Iodine and doxorubicin, a good combination for mammary cancer treatment: antineoplastic adjuvancy, chemoresistance inhibition, and cardioprotection

Abstract

Background: Although mammary cancer (MC) is the most common malignant neoplasia in women, the mortality for this cancer has decreased principally because of early detection and the use of neoadjuvant chemotherapy. Of several preparations that cause MC regression, doxorubicin (DOX) is the most active, first-line monotherapeutic. Nevertheless, its use is limited due to the rapid development of chemoresistance and to the cardiotoxicity caused by free radicals. In previous studies we have shown that supplementation with molecular iodine (I2) has a powerful antineoplastic effect in methylnitrosourea (MNU)-induced experimental models of MC. These studies also showed a consistent antioxidant effect of I2 in normal and tumoral tissues.

Methods: Here, we analyzed the effect of I2 in combination with DOX treatment in female Sprague Dawley rats with MNU-induced MC. In the first experiment (short) animals were treated with the therapeutic DOX dose (16 mg/kg) or with lower doses (8 and 4 mg/Kg), in each case with and without 0.05% I2 in drinking water. Iodine treatment began on day 0, a single dose of DOX was injected (ip) on day 2, and the analysis was carried out on day 7. In the second experiment (long) animals with and without iodine supplement were treated with one or two injections of 4 mg/kg DOX (on days 0 and 14) and were analyzed on day 56.

Results: At all DOX doses, the short I2 treatment induced adjuvant antineoplastic effects (decreased tumor size and proliferating cell nuclear antigen level) with significant protection against body weight loss and cardiotoxicity (creatine kinase MB, cardiac lipoperoxidation, and heart damage). With long-term I2, mammary tumor tissue became more sensitive to DOX, since a single injection of the lowest dose of DOX (4 mg/Kg) was enough to stop tumor progression and a second DOX4 injection on day 14 caused a significant and rapid decrease in tumor size, decreased the expression of chemoresistance markers (Bcl2 and survivin), and increased the expression of the apoptotic protein Bax and peroxisome proliferator-activated receptor type gamma.

Conclusions: The DOX-I2 combination exerts antineoplastic, chemosensitivity, and cardioprotective effects and could be a promising strategy against breast cancer progression.

Figures

Figure 1

Effect of molecular iodine(I2) and different doses of doxorubicin (DOX) on body weight and tumor size in MNU-induced rats. Animals with tumors (2–3 cm3) received single injections of DOX and 0.05% I2 treatment (drinking water; beginning 2 days before the DOX injections). Data are expressed as the mean ± SD (n = 10). * Indicates p < 0.05 compared to control or **compared with DOX group.

Figure 2

Proliferation rate. Immunohistochemical presence of PCNA-positive cells in tumors from control and DOX16-treated animals with and without iodine supplement for one week. PCNA-positive cells were revealed with diaminobenzidine (brown stain) and counterstained with hematoxylin (purple stain). Data are expressed as the mean ± SD (n = 6). Means with different letters indicate statistically significant differences (p < 0.05).

Figure 3

Short-term effect on gene expression. Animals with tumors (2–3 cm3) received a single injection of DOX16 and were treated with 0.05% I2 for one week. mRNA expression was measured by qPCR. β-actin mRNA was amplified to check for RNA quantity and integrity. The experiments were repeated three times with independent RNA samples. Values are expressed as mean ± SD. Means with different letters indicate statistically significant differences (p < 0.05).

Figure 4

Cardiac responses. CK-MB serum concentration (A), cardiac lipoperoxidation [malondialdehyde (MDA); B] and catalase activity [U* = μmoles of H2O2 consumed/min; C] were measured in MNU-treated animals injected with saline (Control) or DOX exposed or not to 0.05% I2 for one week. Values are expressed as mean ± SD. Means with different letters indicate statistically significant differences (p < 0.05). Basal group in CK-MB corresponds to control animals without MNU treatment.

Figure 5

Iodine antioxidant power. The antioxidant capacity of different chemical forms of iodine was analyzed with the ferric reducing/antioxidant power assay (FRAP). Lugol: solution of I2:KI (1:3). Ascorbic acid was used as positive control. Values are expressed as mean ± SD (n = 4).

Figure 6

Long-term effect on tumor size (% change). Animals with tumors (2–3 cm3) received one (day 0) and/or two (days 0 and 14) injections of saline (Control) or DOX4 with and without I2 supplement, and tumor size was measured at 56 days. Single injection on day 0 of DOX4 (DOX4); I2 alone (I2); DOX4 + I2 (DOX4/I2); DOX4 + second injection (day 14) of DOX4 (DOX4 + DOX4); I2 with only the day 14 injection of DOX4 (I2 + DOX4), and DOX/I2 with the second injection of DOX4 on day 14 (DOX4/I2 + DOX4). Vertical arrows indicated DOX4 injections days. * Indicates p < 0.05 compared to control, **compared with I2 and DOX4/I2 groups.

Figure 7

Long-term I2 effect on gene expression. Animals with tumors (2–3 cm3) received two injections of saline (Control) or DOX4 without (DOX4 + DOX4) or with continuous I2 supplement (DOX4/I2 + DOX4) for 56 days. mRNA expression was measured by qPCR. β-actin mRNA was amplified to check for RNA quantity and integrity. The experiments were repeated three times with independent RNA samples. Values are expressed as mean ± SD. Means with different letters are statistically significantly (p < 0.05).