Antineoplastic effect of iodine in mammary cancer: participation of 6-iodolactone (6-IL) and peroxisome proliferator-activated receptors (PPAR)

Abstract

Introduction: Studies in mammary cancer demonstrated that moderately high concentrations of molecular iodine (I2) have a antiproliferative and apoptotic effect either in vivo as in vitro, however the cellular intermediated involved in these effects has not been elucidated.

Methods: Virgin Sprague-Dawley rats were treated with methyl-nitrosourea (MNU: single dose ip, 50 mg/Kg bw) and the participation of arachidonic acid (AA) and PPAR receptors in the antineoplasic effect of I2 where analyzed.

Results: I2-treated rats for four weeks exhibited a significant reduction in the incidence (62.5 vs. 100%) and size (0.87 +/- 0.98 vs 1.96 +/- 1.5 cm3) of mammary tumors. HPLC analysis showed that tumoral but not normal mammary tissue contained an elevated basal concentration of AA and significantly more AA-iodinated called 6-iodolactone (6-IL) after chronic I2 treatment. Tumors from I2-treated rats showed fewer cells positive to proliferating cell nuclear antigen, lower blood vessel density, as well as decreases in vascular endothelial growth factor, urokinase-type plasminogen activator, and PPAR type alpha (PPARalpha). These same tumors showed increases in the cell death markers, TUNEL-positive cells (p < 0.05) and the enzyme caspase-3 (trend), as well as significant induction of PPAR type gamma (PPARgamma).

Conclusion: Together, these data demonstrate that the antineoplasic effect of iodine involves 6-IL formation and PPARgamma induction.

Figure 1

Chromatographic analyzes. Quantification by HPLC of arachidonic acid (AA) content and 6-iodolactone (6-IL) formation in normal (MG) or tumoral (Tumor) mammary glands from rats with or without molecular iodine supplements. A, representative chromatograms; a) base line, b) standards, c) MG control, d) MG +I₂, e) tumor, f) tumor + I₂. B, quantification; data are expressed as the mean ± SD (n = 10). Differences between experimental groups were analyzed using a one-way ANOVA and the Tukey-HSD Test. *indicates significant difference from the appropriate control (p < 0.05)

Figure 2

Proliferative rate. Immunohistochemical presence of PCNA-positive cells in tumors from control (A) and I₂-supplemented rats (B); PCNA-positive cells were revealed with diaminobenzidine (brown stain) and counterstained with hematoxylin (purple stain). C, quantification; D, size of tumors (Volume). Differences between experimental groups were analyzed using an unpaired t test. *indicates significant difference from the appropriate control (p < 0.05).

Figure 3

Apoptotic rate. Representative sections of TUNEL-positive cells in tumors from control (A) or I₂-treated rats (B), magnification 20×. C, Section of rat tumor treated with I₂ that shows an extensive zone of TUNEL-positive cells (white arrows), magnification 10×. TUNEL-positive cells were revealed with diaminobenzidine (brown stain) and counterstained with hematoxylin (purple stain). D, TUNEL-positive cell quantification. The difference between experimental groups was analyzed using an unpaired t test. *indicates significant difference from the control (p < 0.05)

Figure 4

Caspase-3 activity. Individual tumors from control or I₂-treated rats were assayed; horizontal line represents the mean of each group. Differences between experimental groups were analyzed using an unpaired t test. A tendency toward higher caspase-3 activity (p = 0.246) in I₂-treated tumors was observed.

Figure 5

Vascular area. Immunofluorescence blood vessels in tumors from control (A) and I₂-supplemented rats (B). Vessel area was calculated as the total field area positively stained for the vascular marker (μm²). C, quantification. Differences between experimental groups were analyzed using an unpaired t test. *indicates significant difference from the control (p < 0.05)

Figure 6

Vascular Endothelial Growth Factor (VEGF) expression. Isoforms of VEGF mRNA in normal (MG) or tumoral (tumor) mammary glands from control or I₂-treated rats were measured by the real time PCR method. A. Representative gel of amplicon of Cyclophilin (Cyc) and 120 and 164 isoforms of VEGF mRNA in 2% agarose gel stained with ethidium bromide. 1, ladder; 2, MG; 3, MG + I₂., 4, Tumor; 5, Tumor + I₂; 6, all the PCR reagents without RT sample. B. VEGF mRNA quantification. Cyclophilin (Cyc) served as internal control and was used to normalize for differences in input RNA. Data are expressed as the mean ± SD. Differences between experimental groups were analyzed using a one-way ANOVA and the Tukey-HSD Test. Means with different letters are significantly different (p < 0.05)

Figure 7

Urokinase Plasminogen Activator (uPA) expression. uPA mRNA in normal (MG) or tumoral (tumor) mammary glands from control or I₂-treated rats were measured by the real time PCR method, and Cyclophilin (Cyc) served as internal control. UD, undetectable levels. Data are expressed as the mean ± SD. Differences between experimental groups were analyzed using a one-way ANOVA and the Tukey-HSD Test. Means with different letters are significantly different (p < 0.05)

Figure 8

PPAR expression. Isoforms of PPAR mRNA in normal (MG) or tumoral (tumor) mammary glands from control or I₂-treated rats were measured by the real time PCR method. A, PPAR type alpha (PPARα). B, PPAR type gamma (PPARγ). Cyclophilin (Cyc) served as internal control and was used to normalize for differences in input RNA. Data are expressed as the mean ± SD. Differences between experimental groups were analyzed using a one-way ANOVA and the Tukey-HSD Test. Means with different letters are significantly different (p < 0.05)