Histamine prevents radiation-induced mesenchymal changes in breast cancer cells.

Radiotherapy is a prime option for treatment of solid tumors including breast cancer though side effects are usually present. Experimental evidence shows an increase in invasiveness of several neoplastic cell types through conventional tumor irradiation. The induction of epithelial to mesenchymal transition is proposed as an underlying cause of metastasis triggered by gamma irradiation. Experiments were conducted to investigate the role of histamine on the ionizing radiation-induced epithelial to mesenchymal transition events in breast cancer cells with different invasive phenotype. We also evaluated the potential involvement of Src phosphorylation in the migratory capability of irradiated cells upon histamine treatment. MCF-7 and MDA-MB-231 mammary tumor cells were exposed to a single dose of 2Gy of gamma radiation and five days after irradiation mesenchymal-like phenotypic changes were observed by optical microscope. The expression and subcellular localization of E-cadherin, ß-catenin, vimentin and Slug were determined by immunoblot and indirect immunofluorescence. There was a decrease in the epithelial marker E-cadherin expression and an increase in the mesenchymal marker vimentin after irradiation. E-cadherin and ß-catenin were mainly localized in cytoplasm. Slug positive nuclei, matrix metalloproteinase-2 activity and cell migration and invasion were significantly increased. In addition, a significant enhancement in Src phosphorylation/activation could be determined by immunoblot in irradiated cells. MCF-7 and MDA-MB-231 cells also received 1 or 20µM histamine during 24h previous to be irradiated. Notably, pre-treatment of breast cancer cells with 20µM histamine prevented the mesenchymal changes induced by ionizing radiation and also reduced the migratory behavior of irradiated cells decreasing phospho-Src levels. Collectively, our results suggest that histamine may block events related to epithelial to mesenchymal transition in irradiated mammary cancer cells and open a perspective for the potential use of histamine to improve radiotherapy efficacy.

Effects of oligoelements Se, Zn, and Mn plus Lachesis muta venom in experimental scleroderma.

Scleroderma, sclerosis of the skin, is a severe autoimmune disease refractant to all kind of treatments. To study the in vivo effects of a combination of three oligoelements selenium (Se), zinc (Zn), and manganese (Mn) plus Lachesis muta venom (O-LM) on the bleomycin (BLM)-induced scleroderma mouse experimental model. C3H mice were randomly divided into four groups: control (phosphate-buffered saline (PBS)), O-LM, BLM, and BLM + O-LM. All administrations were performed subcutaneously into the back of mice. BLM was injected 5 days per week for three consecutive weeks and O-LM was administered simultaneously with BLM from the beginning of the experiments and lasted for 3 weeks after the final BLM or PBS injection (for O-LM and BLM + O-LM groups), when animals were sacrificed and histopathological, immunohistochemical, thiobarbituric acid reactive species (TBARS) evaluation, and autoantibodies detection were determined. O-LM significantly reduced BLM-induced enhanced dermal thickness (605 ± 47 vs. 956 ± 59 µm, P < 0.01), collagen deposition, and mast cells infiltration (43.1 ± 1.0 vs. 102 ± 14.1 mast cells, P < 0.05). O-LM administration significantly blocked BLM-induced oxidative damage and the enhanced immunoreactive fibroblasts for α-smooth muscle actin while reduced BLM-induced autoantibodies that strongly react mainly with skin and spleen. O-LM significantly reduced BLM-induced scleroderma through the modulation of antioxidant and immunological pathways.

Antitumor activity of histamine and clozapine in a mouse experimental model of human melanoma.

BACKGROUND: Functional presence of histamine H4 receptor (H4R) was demonstrated in human melanoma cell lines and biopsies. OBJECTIVE: The purposes of this work were to investigate signal transduction pathways and biological responses triggered by the activation of H4R in human primary (WM35) and metastatic (M1/15) melanoma cell lines and to evaluate the in vivo antitumor activity of histamine (HA) and clozapine (CLZ) on human M1/15 melanoma xenografts. METHODS: Clonogenic assay, incorporation of BrdU, cell cycle distribution, phosphorylation levels of ERK1/2 and cAMP production were evaluated in vitro. An experimental human melanoma model was developed into athymic nude mice. Tumor growth, survival and histochemical studies were performed in order to investigate the expression levels of H4R, HA, PCNA, mitotic index (MI), and angiogenesis. RESULTS: The results indicate that H4R agonists inhibited forskolin-induced cAMP levels only in M1/15 cells while increased phosphorylation levels of ERK1/2 and decreased proliferation in both cell types. In vivo studies show that HA and CLZ (1mgkg(-1), sc) significantly increased median survival and decreased tumor volume. These effects were associated to a reduction in MI, in the expression of proliferation marker and in intratumoral neovascularization. CONCLUSIONS: We conclude that HA and CLZ exhibit an antitumoral effect in vitro and in vivo on human melanoma, suggesting the therapeutic potential of these compounds for the treatment of malignant melanoma.

Protection of radiation-induced damage to the hematopoietic system, small intestine and salivary glands in rats by JNJ7777120 compound, a histamine H4 ligand.

Based on previous data on the histamine radioprotective effect on highly radiosensitive tissues, in the present work we aimed at investigating the radioprotective potential of the H4R ligand, JNJ7777120, on ionizing radiation-induced injury and genotoxic damage in small intestine, salivary glands and hematopoietic tissue. For that purpose, rats were divided into 4 groups. JNJ7777120 and JNJ7777120-irradiated groups received a daily subcutaneous JNJ7777120 injection (10 mg/kg) starting 24 h before irradiation. Irradiated groups received a single dose of 5 Gy on whole-body using Cesium-137 source and were sacrificed 3 or 30 days after irradiation. Tissues were removed, fixed, stained with hematoxylin and eosin or PAS staining and histological characteristics were evaluated. Proliferative and apoptotic markers were studied by immunohistochemistry, while micronucleus assay was performed to evaluate DNA damage. Submandibular gland (SMG) function was evaluated by methacholine-induced salivation. Results indicate that JNJ7777120 treatment diminished mucosal atrophy and preserved villi and the number of crypts after radiation exposure (240±8 vs. 165±10, P<0.01). This effect was associated to a reduced apoptosis and DNA damage in intestinal crypts. JNJ7777120 reduced radiation-induced aplasia, preserving medullar components and reducing formation of micronucleus and also it accelerated bone marrow repopulation. Furthermore, it reduced micronucleus frequency in peripheral blood (27±8 vs. 149±22, in 1,000 erythrocytes, P<0.01). JNJ7777120 completely reversed radiation-induced reduced salivation, conserving glandular mass with normal histological appearance and reducing apoptosis and atrophy of SMG. JNJ7777120 exhibits radioprotective effects against radiation-induced cytotoxic and genotoxic damages in small intestine, SMG and hematopoietic tissues and, thus, could be of clinical value for patients undergoing radiotherapy.

Therapeutic potential of histamine H4 receptor agonists in triple-negative human breast cancer experimental model.

BACKGROUND AND PURPOSE: The presence of the histamine H4 receptor (H4R) was previously reported in benign and malignant lesions and cell lines derived from the human mammary gland. The aim of this work was to evaluate the effects of H4R ligands on the survival, tumour growth rate and metastatic capacity of breast cancer in an experimental model. EXPERIMENTAL APPROACH: Xenograft tumours of the highly invasive human breast cancer cell line MDA-MB-231 were established in immune deficient nude mice. The following H4R agonists were employed: histamine (5 mg kg⁻¹), clozapine (1 mg kg⁻¹) and the experimental compound JNJ28610244 (10 mg kg⁻¹). RESULTS: Data indicate that developed tumours were highly undifferentiated, expressed H4R and exhibited high levels of histamine content and proliferation marker (PCNA) while displaying low apoptosis. Mice of the untreated group displayed a median survival of 60 days and a tumour doubling time of 7.4 ± 0.6 days. A significant decrease in tumour growth evidenced by an augment of the tumour doubling time was observed in the H4R agonist groups (13.1 ± 1.2, P < 0.01 in histamine group; 15.1 ± 1.1, P < 0.001 in clozapine group; 10.8 ± 0.7, P < 0.01 in JNJ28610244 group). This effect was associated with a decrease in the PCNA expression levels, and also reduced intratumoural vessels in histamine and clozapine treated mice. Histamine significantly increased median survival (78 days; Log rank Mantel-Cox Test, P = 0.0025; Gehan-Breslow-Wilcoxon Test, P = 0.0158) and tumoural apoptosis. CONCLUSIONS AND IMPLICATIONS: Histamine through the H4R exhibits a crucial role in tumour progression. Therefore, H4R ligands offer a novel therapeutic potential as adjuvants for breast cancer treatment.

PANC-1 cells proliferative response to ionizing radiation is related to GSK-3ß phosphorylation.

Radiotherapy may be used to treat pancreatic cancer and relieve pain. We have previously reported that histamine modulates pancreatic adenocarcinoma PANC-1 cell proliferation. This work was aimed to evaluate whether histamine improves radiosensitivity of PANC-1 cells in relation to phosphorylation/inhibition of glycogen synthase kinase-3ß (GSK-3ß). Immediately after γ irradiation, intracellular hydrogen peroxide was markedly decreased together with a rapid increase in catalase activity. Although histamine diminished catalase activity in nonirradiated cells, it only partially hindered the increase observed in irradiated cells and could not modify radiosensitivity. In control cells, a high expression of total and a very low expression of phosphorylated/inactive GSK-3ß were found. An increment in reactive oxygen species levels produced an augmentation in GSK-3ß phosphorylation and suppressed cell proliferation. In both control and histamine-treated irradiated cells, the rise in catalase activity lowered reactive oxygen species levels and only a small increase in phosphorylated GSK-3ß was detected. Alternatively, 3-aminotriazole, an irreversible inhibitor of catalase, reduced the survival fraction in irradiated control cells along with an increment in phosphorylated GSK-3ß. These results suggest that upon irradiation, early catalase activation may be responsible for keeping GSK-3ß active conceding cells a survival advantage toward cytotoxic effects of ionizing radiation.

Radioprotection of sensitive rat tissues by oligoelements Se, Zn, Mn plus Lachesis muta venom.

In this study we first evaluated the general radioprotective efficacy of Se, Zn and Mn (4 µg/ml each) plus Lachesis muta venom (4 ng/ml) combination (O-LM) by determining survival on rats irradiated with lethal doses of gamma-rays. The aim of the second part of the study was to investigate the O-LM ability to prevent ionizing radiation-induced damage on small intestine, bone marrow and submandibular glands. Hence, histological characteristics and functional studies, together with proliferation and apoptotic marker levels on whole body irradiated rats with a 5 Gy dose were evaluated. Results show that all animals of the untreated group died after whole body irradiation with 8 and 10 Gy while 60 day-survival was more than 80% and 40% in O-LM-treated animals, respectively. Histopathological examinations revealed a high degree of small intestine and submandibular gland radioprotection 3 days post-irradiation. O-LM inhibited histological damage on small intestine, restoring the radiation-induced reduction in villous height and crypt number. O-LM prevented radiation-induced loss of salivary gland function and morphological alterations. These effects were associated to a complete inhibition of radiation-induced apoptosis. Furthermore, studies performed 30 days post-irradiation revealed that O-LM significantly improved bone marrow repopulation, increasing all medullar progenies to the extent of the non-irradiated animals, and completely prevented permanent submandibular gland alterations. Based on the present results and taking into account that O-LM is being safely administered in phase I clinical trial as an immunomodulator, we conclude that O-LM is a non-toxic promising approach to achieve radioprotection for patients undergoing radiotherapy.

Role of H4 receptor in histamine-mediated responses in human melanoma.

We have previously reported that histamine at micromolar concentrations reduces the proliferation of melanoma cell lines. It is also known that melanoma cells express histamine H1, H2, and H3 receptors. The aim of this study was to investigate the presence of histamine H4 receptor (H4R) in human melanoma cells and its associated biological processes. To better understand the importance of histamine in tumor development, we explored the expression of H4R in human melanoma tissue biopsies. The expression of H4R in WM35 and M1/15 cells was analyzed by reverse-transcription-PCR, western blot, and immunocytochemistry. To characterize the biological responses we evaluated cell proliferation by clonogenic assay and 5-bromo-2'-deoxyuridine incorporation. In addition, cell senescence and differentiation were determined by ß-galactosidase enzyme assay and dopa oxidase activity, respectively. The expression levels of H4R were determined by immunohistochemistry in 19 samples of human malignant lesions. Results indicate that melanoma cells express H4R at the messenger RNA and protein levels. By using histamine agonists, antagonists, and H4R small-interfering RNA we showed that the inhibitory effect of histamine on proliferation was in part mediated through the stimulation of the H4R. The decrease in proliferation was associated with an induction of cell senescence and an increase in melanogenesis, which is a differentiation marker of these cells. Furthermore, H4R was expressed in 42% of human melanoma biopsies. To our knowledge, this is the first report that describes the presence of the H4R in melanoma cells and tissue, suggesting a potential therapeutic application of H4R ligands.

Histamine prevents functional and morphological alterations of submandibular glands induced by ionising radiation.

PURPOSE: Xerostomia is a common, disturbing side-effect among patients treated with radiotherapy for head-and-neck cancer. The aim of the present work was to investigate whether histamine could prevent salivary gland dysfunction and histological alterations exerted by ionising radiation. MATERIALS AND METHODS: Forty-eight rats were divided into four groups. Histamine and histamine-5 Gy groups received a daily subcutaneous histamine injection (0.1 mg/kg) starting 24 h before irradiation. Histamine-5 Gy and untreated-5 Gy groups were irradiated with a single dose of whole-body Cesium-137 irradiation. Control and untreated-5 Gy groups were given daily saline injections. Three days post irradiation metacholine-induced salivary secretion was measured or animals were sacrificed and submandibular gland (SMG) removed, stained and histological characteristics were evaluated. Proliferation and apoptosis markers were studied by immunohistochemistry. RESULTS: Radiation decreased salivary secretion by 40% in comparison to untreated rats, which was associated with loss of SMG mass, alteration of epithelial architecture, partial loss of secretor granular material, diminished proliferation and a remarkable apoptotic response. In contrast, histamine completely reversed the reduced salivation induced by radiation, conserved glandular mass with normal appearance and preserved the structural organisation of secretor granules. Radiation-induced toxicity is prevented by histamine essentially by suppressing apoptosis of ductal and acinar cells, reducing the number of apoptotic cells per field (19.0 ± 3.8 vs. 106.0 ± 12.0 in untreated animals, P < 0.001), and also by preventing the radiation-induced decrease in cell proliferation. CONCLUSIONS: Histamine prevents morphological and functional radiation-induced damage on SMG, representing a potential radioprotector for treatment of patients undergoing radiotherapy for head and neck malignancies.

Histamine protects bone marrow against cellular damage induced by ionising radiation.

PURPOSE: Based on our previous data on the histamine radioprotective effect on small intestine, in the present work we aimed to determine whether histamine is able to protect bone marrow cells against ionising radiation damage. MATERIALS AND METHODS: 56 mice and 40 rats were divided into four groups. Histamine and histamine-irradiated groups received a daily subcutaneous histamine injection (0.1 mg/kg) starting 24 h before irradiation. Irradiated groups received a single dose on whole-body using Cesium-137 source and were sacrificed three days after irradiation. We evaluated the number of medullar components, bone marrow trophism, oedema, vascular damage, and other histological characteristics and also proliferation markers by immunohistochemistry. RESULTS: Histamine treatment substantially reduced the grade of aplasia, the oedema and vascular damage induced by ionising radiation on bone marrow of mice and rats. Additionally, histamine preserved medullar components increasing the number of megakaryocytes (14.0 +/- 1.0 vs. 7.3 +/- 1.0 in mice; and 9.9 +/- 1.3 vs. 4.1 +/- 1.0 in rats, P < 0.01) and also myeloid (253.4 +/- 37.6 vs. 7.8 +/- 1.5 in mice; and 52.0 +/- 3.7 vs. 31.8 +/- 3.1 in rats, P < 0.01), lymphoid (97.4 +/- 6.5 vs. 19.8 +/- 1.6 in mice; and 23.4 +/- 0.9 vs. 11.7 +/- 2.5 in rats, P < 0.01) and erythroid cells (165.0 +/- 9.1 vs. 8.8 +/- 2.8 in mice; and 27.3 +/- 2.3 vs. 15.6 +/- 3.5 in rats, P < 0.01) per mm(2). This effect was associated with an increased proliferation rate of bone marrow cells. CONCLUSIONS: Histamine reduces ionising radiation toxicity on bone marrow cells being a suitable candidate for use as radioprotector, especially for patients undergoing radiotherapy who are at the risk of bone marrow or small intestine damage.

Involvement of hydrogen peroxide in histamine-induced modulation of WM35 human malignant melanoma cell proliferation.

Histamine is a recognized growth factor in melanoma, and exogenous histamine produces a dual effect on proliferation. We have previously reported that histamine at micromolar concentrations reduces the proliferation of melanoma cell lines. To investigate the mechanism by which histamine inhibits proliferation of WM35 human melanoma cells, we have studied the involvement of histamine in reactive oxygen species production and antioxidant enzyme regulation in these cells. Results indicate that histamine treatment (10 microM) significantly increased hydrogen peroxide levels, whereas it slightly decreased superoxide levels associated with an enhancement of superoxide dismutase and a reduction in catalase activity. Additionally, catalase treatment reversed the inhibitory effect of histamine on proliferation, and various treatments that reduce hydrogen peroxide formation increased proliferation of these cells. Furthermore, we demonstrate that the inhibition of proliferation produced by histamine was mediated at least in part by an induction of cell senescence. We conclude that hydrogen peroxide is involved in histamine-mediated modulation of proliferation in malignant melanoma cells.

Aminoguanidine impedes human pancreatic tumor growth and metastasis development in nude mice.

AIM: To study the action of aminoguanidine on pancreatic cancer xenografts in relation to cell proliferation, apoptosis, redox status and vascularization. METHODS: Xenografts of PANC-1 cells were developed in nude mice. The animals were separated into two groups: control and aminoguanidine treated. Tumor growth, survival and appearance of metastases were determined in vivo in both groups. Tumors were excised and ex vivo histochemical studies were performed. Cell growth was assessed by Ki-67 expression. Apoptosis was studied by intratumoral expression of B cell lymphoma-2 protein (Bcl-2) family proteins and Terminal deoxynucleotidyl transferase biotin-dUTP Nick End Labeling (Tunel). Redox status was evaluated by the expression of endothelial nitric oxide synthase (eNOS), catalase, copper-zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD) and glutathione peroxidase (GPx). Finally, vascularization was determined by Massons trichromic staining, and by VEGF and CD34 expression. RESULTS: Tumor volumes after 32 d of treatment by aminoguanidine (AG) were significantly lower than in control mice (P < 0.01). Median survival of AG mice was significantly greater than control animals (P < 0.01). The appearance of both homolateral and contralateral palpable metastases was significantly delayed in AG group. Apoptotic cells, intratumoral vascularization (trichromic stain) and the expression of Ki-67, Bax, eNOS, CD34, VEGF, catalase, CuZnSOD and MnSOD were diminished in AG treated mice (P < 0.01), while the expression of Bcl-2 and GPx did not change. CONCLUSION: The antitumoral action of aminoguanidine is associated with decreased cell proliferation, reduced angiogenesis, and reduced expression of antioxidant enzymes.

Mechanisms underlying the radioprotective effect of histamine on small intestine.

PURPOSE: To examine the protective effects of histamine on intestinal damage produced by gamma-radiation. MATERIALS AND METHODS: 56 mice were divided into 4 groups. Histamine and Histamine-10 Gy groups received a daily subcutaneous histamine injection (0.1 mg/kg) starting 20 hours before irradiation and continued until the end of the experimental period; the untreated group received saline. Histamine-10 Gy and untreated-10 Gy groups were irradiated with a single dose on whole-body using Cesium-137 source (7 Gy/min) and were sacrificed 3 days after irradiation. Small intestine was removed, fixed and stained with hematoxylin and eosin. The number of intestinal crypts per circumference, and other histological characteristics of intestinal cells were evaluated. We further determined by immunohistochemistry the expression of proliferating cell nuclear antigen (PCNA), Bax, Bcl-2 (pro- and anti-apoptotic protein, respectively), antioxidant enzymes (Superoxide dismutase (SOD), Catalase and Glutathione peroxidase), histamine content and apoptosis by terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate biotin nick end labeling (TUNEL) assay. Cells in the S phase of the cell cycle were identified by immunohistochemical detection of 5-bromo-2'-deoxyuridine (BrdU) incorporation. RESULTS: Histamine treatment reduced mucosal atrophy, edema and preserved villi, crypts and nuclear and cytoplasmic characteristics of small intestine after radiation exposure. Additionally, histamine treatment increased PCNA expression and the BrdU-positive cell number, histamine content, decreased the number of apoptotic cells and significantly increased Catalase and copper-zinc-containing SOD of irradiated mice. CONCLUSIONS: Histamine prevents radiation-induced toxicity by increasing proliferation of damaged intestinal mucosa and suppressing apoptosis that was associated with an increase in SOD and Catalase levels. This effect might be of clinical value in patients undergoing radiotherapy.

Antitumoral activity of a new series of 5,6-dihydrobenzo(a)carbazoles.

The overlapping of three-dimensional structures of 5,6-dihydrobenzo(a)carbazole (DHBC) derivatives over the structure of 4-hydroxytamoxifen (4-OH-TAM), by means of the MDL CHEMLAB 11.0 computational program, shows a reasonable structural and spatial resemblance. This finding raised the hypothesis of their possible antitumoral activity, similar to that of tamoxifen (TAM). A number of DHBCs with an alkyl chain and a second basic nitrogen as substituent were synthesized in our laboratory and their possible antitumoral activity was tested by means of: 1) competitive radioligand assays to determine relative drug affinity for the estrogen receptor (ER); 2) in vivo studies, giving the synthetic drugs subcutaneously (1 mg kg-1 day-1) to Sprague-Dawley rats with N-nitroso-N-methylurea (NMU)-induced mammary tumors; and 3) in vitro cell proliferation experiments employing the soft agar clonogenic technique. Besides, studies on toxicity and histopathological analyses of organs and tumors from treated animals were performed. Results obtained showed that: 1) relative binding affinities (RBA) for the ER were similar to that of TAM; 2) some structures showed significant antitumoral activity and induced tumoral regression similar to TAM; and 3) these compounds had in vitro inhibitory effect on cell proliferation. Even though all the compounds of the series of synthesized DHBCs showed affinity for the ER similar to TAM, the results of in vivo experiments confirmed the crucial role of hydroxyl groups in the molecule and of the interatomic distance between them, similar to that of estradiol, as well as the necessary presence of the aminoalkyl chain on the annular N atom. However, the effect of alkyl chain enlargement in the nitrogen substituent on the biological activity of those drugs is as yet unclear.