Figure 5

Gefitinib reduced plasma total calcium concentrations and urinary cAMP creatinine ratios in nude mice with RWGT2 xenografts and hypercalcaemia. (A) Hypercalcaemic mice with RWGT2 xenografts (n=9) were treated daily for 3 days with gefitinib (200 mg kg⁻¹, per os (p.o.)). The first treatment occurred at the onset of hypercalcaemia. Treatment resulted in a statistically significant decrease in total plasma calcium concentrations compared to pretreatment values at all time points (6, 24, 52 and 78 h) and compared to untreated mice (n=8). Repeated measures one-way ANOVA, mean±s.e.m., ^*P<0.001. Normal nude mice (n=5) were treated daily for 3 days with gefitinib (200 mg kg⁻¹, p.o.) or untreated (n=5). Gefitinib did not decrease total plasma calcium concentrations compared to pretreatment values at all time points and compared to untreated mice. Repeated measures one-way ANOVA, mean±s.e.m. ^*P=0.001 relative to treated; ^§P=0.001 relative to baseline values. (B) Spontaneously voided urines were collected at the indicated times after the first gefitinib treatment (200 mg kg⁻¹). Cyclic AMP and creatinine concentrations were measured. The cAMP and creatinine ratio was expressed as nM mg⁻¹. Mice received additional doses of gefitinib at 24 and 48 h. Values at each time point represent the mean of two measurements from eight tumour-bearing mice from both the gefitinib-treated and untreated groups, as well as, 16 nontumour-bearing mice at baseline. Cyclic AMP levels were decreased in the gefitinib-treated mice when compared to untreated mice at all time points. Repeated measures one-way ANOVA, Mean±s.e.m., ^*P=0.02 relative to treated; ^§P=0.004 relative to baseline values. In all figures, the baseline and pretreatment groups are represented by the columns labelled base and pretreat, respectively. (C) Plasma PTHrP concentrations were measured using plasma samples collected before tumour injection (base) and at 6 and 78 h after gefitinib or placebo treatment. Experiments included nine gefitinib-treated mice and eight untreated mice. Baseline PTHrP values were from the same 17 mice. In the gefitinib-treated mice (black column), there was a mild reduction at 6 h followed by a marked reduction (50%) in plasma PTHrP concentrations when compared to untreated mice (white columns) at the respective time points. Untreated and gefitinib-treated mice with RWGT2 xenografts had PTHrP concentrations that were significantly greater than baseline at both 6 and 78 h. ^*P=0.02 relative to treated; ^§P=0.006 relative to baseline values. (D) Gefitinib treatment reduced PTHrP mRNA levels in RWGT2 tumours. RNA was extracted from tumours that were removed 78 h after hypercalcaemia was identified. The ratio of PTHrP to GAPDH mRNA was assayed by QRT-PCR. The ratio of PTHrP to GAPDH mRNA in the gefitinib-treated tumours (n=6) was decreased 60% as compared to untreated (n=6). The QRT-PCR was repeated twice with similar results. P=0.014.