Cell Line Characterization and Compound Evaluation in Head and Neck Cancer

UM-SCC-74A, UM-SCC-1, UM-SCC-74B, UM-SCC-38 and UM-SCC-47 were obtained from the laboratory of Dr. Thomas E. Carey at the University of Michigan [26] (link). CAL27 was obtained from ATCC (Manassas, VA). Cisplatin resistant cell line (CAL27-CisR) was generated from its parental CAL27 cell line (ATCC) as previously described [27] (link). The identity of all cell lines was authenticated by STR genotyping (AmpFlSTR Identifiler PCR Amplification Kit, Applied Biosystems, Carlsbad, CA). All the head and neck cancer cell lines were cultured in Dulbecco's modified Eagle medium (DMEM, Invitrogen, Carlsbad, CA), 10% fetal bovine serum, 1% penicillin/streptomycin and 1% non-essential amino acids (Invitrogen). H-4073 was synthesized as described previously [24] (link). The stock compound was freshly dissolved in DMSO for in vitro work. For in vivo work, H-4073 was mixed with animal feed (50 ppm dose by Harlan Tekland). Antibodies against pSTAT3 (Tyr705), pAkt (Ser473), pp38 (Thr180/Tyr182), pERK1/2 (Thr202/Tyr204) and GAPDH were obtained from Cell Signaling Technology (Danvers, MA) and pFAK (Tyr397) antibody was from Abcam (Cambridge, MA). CD31 antibody was from Dianova (Hamburg, Germany) and p21 antibody was obtained from Calbiochem (EMD Millipore, Billerica, MA).