Nitroxide tempo, a small molecule, induces apoptosis in prostate carcinoma cells and suppresses tumor growth in athymic mice
Authors: Suy Simeng , Mitchell James B. , Samuni Ayelet , Mueller Susette , Kasid Usha 
Published: January 2005

BACKGROUND

In previous studies, nitroxide tempo (2, 2, 6, 6‐tetramethyl‐piperidine‐1‐oxyl), a small molecule, induced cell death in cancer cells. The current study examined the antineoplastic properties of tempo in the human hormone‐dependent/hormone‐independent prostate carcinoma models (LNCaP, DU‐145, and PC‐3).

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. Cancer 2005. © 2005 American Cancer Society.