Phenothiazines suppress proliferation and induce apoptosis in cultured leukemic cells without any influence on the viability of normal lymphocytes. Phenothiazines and leukemia.
OBJECTIVE
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 micro M) 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 micro M 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 micro M. 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 Ca(2+)-induced cytochrome c release in isolated mitochondria of leukemic cells.


CONCLUSIONS
The results suggest that in clinically relevant doses (up to 20 micro M) 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.
Zhelev
Zhivko
Z
National Institute for Advanced Industrial Science and Technology, AIST-Shikoku, 2217-14 Hayashi-cho, 761-0395 Takamatsu, Kagawa, Japan.
Ohba
Hideki
H
Bakalova
Rumiana
R
Hadjimitova
Vera
V
Ishikawa
Mitsuru
M
Shinohara
Yasuo
Y
Baba
Yoshinobu
Y
D000255:Adenosine Triphosphate / Q000032:analysis; D000970:Antineoplastic Agents / Q000627:therapeutic use / Q000633:toxicity*; D017209:Apoptosis; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D045304:Cytochromes c / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007938:Leukemia / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D008163:Luminescent Measurements; D008214:Lymphocytes / Q000187:drug effects; D010640:Phenothiazines / Q000627:therapeutic use / Q000633:toxicity*
D000970:Antineoplastic Agents; D010640:Phenothiazines; D000255:Adenosine Triphosphate; D045304:Cytochromes c