Fig. 4

Zinc supplementation improves the antitumor effects of low-dose ADR. a RKO cells (2x10⁵) were plated at subconfluence in 60-mm Petri dish and the day after treated with ADR (0.1 μg/ml) and ZnCl₂ (100 μM) alone or in combination for 24 h. Cell death was measured by trypan blue exclusion assay and expressed as percentage ± SD of three independent experiments performed in duplicate. Paired student’s t test was used for the statistical analysis of cell death percentages in the presence of ADR alone or in combination with ZnCl₂ (*: P < 0.01). b RKO cells were injected i.m. into the right flanks of nude mice. Drug and ZnCl₂ treatments were started when the tumors became palpable, and the size of tumors was measured twice a week and plotted as the average tumor volumes versus number of days post-treatment. Paired student’s t test was used for the statistical analysis of tumor volumes in the presence of ADR alone (black square) or in combination with ZnCl₂ (red square) (*: P < 0.01). c Percentage change of mouse body weight measured throughout the experiment. d Tumors were explanted at day 12 after treatments and used for total mRNA extraction and RT-PCR analysis of p53 target genes. 28S was used as a control for efficiency of RNA extraction and transcription. RNA samples from explanted tumors show respectively one Mock tumor, two ADR-treated tumors and two ADR/ZnCl₂-treated tumors. One representative experiment, out of two independent experiments generating similar results, is shown here. e Densitometric analysis of gene expression was plotted as expression ratio to 28S. The data represent the mean of two independent experiments ± S.D. Paired student’s t test was used for the statistical analysis of the normalized gene expression (Gene/28S ratios) at different ADR doses, with or without ZnCl₂ (*: P < 0.01)