion rates were observed after culturing the metastatic cells under hypoxic conditions. Treatment with NQC inhibited the nuclear translocation of Nectin-4 and decreased the proliferation and invasion rates of the cells by inhibiting the induction of base excision repair (BER) pathway components and ADAM-17 expression levels. After combination treatment of Nectin-4 overexpressing metastatic CC cells with a specific ADAM-17 inhibitor (GW280264) and NQC, a decreased Nectin-4 expression, without alterations in BER and/or other NOTCH pathway components, was noted.


Our data indicate that Nectin-4 may play a prominent role in 5-FU resistance of metastatic CC cells and that NQC sensitizes these cells by Nectin-4 deregulation through ADAM-17 inhibition, a major component of the NOTCH signalling pathway.


Cervical cancer Cancer stem cells 5-FU resistance Nectin-4 Metastasis 



We are very thankful to the Indian Council of Medical Research (ICMR), Government of India for providing a fellowship to AN. The authors sincerely thank Mark Zakshevsky, Department of Anatomy and Cell Biology, University of Florida, Gainesville, Florida and Dr. C. R. Patil, Professor and Head, Department of Pharmacology, R. C. Patel Institute of Pharmaceutical Education and Research, India for carefully proofreading the manuscript.

Author contribution

AN performed most experiments and analysed the data. SD, DN and CS performed the statistical analyses. CNK conceived the hypothesis, analysed the data and wrote the manuscript. SN modified the manuscript and edited the English grammar.


This study was funded by the Indian Council of Medical Research, Govt. of India (ref number- ICMR-35/22/2012-BMS).

Compliance with ethical standards

Conflict of interest

All authors declare no conflict of interest.


  1. 1.
    L. Wasim, M. Chopra, Synergistic anticancer effect of panobinostat and topoisomerase inhibitors through ROS generation and intrinsic apoptotic pathway induction in cervical cancer cells. Cell Oncol 41, 201–212 (2018)CrossRefGoogle Scholar
  2. 2.
    A. Sathyanarayanan, K.S. Chandrasekaran, D. Karunagaran, microRNA-145 modulates epithelial-mesenchymal transition and suppresses proliferation, migration and invasion by targeting SIP1 in human cervical cancer cells. Cell Oncol 40, 119–131 (2017)CrossRefGoogle Scholar
  3. 3.
    Y. Osaka, M. Shinohara, S. Hoshino, T. Ogata, Y. Takagi, A. Tsuchida, T. Aoki, Phase II study of combined chemotherapy with docetaxel, CDDP and 5-FU for highly advanced esophageal cancer. Anticancer Res 31, 633–638 (2011)Google Scholar
  4. 4.
    S. Hemaiswarya, M. Doble, Combination of phenylpropanoids with 5-fluorouracil as anti-cancer agents against human cervical cancer (HeLa) cell line. Phytomedicine 20, 151–158 (2013)CrossRefGoogle Scholar
  5. 5.
    M. Nagata, H. Nakayama, T. Tanaka, R. Yoshida, Y. Yoshitake, D. Fukuma, K. Kawahara, Y. Nakagawa, K. Ota, A. Hiraki, M. Shinohara, Overexpression of cIAP2 contributes to 5-FU resistance and a poor prognosis in oral squamous cell carcinoma. Br J Cancer 105, 1322–1330 (2011)CrossRefGoogle Scholar
  6. 6.
    R. Mori, M. Futamura, T. Tanahashi, Y. Tanaka, N. Matsuhashi, K. Yamaguchi, K. Yoshida, 5FU resistance caused by reduced fluoro-deoxyuridine monophosphate and its reversal using deoxyuridine. Oncol Lett 14, 3162–3168 (2017)CrossRefGoogle Scholar
  7. 7.
    L. Zhang, R. Song, D. Gu, X. Zhang, B. Yu, B. Liu, J. Xie, The role of GLI1 for 5-Fu resistance in colorectal cancer. Cell Biosci 7, 17 (2017)CrossRefGoogle Scholar
  8. 8.
    A. Rengaraj, B. Subbiah, Y. Haldorai, D. Yesudhas, H.J.G. Yun, S. Kwon, Y.K. Sangdun Choi, E.S. Han, H.S.N. Kim, Y.S. Huh, PAMAM/5-fluorouracil drug conjugate for targeting E6 and E7 oncoproteins in cervical cancer: A combined experimental/in silico approach. RSC Adv 7, 5046–5054 (2017)Google Scholar
  9. 9.
    P. Dhawan, R. Ahmad, R. Chaturvedi, J.J. Smith, R. Midha, M.K. Mittal, M. Krishnan, X. Chen, S. Eschrich, T.J. Yeatman, R.C. Harris, M.K. Washington, K.T. Wilson, R.D. Beauchamp, A.B. Singh, Claudin-2 expression increases tumorigenicity of colon cancer cells: Role of epidermal growth factor receptor activation. Oncogene 30, 3234–3247 (2011)CrossRefGoogle Scholar
  10. 10.
    Y. Takai, J. Miyoshi, W. Ikeda, H. Ogita, Nectins and nectin-like molecules: Roles in contact inhibition of cell movement and proliferation. Nat Rev Mol Cell Biol 9, 603–615 (2008)CrossRefGoogle Scholar
  11. 11.
    M.S. Derycke, S.E. Pambuccian, C.B. Gilks, S.E. Kalloger, A. Ghidouche, M. Lopez, R.L. Bliss, M.A. Geller, P.A. Argenta, K.M. Harrington, A.P. Skubitz Nectin, 4 overexpression in ovarian cancer tissues and serum: Potential role as a serum biomarker. Am J Clin Pathol 134, 835–845 (2010)CrossRefGoogle Scholar
  12. 12.
    A. Takano, N. Ishikawa, R. Nishino, K. Masuda, W. Yasui, K. Inai, H. Nishimura, H. Ito, H. Nakayama, Y. Miyagi, E. Tsuchiya, N. Kohno, Y. Nakamura, Y. Daigo, Identification of nectin-4 oncoprotein as a diagnostic and therapeutic target for lung cancer. Cancer Res 69, 6694–6703 (2009)CrossRefGoogle Scholar
  13. 13.
    S. Fabre-Lafay, S. Garrido-Urbani, N. Reymond, A. Goncalves, P. Dubreuil, M. Lopez, Nectin-4, a new serological breast cancer marker, is a substrate for tumor necrosis factoralpha-converting enzyme (TACE)/ADAM-17. J Biol Chem 280, 19543–19550 (2005)CrossRefGoogle Scholar
  14. 14.
    D. Das, S.R. Satapathy, S. Siddharth, A. Nayak, C.N. Kundu, NECTIN-4 increased the 5-FU resistance in colon cancer cells by inducing the PI3K–AKT cascade. Cancer Chemother Pharmacol 76, 471–479 (2015)CrossRefGoogle Scholar
  15. 15.
    S. Nishiwada, M. Sho, S. Yasuda, K .Shimada, I. Yamato, T. Akahori, S. Kinoshita, M. Nagai, N. Konishi, Y. Nakajima, Nectin-4 expression contributes to tumor proliferation, angiogenesis and patient prognosis in human pancreatic cancer. J Exp Clin Cancer Res 34, 30 (2015)Google Scholar
  16. 16.
    Y. Zhang, S. Liu, L. Wang, Y. Wu, J. Hao, Z. Wang, W. Lu, X.A. Wang, F. Zhang, Y. Cao, H. Liang, H. Li, Y. Ye, Q. Ma, S. Zhao, Y. Shu, R. Bao, L. Jiang, Y. Hu, J. Zhou, L. Chen, Y. Liu, A novel PI3K/AKT signalling axis mediates Nectin-4-induced gallbladder cancer cell proliferation, metastasis and tumor growth. Cancer Lett 375, 179–189 (2016)CrossRefGoogle Scholar
  17. 17.
    S. Siddharth, K. Goutam, S. Das, A. Nayak, D. Nayak, C. Sethy, M.D. Wyatt, C.N. Kundu, Nectin-4 is a breast cancer stem cell marker that induces WNT/β-catenin signalling via Pi3k/Akt axis. Int J Biochem Cell Biol 89, 85–94 (2017)CrossRefGoogle Scholar
  18. 18.
    E.S. Nabih, F.I. Abdel Motaleb, F.A. Salama, The diagnostic efficacy of nectin 4 expression in ovarian cancer patients. Biomarker 19, 498–504 (2014)CrossRefGoogle Scholar
  19. 19.
    D. Das, R. Preet, P. Mohapatra, S.R. Satapathy, C.N. Kundu, 1, 3-Bis (2-chloroethyl)-1-nitrosourea enhances the inhibitory effect of resveratrol on 5-fluorouracil sensitive/resistant colon cancer cells. World J Gastroenterol 19, 7374–7388 (2013)CrossRefGoogle Scholar
  20. 20.
    Q.E. Wang, DNA damage responses in cancer stem cells: Implications for cancer therapeutic strategies. World J Biol Chem 6, 57–64 (2015)CrossRefGoogle Scholar
  21. 21.
    M. Maugeri-Saccà, M. Bartucci, R. De Maria, DNA damage repair pathways in cancer stem cells. Mol Cancer Ther 11, 1627–1636 (2012)CrossRefGoogle Scholar
  22. 22.
    A.J. Groot, M.A. Vooijs, The role of Adams in notch signalling. Adv Exp Med Biol 727, 15–36 (2012)CrossRefGoogle Scholar
  23. 23.
    E.R. Andersson, R. Sandberg, U. Lendahl, Notch signalling: Simplicity in design, versatility in function. Development 138, 3593–3612 (2011)CrossRefGoogle Scholar
  24. 24.
    Z. Guo, X. Jin, H. Jia, Inhibition of ADAM-17 more effectively down-regulates the notch pathway than that of γ-secretase in renal carcinoma. J Exp Clin Cancer Res 32, 26 (2013)CrossRefGoogle Scholar
  25. 25.
    A. Baumgart, S. Seidl, P. Vlachou, L. Michel, N. Mitova, N. Schatz, K. Specht, I. Koch, T. Schuster, R. Grundler, M. Kremer, F. Fend, J.T. Siveke, C. Peschel, J. Duyster, T. Dechow, ADAM17 regulates epidermal growth factor receptor expression through the activation of Notch1 in non-small cell lung cancer. Cancer Res 70, 5368–5378 (2010)CrossRefGoogle Scholar
  26. 26.
    A. Sommer, F. Kordowski, J. Büch, T. Maretzky, A. Evers, J. Andrä, S. Düsterhöft, M. Michalek, I. Lorenzen, P. Somasundaram, A. Tholey, F.D. Sönnichsen, K. Kunzelmann, L. Heinbockel, C. Nehls, T. Gutsmann, J. Grötzinger, S. Bhakdi, K. Reiss, Phosphatidylserine exposure is required for ADAM17 sheddase function. Nat Commun 7, 11523 (2016)Google Scholar
  27. 27.
    A. Nayak, S. Siddharth, S. Das, D. Nayak, C. Sethy, C.N. Kundu, Nanoquinacrine caused apoptosis in oral cancer stem cells by disrupting the interaction between GLI1 and β catenin through activation of GSK3β. Toxicol Appl Pharmacol 330, 53–64 (2017)CrossRefGoogle Scholar
  28. 28.
    R. Preet, P. Mohapatra, D. Das, S.R. Satapathy, T. Choudhuri, M.D. Wyatt, C.N. Kundu, Lycopene synergistically enhances quinacrine action to inhibit Wnt-TCF signalling in breast cancer cells through APC. Carcinogenesis 34, 277–286 (2013)CrossRefGoogle Scholar
  29. 29.
    S.R. Satapathy, S. Siddharth, D. Das, A. Nayak, C.N.Kundu enhancement of cytotoxicity and inhibition of angiogenesis in Oral Cancer stem cells by a hybrid nanoparticle of bioactive Quinacrine and silver: Implication of base excision repair Cascade. Mol Pharm 12, 4011–4025 (2015)CrossRefGoogle Scholar
  30. 30.
    S. Siddharth, S. Das, A. Nayak, C.N. Kundu, SURVIVIN as a marker for quiescent-breast cancer stem cells-an intermediate, adherent, pre-requisite phase of breast cancer metastasis. Clin Exp Metastasis 33, 661–675 (2016)CrossRefGoogle Scholar
  31. 31.
    A. Nayak, S.R. Satapathy, D. Das, S. Siddharth, N. Tripathi, P.V. Bharatam, C. Kundu, Nanoquinacrine induced apoptosis in cervical cancer stem cells through the inhibition of hedgehog-GLI1 cascade: Role of GLI-1. Sci Rep 6, 20600 (2016)Google Scholar
  32. 32.
    S.S. Virtanen, T. Ishizu, J.A. Sandholm, E. Löyttyniemi, H.K. Väänänen, J.M. Tuomela, P.L. Härkönen, Alendronate-induced disruption of actin cytoskeleton and inhibition of migration/invasion are associated with cofilin downregulation in PC-3 prostate cancer cells. Oncotarget 9, 32593–32608 (2018)CrossRefGoogle Scholar
  33. 33.
    C.N. Kundu, R. Balusu, A.S. Jaiswal, C.G. Gairola, S. Narayan, Cigarette smokecondensate-induced level of adenomatous polyposis coli blocks long-patchbase excision repair in breast epithelial cells. Oncogene 26, 1428–1438 (2007)CrossRefGoogle Scholar
  34. 34.
    K.L. Boylan, P.C. Buchanan, R.D. Manion, D.M. Shukla, K. Braumberger, C. Bruggemeyer, A.P. Skubitz, The expression of Nectin-4 on the surface of ovarian cancer cells alters their ability to adhere, migrate, aggregate, and proliferate. Oncotarget 8, 9717–9738 (2017)CrossRefGoogle Scholar
  35. 35.
    S. Fabre-Lafay, F. Monville, S. Garrido-Urbani, C. Berruyer-Pouyet, C. Ginestier, N. Reymond, P. Finetti, R. Sauvan, J. Adélaïde, J. Geneix, E. Lecocq, C. Popovici, P. Dubreuil, P. Viens, A. Gonçalves, E. Charafe-Jauffret, J. Jacquemier, D. Birnbaum, M. Lopez, Nectin-4 is a new histological and serological tumor associated marker for breast cancer. BMC Cancer 7, 73 (2007)Google Scholar
  36. 36.
    Y. Huang, N. Benaich, C. Tape, H.F. Kwok, G. Murphy, Targeting the sheddase activity of ADAM17 by an anti-ADAM17 antibody D1(A12) inhibits head and neck squamous cellcarcinoma cell proliferation and motility via blockage of bradykinin induced HERs transactivation. Int J Biol Sci 10, 702–714 (2014)CrossRefGoogle Scholar
  37. 37.
    T. Rzymski, A. Petry, D. Kračun, F. Rieß, L. Pike, A.L. Harris, A. Görlach, The unfolded protein response controls induction and activation of ADAM17/TACE by severe hypoxia and ER stress. Oncogene 31, 3621–3634 (2012)CrossRefGoogle Scholar
  38. 38.
    K.M. Capaccione, S.R. Pine, The notch signalling pathway as a mediator of tumorsurvival. Carcinogenesis 34, 1420–1430 (2013)CrossRefGoogle Scholar
  39. 39.
    N.N. Pavlova, C. Pallasch, A.E. Elia, C.J. Braun, T.F. Westbrook, M. Hemann, S. Elledge, A role for PVRL4-driven cell-cell interactions in tumorigenesis. eLife 2, e00358 (2013)CrossRefGoogle Scholar
  40. 40.
    W.H. Matsui, Cancer stem cell signalling pathways. Medicine (Baltimore) 95, S8–S19 (2016)CrossRefGoogle Scholar

Copyright information

© International Society for Cellular Oncology 2019

Authors and Affiliations

  • Anmada Nayak
    • 1
  • Sarita Das
    • 1
  • Deepika Nayak
    • 1
  • Chinmayee Sethy
    • 1
  • Satya Narayan
    • 2
  • Chanakya Nath Kundu
    • 1
    Email author
  1. 1.Cancer Biology Division, KIIT School of BiotechnologyKIIT UniversityBhubanesarIndia
  2. 2.Department of Anatomy and Cell Biology, College of MedicineUniversity of FloridaGainesvilleUSA

Personalised recommendations