Cancer Chemotherapy and Pharmacology

, Volume 56, Issue 6, pp 615–622 | Cite as

Micromolar taxol, with or without hyperthermia, induces mitotic catastrophe and cell necrosis in HeLa cells

  • John Michalakis
  • Spyros D. Georgatos
  • John Romanos
  • Helen Koutala
  • Vassilis Georgoulias
  • Dimitris Tsiftsis
  • Panayiotis A. Theodoropoulos
Original Article

Abstract

Purpose: Although the mode of action of taxol, when used in nanomolar or micromolar concentrations during long periods, is extensively studied, there are few data available on taxol-mediated cytotoxicity when the drug is applied for a short time alone or in combination with hyperthermia. We studied the effect of taxol and hyperthermia on cell cycle kinetics, proliferation, and mode of cell death in human cervical carcinoma HeLa cells, following a scheme which resembles the one currently used in regional chemotherapy. methods: Cells were incubated with micromolar doses of taxol for two h under normothermic or hyperthermic conditions and then cultured in drug-free medium for several days. Cell viability was assessed via an MTT assay. Necrotic and apoptotic cell death was determined using Trypan blue staining and TUNNEL assay, respectively. Flow cytometry was used for the analysis of cell cycle kinetics and the counting of apoptotic cells. Mitotic index, nuclear morphology and nuclear envelope organization were analyzed by fluorescence microscopy. results: Cells exposed to micromolar doses of taxol for 2 h and then transferred to a drug-free medium for 24 h were arrested at G2/M or M phase. When treated cells were cultured in normal media for longer periods, most of them remained in a tetraploid state, became multinucleated without properly completing cytokinesis and died mostly by necrosis. Hyperthermia alone exerted a cytotoxic effect, inhibited proliferation and caused minor changes in cell cycle kinetics. When combined with taxol treatment, hyperthermia modified the cell cycle-arresting effects of the drug, but did not alter significantly taxol-mediated cytotoxicity. conclusions: From these data we conclude that short time incubation of HeLa cells under normothermic or hyperthermic conditions with micromolar concentrations of taxol is sufficient enough to induce extended cell growth arrest and cell death by necrosis.

Keywords

Taxol Hyperthermia Cell necrosis Cell cycle 

Notes

Acknowledgements

This work was supported by PENED 2002 from the Greek Secretariat of Research and Technology (P.A.T), the Cretan Association for Biomedical Research, PEVE (P.A.T. and S.D.G.) and the fifth Framework project “Kinesins and disease” (S.D.G.). We thank Prof. Elias Castanas for helpful suggestions and comments on the manuscript.

References

  1. 1.
    Abal M, Souto AA, Amat-Guerri F, Acuna AU, Andreu JM, Barasoain I (2001) Centrosome and spindle pole microtubules are main targets of a fluorescent taxoid inducing cell death. Cell Motil Cytoskeleton 49:1–15Google Scholar
  2. 2.
    Arnal I, Wade RH (1995) How does taxol stabilize microtubules? Curr Biol 5:900–908Google Scholar
  3. 3.
    Blajeski AL, Kottke TJ, Kaufmann SH (2001) A multistep model for paclitaxel-induced apoptosis in human breast cancer cell lines. Exp Cell Res 270:277–288Google Scholar
  4. 4.
    Bogdan C, Ding A (1992) Taxol, a microtubule-stabilizing antineoplastic agent, induces expression of tumor necrosis factor alpha and interleukin-1 in macrophages. J Leukoc Biol 52:119–121Google Scholar
  5. 5.
    Chan TA, Hermeking H, Lengauer C, Kinzler KW, Vogelstein B (1999) 14–3-3Sigma is required to prevent mitotic catastrophe after DNA damage. Nature 401:616–620Google Scholar
  6. 6.
    Chen JG, Yang CP, Cammer M, Horwitz SB (2003) Gene expression and mitotic exit induced by microtubule-stabilizing drugs. Cancer Res 63:7891–7899Google Scholar
  7. 7.
    Cividalli A, Cruciani G, Livdi E, Pasqualetti P, Tirindelli Danesi D (1999) Hyperthermia enhances the response of paclitaxel and radiation in a mouse adenocarcinoma. Int J Radiat Oncol Biol Phys 44:407–412Google Scholar
  8. 8.
    Cividalli A, Livdi E, Ceciarelli F, Piscitelli M, Pasqualetti P, Cruciani G, Danesi DT (2000) Hyperthermia and paclitaxel–epirubicin chemotherapy: enhanced cytotoxic effect in a murine mammary adenocarcinoma. Int J Hyperthermia 16:61–71Google Scholar
  9. 9.
    Danesi R, Figg WD, Reed E, Myers CE (1995) Paclitaxel (taxol) inhibits protein isoprenylation and induces apoptosis in PC-3 human prostate cancer cells. Mol Pharmacol 47:1106–1111Google Scholar
  10. 10.
    de Bree E, Romanos J, Michalakis J, Relakis K, Georgoulias V, Melissas J, Tsiftsis DD (2003) Intraoperative hyperthermic intraperitoneal chemotherapy with docetaxel as second-line treatment for peritoneal carcinomatosis of gynaecological origin. Anticancer Res 23:3019–3027Google Scholar
  11. 11.
    de Bree E, Rosing H, Beijnen JH, Romanos J, Michalakis J, Georgoulias V, Tsiftsis DD (2003) Pharmacokinetic study of docetaxel in intraoperative hyperthermic i.p. chemotherapy for ovarian cancer. Anticancer Drugs 14:103–110Google Scholar
  12. 12.
    Ding AH, Porteu F, Sanchez E, Nathan CF (1990) Shared actions of endotoxin and taxol on TNF receptors and TNF release. Science 248:370–372Google Scholar
  13. 13.
    Erenpreisa JE, Ivanov A, Dekena G, Vitina A, Krampe R, Freivalds T, Selivanova G, Roach HI (2000) Arrest in metaphase and anatomy of mitotic catastrophe: mild heat shock in two human osteosarcoma cell lines. Cell Biol Int 24:61–70Google Scholar
  14. 14.
    Jordan MA, Toso RJ, Thrower D, Wilson L (1993) Mechanism of mitotic block and inhibition of cell proliferation by taxol at low concentrations. Proc Natl Acad Sci USA 90:9552–9556Google Scholar
  15. 15.
    Jordan MA, Wendell K, Gardiner S, Derry WB, Copp H, Wilson L (1996) Mitotic block induced in HeLa cells by low concentrations of paclitaxel (Taxol) results in abnormal mitotic exit and apoptotic cell death. Cancer Res 56:816–825Google Scholar
  16. 16.
    Lane HA, Nigg EA (1996) Antibody microinjection reveals an essential role for human polo-like kinase 1 (Plk1) in the functional maturation of mitotic centrosomes. J Cell Biol 135:1701–1713Google Scholar
  17. 17.
    Leal BZ, Meltz ML, Mohan N, Kuhn J, Prihoda TJ, Herman TS (1999) Interaction of hyperthermia with Taxol in human MCF-7 breast adenocarcinoma cells. Int J Hyperthermia 15:225–236Google Scholar
  18. 18.
    Lee LF, Schuerer-Maly CC, Lofquist AK, van Haaften-Day C, Ting JP, White CM, Martin BK, Haskill JS (1996) Taxol-dependent transcriptional activation of IL-8 expression in a subset of human ovarian cancer. Cancer Res 56:1303–1308Google Scholar
  19. 19.
    Mickey B, Howard J (1995) Rigidity of microtubules is increased by stabilizing agents. J Cell Biol 130:909–917Google Scholar
  20. 20.
    Mohamed F, Marchettini P, Stuart OA, Urano M, Sugarbaker PH (2003) Thermal enhancement of new chemotherapeutic agents at moderate hyperthermia. Ann Surg Oncol 10:463–468Google Scholar
  21. 21.
    Moos PJ, Fitzpatrick FA (1998) Taxane-mediated gene induction is independent of microtubule stabilization: induction of transcription regulators and enzymes that modulate inflammation and apoptosis. Proc Natl Acad Sci USA 95:3896–3901Google Scholar
  22. 22.
    Othman T, Goto S, Lee JB, Taimura A, Matsumoto T, Kosaka M (2001) Hyperthermic enhancement of the apoptotic and antiproliferative activities of paclitaxel. Pharmacology 62:208–212Google Scholar
  23. 23.
    Rietbroek RC, Katschinski DM, Reijers MH, Robins HI, Geerdink A, Tutsch K, d’Oleire F, Haveman J (1997) Lack of thermal enhancement for taxanes in vitro. Int J Hyperthermia 13:525–533Google Scholar
  24. 24.
    Rowinsky EK, Donehower RC (1995) Paclitaxel (taxol). N Engl J Med 332:1004–1014Google Scholar
  25. 25.
    Schrump DS, Zhai S, Nguyen DM, Weiser TS, Fisher BA, Terrill RE, Flynn BM, Duray PH, Figg WD (2002) Pharmacokinetics of paclitaxel administered by hyperthermic retrograde isolated lung perfusion techniques. J Thorac Cardiovasc Surg 123:686–694Google Scholar
  26. 26.
    Sharma D, Chelvi TP, Kaur J, Ralhan R (1998) Thermosensitive liposomal taxol formulation: heat-mediated targeted drug delivery in murine melanoma. Melanoma Res 8:240–244Google Scholar
  27. 27.
    Theodoropoulos PA, Polioudaki H, Kostaki O, Derdas SP, Georgoulias V, Dargemont C, Georgatos SD (1999) Taxol affects nuclear lamina and pore complex organization and inhibits import of karyophilic proteins into the cell nucleus. Cancer Res 59:4625–4633Google Scholar
  28. 28.
    van Bree C, Savonije JH, Franken NA, Haveman J, Bakker PJ (2000) The effect of p53-function on the sensitivity to paclitaxel with or without hyperthermia in human colorectal carcinoma cells. Int J Oncol 16:739–744Google Scholar
  29. 29.
    Woods CM, Zhu J, McQueney PA, Bollag D, Lazarides E (1995) Taxol-induced mitotic block triggers rapid onset of a p53-independent apoptotic pathway. Mol Med 1:506–526Google Scholar
  30. 30.
    Yeung TK, Germond C, Chen X, Wang Z (1999) The mode of action of taxol: apoptosis at low concentration and necrosis at high concentration. Biochem Biophys Res Commun 263:398–404Google Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • John Michalakis
    • 1
  • Spyros D. Georgatos
    • 2
  • John Romanos
    • 1
  • Helen Koutala
    • 3
  • Vassilis Georgoulias
    • 4
  • Dimitris Tsiftsis
    • 1
  • Panayiotis A. Theodoropoulos
    • 3
  1. 1.Department of Surgical Oncology, School of MedicineThe University of CreteHeraklionGreece
  2. 2.Laboratory of Biology, School of MedicineThe University of IoanninaIoanninaGreece
  3. 3.Department of Biochemistry, School of MedicineThe University of Crete71 003HeraklionGreece
  4. 4.Department of Medical Oncology, School of MedicineThe University of CreteHeraklionGreece

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