Nitric oxide contributes to adriamycin's antitumor effect.
Recently, several antitumor drugs have been shown to stimulate nitric oxide (NO) production.


OBJECTIVE
To determine if adriamycin induces NO production in breast cancer cells in vitro and whether NO contributes to adriamycin's antitumor effect in vivo.


METHODS
Murine breast cancer cells (EMT-6) were incubated with adriamycin (ADRIA, 0, 10, 100, 1000 microM) in the presence or absence of the NO synthase inhibitor aminoguanidine (AG, 1 mM). Twenty-four hours later nitrite accumulation (Greiss reagent) and cell viability (MTT assay) were assessed. Supernatants from adriamycin-stimulated cells were also analyzed at 6, 8, and 24 hr for TNF, IL-1, and IFN gamma (ELISA). For in vivo experiments, 10(5) EMT-6 cells were injected into the flank of BALB/c mice (n = 20) and 1 hr later mice received one of four treatments: (1) saline, (2) ADRIA (10 mg/kg ip), (3) AG (100 mg/kg sc BID), or (4) ADRIA (10 mg/kg ip) and AG (100 mg/kg sc BID). Two weeks later tumor size was measured and in situ tumor cell apoptosis was determined by fluorescent microscopy and flow cytometry.


RESULTS
Adriamycin was cytotoxic to EMT-6 cells with 100 microM resulting in nearly 100% killing (P < 0.01). Adriamycin also stimulated nitrite accumulation with 100 microM producing 6.5 +/- 0.26 microM nitrite (P < 0.001). AG blocked adriamycin-stimulated nitrite accumulation (P < 0.05), but did not inhibit cytotoxicity in vitro. In vivo, adriamycin inhibited tumor size by nearly 400% (P < 0.001), while AG attenuated adriamycin's effect on tumor growth (P < 0.05). There was no difference in the detection of apoptotic tumor cells between the adriamycin and adriamycin and AG groups as determined by immunohistochemistry and flow cytometry.


CONCLUSIONS
These findings suggest that adriamycin stimulated NO production in EMT-6 cells, but adriamycin's cytotoxicity in vitro was NO-independent. In vivo, adriamycin inhibited tumorigenesis partially via an NO-dependent, nonapoptotic mechanism.
Lind
D S
DS
Department of Surgery, University of Florida, Gainesville 32610, USA.
Kontaridis
M I
MI
Edwards
P D
PD
Josephs
M D
MD
Moldawer
L L
LL
Copeland
E M
EM
D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D004317:Doxorubicin / Q000494:pharmacology*; D007371:Interferon-gamma / Q000502:physiology; D007375:Interleukin-1 / Q000502:physiology; D008325:Mammary Neoplasms, Experimental; D051379:Mice; D009569:Nitric Oxide / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology
D000903:Antibiotics, Antineoplastic; D007375:Interleukin-1; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D004317:Doxorubicin; D007371:Interferon-gamma; D019001:Nitric Oxide Synthase