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Cancer Chemotherapy and Pharmacology

, Volume 53, Issue 3, pp 267–275 | Cite as

Phenothiazines suppress proliferation and induce apoptosis in cultured leukemic cells without any influence on the viability of normal lymphocytes

Phenothiazines and leukemia
  • Zhivko Zhelev
  • Hideki Ohba
  • Rumiana Bakalova
  • Vera Hadjimitova
  • Mitsuru Ishikawa
  • Yasuo Shinohara
  • Yoshinobu Baba
Original Article

Abstract

Purpose

The purpose of the present study was to investigate the effects of phenothiazines (at clinically relevant doses) on the viability and proliferation of leukemic cell lines and normal lymphocytes, and to investigate the possibility of specific induction of apoptosis in leukemic cells.

Methods

Phenothiazines with different chemical structure and hydrophobicity were used: chlorpromazine (CPZ); levomepromazine (LVPZ); prometazine (PMZ); trifluoperazine (TFPZ); thioridazine (TRDZ). The leukemic cell lines used were: Daudi and Raji (derived from Burkitt’s lymphoma), K-562 (derived from myelogenous leukemia), and BALL-1, MOLT-4, HPB-ALL and CCRF-HSB-2 (derived from acute lymphoblastic leukemia). The cytotoxicity of the phenothiazines was determined by a CellTiter-Glo luminescent cell viability assay, using ATP bioluminescence as a marker of cell viability as well as a marker of mitochondrial activity. The proliferation of leukemic cells was determined using a CellTiter-AQ cell proliferation assay which is based on the reduction of a methyl-tetrazolium compound to the formazan product. Apoptosis induction was estimated using phosphatidylserine (PSer) translocation to the cell surface and DNA fragmentation as characteristics of the process.

Results

Phenothiazines (at concentrations in the range 0.1–10 μM) did not affect the viability of normal lymphocytes during a 24-h incubation. Moreover, about 15–20% increase in ATP bioluminescence was observed in normal cells during treatment with 40 μM phenothiazines. In contrast, the phenothiazines manifested strong cytotoxicity and antiproliferative activity against leukemic cells. The most powerful drugs were TFPZ and TRDZ, followed by CPZ. They showed a significant cytotoxic effect against leukemic cells even at 5–10 μM. The most sensitive cell lines were MOLT-4 and Raji, and the most resistant were HPB-ALL and CCRF-HSB-2. All phenothiazines induced PSer exposure on the surface of leukemic cells, but not of normal lymphocytes. TFPZ, TRDZ and CPZ also induced DNA fragmentation in almost all leukemic cell lines during a 48-h incubation. The strongest apoptotic agent was TRDZ. The apoptosis induction was not accompanied by a significant release of cytochrome c from the mitochondria into the cytoplasm of native cells. Moreover, the drugs markedly suppressed Ca2+-induced cytochrome c release in isolated mitochondria of leukemic cells.

Conclusions

The results suggest that in clinically relevant doses (up to 20 μM) some phenothiazines (TFPZ, TRDZ, CPZ) expressed a selective cytotoxicity and antiproliferative activity, and induced apoptosis in leukemic cells without any influence on the viability of normal lymphocytes. It is considered that the mechanism of apoptosis induction in phenothiazine-treated leukemic cells is associated with inhibition of mitochondrial DNA polymerase and decreased ATP production, which are crucial events for the viability of cancer cells.

Keywords

Cultured leukemic cells Normal lymphocytes Phenothiazines Cytotoxicity Proliferation Apoptosis 

Abbreviations

CPZ

Chlorpromazine

LVPZ

Levomepromazine

PBS(−)

Phosphate-buffered saline (Ca2+- and Mg2+-free)

PMZ

Prometazine

PSer

Phosphatidylserine

ROS

Reactive oxygen species

TFPZ

Trifluoperazine

TRDZ

Thioridazine

Notes

Acknowledgements

The technical assistance of Mrs Kawahara of the AIST-Kyushu is gratefully acknowledged. This study was supported in part by the JSPS Invitation Fellowship Program for Research in Japan.

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Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Zhivko Zhelev
    • 1
  • Hideki Ohba
    • 1
  • Rumiana Bakalova
    • 1
  • Vera Hadjimitova
    • 2
  • Mitsuru Ishikawa
    • 1
  • Yasuo Shinohara
    • 1
    • 3
  • Yoshinobu Baba
    • 1
    • 3
  1. 1.Single-Molecule Bioanalysis LaboratoryNational Institute for Advanced Industrial Science and Technology, AIST-ShikokuTakamatsuJapan
  2. 2.Department of Medical Physics and BiophysicsMedical UniversitySofiaBulgaria
  3. 3.Faculty of Pharmaceutical SciencesTokushima UniversityTokushimaJapan

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