The combination of perifosine and TRAIL exhibits enhanced effects on decreasing cell survival ( A ), inducing apoptosis ( B ) and inhibiting colony formation ( C ) in HNSCC cell lines and augments xenograft growth inhibition ( D ). A, The indicated cell lines were seeded in 96-well plates and the next day treated with the indicated concentrations of TRAIL alone, perifosine (PRFS) alone, or their respective combinations (P + T). After 24 h, the cells were subjected to estimation of cell number using the SRB assay. The data are means ± SD of four replicate determinations. B, M4e cells were treated with PBS control, 5 μM perifosine, 10 ng/ml TRAIL and the combination of perifosine and TRAIL. After 24 h, apoptotic (sub-G1) cells were measured by flow cytometry. C, M4e cells plated in 12-well cell culture plates were treated with 1 μM perifosine, 10 ng/ml TRAIL, or their combination. The same treatments were repeated every 3 days. After 12 days, the plates were stained for cell colonies with crystal violet dye, and photographs of colonies taken using a digital camera. D, Four groups of mice were treated with PBS (n = 11), perifosine (n = 11), TRAIL (n = 13) and perifosine combined with TRAIL (n = 13), respectively, as described in "Materials and Methods". After about 3 weeks, the tumors were removed from the sacrificed mice and weighed. The data are means ± SD.