Growth inhibitory effect of a combined treatment of 13-cis-retinoic acid, interferon-α2a and α-tocopherol on squamous cell carcinoma of the head and neck

Proc Amer Assoc Cancer Res, Volume 46, 2005 2466 Our previous phase II clinical trails with the combination of 13-cis-retinoic acid (R), interferon-α2a (I), and α-tocopherol (T) as a biochemoprevention regimen in patients with advanced oral premalignant lesions and as a bioadjuvant treatment to patients with locally advanced head and neck cancer have shown promising outcomes. The purpose of this study is to elucidate the anti-cancer mechanism of these three agents in squamous cell carcinoma of the head and neck (SCCHN) in vitro . Five head and neck cancer cell lines (886LN, SQCCY1, 38, Tu212, and Tu177) were used for this study. Cell growth inhibition assay showed that three drugs in single agent treatment for 72 hours inhibited growth of all five SCCHN cell lines in a dose dependent manner. The combination treatment of R (1μM), I (200 IU/ml) and T (15 μM) for 72 hours achieved cooperative effects of cell growth inhibition as compared with any two-drug combinations or single agents in the same dosage on all five cell lines. Tu212 and 886LN were then selected to further investigate the underlying mechanisms of the three-drug combination using the same dosages on SCCHN. Annexin-V binding assay by flow cytometry demonstrated that two-drug combined treatments of R-I, R-T, and I-T for 72 hours, in 886LN cell line, more effectively induced apoptosis (22.5 ± 6.3%, 27.5 ± 8.1%, and 18.4 ± 5.3 %, respectively) than the single agents of R (15.8 ± 5.4%), I (16 ± 3.4%), T (14.3 ± 5.3 %) or control group (8.2 ± 0.3%). However, the three-drug combination even further induced apoptosis (35.8 ± 8.2%). Similar result was also observed in Tu212 cell line. In addition, cell cycle analysis showed apparent S phase arrest after treatment of Tu212 cells with the three-drug combination for 24 hours (42.7%) and 48 hours (43.7%) as compared with that in control group (34.6%). Apoptosis related proteins were also examined by immunoblotting analysis. It was shown that even though two-drug combinations or single agent treatments for 72 hours induced cleavages of caspase-8, -9, -3, and PARP in both Tu212 and 886LN cell lines; the three-drug combination further increased the cleavages of these proteins. Caspase-3 activity assay was performed to confirm these observations using 886LN cell line. Each two-drug combination moderately induced caspase-3 activation as compared with either control or single agent treatments. More importantly, the three-drug combination showed much stronger caspase-3 activity than the two-drug combinations of R-I (p = 0.007), R-T (p = 0.08), I-T (p = 0.001). Taken together, our results suggest that the three-drug combination has a cooperative effect on tumor cell inhibition compared to single agents or any two-drug combinations. The anti-tumor activity may be exerted through both cell cycle arrest at S phase and apoptosis related pathways. (Supported by NCI 2 RO1 75603 to D.M.S)