Induction of caspase 3 activity, bcl-2 bax and p65 gene expression modulation in human acute promyelocytic leukemia HL-60 cells by doxorubicin with amifostine.
The influence of amifostine alone and in combination with doxorubicin, cytarabine, and etoposide on the cell growth and on bcl-2, bax and p65 gene expression was investigated in human acute promyelocytic leukemia cell line HL-60. No or very little influence of the exposure of HL-60 cells to amifostine (10(-6) to 10(-2) M) on cell proliferation was shown. Proliferation of HL-60 cells exposed to doxorubicin, cytarabine, or etoposide dropped down with increasing doses of these drugs. Only in the case of doxorubicin, more effective inhibition of HL-60 cell growth was observed when combination of doxorubicin, cytarabine or etoposide with amifostine was used. Cytotoxic effect of cytarabine or etoposide was not reduced by amifostine. The lowering of the cytotoxic index (IC50) was observed only when HL-60 cells were preincubated with amifostine followed by doxorubicin treatment. IC50 was estimated as 2.1 x 10(-7) M and 0.9 x 10(-7) M for doxorubicin and doxorubicin with amifostine, respectively. This effect was accompanied by the induction of caspase 3 activity. HL-60 cells treated with doxorubicin alone showed about 35-fold increase in caspase 3 activity. The enzyme activity was stimulated by combination of doxorubicin with amifostine up to 94 times. Furthermore, the expression of bcl-2 and bax genes involved in apoptosis as well as tumor-associated p65 gene were determined. Semiquantitative reverse transcriptase polymerase chain reaction showed a decrease in bcl-2 and an increase in bax and p65 expression in HL-60 cells treated with doxorubicin in combination with amifostine when compared with the cells treated only with doxorubicin. Amifostine may potentiate doxorubicin therapeutic efficiency in human acute promyelocytic leukemia cells.
Department of Pharmaceutical Biochemistry, Molecular Biology Laboratory, Medical University of Łódź, Muszyńskiego 1, PL 90-151 Łódź, Poland.
Marek
M
Rózalski
Marek
M
Mirowski
Ewa
E
Balcerczak
Urszula
U
Krajewska
Wojciech
W
Młynarski
Ryszard
R
Wierzbicki
D004999:Amifostine / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D003561:Cytarabine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000494:pharmacology*; D004357:Drug Synergism; D005786:Gene Expression Regulation / Q000187:drug effects*; D018922:HL-60 Cells / Q000187:drug effects; D006801:Humans; D020128:Inhibitory Concentration 50; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D051996:Transcription Factor RelA / Q000235:genetics / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism
D000970:Antineoplastic Agents; C494826:BAX protein, human; D019253:Proto-Oncogene Proteins c-bcl-2; D051996:Transcription Factor RelA; D051028:bcl-2-Associated X Protein; D003561:Cytarabine; D004317:Doxorubicin; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D004999:Amifostine