Figure 2. Rigosertib reduces viability and enhances apoptosis in HNSCC cell lines.

A. Evaluating HPV status in HNSCC cell lines by PCR. Total DNA from each cell line was amplified with primers to the HPV-16 E6 gene and run on a DNA gel. B. Representative Western blot image of HPV E6 analysis in HNSCC cancer cell lines. GAPDH used as loading control. C. Cell viability as measured by MTS. FaDu, Detroit 562, UMSCC 1, UMSCC 47 and UMSCC 104 cells were treated with increasing concentrations of rigosertib for 48 h, and cell viability was assessed. 50% growth inhibition (IC₅₀) is recorded for each cell line in μM in the legend. Untreated cells were considered 100% viable and percent viability of cells treated with rigosertib was calculated vs. this control. Data represent the mean +/− SD of 3 independent experiments. D and E. Apoptosis as measured by DNA fragmentation (TUNEL). FaDu and Detroit 562 cells were treated with DMSO (Ctrl), ON 01911.Na (inactive control compound) and increasing concentrations of rigosertib for 48 h. Apoptosis was assessed by TUNEL and flow cytometry and data displayed as number of apoptotic cells/total cells. Data represent the mean +/− SD of 3 independent experiments (***p ≤ 0.001). F. FaDu cells were treated with DMSO (UN) or rigosertib with or without Z-VAD-FMK for 48 h. Apoptosis was assessed by TUNEL and flow cytometry and data displayed as number of apoptotic cells/total cells. Data represent the mean +/− SD of 3 independent experiments (***p ≤ 0.001). G. Representative fluorescent microscopic images of TUNEL assay. FaDu and Detroit 562 cells incubated with 1.0 μM ON 01911.Na (control compound) or 1.0 μM rigosertib for 48 h. before fixing, performing TUNEL assay, and capturing images. Blue = DAPI. Green = Fragmented DNA. H. Representative Western blot analysis of the effects of rigosertib on apoptotic protein cleavage. FaDu cells were incubated with DMSO (Ctrl), or increasing concentrations of ON 01911.Na (control compound) or rigosertib for 24 h before Western blot evaluation of PARP, caspase-3, and caspase-9 cleavage, and Mcl-1. GAPDH used as loading control.