cytochrome-c-t and 2--nitroflavone

We explored the in vitro and in vivo mechanism of antitumor action of the synthetic flavonoid 2'-nitroflavone on LM3 murine mammary adenocarcinoma cells. In vitro assays showed that 2'-nitroflavone increased the population of LM3 hypodiploid cells and produced a typical ladder of DNA fragmentation. Apoptotic cell death was also characterized by the activation of caspase-8, -9 and -3, by an increment in the expression levels of the proapoptotic protein Bax and by the release of cytochrome c to cytosol. The in vivo effect of 2'-nitroflavone on tumor growth was studied in BALB/c mice injected subcutaneously with LM3 cells. Results showed that tumor volume and weight were significantly reduced at doses of 10 and 40 mg/kg of 2'-nitroflavone, respectively. Apoptotic cells were identified by TUNEL assay in tumor slices from mice treated with 10 mg/kg of 2'-nitroflavone. Western blot analysis of tumor lysate supernatants from treated mice revealed an upregulation of the total levels of Bax and Fas receptor. In addition, administration of 40 mg/kg of 2'-nitroflavone to nontumor-bearing mice showed no histopathological effects on different organ tissues. This is the first report of the in vivo growth inhibitory effect of 2'-nitroflavone as an apoptotic agent likely useful for mammary adenocarcinoma treatment.

Topics: Adenocarcinoma; Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Caspases; Cell Line, Tumor; Cytochromes c; Female; Flavones; Flow Cytometry; In Situ Nick-End Labeling; In Vitro Techniques; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Proto-Oncogene Proteins c-bcl-2; Xenograft Model Antitumor Assays

The mechanism of antitumor action of a synthetic nitroflavone derivative, 2'-nitroflavone, was evaluated in vitro in HeLa human cervix adenocarcinoma cells. We showed that the nitroflavone derivative slowed down the cell cycle at the S phase and increase the population of cells at the G2/M phase after 24h of incubation. The treatment with 2'-nitroflavone also induced an apoptotic response, characterized by an increase of the sub-G1 fraction of cells, by cells with chromatin condensation and membrane blebbing, by a typical ladder of DNA fragmentation and by detection of apoptotic cells stained with Annexin V. The observed apoptosis was regulated by caspase-8 and -9, both contributing to the activation of the effector caspase-3. In addition, inhibitors of caspase-8 or -9 partially protected HeLa cells from 2'-nitroflavone-induced cell death. We also found that 2'-nitroflavone did not affect the total amount of Bax and Bcl-2 proteins, although a translocation of Bax from cytosol to mitochondria was evident after 6h of exposure. Furthermore, 2'-nitroflavone decreased the expression of the anti-apoptotic Bcl-XL protein, induced the release of cytochrome C to cytosol and increased the levels of Fas and Fas-L. Our results indicated that both death receptor and mitochondria-dependent pathways are involved in the apoptotic cell death triggered by 2'-nitroflavone and suggest that this derivative could be a potentially useful agent for the treatment of certain malignancies.

Topics: Apoptosis; Caspases; Cell Cycle; Cytochromes c; Fas Ligand Protein; Female; Flavones; HeLa Cells; Humans; Oligopeptides; Proto-Oncogene Proteins c-bcl-2; Uterine Cervical Neoplasms