METHODS: The apoptotic effects of
tempo were examined by the flow cytometric analysis of cells labeled with
fluorescein isothiocyanate-conjugated
annexin-V, and by electron microscopy. Enzymatic assays were performed to measure the activities of 2
cysteine proteases, i.e.,
caspase-9 and
caspase-3, in
tempo-treated cells. The effects of
tempo on cell proliferation and on cell cycle distribution profiles were measured by the flow cytometric assay using immunofluorescent staining of incorporated 5'-bromo-2'-deoxyuridine (
BrdU) coupled with
7-amino-actinomycin D (7-AAD) staining of total
DNA. The number of proliferating cells was also determined independently by
enzyme-linked
immunosorbent assay using chemiluminescent detection of incorporated
BrdU. Subcutaneous growth of human prostate
carcinoma in athymic mice was monitored after intratumoral administration of
tempo into
tumor-bearing mice. In addition, cell viability assays were performed to compare the cytotoxic effect of a combination of
doxorubicin or
mitoxantrone and
tempo with single agents.
RESULTS:
Tempo treatment of prostate
carcinoma cells caused a significant increase in the number of apoptotic cells compared with control groups (
tempo, 2.5 mM, 24 hours: DU-145, approximately 3.4-fold; PC-3, approximately 6-7-fold;
tempo 1 mM, 24 hours: LNCaP, approximately 12-fold).
Tempo-induced loss of cell viability was blocked partially or completely after pretreatment of cells with
actinomycin-D or
cycloheximide, suggesting a de novo macromolecule synthesis-dependent mechanism of cell death. Electron microscopy revealed aggregation and marginalization of
chromatin in the nuclei of a large number of
tempo-treated LNCaP cells.
Tempo treatment of LNCaP cells resulted in enhanced activities of
caspase-9 (
tempo, 5 mM, 15 hours: approximately 2-fold) and
caspase-3 (
tempo, 2.5 mM, 24 hours: approximately 12-fold).
Tempo treatment also led to an enhanced number of cells in G2/M phase of the cell cycle (
tempo, 5.0 mM, 24 hours: DU-145, approximately 1.6-fold; PC-3, approximately 1.5-fold; LNCaP, approximately 5.3-fold), and decreased
BrdU incorporation indicative of a decline in the number of proliferating cells (
tempo, 2.5 mM, 24 or 48 hours; DU-145, approximately 2-3-fold; PC-3, approximately 1.2-fold; LNCaP, approximately 5-10-fold). Administration of
tempo into LNCaP
tumor-bearing mice resulted in a significant inhibition of
tumor growth (percent initial
tumor volume [Day 30, n = 4]: vehicle, 845.35 +/- 272.83;
tempo, 9.72 +/- 9.72;
tempo vs. vehicle, P < 0.02). In
hormone-refractory prostate
carcinoma cells, a combination of relatively low doses of
tempo and
doxorubicin or
mitoxantrone caused enhanced cytotoxicity as compared with single agents.
CONCLUSIONS: These data demonstrated that
nitroxide tempo induced apoptosis and activated a
caspase-mediated signaling pathway in prostate
carcinoma cells.
Tempo treatment also caused cell cycle arrest in G2/M phase and decreased the number of proliferating cells (S phase).
Tempo treatment of
tumor-bearing mice led to inhibition of
tumor growth, suggesting that
tempo is a novel member of the small-molecule family of
antineoplastic agents.