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Mammary cancer is the world's most common malignant neoplasia for women. Recently, the mortality for breast cancer has fallen due principally to early detection and to use of neoadjuvant chemotherapy. Anthracycline antibiotics are the most commonly used anticancer drugs, and Doxorubicin (DOX) is the first choice to treat mammary cancer. Nevertheless, its use has limited by its cadiotoxic side effects, which are generated by free radicals. Our studies in methylnitrosourea (MNU)-induced mammary cancer models have shown that supplementation with moderately high doses of molecular iodine (I2) exhibits two distinctive and beneficial effects: antineoplastic and antioxidant. In the present work we explored these dual characteristic of I2 (0.05%) as an adjuvant to DOX16 (therapeutic dose of 16 mg/kg) and DOX8 (8 mg/kg) in female Sprague Dawley rats (200 g) with tumors induced by a single dose of 50 mg MNU per Kg. Iodine intake is initiated 2 days before DOX application and continues up to sacrifice 7 days later. The following parameters were registered: body weight, tumor size, tumor proliferation rate (proliferating cell nuclear antigen, PCNA), serum creatinine kinase-MB activity (CK-MB), cardiac lipoperoxidation (cLPO), and cardiac catalase activity (cCAT). Values correspond to mean± SD. Different superscripts represent significant (p≥0.05). Tumor reduction was greater in the DOX16 + I2 group than in the DOX 16 group (75% vs. 50%, respectively), and was associated with a significantly greater decrease in the rate of tumor cell proliferation (25% vs 10% decrease in PCNA-positive cells), showing the synergistic effect of I2. In contrast, at both DOX doses, the DOX + I2, groups had lower body weight loss and less cardiac damage (CK-MB; 280% vs 100%, cLPO 100% vs 0%) than with DOX alone. cCAT was not changed by any of the treatments.Conclusions: Iodine acts as an adjuvant by significantly enhancing the antiproliferative effect of DOX at a therapeutic dose (DOX16), and it exerts significant general and cardiac protective effects that do not involve catalase activation. Cellular and molecular mechanisms involved in these dual effects of iodine are currently being analyzed. The authors thank Drs. Mauricio Diaz, Marcela Lizano, and Brenda Anguiano as well as Biol. Felipe Ortiz for their technical assistance and Leonor Casanova for academic supports. We also thank Dr. Dorothy Pless for proof-reading. Supported in part by PAPIIT/UNAM 201210 and CONACYT 78955 and 47928/47928. Figure available in online version. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-10-16.

Several laboratories including ours have shown that iodine (I2) intake exhibits potent antiproliferative and apoptotic effects in vivo and in vitro models of mammary cancer. Molecular analysis of these effects indicates that antioxidant; cellular arrest and apoptotic pathways are involved. As the cellular mechanism that triggers these antineoplastic effects we have proposed that I2 could act in 2 possible ways, directly as antioxidant or indirectly by generating an iodinated arachidonic acid (6-iodoloctone) which exhibits apoptotic effects and is a specific ligand of proliferative peroxisome activated receptor gamma (PPARg). In previous preliminary reports, we corroborated an antiproliferative I2 effect (decreased PCNA) in premenopausal (pre-M) and an apoptotic action (triggering the Bax-Caspase pathway) in both pre-M and post menopausal (post-M) women with early stage breast cancer. In the present report we raise the casuistic and deeply in the analysis of mechanisms involved in this antineoplastic effect of I2. This study includes 22 women (average age 52.9 ± 12.6) diagnosed with early stage breast cancer (IIa and IIb). Patients were divided according to hormonal status into pre-M and post-M groups and were supplemented with I2 (5 mg daily) or placebo (vegetable dye) for2-5 weeks before surgery. Thyroid status (T3 and TSH; RIA) and iodine intake (urine, HPLC) were analyzed. In biopsies (beginning) and/or tumors (final), the level and location (cytosol or nuclear) of estrogen receptor alfa (ERα) and beta (ERb) were analyzed by immunohistochemistry. In tumors, we measured PPARg expression, invasive potential [hypoxia-inducible factor (HIF) and vascular endothelial growth factor (VEGF) by real time PCR] and fibrosis content (Masson's trichromic method). The results show that I2 supplementation in pre-M women is accompanied by a significant increase in the concentration of total ERα and PPARg and no change in total ERb; however, the fraction of ERα in the nucleus was significant lower, and the fibrosis content was higher (70%) than in the placebo group (40%). In contrast, I2 supplements significantly decreased HIF and VEGF expression in both pre-and post-M women. In conclusion, our data show that, in addition to the apoptotic and anti-invasive effects on mammary cancer exhibited by I2 that are independent of hormonal status, in pre-M women, I2 also acts to diminish the effects of estrogen (decreases nuclear translocation of ERα). Experiments that analyze the possible role of PPARg in these actions are in progress. We thank Dr. Jorge Alvarez-Aguirre2,3, Dr. Alonso Gallegos-Corona2, Lic. Concepción Correa-Tinajero3, Alejandro Nuñez-Nolasco2,3, Dra. Norma Uribe Uribe4, Jaime Ayala García4 and Dr. Dorothy Pless1. This work was supported by in part by PAPIIT-UNAM IN201210 and CONACyT 78955 and 85952. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-14-15.

There are multiple pieces of evidence that the sympathetic nervous system and thyroid hormones play crucial roles in normal and cancerous development of tissues. It is well established that chronic β-adrenergic stimulation (cAMP-PKA-CREB pathway) is a risk factor for progression of prostate cancer because it promotes cell migration, tumor invasion, and metastasis. Moreover, studies in human prostate cancer cells (LNCaP) show that a high dose of adrenaline induces transdifferentiation of secretor epithelium into a neuroendocrine phenotype with high invasive potential. Although studies from our group have shown a direct relation between sympathetic-input and prostate T3 generation (which seems to be associated with epithelial differentiation that occurs at puberty), there is no evidence about T3 effects on the progression of prostate cancer. Studies in hepatic cancer show that T3 administration reverses the neoplastic lesions induced by a chemical carcinogen (differentiating effects). The aim of this study was to analyze, in models of prostate cancer (in vivo and in vitro), the effects of β-adrenergic stimulation, T3, or both on tumor progression. Nude mice (nu/nu, 8 weeks old) were subcutaneously transplanted with human prostate cancer cells (LNCaP). Two days later, the mice were implanted in the back with a controlled-release pellet of isoproterenol (ISO, β-adrenergic agonist, 200 μg/day), and T3 was administrated in the drinking water (2.5 μg/day). Single (ISO or T3) or combined treatments (ISO+T3) were maintained for 6 weeks, and body weight, tumor incidence, and tumor volume were registered every week. In prostate tumor, qRT-PCR was used to analyze the expression of genes related to angiogenesis (vascular endothelial growth factor, VEGF), invasion (urokinase plasmin activator, uPA; metalloproteinase 9, MMP9), and neuroendocrine phenotype (chromogranin A, CgA). The results showed that none of the treatments affected body weight. In the ISO and ISO + T3 groups, tumors appeared in week 3, while in control and T3 groups the onset of the tumors occurred by week 5. In comparison with the control group, ISO treatment decreased tumor growth, but increased the expression of VEGF, uPA, MMP9, and CgA (2-3 fold). These effects are consistent with the well known effects of β-adrenergic stimulation on the acquisition of the neuroendocrine phenotype (slowly proliferating but highly invasive tumors). T3 administration prevents the increase of VEGF, uPA, MMP9, and CgA expression induced by ISO. Administration of T3 alone did not modify the tumoral growth or gene expression mentioned above. Migration assays in vitro of LNCaP cells showed that ISO increased cell migration, and T3 prevented the ISO effect (ISO + T3). These findings suggest that T3 might inhibit the transcription of genes dependent on β-adrenergic stimulation in prostate cancer. Current studies are analyzing if the protective effects of T3 might be associated with CREB inactivation. The authors are grateful to Guadalupe Delgado, Felipe Ortiz, and Martín García for their technical assistance. We thank Dr. Erika Rendón (Facultad de Medicina, UNAM) for her advice for the in vitro assays. Supported by CONACYT (78955, 87196, 127368), PAPIIT (IN201210). Citation Format: Evangelina Delgado-González, Carmen Aceves, Brenda Anguiano. Triiodothyronine (T3) supplementation prevents the overexpresion of invasion factors induced by β-adrenergic stimulation in prostate cancer models [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C62.

Prostate cancer is the second most frequently diagnosed cancer in men worldwide, but incidence rates vary, being lower in Japan than in the USA. This low incidence in Japanese populations has been explained by the high intake of iodine (5280 μg/day in Japan vs 209 μg in USA), among other factors. Studies in thyroid and breast cancer have shown that iodine supplementation induces apoptosis, reduces cell proliferation, and decreases the expression of cell invasion factors (VEGF, uPa, uPAR). In both tissues, iodide (I−) uptake is mediated by the Na+/I− symporter (NIS), whereas iodine (I2) uptake depends on a facilitated diffusion mechanism. Our group showed that I− or I2 supplementation of rats with prostate hyperplasia (induced with sex hormones) decreases the DNA content and has no harmful effect on normal tissue. In human prostate cancer cells (LNCaP), I− uptake is mediated via NIS, while I2 uptake is independent from NIS. Both I− and I2 treatment activated the apoptotic pathway mediated by Bax/Bcl2, and caspases, suggesting that either form of iodine might be used as an antineoplastic agent against prostate cancer. Here, we determined the uptake of I− and I2 in normal (WT) and tumoral mouse prostate, and analyzed the effect of a mixed iodide/iodine (0.5 mg I−+ 0.25 mg I2) supplement (starting at 6 weeks of age) on the progression of prostate cancer in a transgenic model (Transgenic Adenocarcinoma of Mouse Prostate, TRAMP). The TRAMP mice developed intraepithelial neoplastic lesions (PIN) and cancer around 12 and 24 weeks of age, respectively. Iodine uptake was analyzed with radioactive tracers (125I− and 125I2), which were ip injected into WT and TRAMP mice at these two ages. The radiolabel was quantified at 120 min post-injection in a gamma counter. The results showed that normal and tumoral prostates take up both chemical forms of iodine, but only I− uptake is blocked by the classic NIS inhibitor perchlorate. These data corroborate NIS participation in I− uptake and support the notion that another transporter mediates I2 uptake. Iodine supplementation had no effect on the genitourinary tract weight, histopathological acini aspect (hematoxylin-eosina stain), or proliferative rate (immunoreactivity to proliferating cell nuclear antigen) of any groups. Currently, we are analyzing the effect of iodine on tumor apoptosis and the expression of molecular factors associated with cell invasion and metastasis (epithelial-mesenchymal transition). The authors are grateful to Felipe Ortiz and Martín García for their technical assistance. Supported by CONACYT (78955, 87196, 127368), PAPIIT (IN201210). Citation Format: Paloma Olvera, Guadalupe Delgado, Carmen Aceves, Brenda Anguiano. Uptake and potential antineoplasic effects of iodine on prostate cancer in the TRAMP model [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B40.

In spite of advances in therapeutics and screening programs, breast cancer metastasis to vital organs (brain, lungs, and bone) is the main cause of death of the patients diagnosed with this disease. The process of metastasis requires a series of steps including the expression of several molecular factors that permit the invasion, angiogenesis, and colonization of other organs. Data generated in different laboratories including ours have shown that molecular iodine (I2) exerts a potent antineoplastic effect on in vitro and in vivo cancer models. Molecular analyses of this effect in methylnitrosourea-induced mammary cancer showed that, besides activating the Bax-caspase apoptotic pathway, I2 also interfered with the metastatic potential by decreasing the expression of urokinase-type plasminogen activator (uPA) and vascular endothelial growth factor (VEGF), proteins intimately related to invasion and vascularization, respectively. To determine the role of I2 in the implantation and metastatic potential of human mammary cancer, we used xenografts of two human breast cancer cell lines with different metastatic potential (MCF-7 and MDA-MB231). Female athymic homozygotic (Foxn1 nu/nu) mice (8 weeks old) were injected subcutaneously with 5 million parental cells of each cell line in 50 µl PBS and 50 µl matrigel. Drinking water for the control group was deionized water, which was supplemented with 0.025% I2 for the iodine group. All animals were monitored for 6 weeks at weekly intervals for the development of tumors. Tumor sizes were calculated by the ellipsoid formula, proliferation rate was measured by Proliferating Cell Nuclear Antigen (PCNA) immunohistochemistry, and the metastatic potential was estimated by uPA activity (enzymatic assay). The results showed that the I2 supplement decreases the implantation capacity (incidence), tumoral growth, and cell proliferation of both cell lines, MDA-MB231 (20%; 0.18 ± 0.08 cm3; 34.8% PCNA-positive cells/field) and MCF-7 (50%; 0.5 ± 0.07 cm3; 34.3% PCNA-positive cells/field) in comparison with the control (95.1%; 2.2 ± 0.3 cm3; 42.8% PCNA-positive cells/field). In contrast, I2 decreased the metastatic potential (uPA activity) only in those tumors that had been generated from MCF-7 (32 ± 5.6 vs 52 ± 2.8 IU/mg protein control). In conclusion, our data suggest that I2 supplementation interferes primarily with the implantation and proliferation of mammary cancer cells, but it may be ineffective in decreasing the metastatic potential of those cancer cells with a more aggressive phenotype. Acknowledgements to Biol. Felipe Ortíz, Dr. Laura Vega for the technical support, and Dr. Dorothy Pless for proofreading. Study partially supported by UNAM/DGAPA IN201210 and CONACYT 78955. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4224. doi:10.1158/1538-7445.AM2011-4224

Breast cancer (BC) is the most common type of cancer worldwide in reproductive-age women. Genetic (mutations in the gene BRCA), reproductive (menarche, pregnancy, and menopause), and environmental factors (lipids, alcohol, iodine, etc.) are known to be involved in BC etiology. Previous studies have demonstrated that molecular iodine (I2) has an antineoplastic effect on mammary cancer cells in vivo as well as in vitro through activation of caspase-dependant apoptotic pathways. It has also been demonstrated that this element has a powerful antioxidant effect, even greater than that of vitamin E. These dual beneficial effects of I2 were corroborated in rats with methyl-nitrosourea-induced mammary cancers treated with anthracyclines (AC) – an antineoplastic drug that also has a free radical cardiopathic secondary effect but is commonly used in humans. In combination with AC, I2 acted as an adjuvant, decreasing tumoral size and providing significant cardioprotection. In the present study we did a clinical evaluation of the additive effect of I2 (5 mg/day) or placebo (vegetable dye) with a common antineoplastic cocktail containing AC (Fluorouracil, Epirubicyn, Cyclophosphamide, Taxotere [FEC/TE], four to six cycles) in advanced BC patients (stages III and IV). Preliminary results (N=14) show a significant antineoplastic adjuvant effect of I2 when administered together with FEC/TE, and complete remission in 33 % of the cases. This result permitted conservative surgery on 3 of the 14 patients. Molecular analysis showed that in the tumoral mass of I2-supplemented patients, there was a significant decrease of the cell proliferation rate, as tested with Proliferating Cell Nuclear Antigen, PCNA immunohistochemistry (98.6% vs 80.8%), and of cyclin D1 expression (1.2 ±0.6 vs 0.3 ± 0.06; real time PCR) when compared with the control group (FEC/TE+ placebo). We also found a significant increase in the apoptosis rate (Terminal deoxynucleotidyl transferase dUTP nick end labeling; TUNEL) in the FEC/TE + I2 group (42.2 ± 4.8 vs 73.2 ± 1.8) Significantly lower serum levels of the cardiac kinase CK-MB were found in FEC/TE +I2 patients (21.5 ± 3.8 vs 8.0 ± 2.6 UI/ml) indicating a protective effect against myocardial damage. We confirmed the iodine intake of our patients by measuring the levels of this molecule in urine samples (44 ± 4.5 vs 256 ± 13 ug/dl). In conclusion, our results show a significant dual effect of iodine with FEC/TE that lead us to propose its implementation as a novel treatment for BC that could, in the future, allow lower doses of standard chemotherapy, reducing adverse effects and cost. Acknowledgements to Biol. Felipe Ortíz and Biol. Paloma Olvera for the technical support, and Dr. Dorothy Pless for proofreading. Study partially supported by UNAM/DGAPA IN201210 and CONACYT 78955 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3509. doi:10.1158/1538-7445.AM2011-3509

Abstract #3082 Breast cancer is the most frequent malignant neoplasia in women. One factor thought to influence the incidence of breast cancer is diet. Japanese women, who have a high dietary intake of iodine (5280 vs 209 µg/day in western population) due to ingestion of seaweed, have the lowest incidence of breast cancer in the world. In normal mammary gland, iodine deficiency is involved in dysplasias, which are reversible with I2 but not with iodide administration. Recent data generated in our laboratory showed that continuous treatment with I2, but not iodide, has a potent antineoplastic effect on tumoral progression in N-methyl-N-nitrosourea-treated virgin rats (50-70%). Also I2 treatment in MCF-7 human breast cancer cells, but not in normal mammary cells (MCF-12F), shows a significant apoptotic effect induced by activation of Bax-caspases and AIF-PARP-1 pathways. Based on these findings, a study of women diagnosed with cancer (mammography and tru-cut biopsy) was initiated. Patients were divided into placebo or I2 (5 mg/day) groups for 2 to 5 weeks before surgery. One day before beginning the I2 treatment and the day of the surgery, urine and blood samples were collected. The day before surgery, a second mammography was obtained. In the tumor samples proliferation (PCNA), apoptosis (TUNEL), and vasculature area (CD-31) were analyzed by immunohistochemistry. Bax, Bcl2, p53, and VEGF proteins were analyzed by Western blot. Circulating TSH and T3 levels were measured by RIA and urinary iodide by HPLC. The results showed that although tumor size did not differ between groups, a significantly lower proliferation and increased apoptotic rate were observed in tumors from women supplemented with iodine. In these tumors the ratio Bax/Bcl2 increased, p53 levels were unchanged, and VEGF levels and vasculature area diminished significantly. Serum TSH and T3 showed no changes in any group, indicating that I2 treatment had not compromised the thyroid status. We propose that I2 supplements should be considered for use in clinical trials of breast cancer therapies. We thank Dr. Jorge Álvarez-Aguirre2,3, Dr. Alonso Gallegos-Corona2, Lic. Concepción Correa-Tinajero3, Alejandro Núñez-Nolasco2,3, and Silvia Serrato-Águila2. This work was partially supported by: PAPIIT-UNAM IN201207, and CONACyT 44976-M and 85952. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3082.