Induction of apoptosis in vitro and in vivo effects of a synthetic nitroflavone derivative on murine mammary adenocarcinoma cells

CÁRDENAS, M; BLANK, V; ZOTTA, E; MARDER, M; ROGUIN, L

San Antonio, Texas, USA

Simposio; 31st Annual CTRC-AACR San Antonio Breast Cancer Symposium; 2008

American Asociation of Cancer Research

Induction of apoptosis in vitro and in vivo effects of a synthetic nitroflavone derivative on murine mammary adenocarcinoma cells. Cárdenas, M.*; Blank, V.*; Zotta, E.#; Marder, M.*; Roguin, L.* *IQUIFIB (UBA-CONICET), School of Pharmacy and Biochemistry, University of Buenos Aires and #, Department of Physiology, School of Medicine, University of Buenos Aires, Argentina. Background: Flavonoids are polyphenolic compounds found in dietary components of vegetable origin. They exhibit multiple biological activities, such as antiproliferative, anxiolytic, antiallergic, antiangiogenic and antioxidant actions. We have previously demonstrated that the synthetic flavone 2´-nitroflavone (2´-NF) exerts a strong antimitogenic activity in various human and murine tumor cell lines, without disturbing the proliferation of non-neoplasic cells. In order to elucidate the mechanism of antitumor action, we first studied the in vitro ability of 2´-NF to induce apoptosis in a cell line derived from a murine mammary tumor (LM3), and then investigated its in vivo growth inhibitory effects. Methods and results: Different experimental assays were performed to evaluate the in vitro apoptotic response of 2´-NF. Thus, when LM3 cells were treated in the presence or absence of 20 µM of 2´-NF, an increment in the proportion of hypodiploid cells was evident after 24 and 48 h of incubation. A typical DNA ladder fragmentation pattern was visualized by agarose gel electrophoresis and an increment in the total expression of the pro-apoptotic Bax protein was obtained by western blot after 24 hours of exposure to 2´-NF. Caspase 3 activity was also determined by fluorescence detection of the cleaved substrate Ac-DEVD-AMC after 24 hours of treatment. In order to study the in vivo antitumor action, female BALB/c mice were first injected subcutaneously with 300000 LM3 cells. After 7 days, 1, 10 or 40 mg/kg of 2´-NF dissolved in 0.2 ml of vehicle (5% DMSO, 0.05% Tween-80, NaCl 0.15 M) were administrated i.p. twice a week for 3 weeks. During the treatment, tumor sizes were measured every 3 days and, at the end of the treatment, tumors were excised, measured and weighted. On average, treated-mice tumor volume was reduced between 70% (p<0.01) and 43% (p<0.05) with 10 and 40 mg/kg doses, respectively. No significant volume reduction was observed with 1mg/kg dose. Besides, tumors weight decreased approximately 50% (p<0.05) both with 10 and 40 mg/kg doses. TUNEL assay performed on tumor slices from treated (10mg/kg dose) and non treated mice showed that 2´NF induces apoptosis on mammary tumors. In addition, western blot analyses of tumor lysate supernatants revealed an upregulation of the total levels of Bax and Fas receptor proteins in 2´-NF treated tumors with a concomitant downregulation of the antiapoptotic protein Bcl-2. Finally, toxicity studies performed in different organ-tissues from animals injected with 40 mg/kg of 2´-NF showed no histological alterations. Conclusion: Taken together, our results suggest the potential therapeutic use of 2´-NF as an antitumor agent, capable of inducing apoptosis both in vitro and in vivo in breast cancer cells without generating systemic toxic effects.