A, MSTO-211H cells were exposed to cisplatin or to piroxicam and cisplatin and DNA content was analyzed by flow cytometry analysis. After 24 hours untreated (Ctrl) and treated cells (C, P/C) were collected, labeled with propidium iodide (PI) and analyzed. B, Cell viability analysis with the trypan blue. C, Apoptosis analysis by Annexin V-FITC/PI (Q1: necrosis; Q2: late apoptosis; Q3: healthy cells; Q4: early apoptosis). D, data summary of the apototic index. Data were performed on three independent experiments with comparable results. Ctrl: cells, C: cisplatin, P/C: piroxicam and cisplatin.

A and B, Apoptosis analysis by Annexin V-FITC/PI in NCI (A) and Mes1 (B) (Q1: necrosis; Q2: late apoptosis; Q3: healthy cells; Q4: early apoptosis). C and D, data summary of the apototic index. Cells were treated as described above for the MSTO-211H. Data were performed on three independent experiments with comparable results. Ctrl: cells, C: cisplatin, P/C: piroxicam and cisplatin.

Gene numbers after different time and drug treatments are shown. It is evident that only a 24 hour treatment reveals a consistent number of genes both in single piroxicam treatment and in the combined one. C: cisplatin; P: piroxicam; P/C: piroxicam and cisplatin.

p21 functional relationship with other differentially expressed genes involved in cell cycle progression detected in this work. A, Expression after 24 hours treatments with piroxicam. B, Expression after 24 hours treatments with piroxicam/cisplatin. For each gene the relationship and the expression (red up-regulated, green down-regulated) are shown. Arrows indicate the direction of the relationship.

A, Western blot analysis and relative expression level on p53 and p21 proteins after 24 hours P, C or P/C treatment in MSTO11H. The analysis reveals an increase of p53 levels after C treatment probably related to the cisplatin-induced cellular stress. Indeed p21 levels appear decreased in the P/C combined treatment. Total proteins were incubated with p21 antibody, or p53 antibody. B, Western blot analysis and relative expression level on p53 and p21 proteins in cytoplasmic and nuclear subcellular fractions. Most of the p53 protein is localized in the nucleus and there is a similar result for p21. In addition the p21 nucleus/cytoplasm ratio increases in the prolonged piroxicam pre-treatment before adding cisplatin (lanes P24h). Proteins were probed with specific cytoplasmic (tubulin) or nuclear (RCC1) antibodies to exclude fractions cross-contamination. In all the experiments, actin was used as loading control. Histograms of relative expression level refer to p53 and p21 normalized expression and derived by the analysis of three independent experiments. Statistical analysis was done as indicated in Material and Methods. -: untreated cells, P: piroxicam; C: cisplatin; P/C: piroxicam and cisplatin P24h: piroxicam and cisplatin after piroxicam pretreatment.

A, Western blot analysis and relative expression level on p21 expression after p21 siRNA transient experiment. MSTO cells transfected with control or p21 siRNA were harvested at 24 or 48 hours after transfection. Total proteins were incubated with p21 antibody, or p42 antibody as internal control. Complete silencing was detected both at 24 and 48 hours. B, Western blot analysis and relative expression level on p53 expression after 24 hours, C or P/C combined treatment of MSTO-211H p21 silenced cells. 24 hours after silencing, cells were exposed to different drugs as indicated before protein extraction. Total proteins were incubated with p21 antibody, or p53 antibody. In all the experiments, actin was used as loading control. Histograms of relative expression level refer to p53 and p21 normalized expression and derived by the analysis of three independent experiments. Statistical analysis was done as indicated in Material and Methods. Cells: untreated cells; Ctrl: cells transfected with control siRNA; C: cisplatin; P/C: piroxicam and cisplatin.

MSTO-211H cells were exposed to cisplatin or to piroxicam and cisplatin after 24 hours transfection with p21 siRNA or with the control siRNA. After transfection cells were drug-treated for additional 24 hours, then DNA content was analyzed by flow cytometry analysis with propidium iodide staining treating cells. A, p21 silenced cells show a marked apoptosis reduction after the combined treatment. B, Apoptosis was completely restored in the same experiments using control siRNA. C and D, Cell viability analysis with the trypan blue showed a reduced apoptosis only in p21 silenced cells. Data were performed on three independent experiments with comparable results. Ctrl: cells, C:cisplatin, P/C: piroxicam and cisplatin.