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Cyclin-dependent kinase inhibitors, roscovitine and purvalanol, induce apoptosis and autophagy related to unfolded protein response in HeLa cervical cancer cells

  • Pelin Ozfiliz-Kilbas
  • Bahar Sarikaya
  • Pinar Obakan-Yerlikaya
  • Ajda Coker-Gurkan
  • Elif Damla Arisan
  • Benan Temizci
  • Narcin Palavan-Unsal
Original Article

Abstract

Roscovitine (Rosc) and purvalanol (Pur) are competitive inhibitors of cyclin-dependent kinases (CDKs) by targeting their ATP-binding pockets. Both drugs are shown to be effective to decrease cell viability and dysregulate the ratio of pro- and anti-apoptotic Bcl-2 family members, which finally led to apoptotic cell death in different cancer cell lines in vitro. It was well established that Bcl-2 family members have distinct roles in the regulation of other cellular processes such as endoplasmic reticulum (ER) stress. The induction of ER stress has been shown to play critical role in cell death/survival decision via autophagy or apoptosis. In this study, our aim was to investigate the molecular targets of CDK inhibitors on ER stress mechanism related to distinct cell death types in time-dependent manner in HeLa cervical cancer cells. Our results showed that Rosc and Pur decreased the cell viability, cell growth and colony formation, induced ER stress-mediated autophagy or apoptosis in time-dependent manner. Thus, we conclude that exposure of cells to CDK inhibitors induces unfolded protein response and ER stress leading to autophagy and apoptosis processes in HeLa cervical cancer cells.

Keywords

Roscovitine Purvalanol Cervical cancer ER stress Autophagy Apoptosis 

Notes

Acknowledgements

This study was supported by Istanbul Kultur University Scientific Projects Support Center and TUBITAK (The Scientific and Technological Research Council of Turkey) 2209 Program. The team was Gozde Sukur, Bahar Sarıkaya, Kubra Tugtekin and Ezgi Gultekin. We are thankful for technical assistance to Gozde Sukur, Kubra Tugtekin and Ezgi Gultekin for Figs. 1a, b and 3b, c. Pelin Ozfiliz-Kilbas and Bahar Sarıkaya were involved in all figures. The data obtained by Benan Temizci was repeated by Bahar Sarıkaya. The project was designed by Elif Damla Arisan and Narcin Palavan-Unsal. Ajda Coker-Gurkan and Pınar Obakan-Yerlikaya interpreted the data and were involved in the writing of the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical statement

We declare that this article does not involve any studies with human participants or animals performed by any of the authors. The research has been performed on commercially available cell lines. All authors read and confirmed the final version of the manuscript.

Supplementary material

11033_2018_4222_MOESM1_ESM.tif (429 kb)
Supplemental figure. CHOP promoter (-649/+136) pmCherry-1 plasmid was visualized on 0.8% agarose gel. M: 1kb marker, 1: (-649/+136) pmCherry-1 plasmid (TIF 428 KB)

References

  1. 1.
    Visagie MH, Jaiswal SR, Joubert AM (2016) In vitro assessment of a computer-designed potential anticancer agent in cervical cancer cells. Biol Res 49(1):43CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Malumbres M, Barbacid M (2005) Mammalian cyclin-dependent kinases. Trends Biochem Sci 30(11):630–641CrossRefPubMedGoogle Scholar
  3. 3.
    Bhattacharya S, Ray RM, Johnson LR (2014) Cyclin-dependent kinases regulate apoptosis of intestinal epithelial cells. Apoptosis 19(3):451–466CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Whittaker SR, Te Poele RH, Chan F, Linardopoulos S, Walton MI, Garrett MD, Workman P (2007) The cyclin-dependent kinase inhibitor seliciclib (R-roscovitine; CYC202) decreases the expression of mitotic control genes and prevents entry into mitosis. Cell Cycle 6(24):3114–3131CrossRefPubMedGoogle Scholar
  5. 5.
    Wesierska-Gadek J, Wandl S, Kramer MP, Pickem C, Krystof V, Hajek SB (2008) Roscovitine up-regulates p53 protein and induces apoptosis in human HeLaS(3) cervix carcinoma cells. J Cell Biochem 105(5):1161–1171CrossRefPubMedGoogle Scholar
  6. 6.
    Coker-Gurkan A, Arisan ED, Obakan P, Ozfiliz P, Kose B, Bickici G, Palavan-Unsal N (2015) Roscovitine-treated HeLa cells finalize autophagy later than apoptosis by downregulating Bcl2. Mol Med Rep 11(3):1968–1974CrossRefPubMedGoogle Scholar
  7. 7.
    Bach S, Knockaert M, Reinhardt J, Lozach O, Schmitt S, Baratte B, Koken M, Coburn SP, Tang L, Jiang T, Liang DC, Galons H, Dierick JF, Pinna LA, Meggio F, Totzke F, Schachtele C, Lerman AS, Carnero A, Wan Y, Gray N, Meijer L (2005) Roscovitine targets, protein kinases and pyridoxal kinase. J Biol Chem 280(35):31208–31219CrossRefPubMedGoogle Scholar
  8. 8.
    Kolodziej M, Goetz C, Di Fazio P, Montalbano R, Ocker M, Strik H, Quint K (2015) Roscovitine has anti-proliferative and pro-apoptotic effects on glioblastoma cell lines: a pilot study. Oncol Rep 34(3):1549–1556CrossRefPubMedGoogle Scholar
  9. 9.
    Gary C, Hajek M, Biktasova A, Bellinger G, Yarbrough WG, Issaeva N (2016) Selective antitumor activity of roscovitine in head and neck cancer. Oncotarget 7(25):38598–38611CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Obakan P, Arisan ED, Ozfiliz P, Coker-Gurkan A, Palavan-Unsal N (2014) Purvalanol A is a strong apoptotic inducer via activating polyamine catabolic pathway in MCF-7 estrogen receptor positive breast cancer cells. Mol Biol Rep 41(1):145–154CrossRefPubMedGoogle Scholar
  11. 11.
    Coker-Gurkan A, Arisan ED, Obakan P, Akalin K, Ozbey U, Palavan-Unsal N (2015) Purvalanol induces endoplasmic reticulum stress-mediated apoptosis and autophagy in a time-dependent manner in HCT116 colon cancer cells. Oncol Rep 33(6):2761–2770CrossRefPubMedGoogle Scholar
  12. 12.
    Berrak O, Arisan ED, Obakan-Yerlikaya P, Coker-Gurkan A, Palavan-Unsal N (2016) mTOR is a fine tuning molecule in CDK inhibitors-induced distinct cell death mechanisms via PI3K/AKT/mTOR signaling axis in prostate cancer cells. Apoptosis 21(10):1158–1178CrossRefPubMedGoogle Scholar
  13. 13.
    Obakan-Yerlikaya P, Arisan ED, Coker-Gurkan A, Adacan K, Ozbey U, Somuncu B, Baran D, Palavan-Unsal N (2017) Calreticulin is a fine tuning molecule in epibrassinolide-induced apoptosis through activating endoplasmic reticulum stress in colon cancer cells. Mol Carcinog 56(6):1603–1619CrossRefPubMedGoogle Scholar
  14. 14.
    Rashid HO, Yadav RK, Kim HR, Chae HJ (2015) ER stress: Autophagy induction, inhibition and selection. Autophagy 11(11):1956–1977CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Luo B, Lee AS (2013) The critical roles of endoplasmic reticulum chaperones and unfolded protein response in tumorigenesis and anticancer therapies. Oncogene 32(7):805–818CrossRefPubMedGoogle Scholar
  16. 16.
    Deldicque L (2013) Endoplasmic reticulum stress in human skeletal muscle: any contribution to sarcopenia? Front Physiol 4:236CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Cuervo AM (2004) Autophagy: in sickness and in health. Trends Cell Biol 14(2):70–77CrossRefPubMedGoogle Scholar
  18. 18.
    Mizushima N, Klionsky DJ (2007) Protein turnover via autophagy: implications for metabolism. Annu Rev Nutr 27:19–40CrossRefPubMedGoogle Scholar
  19. 19.
    Huang J, Dibble CC, Matsuzaki M, Manning BD (2008) The TSC1-TSC2 complex is required for proper activation of mTOR complex 2. Mol Cell Biol 28(12):4104–4115CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    McManus S, Bisson M, Chamberland R, Roy M, Nazari S, Roux S (2016) Autophagy and 3-phosphoinositide-dependent kinase 1 (PDK1)-related kinome in pagetic osteoclasts. J Bone Miner Res 31(7):1334–1343CrossRefPubMedGoogle Scholar
  21. 21.
    He Q, Lee DI, Rong R, Yu M, Luo X, Klein M, El-Deiry WS, Huang Y, Hussain A, Sheikh MS (2002) Endoplasmic reticulum calcium pool depletion-induced apoptosis is coupled with activation of the death receptor 5 pathway. Oncogene 21(17):2623–2633CrossRefPubMedGoogle Scholar
  22. 22.
    Yoshida T, Shiraishi T, Horinaka M, Wakada M, Sakai T (2007) Glycosylation modulates TRAIL-R1/death receptor 4 protein: different regulations of two pro-apoptotic receptors for TRAIL by tunicamycin. Oncol Rep 18(5):1239–1242PubMedGoogle Scholar
  23. 23.
    Wu Y, Fabritius M, Ip C (2009) Chemotherapeutic sensitization by endoplasmic reticulum stress: increasing the efficacy of taxane against prostate cancer. Cancer Biol Ther 8(2):146–152CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Coker-Gurkan A, Arisan ED, Obakan P, Guvenir E, Unsal NP (2014) Inhibition of autophagy by 3-MA potentiates purvalanol-induced apoptosis in Bax deficient HCT 116 colon cancer cells. Exp Cell Res 328(1):87–98CrossRefPubMedGoogle Scholar
  25. 25.
    Sutherland RL, Musgrove EA (2009) CDK inhibitors as potential breast cancer therapeutics: new evidence for enhanced efficacy in ER + disease. Breast Cancer Res 11(6):112CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Appleyard MV, O’Neill MA, Murray KE, Paulin FE, Bray SE, Kernohan NM, Levison DA, Lane DP, Thompson AM (2009) Seliciclib (CYC202, R-roscovitine) enhances the antitumor effect of doxorubicin in vivo in a breast cancer xenograft model. Int J Cancer 124(2):465–472CrossRefPubMedGoogle Scholar
  27. 27.
    Cihalova D, Hofman J, Ceckova M, Staud F (2013) Purvalanol A olomoucine II and roscovitine inhibit ABCB1 transporter and synergistically potentiate cytotoxic effects of daunorubicin in vitro. PLoS ONE 8(12):e83467CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Wesierska-Gadek J, Borza A, Walzi E, Krystof V, Maurer M, Komina O, Wandl S (2009) Outcome of treatment of human HeLa cervical cancer cells with roscovitine strongly depends on the dosage and cell cycle status prior to the treatment. J Cell Biochem 106(5):937–955CrossRefPubMedGoogle Scholar
  29. 29.
    Cui C, Wang Y, Wang Y, Zhao M, Peng S (2013) Exploring the relationship between the inhibition selectivity and the apoptosis of roscovitine-treated cancer cells. J Anal Methods Chem 2013:389390PubMedPubMedCentralGoogle Scholar
  30. 30.
    Song H, Vita M, Sallam H, Tehranchi R, Nilsson C, Siden A, Hassan Z (2007) Effect of the Cdk-inhibitor roscovitine on mouse hematopoietic progenitors in vivo and in vitro. Cancer Chemother Pharmacol 60(6):841–849CrossRefPubMedGoogle Scholar
  31. 31.
    Nair BC, Vallabhaneni S, Tekmal RR, Vadlamudi RK (2011) Roscovitine confers tumor suppressive effect on therapy-resistant breast tumor cells. Breast Cancer Res 13(3):R80CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Du J, Widlund HR, Horstmann MA, Ramaswamy S, Ross K, Huber WE, Nishimura EK, Golub TR, Fisher DE (2004) Critical role of CDK2 for melanoma growth linked to its melanocyte-specific transcriptional regulation by MITF. Cancer Cell 6(6):565–576CrossRefPubMedGoogle Scholar
  33. 33.
    Hikita T, Oneyama C, Okada M, Purvalanol A (2010) a CDK inhibitor, effectively suppresses Src-mediated transformation by inhibiting both CDKs and c-Src. Genes Cells 15(10):1051–1062CrossRefPubMedGoogle Scholar
  34. 34.
    Rodic S, Vincent MD (2018) Reactive oxygen species (ROS) are a key determinant of cancer’s metabolic phenotype. Int J Cancer 142(3):440–448CrossRefPubMedGoogle Scholar
  35. 35.
    Franco J, Balaji U, Freinkman E, Witkiewicz AK, Knudsen ES (2016) Metabolic reprogramming of pancreatic cancer mediated by CDK4/6 inhibition elicits unique vulnerabilities. Cell Rep 14(5):979–990CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Arisan ED, Obakan P, Coker-Gurkan A, Calcabrini A, Agostinelli E, Unsal NP (2014) CDK inhibitors induce mitochondria-mediated apoptosis through the activation of polyamine catabolic pathway in LNCaP, DU145 and PC3 prostate cancer cells. Curr Pharm Des 20(2):180–188CrossRefPubMedGoogle Scholar
  37. 37.
    Zeeshan HM, Lee GH, Kim HR, Chae HJ (2016) Endoplasmic reticulum stress and associated ROS. Int J Mol Sci 17(3):327CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Kramer B, Ferrari DM, Klappa P, Pohlmann N, Soling HD (2001) Functional roles and efficiencies of the thioredoxin boxes of calcium-binding proteins 1 and 2 in protein folding. Biochem J 357(Pt1):83–95CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Walter P, Ron D (2011) The unfolded protein response: from stress pathway to homeostatic regulation. Science 334(6059):1081–1086CrossRefPubMedGoogle Scholar
  40. 40.
    Zhong F, Xie J, Zhang D, Han Y, Wang C (2015) Polypeptide from Chlamys farreri suppresses ultraviolet-B irradiation-induced apoptosis through restoring ER redox homeostasis, scavenging ROS generation, and suppressing the PERK-eIF2a-CHOP pathway in HaCaT cells. J Photochem Photobiol B 151:10–16CrossRefPubMedGoogle Scholar
  41. 41.
    Schroder M, Kaufman RJ (2005) ER stress and the unfolded protein response. Mutat Res 569(1–2):29–63CrossRefPubMedGoogle Scholar
  42. 42.
    Bravo R, Parra V, Gatica D, Rodriguez AE, Torrealba N, Paredes F, Wang ZV, Zorzano A, Hill JA, Jaimovich E, Quest AF, Lavandero S (2013) Endoplasmic reticulum and the unfolded protein response: dynamics and metabolic integration. Int Rev Cell Mol Biol 301:215–290CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Dkhissi F, Raynal S, Lawrence DA (1999) Altered complex formation between p21waf, p27kip and their partner G1 cyclins determines the stimulatory or inhibitory transforming growth factor-beta1 growth response of human fibroblasts. Int J Oncol 14(5):905–910PubMedGoogle Scholar
  44. 44.
    Han C, Jin L, Mei Y, Wu M (2013) Endoplasmic reticulum stress inhibits cell cycle progression via induction of p27 in melanoma cells. Cell Signal 25(1):144–149CrossRefPubMedGoogle Scholar
  45. 45.
    Kitzmann M, Fernandez A (2001) Crosstalk between cell cycle regulators and the myogenic factor MyoD in skeletal myoblasts. Cell Mol Life Sci 58(4):571–579CrossRefPubMedGoogle Scholar
  46. 46.
    Shenkman M, Tolchinsky S, Kondratyev M, Lederkremer GZ (2007) Transient arrest in proteasomal degradation during inhibition of translation in the unfolded protein response. Biochem J 404(3):509–516CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Li Y, Guo Y, Tang J, Jiang J, Chen Z (2015) New insights into the roles of CHOP-induced apoptosis in ER stress. Acta Biochim Biophys Sin (Shanghai) 47(2):146–147CrossRefGoogle Scholar
  48. 48.
    Tabas I, Ron D (2011) Integrating the mechanisms of apoptosis induced by endoplasmic reticulum stress. Nat Cell Biol 13(3):184–190CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Mihailidou C, Papavassiliou AG, Kiaris H (2014) A crosstalk between p21 and UPR-induced transcription factor C/EBP homologous protein (CHOP) linked to type 2 diabetes. Biochimie 99:19–27CrossRefPubMedGoogle Scholar
  50. 50.
    Rasheva VI, Domingos PM (2009) Cellular responses to endoplasmic reticulum stress and apoptosis. Apoptosis 14(8):996–1007CrossRefPubMedGoogle Scholar
  51. 51.
    Chandrika BB, Yang C, Ou Y, Feng X, Muhoza D, Holmes AF, Theus S, Deshmukh S, Haun RS, Kaushal GP (2015) Endoplasmic reticulum stress-induced autophagy provides cytoprotection from chemical hypoxia and oxidant injury and ameliorates renal ischemia-reperfusion injury. PLoS ONE 10(10):e0140025CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Zhang N, Ji N, Jiang WM, Li ZY, Wang M, Wen JM, Li Y, Chen X, Chen JM (2015) Hypoxia-induced autophagy promotes human prostate stromal cells survival and ER-stress. Biochem Biophys Res Commun 464(4):1107–1112CrossRefPubMedGoogle Scholar
  53. 53.
    B’Chir W, Maurin AC, Carraro V, Averous J, Jousse C, Muranishi Y, Parry L, Stepien G, Fafournoux P, Bruhat A (2013) The eIF2alpha/ATF4 pathway is essential for stress-induced autophagy gene expression. Nucleic Acids Res 41(16):7683–7699CrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    White E, DiPaola RS (2009) The double-edged sword of autophagy modulation in cancer. Clin Cancer Res 15(17):5308–5316CrossRefPubMedPubMedCentralGoogle Scholar
  55. 55.
    Kulkarni YM, Kaushik V, Azad N, Wright C, Rojanasakul Y, O’Doherty G, Iyer AK (2016) Autophagy-induced apoptosis in lung cancer cells by a novel digitoxin analog. J Cell Physiol 231(4):817–828CrossRefPubMedPubMedCentralGoogle Scholar
  56. 56.
    B’Chir W, Chaveroux C, Carraro V, Averous J, Maurin AC, Jousse C, Muranishi Y, Parry L, Fafournoux P, Bruhat A (2014) Dual role for CHOP in the crosstalk between autophagy and apoptosis to determine cell fate in response to amino acid deprivation. Cell Signal 26(7):1385–1391CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.Department of Molecular Biology and GeneticsIstanbul Kultur UniversityIstanbulTurkey

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