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Conceived and designed the experiments: The tumor microenvironment encompasses several stressful conditions for cancer cells such as hypoxia, oxidative stress and pH alterations. Galectin-3, a well-studied member of the beta-galactoside-binding animal family of lectins has been implicated in multiple steps of metastasis as cell-cell and cell-ECM adhesion, promotion of angiogenesis, cell proliferation and resistance to apoptosis.

However, both its aberrantly up- and down-regulated expression was observed in several types of cancer. Thus, the mechanisms that regulate galectin-3 expression in neoplastic settings are not clear. In order to demonstrate the putative role of hypoxia in regulating galectin-3 expression in canine mammary tumors CMTin vitro and in vivo studies were performed.

In malignant CMT cells, hypoxia was observed to induce expression of galectin-3, a phenomenon that was almost completely prevented by catalase treatment of CMT-U27 cells.

Increased galectin-3 expression was confirmed at the mRNA level. Under hypoxic conditions the expression of galectin-3 shifts from a predominant nuclear location to cytoplasmic and membrane expressions.

In in vivo studies, galectin-3 was overexpressed in hypoxic areas of primary tumors and well-established metastases.

Tumor hypoxia thus up-regulates the expression of galectin-3, which may in turn increase tumor aggressiveness. Galectin-3 is a unique member of decrefo family of galectins. It is a carbohydrate-binding le which mediates cell—cell and cell—extracellular matrix ECM interactions and that has been implicated in several key steps of the cancer metastatic process [ 12 ] and drug resistance [ 34 ]. The relationship between the expression of galectin-3 and cancer behavior is controversial and the mechanisms controlling its expression remain unclear [ 56 ].

Our previous studies demonstrated that the expression of galectin-3 and galectinbinding sites is dynamic and seems to be, at least in part, microenvironment-related [ 7 ].

Altered glycan-galectin dynamics is likely to facilitate the lwi of tumor cells from primary sites and thus increase their invasive and metastatic capabilities [ 8 — 11 ]. Deceto was shown to be down-regulated in primary canine mammary carcinomas when compared to adenomas [ 12 ] suggesting a possible selective advantage for malignant growth when the level of this lectin is decreased [ 1314 ].

Despite the low expression of galectin-3 in most malignant tumor areas [ 1215 — 17 ] tumor cells surrounding necrotic areas are found to express more galectin This suggests that a hypoxic microenvironment might increase its expression [ 1218 — 20 ], which in turn might be related to lfi aggressiveness of tumor cells [ 21 ]. Galectin-3 has also been found to act as a chemo-attractant to endothelial 57707 and to stimulate neovascularization through vascular endothelial growth factor VEGF in the tumor stroma [ 22 ], thus contributing to the establishment of an escape route for metastatic cells [ 5707722 ].

Furthermore, galectin-3 confers resistance to anoikis [ 23 ] to these metastatic cells, hence contributing to their survival in the blood flow, a crucial rate-limiting step of metastasis [ 12 ]. Tumor hypoxic regions are those in which cells suffer not only from lack of oxygen but also from glucose and amino acids deprivation, high lactate concentration and oxidative 577.

In solid tumors, hypoxia is primarily a pathophysiological consequence of the high tumor growth with lagging angiogenesis, and is one of the major stress sources for both cancer and normal cells [ 182425 ]. It has been demonstrated that hypoxia-related changes are associated to poor prognosis and to increased chemo and radiotherapy resistance [ 18 ].

In accordance with the above, in breast cancer, the presence of tumor necrosis has been associated to a decreased survival rate [ 2930 ]. This may imply that galectin-3 expression is regulated both at the transcriptional and post-transcriptional levels [ 732 ].


Little is known about the mechanisms underlying these phenomena and the direct role of hypoxia remains unknown. In this study we report that galectin-3 expression is associated with tumor microenvironment hypoxia in canine mammary cancer cells.

Interestingly, hypoxia also leads to a shift in galectin-3 subcellular localization, and its effects are precluded by detoxification. Based on these results, we propose a model to reconcile the overexpression of galectin-3 in necrosis surrounding areas of canine mammary cancer lesions and suggest that this might be a critical player in metastasis.

Hypoxia Up-Regulates Galectin-3 in Mammary Tumor Progression and Metastasis

For hypoxic stimulation, the same CMT-U27 cell line was cultured for 6, 12 and 24 hours in a modulator incubator Binder with a gas mixture of 0. After that, monoclonal rat anti-galectin-3 antibody Bioscience 1: After three washes with PBS, cells were incubated with Alexa conjugated with donkey anti-rat antibody Invitrogen and Alexa conjugated with goat anti-rabbit antibody 1: Nuclei were stained with DAPI for 15 minutes.

Dcereto experiments were performed three independent times. The primary antibodies were revealed using the appropriate peroxidase-conjugated secondary antibodies 1: Actin levels were used to normalize protein amount, and quantification of western blots was performed using Gs calibrated dimensitometer Bio Rad.

These experiments were repeated 3 times. Two microliters deccreto a 1: Each sample was amplified in triplicate and specificity confirmed by dissociation 55707. The level of 18S RNA in each sample was measured and used for normalization of target gene abundance. Primer sequences are listed below. Formalin-fixed and paraffin-embedded tissue samples were deparaffinised in xylene and rehydrated in a series of alcohols. Experimental nude mice, N: Once the primary tumor xenograft attained the volume of 1 cm 3it was excised under general anesthesia.

The animals were humanely euthanized whenever their body weight started to decrease or any signs of poor body condition were shown. Antigen pei was performed with 0. After incubation with the appropriate secondary antibodies for 30 minutes at room temperature, the Avidin-Biotin Decrto Vector Labs amplification system was applied prior to detection.

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Slides were developed with DAB substrate Sigma. To identify hypoxic areas in vivowe used the Hypoxyprobe-1 Kit Chemicon International diluted 1: Slides were lightly counterstained with hematoxylin, dehydrated, and mounted using histologic mounting media, Histomount National Diagnostics.

A negative control, without primary antibody, was included. All stained sections were examined under a light microscopy by three observers de Oliveira JT. For simultaneous visualization ,ei galectin-3 with GLUT-1 on the same tissue section, a double-labeling immunofluorescence was performed. Necrotic tissue exhibiting sections were chosen by the pathologist Gartner, F. All sections were then incubated with 1: Slides were analyzed with a Zeiss fluorescence microscope.

Up-regulation of galectin-3 under hypoxia has been described in a non-neoplastic context [ 20 ].

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In order to assess a possible regulation of galectin-3 by hypoxia in neoplastic settings, we used a highly metastatic canine mammary cancer cell line, CMT-U CMT-U27 cells were cultured for 12 hours in hypoxia 0.

Galectin-3 expression was also increased in protein extracts of hypoxic CMT-U27 cells after 12 hours but not after 24 hours of 0. Despite galectin-3 may be secreted to the extracellular space in an energy-independent manner [ 36 ], our present data seem not to support any additional differences in the secretion of the lectin at different points of hypoxia exposure S2 Fig. Likewise, no differences were found in the expression of galectin-3 when CMT-U27 cells were treated with a proteasome inhibitor, either in normoxic or in hypoxic conditions S3 Fig.

Relative intensity of the indicated protein level bands were normalized to actin. Galectin-3 expression was measured by florescence intensity of each cell, representative cells correspond statistically to median fluorescence of all cells. Galectin-3 was increased in CMT-U27 cells under hypoxia 1. One of the cellular outcomes of hypoxic conditions is oxidative stress, namely the increase in reactive oxygen species ROS production. We hypothesized that the increase in galectin-3 expression in hypoxia could, at least to some extent be, due to oxidative stress in CMT.

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Double-labeling immunofluorescence analyses were performed to evaluate simultaneous expression of galectin-3 and GLUT-1 in normoxic and hypoxic catalase-treated CMT-U27 cells.

Galectin-3 and GLUT-1 presence, as evidenced by fluorescent immunostaining, was simultaneously increased in hypoxic CMT-U27 cells when compared with normoxic controls. An increased heavier form of galectin-3 was observed in hydrogen peroxide-treated cells kept under normoxic conditions.

Next, to detoxify the culture medium, catalase was used to counteract the effect of endogenous H 2 O 2under otherwise hypoxic and normoxic conditions. In hypoxic conditions, upon medium detoxification, galectin-3 expression decreased when compared with hypoxic untreated CMT-U27 cells Fig 2Bcorroborating decrrto immunofluorescence analyses. A Galectin-3 subcellular expression was assessed by double-labelling immunofluorescence.

Decrego galectin-3 cell expression shows that despite galectin-3 increase under hypoxia, in the presence of catalase this was not verified. GLUT-1, included as a well-known target of hypoxic conditions, was also not increased in the presence of catalase. B To quantitatively evaluate galectin-3 expression under different oxidative stress conditions western blot analyses were performed.

Relative intensity of the indicated protein level bands normalizes to actin were measured. In hypoxic conditions, in the presence of catalase, increased expression of galectin-3 was not observed in CMT-U27 cells.

Galectin-3 expression, decreyo kept, both in levels and quantity, similar to that seen in normoxic cells. However, in normoxia, catalase itself appears to increase the expression of galectin In normoxic conditions, hydrogen peroxide treatment induced an apparent increase of a heavier molecular weight form of galectin-3 but had no additive effect on the increase of galectin-3 under hypoxia. Galectin-3 actions rely on its subcellular location and on the phenotype of the cell itself, ranging from a pro-apoptotic function effect when present in the nucleus, to an anti-apoptotic one when in the cytoplasm [ 37 ].

To assess a putative effect of hypoxia and oxidative stress on the subcellular location of galectin-3, we analyzed galectin-3 and GLUT-1 double-labeled cells under a confocal microscope Fig 3. Untreated normoxic cells expressed galectin-3 in the cytoplasm and to a lesser extent in the nucleus. However when untreated CMT-U27 cells were exposed to hypoxia galectin-3 nuclear expression could no longer be found.

Cells treated with H 2 O 2under normal oxygen conditions, showed increased nuclear galectin-3 expression. However, when cells were exposed to both H 2 O 2 and hypoxia, nuclear galectin-3 expression was lost, leo subcellular localization being again mainly cytoplasmic. Subcellular localization of galectin-3 green color and GLUT-1 red color under normoxic and hypoxic conditions was assessed by double-labeling immunofluorescence and observed by laser scanning confocal microscopy.

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Under normoxic conditions galectin-3 had a nuclear and cytoplasmic localization. However, in hypoxic conditions galectin-3 expression was mainly cytoplasmic.

Galectin-3 localization differed in hydrogen peroxide and catalase treated cells under normoxic and hypoxic decdeto. Following hydrogen peroxide treatment in CMT-U27 cells under normal oxygen conditions, galectin-3 left the cytoplasm and was mainly localized in the nucleus.

Nevertheless, in hydrogen peroxide treated cells under hypoxia galectin-3 was mostly cytoplasmic. Catalase treated CMT-U27 cells under normoxia presented galectin-3 in specific organelles; however in catalase-treated hypoxic cells galectin-3 localization was again mostly cytoplasmic.

In fact, cells displayed higher expression levels of galectin-3 mRNA 4-fold increase upon 24 hours of hypoxia exposure when compared with normoxic controls. To further assess galectin-3 mRNA expression by cells under hypoxic conditions 0. An increase in galectin-3 mRNA was observed only upon 24 hours of hypoxia treatment. GLUT-1 green color was used as a hypoxia control.