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Nitric oxide contributes to adriamycin's antitumor effect
D S Lind1, M I Kontaridis, P D Edwards
1Department of Surgery, University of Florida, Gainesville 32610, USA.
Unlabelled:
Recently, several antitumor drugs have been shown to stimulate nitric oxide (NO) production.
Purpose:
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.
Insights
Adriamycin stimulates nitric oxide (NO) production in breast cancer cells. While this NO production is not essential for adriamycin
Area of Science:
- Cancer Research
- Pharmacology
- Immunology
Background:
- Several anticancer drugs are known to enhance nitric oxide (NO) generation.
- The role of NO in the efficacy of these drugs is an area of active investigation.
Purpose of the Study:
- To investigate whether adriamycin induces NO production in breast cancer cells in vitro.
- To determine if NO contributes to the antitumor effects of adriamycin in vivo.
Main Methods:
- Murine breast cancer cells (EMT-6) were treated with adriamycin (ADRIA) and/or the NO synthase inhibitor aminoguanidine (AG).
- Nitrite accumulation and cell viability were assessed in vitro.
- Tumor growth and apoptosis were evaluated in vivo after ADRIA and/or AG treatment in mice bearing EMT-6 tumors.
Main Results:
- Adriamycin demonstrated significant cytotoxicity against EMT-6 cells in vitro.
- Adriamycin treatment led to increased nitrite accumulation, indicating NO production, which was inhibited by AG.
- In vivo, adriamycin significantly reduced tumor size, an effect partially dependent on NO, but apoptosis levels were similar with or without AG.
Conclusions:
- Adriamycin stimulates NO production in breast cancer cells, but this NO production is not required for its direct cytotoxic effect in vitro.
- The in vivo antitumor activity of adriamycin is partly mediated by an NO-dependent, non-apoptotic pathway.