To determine expression of cell cycle and apoptotic genes, biochemical analysis of CCL23 and antisense cyclin D1–transfected CCL23 (CCL23AS) cells in the presence of cisplatin was performed. In addition, biochemical analysis of CAL27 cells before and after treatment with cisplatin was performed to determine expression of cell cycle genes.
CCL23, CCL23AS, and CAL27 cell lines were treated with cisplatin. Western blot analysis, fluorescence-activated cell sorting, and apoptosis assays were performed.
In vitro study of head and neck cancer cell lines CCL23, CCL23AS, and CAL27.
CCL23, CCL23AS, and CAL27 cells were treated with cisplatin.
Main Outcome Measures
Expression of p16, p21, p53, Bcl-xL, Bcl-xS, p27, DP1, MDM2, Bcl-2, c-Jun, and Jun-D were assessed using Western blot analysis.
There was increased expression of p16, p21, p53, BCLxL, and BCLxS genes with cisplatin treatment in the CCL23 and CCL23AS cells. Expression of p27, DP1, MDM2, BCL2, c-iun, and jun-D remained unaltered after treatment. There was decreased phosphorylation of Rb protein with complete absence of hyperphosphorylated Rb in the maximally sensitized antisense cyclin D1–transfected (CCL23AS) cells. Fluorescence-activated cell sorter analysis revealed a decreased G2 phase of the cell cycle and an increased proportion of apoptotic cells in the CCL23AS cell line compared with parental CCL23 cells. Cell killing also occurred in the presence of caspase-3 inhibitor. While CCL23 cells contain wild-type p53, the CAL27 cells have a point mutation in codon 193 (A→T transversion) of exon 6. However, CAL27 cells still exhibited increased expression of p21 after treatment with cisplatin.
These results, in combination with increased expression of the p53 downstream effecter p21, indicate that the cisplatin-induced cell cycle arrest operates through the p16/p53-dependent pathway, and a caspase-independent pathway may be involved. Combination treatment of head and neck squamous cell carcinoma via cell cycle inhibition and cisplatin holds promise as a potential therapy in the clinical setting.