Induction of cell cycle arrest and apoptosis by a combined treatment with 13-cis-retinoic acid, interferon-α2a, and α-tocopherol in squamous cell carcinoma of the head and neck

Background. We have previously conducted phase 11 trials with a combination of 13-cis-retinoic acid (13-cRA), interferon-alpha 2a (IFN-alpha 2a), and alpha-tocopherol (alpha-TF) in patients with advanced oral premalignant lesions and locally advanced head and neck cancer in the adjuvant settings and achieved promising outcomes. The present study was conducted in vitro to elucidate the mechanisms of anti-tumor activity of this 3-drug combination in squamous cell carcinoma of the head and neck (SCCHN). Methods. Five SCCHN cell lines were treated with 13-cRA, IFN-alpha 2a, and alpha-TF as single agents or 2- to 3-drug combinations for 72 hours. Inhibition of cell growth and cell cycle progression and induction of apoptosis by the treatments were evaluated. Results. Our results demonstrated that although each single-agent and 2-drug combination showed a certain level of cell growth inhibition, the 3-drug combination apparently further inhibited cell growth in comparison to any single agents and 2-drug combinations in the 5 SCCHN cell lines. Cell cycle analysis on Tu212 and 886LN cells by flow cytometry exhibited significant accumulation of the cells at S phase in the 3-drug combination. On the other hand, Annexin-V binding assay demonstrated that the 3-drug combination induced more profound apoptosis than any of the single agents or 2-drug combinations. In parallel, proteolytic cleavages of pro-caspase-8, -9, -3 and poly (ADP ribose) polymerase as well as caspase-3 activity induced by the 3-drug treatment were observed. Conclusions. Our data suggests that 3-drug combination biochemopreventive regimen has cooperative inhibitory effect on the growth of SCCHN cells. Both cell cycle arrest and apoptosis contribute to cell growth inhibition of this 3-drug combination therapy. (c) 2006 Wiley Periodicals, Inc.